• Broderick C. Jones, MD, MSc • 954 262 1327 • Offc 1327 Terry • bjones@nova.edu Lymph node with caseous necrosis Pathology What is Pathology? • Pathology • Study of Disease • Examines – • Structural • Biochemical • Functional Changes in - Cells Tissues Organs - that underlie disease • Pathology • Study of Disease • Tools • Molecular • Microbiologic • Immunologic • Morphologic – Techniques To explain the signs and symptoms manifested by patients Provides a rational basis for clinical care and therapy Pathology Clinical Medicine Basic Sciences Etiology Pathogenesis Morphology Clinical Importance Pathology General Pathology Systemic Pathology Disease Process and Clinical Manifestations Systemic Pathology Cardiovascular Hematopathology Respiratory Gastrointestinal Renal Reproductive Endocrine Dermatopathology Musculoskeletal Central Nervous System Pathology • Diagnosis of Disease • Guide Treatment of Disease • Autopsy • Tissue Examination • Clinical Laboratory Pathology • Study of Disease • Diagnosis of Disease • Guide Treatment of Disease • Autopsy • Tissue Examination • Clinical Laboratory Cell Injury Cell Injury and Death n What causes cell injury? u Injurious Stimulus Cell Response to Injury Cell Adaptations n Adaptations u Response to changes in cell environments t Reversible Changes –Size –Number –Phenotype –Metabolic activity –Functions Cell Adaptations Atrophy n Hypertrophy n Hyperplasia n Metaplasia n Intracellular Accumulations n Aging n Cell Adaptations n n JL is a 89 year old (yo) man who was seen in clinic 1 month ago with a family member with increased forgetfulness, difficulty recalling immediate events. JL fell and broke his hip and was admitted to the hospital where he developed pneumonia and expired. An autopsy was performed. Patient’s brain is shown on the following slide. Cell Adaptations n Which of the following brains is from the 89 yo man? A. A B. B Cell Adaptations n Atrophy Reduced size of an organ u Results from a decrease in cell size and number u Cell Adaptations n Atrophy - Decrease in cell size (reduction in organ and/or tissue size) - JL’s Case - Multi-infarct dementia Alzheimer Disease Parkinson Disease Cell Adaptations n Atrophy (Physiological) – reduction in size of uterus after delivery n Atrophy (Pathological) - Decrease in Cell Size - Decreased workload (atrophy of disuse) Loss of innervation (denervation atrophy) Diminished blood supply Inadequate nutrition Loss of endocrine stimulation Local pressure Denervation Autophagy (Intracellular Accumulations) Cell Adaptations n Atrophy u Mechanisms of Atrophy - Intracellular Factors - Decreased protein synthesis - Increased protein degradation – Cellular protein degradation by ubiquitinproteasome pathways • Nutrient Deficiency and Disuse – Activate ubiquitin ligases – Ubiquitin-tagged structures degraded in proteasomes Cell Adaptations n Atrophy u Cellular protein degradation by ubiquitin-proteasome pathways Cell Adaptations n Atrophy u Mechanisms of Atrophy t Autophagy • Starved cell cannabalism for survival • Undigested cell debris creates lipofuscin granules Cell Adaptations n Autophagy Cell Adaptations n Past Medical History (PMHx) u MH is a 63 year old man diagnosed with essential hypertension 30 years ago. Since that time MH has been noncompliant with medication to control blood pressure. u Recently MH reported funny feelings in chest but did not seek medical attention. MH developed ventricular fibrillation (v-fib) and expired. The patient’s heart is shown on the following slide. Cell Adaptations n What does this patient’s heart show? A. Thickened right ventricle B. Thickened left ventricle C. Left chamber dilation D. Right chamber dilation E. Normal heart Left Ventricular Hypertrophy Cell Adaptations n Hypertrophy u Enlargement of Cells t t Increase in the size of cells • Resulting in increase size of the organ Increased size of the cells • Due to synthesis of more structural components Cell Adaptations n Hypertrophy u Enlargement of Cells Physiological u Pathological u Cell Adaptations n Hypertrophy (Physiological) u Enlargement of cells t Growth Factors • Transforming growth factor (TGF-β) • Insulin-like growth factor-1 [IGF-1] • Fibroblast growth factor • Vasoactive agents – α-adrenergic agonists – Endothelin-1 – Angiotensin II Skeletal Muscle Biochemical mechanisms of myocardial hypertrophy. The major known signaling pathways and their functional effects are shown. Mechanical sensors appear to be the major triggers for physiologic hypertrophy, and agonists and growth factors may be more important in pathologic states. ANF, Atrial natriuretic factor; GATA4, transcription factor that binds to DNA sequence GATA; IGF1, insulin-like growth factor; NFAT, nuclear factor activated T cells; MEF2, myocardial enhancing factor 2. Cell Adaptations n Hypertrophy (Pathological) u Enlargement of cells t Growth Factors • TGF-β • [IGF-1] • Fibroblast growth factor • Vasoactive agents – α-adrenergic agonists – Endothelin-1 – Angiotensin II Left Ventricular Hypertrophy Normal Heart Cell Adaptations n TS is a 62 year old man who came to clinic because of difficulty urinating. Digital rectal exam (DRE) revealed an enlarged boggy prostate gland. Biopsies were collected. n Biopsies revealed increased stromal and glandular cells n Diagnosis u – Benign Prostatic Hyperplasia (BPH) Cell Adaptations n TS is a 62 year old man who came to clinic because of difficulty urinating. Digital rectal exam (DRE) revealed an enlarged boggy prostate gland. Biopsies were collected. n Biopsies revealed increased stromal and glandular cells. n Diagnosis u – Benign Prostatic Hyperplasia Cell Adaptations n Hyperplasia u Increase in Cell Numbers t Hormonal t Compensatory t t Physiological • Breast – Puberty – Pregnancy Pathological • Uterus – Endometrial hyperplasia • Skin - Psoriasis • Prostate - BPH Uterus - Endometrial Hyperplasia Normal Epithelium Psoriasis (Skin) Cell Adaptations n CK is a 35 yo cigarette smoker is concerned because she is experiencing hoarseness in her speech. She read an article in a magazine which indicated this might be an early sign of throat cancer. n A biopsy from the oropharynx was obtained and revealed a squamous poulation of cells Cell Adaptations n Metaplasia u Stem Cell Reprogramming t t Stem cells exist in normal tissues Undifferentiated mesenchymal cells present in connective tissue Reversible u Adaptive Substitution of Cells u Cell Adaptations n Metaplasia t Stimuli promote gene expression • Cells driven toward a specific differentiation pathway • Resistant cell to harsh environment t Differentiation • Cytokines • Growth factors • Extracellular matrix components Cell Adaptations n Metaplasia u u u u Columnar → squamous epithelium Reprogramming of stem cells Response to chronic irritation Resistant population of cells • Nicotene replacement therapy doubles the chance that a cigarette smoker will quit. • True • False Cell Adaptations n Intracellular Accumulations u u Metabolic derangements in a cell leading to accumulation of abnormal amounts of various substances Caused by t t t t Abnormal Metabolism Protein folding and transport Lack of an enzyme Ingestion of indigestible material Cell Adaptations n Intracellular Accumulations u Lipids u Cholesterol u Proteins u Glycogen u Pigments Cell Adaptations n MT is a 23 year-old medical student who just aced the first pathology exam. Later MT went out to celebrate with other students who aced the exam. They had a few drinks and began to wonder what happens in the liver when it metabolizes alcohol. n The affects of the alcohol began to kick in when MT decided to try and answer the question on alcohol metabolism. Which of the following is MT’s answer? A. Alcohol is really good for you B. Alcohol has gotten bad reviews C. Alcohol interferes with lipid metabolism D. Alcohol increases water absorption by the kidneys E. Alcohol is transported into the CNS by a carrier protein Cell Adaptations n Intracellular Accumulations u Result of abnormal metabolism t t t u Triglyceride accumulation ↑ Reduced NAD Defective export Example t Liver / Steatosis Cell Adaptations n Intracellular Accumulations u u Result of abnormal metabolism Example t Steatosis n RG is a 42 yo woman in clinic today because of declining exercise tolerance. She runs 2 miles everyday and works out at the gym. But over the year she began to notice she is becoming short of breath very quickly even when reducing distance running and exercise n Labs u ↑ Hematocrit ↑ Hemoglobin u ↓ α1-antitrypsin (anti-elastase) CXR u Hyperinflation n A/P CXR Hyperinflation Cell Adaptations n Intracellular Accumulations Mutations in proteins can slow folding of protein for transport Example u α-1-antitrypsin deficiency (Antiu n elastase) Cell Adaptations n Intracellular Accumulations (other examples) n n n Lipofuscin granules u In cardiac myocytes Hemosiderin granules u In liver cells Protein reabsorption droplets u In the renal tubular epithelium Cell Adaptations n Intracellular Accumulations (Pathologistical) u u u Accumulations most often are reversible Overload can cause cell injury or cell death Patient death may occur without medical intervention Cell Response to Injury Cell Injury and Death n Causes of Cell Injury u Oxygen deprivation (Hypoxia) u Physical Agents u Chemical Agents and Drugs u Infectious Agents u Immunological Reactions u Genetic Derangements u Nutritional Imbalances Cell Injury and Death n Causes of Cell Injury u u u u u u u Oxygen deprivation (Hypoxia) Physical Agents Chemical Agents and Drugs Infectious Agents Immunological Reactions Genetic Derangements Nutritional Imbalances How do these adverse stimuli injure cells? What is the mechanism? Cell Injury n Mechanisms of Cell Injury u Loss of ATP u Mitochondrial damage u Influx of Calcium u Free radical accumulation u Damage to DNA and proteins u Membrane damage Mechanisms of Cell Injury n Cascade of Events (Ischemia) u ↓ Oxidative Phosphorylation ↓ ATP t ↓ Membrane pumps • Influx – H2O Results in – - Cellular swelling – Ca+2 - Organelle swelling – Na+ • Efflux – K+ Mechanisms of Cell Injury n Cascade of Events (Ischemia) u ↑ Anerobic glycolysis t ↓ Glycogen t ↓ pH • Clumping of chromatin • Enzyme function abnormalities n Mechanisms of Cell Injury n Loss of ATP n Mechanisms of Cell Injury n Mitochondrial Damage u u Inability to generate ATP → Necrosis Membrane leakage n Mechanisms of Cell Injury n Influx of Calcium u Activates intracellular enzymes t Cellular damage SOD; Superoxide Dismutase The generation, removal, and role of reactive oxygen species (ROS) in cell injury. The production of ROS is increased by many injurious stimuli. These free radicals are removed by spontaneous decay and by specialized enzymatic systems. Excessive production or inadequate removal leads to accumulation of free radicals in cells, which may damage lipids (by peroxidation), proteins, and deoxyribonucleic acid (DNA), resulting in cell injury. n Mechanisms of Cell Injury n Membrane Damage Cell Response to Injury n Reversible Cell Injury Cell Injury n Magnitude of Cell Injury u u u Nature of Injury Cell Factors • Type • State • Adaptability Biochemical Mechanisms Determine if cellular damage is Reversible or Irreversible Cell Functions and Response to Injury n Reversible Cell Injury (Cellular Changes) u u Plasma membrane alterations t Blebbing t Blunting t Loss of microvilli Mitochondrial changes t Swelling t Appearance of small amorphous densities Cell Functions and Response to Injury n Reversible Cell Injury u Cellular swelling and ↑ vacuoles • (Hydropic change) u u u u Dilation of the ER t Detachment of polysomes Intracytoplasmic myelin Nuclear alterations Disaggregation of granular and fibrillar elements n Reversible Cell Injury u u u u Plasma membrane alterations Mitochondrial changes Dilation of the ER Nuclear alterations Cell Response to Injury n Irreversible Cell Injury Cell Functions and Response to Injury n Irreversible Cell Injury (Cellular Necrosis) u Increased eosinophilia t u Myelin figures accumulate t u u Denaturation of proteins Damaged cellular membranes Mitochondrial amorphous densities increase Nuclear changes t Karyolysis (Enzymatic degradation by endonucleases) t Pyknosis (Nuclear shrinkage and increased basophilia) t Karyorrhexis (Pyknotic nucleus undergoes fragmentation) Cell Functions and Response to Injury n Irreversible Cell Injury (Cellular Necrosis) u Viable Cardiac Cells Increased eosinophilia or u Decreased basophilia Cardiac Cells Post MI n Irreversible Cell Injury (Cellular Necrosis) u u u u Increased eosinophilia Myelin figure accumulation Mitochondrial densities increase Nuclear changes Cell Functions and Response to Injury / Irreversible Injury Clinical Correlation Blood Vessel Cell Death Extracellular Space Labs Normal Cell Cell Contents Troponin CPK-MB AST ALT n If a cell cannot adapt and becomes irreversibly injured it must _____? A. B. C. D. E. Seek help Keep trying Die or undergo necrosis Give up Recycle itself Cell Response to Stress n Cell Death u Necrosis Necrosis and Cell Death n Coagulative Necrosis Liquefactive Necrosis Caseous Necrosis Fat Necrosis Fibrinous Gangrenous n Apoptosis n n n n n Necrosis and Cell Death n Coagulative Necrosis u u u u Cause / Ischemia Architecture is preserved Affected tissues are firm Proteins and enzymes denatured Coagulative Necrosis of Cardiac Myocytes Normal Cardiac Myocytes Necrosis and Cell Death n Liquefactive Necrosis u u u Occurs in focal bacterial or, occasionally, fungal infections t Accumulation of leukocytes t Liberation of enzymes Tissue → liquid viscous mass t Digestion of dead cells Infarction of brain Liquefactive Necrosis Brain CVA ; Liquefactive Necrosis Necrosis and Cell Death n Caseous Necrosis “Caseation” Lung tissue u u u Foci of tuberculous infection “Caseous” (Cheeselike) t Friable white areas of necrosis Focus of Inflammation t Granuloma • Collection of fragmented cells • Amorphous granular debris • Enclosed within an inflammatory border Granuloma with Langerhan Giant cells (Top and Bottom) Necrosis and Cell Death n Fat Necrosis u u u Occurs with acute pancreatitis Pancreatic lipases released into peritoneal cavity t Liquefy the membranes of fat cells in the peritoneum t Lipases split the triglyceride esters t Fatty acids combine with calcium • Visible chalky-white areas (fat saponification) Result - focal areas of fat destruction t Fatty acids + calcium = fat saponification • Produce chalky-white areas Fat Necrosis involving omentum (Gross) Necrosis and Cell Death n Fibrinous Necrosis u u u u Necrosis involving blood vessels Antigen and antibody deposition in arterial walls t Immune complexes • Pro-inflammatory Fibrin leaks into vessel walls Produces a bright pink amorphous deposit in vessel walls Blood Vessel Fibrinoid Necrosis Fibrinous Necrosis Blood Vessel; Fibrinoid Necrosis Necrosis and Cell Death n Gangrenous Necrosis u u u u Clinical designation Refers to an infarcted limb involving multiple tissue planes Coagulative necrosis has occurred Superimposed bacterial infection t Can produce a liquefactive necrosis Gangrenous Necrosis Cell Response to Stress n Apoptosis Cell Death and Necrosis n Apoptosis u u u Tightly regulated suicide pathway program Elimination of cells that are no longer needed Maintains a steady number of various cell populations in tissues Cell Death and Necrosis n Apoptosis u Physiological t t t t Programmed destruction of cells during embryogenesis Involution of hormone-dependent tissues upon hormone withdrawal Cell loss in proliferating cell populations Maintains a steady number of various cell populations in tissues Cell Death and Necrosis n Apoptosis u Pathological t Eliminates cells injured beyond repair • DNA damage • Accumulation of mis-folded proteins • Cells infected by viruses Cell Death and Necrosis n Morphologic Features u Apoptosis t t t t n Cell shrinkage Chromatin condensation Formation of cytoplasmic blebs and apoptotic bodies Phagocytosis of apoptotic cells or cell bodies, by macrophages Similar to cell death due to irreversible injury Cell Death and Necrosis n Mechanism u Apoptosis t Intrinsic (Mitochondrial Pathway) • Caspases become active t Extrinsic (Death receptor–Initiated Pathway) • Caspases trigger the degradation of critical cellular components Cell Death and Necrosis n Mechanism u Apoptosis t Intrinsic / Mitochondrial Pathway Cell Death and Necrosis n Apoptosis (Mechanism) u Intrinsic / Mitochondrial Pathway 1. Lack of growth and survival signals / injury 2. Activate Bim, Bid, and Bad sensors 3. Bim, Bid, and Bad activate Bax and Bak 4. Bax and Bak insert channels into Mitochondrial leakage of cytochrome C 5. Cytochrome C activation of caspases 9 and others 6. Executioner caspases activation Cell Death and Necrosis n Apoptosis (Mechanism) u Extrinsic / Death Receptor–Initiated Pathway u TNF and Fas related proteins t t t FasL to Fas binding Cytoplasmic domains form a binding site for FADD FADD activates caspase 8 and other caspases Cell Death and Necrosis n Mechanism u Apoptosis • Extrinsic / Death receptor–initiated pathway Apoptosis n Execution Phase n Executioner Caspases u Caspases (3 and 6) t Act on many cellular components • Indirectly activate DNase – Cleavage of DNA into nucleosomesized pieces t Executioner Caspases • Degradation and fragmentation of nuclear matrix Apoptosis u Summary n Necroptosis u u u u u u u n TNF-mediated necroptosis Cross-linking of TNFR1 by TNF Recruitment of RIP1 and RIP3 Inhibition of caspase 8 RIP1 and RIP3 to initiate signals that affect mitochondrial Generation of ATP and ROS This is followed by events typical of necrosis Pyroptosis u u Cells infected by microbes Activation of caspase-1 + caspase-11 = death of the infected cell Necroptosis Apoptosis Pathologic Calcification n Abnormal Tissue Deposition u Calcium salts t Iron t Magnesium • other mineral salts u Types t Dystrophic calcification t Metastatic calcification Pathologic Calcification n Pathogenesis u Membrane Damage +2 t Ca binds to the phospholipids in membrane vesicles t Phosphate groups bind calcium +2 t Ca and phosphate binding cycle repeated +2 t Ca and phosphate groups generate microcrystals +2 t Ca deposition 97 Pathologic Calcification n Dystrophic Calcification u Pathology t Encountered in areas of necrosis t Macroscopic • Fine, white granules or clumps, gritty deposits Pathologic Calcification n Dystrophic Calcification u Pathology t Encountered in areas of necrosis t Macroscopic • White granules • Clumps • Gritty deposits Dystrophic Calcification of Aortic Valve Pathologic Calcification n Metastatic Calcification u n Results from conditions that produce hypercalcemia Principal causes of Hypercalcemia u Increased secretion of Parathyroid Hormone (PTH) t t u Destruction of Bone tissue t t u u Ectopic secretion PTH-related protein by malignant tumors Secondary to primary tumors of bone marrow (e.g., multiple myeloma, leukemia) Diffuse skeletal metastasis (e.g., breast cancer) Vitamin D Related Disorders Renal Failure t Causes retention of phosphate, leading to secondary hyperparathyroidism Pathologic Calcification n Metastatic Calcification u Occur widely throughout the Body t Affects Interstitial Tissues • Gastric mucosa • Kidneys • Lungs • Systemic arteries • Pulmonary veins t These tissues excrete acid which creates an internal alkaline compartment that predisposes them to metastatic calcification Do you want to take a quiz?? YES! Harmless Quiz n Use any of the following terms to answer the questions in the quiz – Hypertrophy – Hyperplasia – Atrophy – Dystrophic calcification – Metaplasia – Coagulative Necrosis – Liquefactive Necrosis – Gangrenous Necrosis – Caseous Necrosis – Intracellular accumulations * * Terms can be used more than once too! Case 1 n A. B. C. D. E. Which of the following best describes the condition of the heart labelled B. (A is a normal heart). Dilated cardiomyopathy Hypertrophy Metaplasia Coagulative necrosis Fibrin deposition B A C Case 2 n Patient TK underwent a complete thyroidectomy. Thyroid gland revealed an increase in number of cells (normal gland and microscopy – 1 and 2, thyroidectomy – 3 and 4. what condition does this represent? 1 2 A. Metaplasia B. Dysplasia 3 C. Hypertrophy D. Hyperplasia E. Storage disease 4 Case 3 • Unfortunately, MT continued to consume large quantities of alcohol throughout his professional career as a doctor and later succumbed to conditions related with liver failure. At autopsy the liver appeared yellow and microscopy of the liver revealed abundant lipid droplets. What does this pathology represent? A. B. C. D. E. Atrophy Liquefactive necrosis Metaplasia Fatty necrosis Intracellular accumulation Case 4 n 53 yo man with PMHx of lung cancer. Presents to clinic with chills and a low-grade fever. Patient expired from myocardial infarction while waiting to see physician. Autopsy revealed the following lesion in the upper L. lobe. Which of the following is this lesion consistent with? A. Dystrophic calcification B. Coagulative Necrosis C. Liquefactive Necrosis D. Gangrenous Necrosis E. Caseous Necrosis The Day the Earth Stood Still (1951) Thank You! CDM 1125 – Pathology I Inflammation I R. Daniel Bonfil, Ph.D. Professor and Chair of Pathology Professor of Medical Education, Dr. Kiran C. Patel College of Allopathic Medicine rbonfil@nova.edu February 24, 2021 Inflammation Protective response of the host that is essential for survival and is intended to eliminate the initial cause of cell injury (e.g., microbes, toxins), followed by removal of resultant necrotic cells and tissues. Key components include: • Cellular responses • Chemical mediators • Vascular responses Typical Inflammatory Response 2. Release of mediators (cytokines, amines) that recruit leukocytes and proteins (antibodies, complement proteins) from circulation to the site of cell/tissue injury (e.g., microbes, toxins; necrotic tissue). 3. Vasodilation and increased vascular permeability that leads to edema. 4. Activation of recruited leukocytes and molecules that destroy and eliminate the offending substance. 5. Cytokines released by leukocytes attract fibroblasts that deposit ECM and repair the damaged tissue. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 1. Recognition of the offending agent located in extravascular tissues by resident cells and molecules. Two Types of Inflammation FEATURE Onset Duration Main Characteristics Nature of the Response Tissue Injury Local and systemic signs ACUTE Immediate/rapid: minutes to hours Shorter: hours to days Neutrophil (= PMN) infiltrate CHRONIC Insidious/delayed: after acute inflammation (if not resolved) or may arise de novo Longer: up to months or years Monocyte/macrophage and lymphocyte infiltrate Accumulation of fluid and plasma proteins (edema) at the affected site “Physiological” (in very general terms is good) Usually mild and selflimited Prominent “Pathological” (in very general terms is bad, causing disease) Often severe and progressive: angiogenesis and fibrosis Less prominent; may be subtle Inflammatory Reactions as the Cause of Disease Inflammation is normally protective, but some disorders may result from damage caused by inflammatory reactions. Disorders Acute Inflammation Response Cells and Molecules Involved in Injury Acute respiratory distress syndrome - ARDS Neutrophils Asthma Eosinophils; IgE antibodies Antibodies and complement; neutrophils, monocytes Cytokines Glomerulonephritis Septic shock Chronic Inflammatory Response Arthritis Asthma Atherosclerosis Pulmonary fibrosis Lymphocytes, macrophages; antibodies? Eosinophils; IgE antibodies Macrophages; lymphocytes Macrophages; fibroblasts Modified from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Five Cardinal Signs of Inflammation Swelling and reddening of the leg in a patient with cellulitis The external / clinical manifestations of inflammation, are: 1. Redness (rubor*) 2. Heat (calor*) 3. Swelling (tumor*) 4. Pain (dolor*) Described more than 2000 years ago in De Medicina by Celsus (a Roman encyclopedist) 5. Loss of function (functio laesa*) (Described by Rudolph Virchow – “father of modern pathology”– in the late 19th century) * Latin words Herrington CS. Muir’s Textbook of Pathology, 15th ed. © 2014 CRC Press by Taylor & Francis Group, LLC. Five Cardinal Signs of Inflammation (cont.) 1. Redness (rubor*) 2. Heat (calor*) Vasodilation and its resulting increased blood flow are the cause of redness (erythema) and heat at the site of inflammation 3. Swelling (tumor*) Due to increased permeability of the microvasculature, with outpouring of protein-rich fluid (exudate) into extravascular spaces, which produces fluid accumulation (edema) that manifests itself as swelling 4. Pain (dolor*) Caused by nervous stimulation and swelling. Some of the released mediators such as bradykinin increase incites pain. 5. Loss of function (functio laesa*) * Latin words May result from pain that inhibits mobility or from severe swelling that prevents movement in the area. Stimuli Causing Inflammatory Reactions • Infective agents. Bacteria, viruses, fungi, parasites, or toxins released by them. Most common and medically important cause of inflammation. From mild acute inflammation to severe systemic reactions, or prolonged chronic inflammation. • Tissue necrosis. Due to ischemia (reduced blood flow), trauma, physical and chemical injury (e.g., burns, frostbite, exposure to acids). Triggered by different molecules released by necrotic cells. • Foreign bodies. Splinters, sutures, dirt that cause traumatic tissue injury or carry microbes. Deposits of large amount of endogenous substances, such as urate or cholesterol crystals (in gout and atherosclerosis, respectively) cause harmful inflammation. • Immune reactions. Excessive immune response against self antigens (autoimmune diseases) or environmental antigens (allergies), which tend to be persistent and often associated with chronic inflammation. The Five Rs (Steps) of Inflammation 1. Recognition of Injurious Agent (microbes and necrotic cells) 2. Recruitment of Leukocytes 3. Removal of Injurious Agent 4. Regulation of Response (Control) 5. Resolution (Repair) Initiation of Inflammation: Recognition of Microbes and Damaged Cells The first step in inflammatory responses involves the recognition of: • Motifs common to many microbes: PAMPs (pathogen-associated molecular patterns) • Molecules shed or altered in injured/stressed host’s cells: DAMPs (damage-associated molecular patterns) Pattern recognition receptors (PRRs) for PAMPs and DAMPs include: a. Toll-Like Receptors (TLRs) b. NOD*-like Receptors an the Inflammasome *nucleotide-binding oligomerization domain Initiation of Inflammation: Recognition of Microbes and Damaged Cells (cont.) Phagocytes, dendritic cells, and many types of epithelial cells, express TLRs. a. TLRs (Toll like receptors) Plasma membrane TLRs Endosomal TLRs Surface moieties of extracellular microbes (e.g., LPS) DNA/RNA from digested viruses/bacteria Other innate immune cytosolic receptors: NOD-like receptors (NLRs) for bacterial peptidoglycan; products of injured cells Activation of Transcription Factors RIG*-like receptors for viral RNA ↑ pro-inflammatory cytokines ↑ antiviral cytokines (e.g., IFN) ↑ membrane proteins that promote lymphocyte activation *retinoic acid-inducible gene Kumar V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed © 2018 by Elsevier Inc. Initiation of Inflammation: Recognition of Microbes and Damaged Cells (cont.) b. NOD-like receptors and Inflammasome Inflammasome: complex formed by multiple copies of NLRP3, an adapter, and the pro-enzyme caspasea, which becomes activated. Active caspase cleaves the precursor form of interleukin-1β (Pro-IL-1β) into its biologically active form (IL-1βb). The inflammasome is implicated in inflammatory reactions to urate crystals (cause of gout), cholesterol crystals (in atherosclerosis), lipids (metabolic syndrome and obesity-associated diabetes), and amyloid deposits in the brain (Alzheimer disease). a Play essential roles in apoptosis. b Mediator of inflammation that recruits leukocytes and induces fever. Kumar V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed© 2018 by Elsevier Inc. NLRP3 (NOD-like receptor protein 3): intracellular sensor that recognizes PAMPs (products of bacteria) and DAMPs (products of necrotic host cells, such as released ATP, uric acid and cholesterol crystals, and reduced cytosolic K+ ion). ACUTE INFLAMMATION Once resident phagocytes and other sentinel cells recognize PAMPs or DAMPs resulting from an injurious agent (initiation of inflammation), these cells release soluble mediators (cytokines, histamines). Some of these soluble mediators lead to: 1. Vascular Events 2. Cellular Events Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation 1. Vascular Events • Begin early after the recognition of the injurious agent • Help to enhance the movement of leukocytes to the site of injury or infection • Have two main components: a. Changes in Vascular Caliber and Flow b. Increased Vascular Permeability Acute Inflammation 1. Vascular Events a.Changes in Vascular Caliber and Flow • Usually, transient vasoconstriction of arterioles (seconds), due to a neurogenic reflex. • Vasodilation of arterioles mainly caused by relaxation of vascular smooth muscle cells by different cell mediators: histamine, nitric oxide (NO), and prostacyclin (PGI2), followed by opening of microcirculation (venules and capillaries) in the area. • Vasodilation results in increased blood volume in the microvascular bed of the area → heat and redness (erythema) at the inflamed area. 1. Vascular Events Acute Inflammation a.Changes in Vascular Caliber and Flow (cont.) Hydrostatic pressure: blood pressure tends to drive fluid out = Colloidal osmotic pressure: interstitial pressure opposes fluid movement out No net fluid or protein leakage Extravascular Fluid Collection (edema) Progressive vasodilation ↑ permeability of microvasculature, resulting in outpouring of protein-rich fluid (exudate) ↑ [RBCs] and viscosity, resulting in stasis of If purulent exudate, rich in leukocytes (mainly PMNs), debris of dead cells, and often microbes. Exudates (a type of edema) have inflammatory origin. Fluid that accumulates in interstitium, with high protein concentration (>1.5 g/dl), has a specific gravity >1.012, and may contain leukocytes and RBCs (cloudy fluid). Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. blood flow and vascular congestion (externally evidenced as erythema). Leukocytes (mainly neutrophils) begin to accumulate along the vascular endothelial surface (margination). Acute Inflammation 1. Vascular Events (cont.) Increased hydrostatic pressure or decreased osmotic (oncotic) pressure cause accumulation of fluid in the interstitial tissue (edema). However, the edema is caused by a transudate, which is not inflammatory in origin. Increased hydrostatic pressure Decreased osmotic pressure Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Transudates are low in protein content, with few or no cells, clear yellow, with protein content <1.5g/dl, and a specific gravity <1.012. 1. Vascular Events Acute Inflammation b. Increased Vascular Permeability Mechanisms of increased vascular permeability (aka vascular leakage): • Endothelial Cell Retraction: • Endothelial Injury: • Transcytosis: ▪ ↑ endothelial gaps mainly in postcapillary venules ▪ Elicited by histamine, bradykinin, leukotrienes ▪ Immediate (within 15-30 min); short-lived response ▪ endothelial cell (EC) necrosis and detachment ▪ Direct damage by severe injuries (e.g., burns, bacteria that target ECs). Activated leukocytes adhered to the site may release toxic mediators that amplify the reaction ▪ Delayed (2-12 hs) and prolonged response (several hours/days), until the damaged vessels are thrombosed or repaired ▪ Appears to be due to opening of intracellular channels that respond to factors that promote vascular leakage (e.g., vascular endothelial growth factor [VEGF], earlier called vascular permeability factor) ▪ Documented in animals, but its role in acute inflammation in humans still unclear Figures adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation 2. Cellular Events • Leukocytes are the major cellular participants in inflammation; those capable of phagocytosis (neutrophils and macrophages [Mφs]) are the main players. • Neutrophils are rapid responders but short-lived in tissues (1-2 days), while Mφs are slow responders but long-lived in tissues (days or weeks for inflammatory Mφs or years for tissue-resident Mφs). • The cellular phase of acute inflammations consists of two main processes: a. Leukocyte Recruitment b. Phagocytosis and Clearance of Offending Agent Acute Inflammation 2. Cellular Events a. Leukocyte Recruitment Leukocyte migration from the blood vessel lumen to the tissue consists of 4 phases: In the lumen i. Margination & Rolling ii. Firm Adhesion to endothelium iii. Migration across the endothelium (diapedesis) iv. Migration into interstitial tissues Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Acute Inflammation a. Leukocyte Recruitment (cont.) i. Margination & Rolling: laminar blood flow from capillaries into postcapillary venules results in faster movement of smaller (RBCs) than larger (WBCs) blood cells. Thus, RBCs are confined to a central column and push WBCs to the periphery of the venule lumen ( “margination”). Leukocytes get closer to endothelial cells (ECs). Cytokines (TNF, IL-1) secreted in response to microbes/injurious agents together with other mediators (e.g., histamine) “activate” ECs. Activated ECs express the cell adhesion molecules (CAMs) selectins that mediate a weak and transient interaction with cell surface molecules expressed by leukocytes, causing them to “roll” (bind-detach, bind-detach) along the endothelium. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events • Selectins contain a chain of transmembrane glycoproteins with an extracellular lectin-binding domain. • Three members : E-selectin (on ECs), P-selectin (on platelets and ECs), and L-selectin (on most leukocytes at the tip of their microvilli). Molecule Distribution L-selectin (CD62L) Neutrophils, monocytes T cells (naïve and central memory) B cells (naïve) E-selectin (CD62E) ECs activated by cytokines (TNF, IL-1) Ligand Sialyl-Lewis X/PNAd on GlyCAM-1, CD34, MAdCAM1, others; expressed on endothelium (HEV) Sialyl-Lewis X (e.g., CLA) on glycoproteins; expressed on neutrophils, monocytes, T cells (effector, memory) P-selectin (CD62P) ECs activated by cytokines (TNF, IL-1), Sialyl-Lewis X on PSGL-1 and other glycoproteins; histamine, or thrombin; platelets. In expressed on neutrophils, monocytes, T cells inactivated ECs, is found in (effector, memory) intracellular Weibel-Palade bodies, which also synthesize vWF. CLA, Cutaneous lymphocyte antigen-1; EC, endothelial cell; GlyCAM-1, glycan-bearing cell adhesion molecule-1; HEV, high endothelial venule; P-selectin glycoprotein ligand1; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule; vWF, von Willebrand factor. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation a. Leukocyte Recruitment (cont.) 2. Cellular Events Acute Inflammation a. Leukocyte Recruitment (cont.) ii. Firm Adhesion to Endothelium Integrins are transmembrane glycoproteins with α and β chains that mediate adhesion of leukocytes to ECs. Integrins are normally expressed in a low affinity form on leukocyte plasma membranes. When rolling WBCs encounter chemokines secreted by different cells at the site of inflammation, they become activated and their integrins undergo a conformational change that convert them in a high-affinity form. Cytokines - mainly TNF and IL-1 - activate ECs that increase their expression of ligands for integrins. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Binding of high affinity integrins on the leukocytes to their ligands expressed on ECs → signals that result in cytoskeletal changes that arrest leukocytes and firmly attached them to the endothelium. Family Molecule Integrin LFA-1 (CD11aCD18) MAC-1 (CD11bCD18) VLA-4 (CD49aCD29) α4β7 (CD49DCD29) Distribution Neutrophils, monocytes, T cells (naïve, effector, memory) Ligand ICAM-1 (CD54), ICAM-2 (CD102); expressed on endothelium (upregulated on activated endothelium) Monocytes, DCs ICAM-1 (CD54), ICAM-2 (CD102); expressed on endothelium (upregulated on activated endothelium) Monocytes VCAM-1 (CD106); expressed on endothelium T cells (naïve, effector, memory) (upregulated on activated endothelium) Monocytes VCAM-1 (CD106), MAdCAM-1; expressed on T cells (gut homing naïve effector, endothelium in gut and gut-associated lymphoid memory) tissues CLA, Cutaneous lymphocyte antigen-1; GlyCAM-1, glycan-bearing cell adhesion molecule-1; HEV, high endothelial venule; ICAM, intercellular adhesion molecule; Ig, immunoglobulin; IL-1, interleukin-1; MAdCAM-1, mucosal adhesion cell adhesion molecule-1; PSGL-1, P-selectin glycoprotein ligand-1; TNF, tumor necrosis factor; VCAM, vascular cell adhesion molecule. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation a. Leukocyte Recruitment (cont.) 2. Cellular Events iii. Migration across the endothelium (diapedesis) Leukocytes extravasate by squeezing between ECs at intercellular junctions. This migration across the endothelium is mediated by homotypic interactions of PECAM-1 (aka CD31) found on neutrophils and ECs. Leukocytes secrete collagenases enable them to degrade and traverse the subendothelial basement membrane to enter the extravascular tissue. EC Subendothelial basement membrane PECAM-1 : platelet endothelial cell adhesion molecule 1 of the Immunoglobulin (Ig) family Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation a. Leukocyte Recruitment (cont.) 2. Cellular Events Acute Inflammation a. Leukocyte Recruitment (cont.) iv. Migration into interstitial tissues (Chemotaxis) Leukocytes move in the extravascular space toward the site of injury or infection along a gradient of chemoattractants (process know as chemotaxis). Substances produced by microbes and by the host in response to the injurious agent that act as chemoattractants for leukocytes: • Bacterial products – mainly peptides with N-formylmethionine termini. • Cytokines – specially of the chemokine family. • Components of the complement system – particularly C5a. • Products of the lipoxygenase pathway of arachidonic acid (AA) metabolism – particularly leukotriene B4 (LTB4). 2. Cellular Events iv. Migration into interstitial tissues (Chemotaxis) (cont.) Chemotactic molecules bind to specific cell Gcoupled surface receptors → signals that induce polymerization of actin at the leading edge of PMNs and localization of myosin filaments at the back. Kumar, V, Abbas AK, Fausto N, Aster JC. Robbins & Cotran Pathologic Basis of Disease. 8th ed., © 2010 by Saunders, and imprint of Elsevier Inc. Acute Inflammation a. Leukocyte Recruitment (cont.) Leukocytes move by extending filopodia that pull the back of the cell (trailing edge) in the direction of the extension in response to the chemoattractant. Scanning electron micrograph of a moving leukocyte in culture. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Acute Inflammation a. Leukocyte Recruitment (cont.) In general, the type of leukocyte recruited depends on the age of the inflammatory response and the type of stimulus. In most forms of acute inflammation: First 6 to 24 hours Gradually replace PMNs, peak ~2 days after inflammation begins Inflammatory reaction in myocardium after infarction (ischemic necrosis) EARLY (6-24 hs) Neutrophils Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. LATER (24-48 hs) Macrophage Acute Inflammation 2. Cellular Events The cellular phase of acute inflammations consists of two main processes: a. Leukocyte Recruitment b. Phagocytosis and Clearance of Offending Agent 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent Leukocyte Activation Once recruited to the site of infection or injury, neutrophils and monocytes are activated to perform their functions. Their activation is mediated by binding of different factors (e.g., bacterial or apoptotic molecules) to various types of leukocyte cell surface receptors. Once the ligands bind to the receptors, cellular responses that mediate the functions of the leukocytes are initiated. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Phagocytosis of microbes by leukocytes involves 3 sequential steps: 1.Recognition and attachment of the particle to be ingested by the leukocyte 2.Engulfment, followed by formation of phagocytic vacuole 3.Degradation of the ingested material by lysosomal enzymes in phagolysosome Leukocyte Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Acute Inflammation b. Phagocytosis and Clearance of Offending Agent 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) 1. Recognition and attachment of the particle to be ingested by the leukocyte Some of the leukocyte receptors recognize molecules found on microbial walls only (mannose receptors), while others bind low-density lipoprotein (LDL) particles and microbes (scavenger receptors). Often, specific host proteins (called opsonins) coat microbes and target them for phagocytosis (opsonization). Main opsonins for which leukocyte have receptors include: • Fc fragment of IgG antibodies • C3b breakdown product of complement activation • Lectins (carbohydrate-binding proteins in plasma) Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) 2. Engulfment, followed by formation of phagocytic vacuole The particle bound to phagocyte receptors is surrounded by extensions of the leukocyte cytoplasm (pseudopods), and the plasma membrane pinches off to form a cytosolic vesicle (phagosome) that encloses the particle. 3. Degradation of the ingested material by lysosomal enzymes in phagolysosome The phagosome fuses with lysosomes (phagolysosome), and the lysosomal enzymes degrade the particle. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) Intracellular Destruction of phagocytosed material The elimination of infectious agents’ and necrotic cells’ materials ingested by leukocytes is accomplished by: i. Reactive oxygen species (ROS, also called reactive oxygen intermediates) II. Reactive nitrogen species, mainly derived from nitric oxide (NO) iii. Granule Enzymes and other Proteins 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) Intracellular Destruction of phagocytosed material (cont.) i. Reactive oxygen species (ROS; e.g., O2- , H2O2 , HOCl, and OH) → attack and damage lipids, proteins and nucleic acids of microbes and host cells. Myeloperoxidase (MPO-dependent killing Superoxide MPO (in azurophilic dismutase granules of neutrophils) NADPH + Cl- + H+ HOCl + H20 oxidase NADP (oxidized) NADPH (reduced) (hypochlorous acid) 2 O-2 H2O2 + + 2H (superoxide ion) Within the phagosome Mature macrophages lack MPO Fe3+ + HO. + OH(hydroxyl radical) (hypoxyl ion) (Fenton reaction) + O2- 2 O2 Membrane of the phagosome + Fe2+ O2 + HO.+ OH(Haber-Weiss reaction) MPO-independent killing NADPH, nicotinamide adenine dinucleotide phosphate; H2O2 , hydrogen peroxide (concentration not high enough to kill bacteria); MPO, myeloperoxidase; Cl-, chloride ion; HOCl, hypochlorous acid (bactericide); HO., hydroxyl radical). 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) Intracellular Destruction of phagocytosed material (cont.) ii. Reactive nitrogen species Nitric oxide (NO) is a soluble gas derived from arginine by action of nitric oxide synthase (NOS). Inducible NOS (iNOS) is expressed by macrophages when activated by cytokines (e.g., IFN-γ ) or microbial products → ↑NO: . -. NO + O2 ONOO (superoxide ion) Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. (peroxynitrite) Peroxynitrite is a highly reactive free radical that attack and damage lipids, proteins and nucleic acids of microbes and host cells. 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) Intracellular Destruction of phagocytosed material (cont.) iii. Granule Enzymes and other Proteins Larger azurophil (or 1ary) granules Neutrophils contain Smaller specific (or 2ary) granules MPO Bactericidal factors (e.g., defensins) Acid hydrolases Neutral proteases(e.g., elastase, cathepsin G) Lysozyme Collagenases Lactoferrin Plasminogen activator Phagocytic vesicles with engulfed material can fuse with these granules packed with enzymes and other products, resulting in intracellular destruction of infectious agents’ and necrotic cells’ materials ingested by neutrophils. 1ary and 2ary granules can also undergo exocytosis (degranulation), releasing their content in the extracellular space (e.g., degradation of ECM components). Normally, anti-proteases (e.g., α1-anti-trypsin) in serum and tissue fluids control the activity of these granules to avoid harmful effects in the host. 2. Cellular Events Acute Inflammation b. Phagocytosis and Clearance of Offending Agent (cont.) Extracellular Destruction of material (independent of phagocytosis) In response to infectious pathogens (e.g., bacteria, fungi) or inflammatory mediators (e.g., chemokines, C’ proteins), neutrophils can produce Neutrophil extracelullar traps (NETs). NETs are the result of neutrophils’ Healthy Neutrophils SEM of staphylococci nuclei that are lost by a distinctive trapped in NETs form of neutrophil death (NETosis). NETs are viscous networks of histones and associated DNA that provide high concentration of antimicrobial substances and prevent spreading of some infectious pathogens. May be the cause of autoreactivity against own DNA (e.g., systemic autoimmune disease such as systemic lupus erythematosus, SLE). NETosis NET Nuclei lost Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Damaging of Normal Tissues Due to Inflammation In some circumstances, mechanisms that function to eliminate microbes and dead cells (physiologic role of inflammation) can turn against normal tissues: • Normal defense reactions against infectious microbes may result in collateral damage in tissues at or near the site of infection suffer (e.g., in tuberculosis and viral hepatitis: a prolonged host response contributes more to the pathology than does the microbe itself). • Inflammatory responses directed against host tissues (e.g., autoimmune diseases). • Hyperreactivity against usually harmless environmental substances (e.g., allergic diseases). Leukocyte-mediated Injury of Normal Tissues Leukocytes can damage normal tissues by releasing the potential toxic contents of granules into the extracellular milieu in different situations: • Frustrated phagocytosis: cells encounter materials that cannot be easily ingested, such as immune complexes deposited on immovable surfaces (e.g., glomerular basement membrane). The unsuccessful attempt to phagocytose these substances triggers a strong leukocyte activation, and lysosomal enzyme release into the surrounding tissue or lumen. • The membrane of the phagolysosome may be damaged if potentially injurious substances, such as urate and silica crystals, are phagocytosed. • Regurgitation during phagocytosis: The phagocytic vacuole may remain transiently open to the outside before complete closure of the phagolysosome. Clinical Examples of Leukocyte-Induced Injury Disorders ACUTE Acute respiratory distress syndrome Acute transplant rejection Asthma Glomerulonephritis Septic shock Lung abscess CHRONIC Rheumatoid Arthritis Asthma Atherosclerosis Chronic transplant rejection Pulmonary fibrosis Cells and Molecules Involved in Injury Neutrophils Lymphocytes; antibodies and complement Eosinophils (major basic protein); Ig E antibodies Antibodies and complement; Neutrophils, monocytes Cytokines Neutrophils (and bacteria) Lymphocytes, macrophages; antibodies? Eosinophils; IgE antibodies Macrophages; lymphocytes? Lymphocytes, macrophages; cytokines Macrophages; fibroblasts These diseases will be discussed in Systemic Pathology Lectures. Defects in Leukocyte Functions Examples of Conditions Associated with Defective Leukocyte Functions: • ACQUIRED (Most common) - Diabetes, Thermal Injury, Malignancies, Sepsis and Immunodeficiencies Defective chemotaxis - Leukemia, anemia, neonates, malnutrition Defective phagocytosis & microbicidal activity - Bone marrow suppression caused by tumors, chemotherapy, radiation • GENETIC (rare) - Chediak-Higashi Syndrome - Myeloperoxidase deficiency - Chronic granulomatous disease of childhood Decreased production of lymphocytes Disorder in which fusion of lysosomes to phagosomes is prevented → no phagolysosomes Absent MPO-H2O2 system X-linked/autosomal recessive disease characterized by absence of NADPH oxidase CDM 1125 – Pathology I Inflammation II R. Daniel Bonfil, Ph.D. Professor and Chair of Pathology Professor of Medical Education, Dr. Kiran C. Patel College of Allopathic Medicine rbonfil@nova.edu February 25, 2021 Mediators of Inflammation: General Features • Substances that initiate and mediate inflammatory responses. • These mediators can be: ▪ ▪ Cell-derived: released by cells at the site of inflammation (local) Plasma-derived: usually synthesized in the liver and found in plasma as inactive precursors that are activated at the site of inflammation • Most: a) exert their biological property by binding to specific receptors on target cells (others direct activity) b) rather short lived Mediators of Inflammations: Some Examples CELL-DERIVED stored in intracellular granules (secreted locally) or synthesized de novo in response to a stimulus. Preformed in secretory granules MEDIATOR SOURCE MAIN ACTION Histamine Mast cells, basophils, platelets Vasodilation, ↑ Venular permeability Prostaglandins Leukocytes, mast cells Synthesized de novo PlateletActivating factor Leukocytes, mast cells Cytokines Macrophages, ECS, mast cells, leukocytes Vasodilation, pain, fever Vasodilation, ↑ Venular permeability EC activation, leukocyte recruitment and activation; fever PLASMA-DERIVED circulating precursor forms that undergo proteolytic cleavage to become biologically active. Complement Anaphylatoxins System (C3a, C5a) Membrane attack Complex (C5b-9) Coagulation/ Fibrin split products Fibrinolysis system Leukocyte chemotaxis and activation Kinin System kinin/bradykinin Increased vascular permeability; pain Direct target killing Increased vascular permeability, affect clotting (promotes or limits it) Chemical Mediators of Inflammation I. Cell-derived Mediators II. Plasma-derived Mediators Chemical Mediators of Inflammation I. Cell-derived Mediators 1. 2. 3. 4. 5. II. Plasma-derived Mediators Vasoactive Amines Arachidonic Acid Metabolites Cytokines Platelet-activating Factor Nitric Oxide I. Cell-derived Mediators 1. Vasoactive Amines The following two are preformed mediators stored in secretory granules, with an important role as first mediators released in acute inflammation: • Histamine ▪ Released mainly from mast cells in connective tissue adjacent to blood vessels, in response to various stimuli: a) Heat, cold, trauma b) Binding of antibodies to mast cells (allergic reactions) c) Anaphylatoxins (products of complement C3a and C5a) ▪ Causes dilation of arterioles, increases permeability of venules by inducing venular endothelial retraction, and causes EC activation. • Serotonin (aka 5-hydroxytryptamine or 5-HT) ▪ Present in platelets and released during platelet aggregation. It is also present in neuroendocrine cells of the GI tract, spleen and nervous system. ▪ Causes less vasodilation and vascular permeability than with histamine. Arachidonic Acid (AA) is a 20-carbon polyunsaturated fatty acid formed from cell membrane phospholipids. Different stimuli activate phospholipases, which release AA from cell membranes. Outside the cell, two classes of enzymes convert AA in bioactive mediators (eicosanoids): • Cyclooxygenases (COX-1 and COX-2) • 5-Lipoxygenase Eicosanoids bind to G protein-coupled receptors on ≠ cells and mediate virtually every step of inflammation. 5-HETE (5-Hydroxyeicosatetraenoic acid) 5-HPETE (5-hydroperoxyeicosatetraenoic acid) Corticosteroids (inhibit genes encoding Cox-2, phospholipase A, proinflammatory cytokines) mechanical, chemical, physical, or other inflammatory mediators (C5a) Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. I. Cell-derived Mediators 2. Arachidonic Acid Metabolites (Eicosanoids) I. Cell-derived Mediators 2. Arachidonic Acid Metabolites (Eicosanoids) AA metabolites (eicosanoids) via Cyclooxygenase pathway ARACHIDONIC ACID Cyclooxygenase inhibitors (e.g., aspirin, ibuprofen, indomethacin) block prostaglandin synthesis. Cyclooxygenase (as COX-1 and COX-2) Prostaglandin G2 (PGG2) (aka ) (by vascular ECs) . Vasodilation . Inhibits platelet aggregation Prevents thrombosis Prostaglandin H2 (PGH2) ( (by mast cells) (by almost any cell) ) (by platelets) . Vasodilation . ↑ vascular permeability . Vasoconstriction . Promotes platelet aggregation Promotes thrombosis In addition to local effects, prostaglandins are involved in pathogenesis of fever and pain. Potentiates exudation and resulting edema. PGD2 chemoattracts neutrophils. Modified from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. I. Cell-derived Mediators 2. Arachidonic Acid Metabolites (Eicosanoids) AA metabolites (eicosanoids) via Lipoxygenase pathway ARACHIDONIC ACID 12-Lipoxygenase 5-Lipoxygenase 5-HPETE (5-hydroperoxyeicosatetraenoic acid) Lipoxygenase inhibitors (e.g., zileuton) (lipoxygenases are NOT affected by NSAIDs) 5-HETE (5-Hydroxyeicosatetraenoic acid) Chemoattracts neutrophils Leukotriene A4 (LTA4) Chemoattracts neutrophils, and causes their aggregation and adhesion to venular endothelium Lipoxin A4 (LXA4) (by mast cells) Lipoxin B4 (LX B4) Suppress Inflammation by inhibiting recruitment of leukocytes Leukotriene B4 (LTB4) (by neutrophils and some macrophages) Leukotriene C4 (LTC4) Vasoconstriction Bronchospasm mast cells Leukotriene D4 (LTD4) ↑(byPermeability of venules (by mast cells) (by mast cells) Leukotriene E4 (LTE4) (by mast cells) Modified from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Leukotriene receptor antagonists (e.g., Montekulast) I. Cell-derived Mediators 3. Cytokines • Polypeptides (~5–20 kDa) secreted by numerous cells (mainly activated lymphocytes, macrophages, and dendritic cells, but also endothelial, epithelial, and fibroblasts) that initiate and mediate immune and inflammatory responses. • They can affect the same cells that produce them (autocrine effect) or nearby cells (paracrine effect), always through binding to cell surface receptors. • Unlike classic hormones, which circulate in nanomolar (10-9 M) concentrations, cytokines are soluble signaling molecules that act locally typically in picomolar (10-12 M) concentrations (up to 1,000-fold increase due to trauma or infection). • Chemokines are cytokines that act as chemoattractants for other cells (mainly leukocytes during inflammatory responses). I. Cell-derived Mediators 3. Cytokines (cont.) Over 200 cytokines have been described. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) are chief cytokines that act in local and systemic manifestations of inflammation: • Different inflammatory stimuli induce the synthesis of TNF and IL-1. • TNF is mainly produced by activated macrophages, T cells and mast cells, whereas IL-1 is produced by activated macrophages and some epithelial cells. TNF and IL-1 are commonly produced by signals through TLRs and other microbial sensors. • Endothelial activation caused by TNF and IL-1: venular ECs synthesize and express endothelial leukocyte adhesion molecules (mainly E- and Pselectins) and ligands for leukocyte integrins. Other cytokines, chemokines, and eicosanoids increase → ↑ vascular permeability (due to retraction of ECs), and ↑ procoagulant activity. • Activation of leukocytes and other cells: TNF boosts responses of neutrophils and microbicidal activity of macrophages. IL-1 stimulates the generation of TH17 (a subset of CD4+ TH cells), activates fibroblasts (↑ collagen synthesis) and stimulates proliferation of synovial and other mesenchymal cells. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. I. Cell-derived Mediators 3. Cytokines (cont.) Protective Effects (acute phase response): IL-1 and TNF (as well as IL-6) stimulate the synthesis of PGE2, which acts on the hypothalamus (thermoregulatory center) causing fever. In response to IL-1 and -6 and TNF, the liver responds by producing many acute-phase proteins, and the bone marrow produce more leukocytes. Pathologic Effects: TNF, IL-1 and IL-6 are involved in the pathogenesis of the systemic inflammatory response syndrome (SIRS), an exaggerated defense response of the body to a noxious stressor. They contribute to cardiac dysfunction and failure, thrombosis, and cachexia, a pathologic state characterized by muscle mass loss that accompanies some chronic infections and cancer. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. I. Cell-derived Mediators 3. Cytokines (cont.) Main Cytokines Involved in Inflammation Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. I. Cell-derived Mediators 3. Cytokines (cont.) Chemokines are Cytokines that Act as Chemoattractants for Specific Types of Leukocytes ~40 chemokines and 20 receptors for them identified. Small proteins (8-10 kDa) classified into 4 major groups based on the arrangement of cysteine (C) residues : • C-X-C chemokines (one amino acid residue between the first two of the four conserved cysteine residues): Most representative of this group is IL-8 (now called CXCL8). They act primarily on neutrophils, having been secreted by activated macrophages, ECs and other cell types and attracting neutrophils to the inflammatory focus. • C-C chemokines (first two conserved cysteine residues adjacent): Comprise the main monocyte chemoattractants MCP-1 (monocyte chemoattractant protein), and RANTES (regulated and normal T-cell expressed and secreted). They mainly chemoattract monocytes, eosinophils, basophils and lymphocytes, but are not as potent chemoattractants for neutrophils. • C chemokines (lack the first and third of the four conserved cysteines): They are relatively specific for lymphocytes (e.g., lymphotactin). • CX3C chemokines (lack the first and third of the four conserved cysteines): Only member is fractalkine, in a cell surface-bound form that promotes adhesion of monocytes and T cells or a soluble form that chemoattracts those same cells. The Complexity of the Cytokine Network Several different cell types coordinate their efforts as part of the immune system, including B cells, T cells, macrophages, mast cells, neutrophils, basophils and eosinophils. Each of these cell types has a distinct role in the immune system and communicates with other immune cells using secreted cytokines. Cytokine Network – From: https://www.thermofisher.com/us I. Cell-derived Mediators 3. Cytokines (cont.) I. Cell-derived Mediators 3. Cytokines (cont.) Clinical Efficacy of Biological Inhibitors of Cytokines Adapted from: Holdsworth SR, Gan PY. Clinical Journal of American Society of Nephrology 10: 2243-2254, 2015. I. Cell-derived Mediators 4. Platelet-activating Factor (PAF) • PAF is a phospholipid-derived factor that was originally discovered as a mediator of platelet aggregation. • It binds to PAF receptors expressed by many other cells. • It can cause vasoconstriction and bronchoconstriction but can also lead to vasodilation and increased venular permeability when at low concentrations. • There are secreted and cell-bound forms of PAF, which are produced by basophils, mast cells, neutrophils, macrophages, endothelial cells, and even platelets themselves. I. Cell-derived Mediators 5. Nitric Oxide (NO) Short-lived soluble gas derived from arginine by action of nitric oxide synthase (NOS) in the presence of O2. NO plays different functions. Three forms of NOS: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). iNOS is expressed by macrophages activated by inflammatory cytokines or microbial products. NO generated by iNOS destroys microbes. eNOS and nNOS are constitutively expressed at low levels by ECs and neurons, respectively. NO generated by eNOS and nNOS functions to maintain vascular tone and as a neurotransmitter, respectively. NO generated by eNOS plays roles in inflammation by: • Mediating vascular smooth relaxation and vasodilation • Preventing platelet adherence and aggregation at sites of vascular injury and reducing leukocyte recruitment at inflammatory sites. Principal Mediators of Inflammation MEDIATOR PRINCIPAL SOURCES CELL-DERIVED PRINCIPAL ACTIONS (Selected) Histamine Mast cells, basophils, platelets Vasodilation, increased vascular permeability, endothelial activation Serotonin Platelets Increased vascular permeability, Vasoconstriction during clotting Vasodilation, pain, fever Increased vascular permeability, chemotaxis, leukocyte adhesion and activation Prostaglandins Mast cells, leukocytes Leukotrienes Mast cells, leukocytes PlateletLeukocytes, mast cells activating factor Vasodilation, increased vascular permeability, leukocyte adhesion, chemotaxis, degranulation, oxidative burst Cytokines (TNF, IL-1, IL-6) Local: Leukocyte Recruitment, endothelial activation (expression of adhesion molecules, EC contraction) Systemic: fever, metabolic abnormalities, hypotension (shock). Rarely cytokine storm Chemotaxis, leukocyte activation Macrophages, endothelial cells, mast cells (NOTE: with the exception of RBCs, every cell can produce and respond to cytokines) Chemokines Leukocytes, activated macrophages Nitric oxide Endothelium, macrophages Vascular smooth muscle relaxation, microbicidal Chemical Mediators of Inflammation I. Cell-derived Mediators II. Plasma-derived Mediators 1. 2. 3. 4. Complement System Coagulation System Fibrinolytic System Kinin System II. Plasma-derived Mediators 1. Complement System Series of proteins and membrane receptors with important functions in both innate and adaptive immunity and in pathologic inflammatory reactions. It consists of more than 20 proteins (some numbered C1 through C9). The complement (C’) proteins are in inactive forms in the plasma, and many of them are activated to become proteolytic enzymes that degrade other C’ proteins (enzymatic “complement cascade” that amplifies). Many cleavage products of C’ proteins cause increased vascular permeability, chemotaxis, and opsonization. The Complement System can be activated through 3 different pathways: a. Classical Pathway: activators are Ag-Ab Complexes Antibody (Ab; IgM or IgG)) Antigen (Ag) (e.g., products of bacteria, apoptotic cells) C1 complex (C1q, C1r, C1s) C4 Plasma C3 C2 C3 convertase (C4b,2b) C3a b. Lectin Pathway: activators are carbohydrates on microbes Mannose-binding lectin (MBL) Microbial sugars (mainly mannose) MASP-1 (MBL-associated serine protease 1) * II. Plasma-derived Mediators 1. Complement System (cont.) MASP-2 C4 C2 C3 convertase (C4b,2b) *MBL can interact with MASP-1 and -2 or with C1r-C1s to activate C’. II. Plasma-derived Mediators 1. Complement System (cont.) c. Alternative Pathway: triggered by microbial surface molecules (e.g., endotoxin or LPS), complex polysaccharides, or other molecules (viruses, tumor cells) in the absence of antibodies. Plasma C3 C3a Carbohydrate or protein (no Abs involved) Bb is a fragment of Factor B Factors D and B C3 convertase (C3bBb) C5 C5 convertase (C3bBb3b) C5a C3 C3a More C3b is generated by C3 convertase C5b II. Plasma-derived Mediators 1. Complement System (cont.) All 3 pathways generate C3a and C3b. , millions of C3b molecules can be deposited on the surface of microbes in 2-3 min. Alternative pathway C3 C5b C3a Opsonization of microbe by C3b for phagocytosis Chemoattraction of leukocytes Mast cell Histamine Bound C3b is recognized by a phagocyte OPSONIZATION & PHAGOCYTOSIS C5 ↑ VASCULAR PERMEABILITY & VASODILATION C6 C7 C9C9 C5a + C8 C9 C9 C9C9 ACTIVATION OF LIPOXYGENASE pathway of AA metabolism in neutrophils and monocytes Formation of membrane attack complex (MAC) on microbial surface H20 Microbe ACTIVATION OF LEUKOCYTES destruction of microbes INFLAMMATION OSMOTIC LYSIS Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. II. Plasma-derived Mediators 1. Complement System (cont.): Regulation “Complement regulatory proteins” prevent healthy tissues from being injured at sites where the C’ system acts against microbes. C1 inhibitor: protease inhibitor that blocks the activation the C1 complex in the classical pathway of complement. Mutation in the C1-inhibitor gene (inherited deficiency of C1 esterase inhibitor) → hereditary angioedema: edema in multiple tissues (usually selflimited; laryngeal involvement may cause fatal asphyxiation). Decay-Accelerating Factor (DAF): protein linked to plasma membranes that prevents the formation of C3 convertases and, thus, of C5 convertase. Acquired deficiency of DAF → excessive C’ activation and lysis of RBCs in paroxysmal nocturnal hemoglobinuria. Factor H: plasma protein that acts as a cofactor to degrade C3 convertase. Deficiency of factor H (due to gene mutation) → excessive C’ activation → hemolytic uremic syndrome (kidney disease) and vascular permeability in macular degeneration of the eye. II. Plasma-derived Mediators 2. Coagulation System Two pathways: intrinsic (contact) and extrinsic pathways. The intrinsic pathway is initiated by the Hageman factor (Factor XII), an enzyme synthesized by the liver that circulates as a zymogen. It becomes activated by collagen in basement membranes or platelets. Factor XIIa (activated form) initiates a proteolytic cascade of factors that leads to the formation of thrombin that cleaves the plasma protein fibrinogen into an insoluble fibrin clot. Different components of the coagulation system participate in inflammation. Cofactor: HMWK (High molecular weight kininogen) Factor Xa increases vascular permeability and leukocyte emigration. Binding of thrombin to receptors on platelets and ECs causes activation and adhesion of leukocytes. Thrombin also cleaves C5 to generate C5a. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. II. Plasma-derived Mediators 3. Fibrinolytic System This system helps to limit the coagulation process, by solubilizing the fibrin clot. Cofactor: HMWK (High molecular weight kininogen) Tissue plasminogen activator (tPA) is a serine protease found on ECs. tPA converts plasminogen (a protein mainly synthesized in the liver that circulates as a zymogen) into plasmin (active enzyme). Plasmin catalyzes the degradation of fibrin polymers → fibrin split products. Components of the fibrinolytic system participate in inflammation. tPA increase vascular permeability and leukocyte emigration Plasmin cleaves C3 to generate C3a Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. II. Plasma-derived Mediators 4. Kinin System Kallikrein is an enzyme formed by the cleavage of prekallikrein by Factor XIIa of the coagulation system HMWK (High molecular weight kininogen) Kallikrein itself is also capable of activating factor XII and cleaves the plasma HMWK (high molecular weight kininogen) to produce several vasoactive low-molecular weight peptides called kinins. Bradykinin (main kinin) increases vascular permeability, smooth muscle contraction, vasodilation, and pain. Prekallikrein Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. Actions of the Principal Mediators of Inflammation Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Anti-inflammatory Mechanisms Inflammatory reactions subside because many of the mediators are shortlived and are destroyed by degradative enzymes. In addition, there are several mechanisms that counteract inflammatory mediators to limit or terminate the inflammatory responses: • Lipoxins (LXA4 and LXB4): endogenous antagonists of Leukotrienes that suppress Inflammation by inhibiting recruitment of leukocytes. • Complement Regulatory Proteins: C1 inhibitor, DAF, Factor H. • Interleukin-10 (IL-10): secreted by activated macrophages and other cells to provide a negative feedback loop (down-regulates the responses of activated macrophages). • Anti-inflammatory cytokines: TGF-β, IL-4, IL-10, & IL-13. • Tyrosine phosphatases produced by different cells to inhibit pro-inflammatory signals triggered by receptors that recognize microbes and cytokines. MORPHOLOGIC PATTERNS OF ACUTE INFLAMMATION a. b. c. d. Serous Inflammation Fibrinous Inflammation Purulent (Suppurative) Inflammation and Abscess Ulcer Morphologic Patterns of Acute Inflammation a. Serous Inflammation Collection of a watery, protein-poor fluid into spaces created by injury to surface epithelia or into body cavities(peritoneal, pleural, or pericardial cavities). Skin blister in thumb: epidermis separated from the dermis by a focal collection of serous effusion • Typically, the fluid accumulated in body cavities (called effusion) derives from the plasma (due to increased vascular permeability) or from secretions of mesothelial cells (due to irritation). • Examples include skin blisters caused by burns or viral infections contain serous fluid within or below the damaged epidermis. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Due to more severe injuries, resulting in greater vascular permeability that allows large molecules (such as fibrinogen) to pass the endothelial barrier. • A fibrinous exudate* is characteristic of inflammation in the lining of body cavities, such as the meninges, pericardium, and pleura. • Histologically: fibrin exudate appears as a pink meshwork of threads or as an amorphous coagulum. • Fibrinous exudates can be dissolved by fibrinolysis and clearing by macrophages (removal → restoration of normal tissue) OR may convert to scar tissue (organization). *Protein and cell-rich fluid with specific gravity > 1.012 Pericardial surface Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Morphologic Patterns of Acute Inflammation b. Fibrinous Inflammation Characterized by the collection of large amounts of purulent exudate (pus) consisting of neutrophils, necrotic cells, and edema fluid. Bacterial abscesses in the lung in a case of bronchopneumonia • Abscesses: focal collections of pus with a central, largely necrotic region rimmed by a layer of preserved neutrophils, surrounded by congested vessels and fibroblast proliferation (indicative of attempted repair). • The usual outcome with abscess formation is scarring. Abscess with necrotic region with an outer layer of preserved neutrophils, surrounded by congested vessels. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Morphologic Patterns of Acute Inflammation c. Purulent (Suppurative) Inflammation, Abscess Local defect, or excavation, of the surface of an organ or tissue resulting from sloughing (shedding) of inflammatory necrotic material. • It can occur only when tissue necrosis and resultant inflammation exist on or near a surface. • Common sites for ulcerations: (1) mucosa of mouth, stomach, intestines, genitourinary tract, and (2) skin and subcutaneous tissues of the lower extremities in older people with circulatory disorders (e.g., diabetic ulcers of legs). • Initial PMN infiltration and vascular dilation in margins. Later, fibroblast proliferation and scarring in margins and base of ulcers, with lymphocytes, macrophages and plasma cells. Duodenal ulcer Duodenal ulcer Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Morphologic Patterns of Acute Inflammation d. Ulcer Outcomes of Acute Inflammation In general, acute Inflammation has one of four outcomes: • • • • 1 Clearance of injurious stimuli (e.g., bacteria). Clearance of mediators and inflammatory cells. Replacement of injured cells (little tissue damage). Normal architecture and function restoration. 2 3 4 • Formation of pus (suppuration): mixture of living and dead neutrophils and bacteria (e.g., Staphylococci). • When pus is walled off by inflammatory cell and fibrosis, PMN products destroy the tissue, leaving an abscess that usually bursts and discharge pus. • The offending agent is eliminated but the tissue is irreversible injured, filled with connective tissue, fibrosis, scar formation and loss of function. • If an insulting agent persists or resolution is incomplete, the acute inflammation can progress to, chronic inflammation. Strayer DS, Saffitz JE, Rubin E. Rubin's Pathology: Mechanisms of Human Disease, 8th ed., Copyright © 2020 Wolters Kluwer. CHRONIC INFLAMMATION Chronic Inflammation Chronic inflammation is a prolonged process (weeks or months) in which continuing inflammation, tissue destruction, and attempts at repair occur simultaneously, in varying combinations. Main characteristics include: • Mononuclear cell infiltration (macrophages, lymphocytes, and plasma cells). • Tissue destruction or necrosis is mainly due to biologically active substances released by inflammatory cells (e.g., NO, proteases, ROS). • Repair, involving new vessel formation (angiogenesis) and fibrosis. Chronic Inflammation What Causes Chronic Inflammation? 1. Persistent infections by microbes that are difficult to eradicate (e.g., Mycobacterium tuberculosis, Treponema pallidum, certain viruses and fungi – all evoke an immune reaction called “delayed-type hypersensitivity”). 2. Hypersensitivity diseases: excessive and inappropriate immune response against a) the affected person’s own tissues (“autoimmune diseases” such as rheumatoid arthritis, inflammatory bowel disease, psoriasis), or b) against common environmental substances that are usually harmless (“allergic diseases”, such as bronchial asthma). 3. Prolonged exposure to potentially toxic agents such as nondegradable materials either: exogenous (inhaled particulate silica → silicosis) or endogenous (build-up of cholesterol crystals on the artery walls→ atherosclerosis). 4. Mild forms of chronic inflammation that are not conventionally thought of as inflammatory disorders such as neurodegenerative disorders (e.g., Alzheimer disease, gout, metabolic syndrome and the associated type 2 diabetes) and certain cancers in which inflammatory reactions promote tumor development. Chronic Inflammation Morphologic Features Unlike acute inflammation, which shows vascular changes, edema, and a predominant neutrophilic infiltration, chronic inflammations is characterized by: • Infiltration with mononuclear cells (macrophages, lymphocytes, and plasma cells). Chronic inflammation in the lung • Tissue destruction (in the example, normal alveoli are replaced by spaces lined by cuboidal epithelium). • Attempts at Healing by replacement of damaged tissue with connective tissue (in the example, some fibrosis). Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Chronic Inflammation Cells Involved in Chronic Inflammation 1. Macrophages • Tissue cells derived in adults from hematopoietic stem cells in the bone marrow (BM), and during fetal development from progenitor cells in the embryonic yolk sac and fetal liver. • Their circulating forms (monocytes), derived from the BM, enter the blood (life span ~1 day) and then migrate into various tissues to differentiate into macrophages (life span months or years). • Macrophages are diffusively spread in most connective tissue, and located in specific organs (e.g., Kupffer cells in liver, sinus histiocytes in spleen and lymph nodes, microglial cells in CNS). Together, they comprise the mononuclear phagocyte system*. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. CNS: central nervous system; * formerly (and erroneously) called reticuloendothelial system Chronic Inflammation Cells Involved in Chronic Inflammation (cont.) 1. Macrophages (cont.) • Macrophages (MØs) are the dominant cells in chronic inflammatory reactions: a. Secreting mediators of inflammation, such as cytokines (TNF, IL-1, chemokines) and eicosanoids → MØs play a role in initiation and propagation of inflammation. b. Phagocytosing and destroying microbes and injured (dead) host cells. c. Activating other cells, mainly by “presenting” antigens to T cells, and responding to signals from T cells → cell mediate-immune response against microbes. • Different stimuli activate monocytes/macrophages to develop into distinct populations of MØs with different functions through two main pathways: a. Classical macrophage activation → M1 MØs → destroy phagocytosed material and stimulate inflammation. b. Alternative macrophage activation → M2 MØs → play a role in tissue repair. Chronic Inflammation Cells Involved in Chronic Inflammation (cont.) 1. Macrophages (cont.) CLASSICAL MACROPHAGE ACTIVATION Microbial products (e.g., endotoxin) bind to TLRs T-cell derived signals (mainly IFN-ɣ) M1 ALTERNATIVE MACROPHAGE ACTIVATION IL-13, IL-4 produced by T cells and other cells M2 Classically activated MØs (M1) play an important role in host defense against microbes and in many inflammatory reactions Alternatively activated MØs (M2) play an important role in tissue repair Once the irritant is eliminated, M1s die or go to lymph nodes. In chronic inflammatory sites, however, more MØs are recruited and IFN-ɣ may stimulate their fusion into large multinucleated giant cells. Microbicidal actions Phagocytosis and killing of bacteria and fungi Anti-inflammatory effects, wound repair, fibrosis Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Cells Involved in Chronic Inflammation (cont.) Chronic Inflammation 2. Lymphocytes • Although activation of T and B lymphocytes is part of adaptive immune response to specific antigens (e.g., microbes), they often participate in some of the strongest chronic inflammatory reactions (e.g., granuloma). • Both classes of lymphocytes migrate into inflammatory sites using the same adhesion molecule pairs & chemokines that recruit other leukocytes. • In the tissues, B lymphocytes may develop into plasma cells (secrete antibodies), and CD4+ T lymphocytes (secrete cytokines). • Three subsets of CD4+ helper T cells (TH Cells) that elicit different types of inflammatory reaction: ▪ TH1 cells secrete IFN-γ → activates macrophages in the classical pathway. ▪ TH2 cells secrete IL-4, IL-5, and IL-13, which recruit and activate eosinophils and activates macrophages through the alternative pathway. ▪ TH17 cells secrete IL-17 and other cytokines, which induce the secretion of chemokines that recruit neutrophils and monocytes into the reaction. Cells Involved in Chronic Inflammation (cont.) • Macrophages activate T cells by presenting antigen and through many cytokines. T lymphocyte Bidirectional interactions between macrophages and lymphocytes (mainly T cells) tend to amplify and prolong the inflammatory reaction. Activated T lymphocytes IL-17, IL-22 Recruitment of PMNs and monocytes TH17 TH1 IFN-γ Recruitment of leukocytes, inflammation Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Chronic Inflammation 2. Lymphocytes (cont.) • Activated TH17 cells recruit PMNs and monocytes using IL-17 and other chemokines, whereas TH1 cells stimulate classical macrophage activation via IFN-γ. The resulting M1s recruit leukocytes to the site of inflammation using TNF, IL-1, IL-6, IL-12. Chronic Inflammation Cells Involved in Chronic Inflammation (cont.) 3. Eosinophils • Characteristically found around parasitic infections and as part of immune responses mediated by IgE (typically associated with allergies). A focus of inflammation containing numerous eosinophils • Eosinophils are recruited to the site of inflammation by specific chemokines (e.g., eotaxin) released by leukocytes and/or respiratory epithelial cells (in allergies). • Eosinophil granules contain major basic protein, a highly charged cationic protein that not only is toxic to parasites but also to mammalian epithelial cells. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Cells Involved in Chronic Inflammation (cont.) • Tissue-resident cells distributed in connective tissues throughout the body that participate in acute and chronic inflammatory responses. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Chronic Inflammation 4. Mast Cells • In atopic persons - prone to develop allergic disordersIgE antibodies against certain environmental antigens (harmless to most people) bind to the receptor FCεR1 on mast cells (“armed” mast cells). Upon re-exposure to an allergen, the antigen binds to the membranebound IgEs causing their cross-linking → “activation of mast cells” (= degranulation and release of mediators, such as histamine and prostaglandins). • Mast cells also are present in chronic inflammatory reactions. inflammatory reactions. Chronic Inflammation Cells Involved in Chronic Inflammation (cont.) 5. Neutrophils • The presence of neutrophils is the hallmark of acute inflammation. • However, many forms of chronic inflammation may continue to show extensive neutrophilic infiltrates, as a result of either persistent microbes or necrotic cells, or mediators elaborated by activated macrophages and T lymphocytes. • This pattern of inflammation is called “acute on chronic” and it is found in some cases such as: ▪ Osteomyelitis (chronic bacterial infection of bone), which shows neutrophilic exudates that may last for months. ▪ Chronic damage to lungs by cigarette smoking and other irritant stimuli. Chronic Inflammation Granulomatous Inflammation • Morphologically specific pattern of chronic inflammation defined by the localized aggregation of macrophages around an inflammatory focus. • Granuloma formation results from a protective response to eradicate an offending agent that an acute inflammatory response cannot eliminate. Two types of granulomas depending on pathogenesis Immune Granulomas Due to chronic infection fungi, tuberculosis). Foreign Body Granulomas (e.g., some Due to inert foreign bodies (e.g., sutures, talc, or large fibers). MØs activate T cells that secrete cytokines (e.g., IL-2) → activation of more T cells, some of which secrete IF-γ → activation of more MØs. Due to its size, the agent precludes phagocytosis or T-cell immune response. “Epithelioid cells” and “giant cells” surround the foreign material. Morphology Activated MØs with pale pink granular cytoplasm and indistinct boundaries, called epithelioid cells Some epithelioid cells (MØs) fuse to form 40-50 µm multinucleated cells: Langhans’ giant cells (commonly with nuclei in the periphery) Epithelioid and giant cells are commonly surrounded by a collar of lymphocytes In Langhans’ giant cells, 20 or more nuclei re often arranged at the periphery like a horseshoe or a ring Strayer DS, Rubin E, Saffitz JE, Schiller AL. Rubin’s Pathology: Clinicopathologic Foundations of Medicine, 7th ed., Copyright © 2015 Wolters Kluwer Health. Chronic Inflammation Granulomatous Inflammation (cont.) Morphology (cont.) Typical tuberculous granuloma Granulomas associated with infectious microbes (e.g., Mycobacterium tuberculosis) typically show a central zone of caseating necrosis (called necrotizing [caseating*] granulomas) *cheese-like appearance Langhans’ giant cells Granulomas in Crohn disease, sarcoidosis or caused by foreign body tend not to have necrotic centers: non-necrotizing (noncaseating) granulomas. In foreign body granulomas, undigested material is often seen within giant cells. Strayer DS, Saffitz JE, Rubin E. Rubin's Pathology: Mechanisms of Human Disease, 8th ed., Copyright © 2020 Wolters Kluwer. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Chronic Inflammation Granulomatous Inflammation (cont.) Chronic Inflammation What is the Outcome of Granulomatous Inflammation? Granulomas tend to persist for long time. In some cases, they may slowly resolve, leaving no signs of tissue injury. In other cases, they may fibrose as a result of proliferating fibroblasts at the periphery of the granuloma (scar). Infectious necrotizing granulomas may coalesce and, due to destruction of tissue, form cavities filled with caseous material. Chronic Inflammation Examples of Diseases with Granulomatous Inflammation Kumar, V, Abbas AK, Fausto N, Aster JC. Robbins and Cotran Pathologic Basis of Disease, 8th ed. Copyright © 2010 by Saunders, an imprint of Elsevier Inc. Systemic Manifestations of Inflammation Even if localized, inflammation can be associated with systemic manifestations collectively known as “acute-phase response” mediated by cytokines. The initial goal of the acute-phase response is enhancing host resistance to infection or damage, to minimize tissue injury and to promote the resolution and repair of the inflammatory lesion. Clinical example: a severe bout of viral illness (such as influenza). The acute-phase response is associated with the following clinical and pathologic features: • Fever • Increased levels of Acute-phase proteins • Leukocytosis • Other manifestations Systemic Effects of Inflammation • Fever: One of the most typical and earlier manifestations of acute-phase response, particularly when inflammation is associated with infections. MØs and PMNs sense bacterial or viral products (e.g., LPS and double-stranded RNA, respectively) - called exogenous pyrogens - through PRRs, and stimulate leukocytes to release cytokines IL-1 and TNF – endogenous pyrogens. IL-1, TNF and IL-6 stimulate COX production that convert AA into PGs (mainly PGE2). PGs act on the hypothalamus (thermoregulatory center) and stimulate the production of neurotransmitters that elevate the body temperature. Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin reduce fever by inhibiting PG synthesis. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Synthesis of these proteins in hepatocytes is stimulated during acute inflammation by cytokines (mainly IL-6) causing hundred-fold increases in their plasma levels. The best known acute-phase proteins include: C-reactive protein (CRP) and Serum amyloid A (SAA) protein: Both bind to microbial walls and may act as opsonins and fix complement, with beneficial effects. If prolonged SAA production (chronic inflammation), may lead to secondary amyloidosis. High levels of CRP serve as a marker of increased risk for myocardial infarction in patients with coronary artery disease. Fibrinogen: when at high levels, it binds to RBCs and causes “rouleaux formation” (stacking of RBCs, as in a stack of coins). These agglutinated RBCs sediment more rapidly than individual RBCs → ↑erythrocyte sedimentation rate (ESR) (not specific, but a good indicator of an acute inflammatory response). Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Systemic Effects of Inflammation • Acute-phase Proteins: Increased numbers of WBCs (leukocytosis) is a common feature in inflammatory reactions, especially when induced by bacterial infections. Leukocyte count usually reaches 15,000 to 20,000 WBCs/mL (normal WBC count: 4,000-10,000/mL). Sometimes, leukemoid reactions (similar to leukemia) with 40,000-100,000 WBCs/mL. Leukocytosis is initially caused by an accelerated release of leukocytes from bone marrow (BM) post mitotic reserve (caused by TNF and IL-1), with an increase in immature neutrophils in blood (shift to the left) . In prolonged infections, colonystimulating factor (CSF) induces proliferation of BM precursors. Neutrophilia (increased blood neutrophil count) is common in bacterial infections, whereas lymphocytosis (increased blood lymphocyte count) is associated with viral infections (e.g., infectious mononucleosis, mumps,& German measles). Eosinophilia (increase in the absolute number of eosinophils) is associated to allergies and parasite infestations. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Systemic Effects of Inflammation • Leukocytosis: Systemic Effects of Inflammation • Other Systemic Manifestations of the Acute-phase Response: ▪ ▪ ▪ ▪ ▪ ▪ ▪ ↑ heart rate ↑ blood pressure ↓ sweating (due to redirection of blood flow from cutaneous to deep vascular beds to reduce heat loss through skin) Rigors (shivering) Chills (search for warmth) Anorexia, somnolence, and malaise (effect of different cytokines on brain cells) Cachexia (aka wasting syndrome) (TNF-mediated appetite suppression and mobilization of fat stores) When too Many Cytokines Harm In severe infections (sepsis), large amounts of bacterial products in blood or extravascular tissue stimulate enormous amounts of cytokines (notably TNF) that are poured into the circulation (cytokine storm),causing an exacerbated inflammation. A late anti-inflammatory response cannot counteract the inflammation, which continues to escalate, but suppresses immune responses against microbes, making the host more susceptible to 2ary or other infections. Sepsis with one or more end-organ failure is called severe sepsis. This can lead to septic shock (a greater risk for mortality) manifested by the triad of: • Disseminated Intravascular Coagulation (DIC) • Metabolic disturbances, such as insulin resistance and hyperglycemia • Hypotensive shock When too Many Cytokines Harm (cont.) Systemic Inflammatory Syndrome (SIRS) Another form of dysregulated inflammation caused large amounts of cytokines (especially TNF), but may occur as a complication of noninfectious disorders, such as pancreatitis, vasculitis, severe burns, trauma, surgery. Two or more of the following defines SIRS: • >100.4oF (38oC) or <96.8oF (36oC) (normal: 98.7oF or 37oC) • Heart rate >90 beats per minute • Respiratory Rate>20 breaths per minute (normal: 12-15 per minute) or PaCO2 (partial pressure of carbon dioxide) <32 (normally 40) • WBC count >12,000 or <4,000 (normal 4,000 to 10,000) Sepsis is often defined as SIRS in the setting of infection. Multiple organ dysfunction syndrome (MODS) is a progressive physiologic dysfunction in ≥2 organs or organ systems, that can occur at the severe end of SIRS or sepsis. CDM 1125 – Pathology I Inflammation III R. Daniel Bonfil, Ph.D. Professor and Chair of Pathology Professor of Medical Education, Dr. Kiran C. Patel College of Allopathic Medicine rbonfil@nova.edu February 25, 2021 Tissue Repair In addition to eliminate the danger imposed by microbes and injured tissues to the host, the inflammatory response serves to initiate the repair of injured tissues. Tissue repair occurs by two main types of reactions: 1. Regeneration: replacement of damaged cells and recovering of normal function by proliferation of residual (uninjured) cells of the same type that retain proliferative capacity or by tissue stem cells. 2. Repair by Scarring: deposition of connective (fibrous) tissue resulting in the formation of a scar that provides structural stability but cannot recover the function of lost parenchymal cells. It occurs in tissues that lack cells of the same type cells with proliferative capacity or stem cells. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Tissue Repair 1. Tissue Regeneration 2. Repair by Scarring 1. Tissue Regeneration: Dependent on: • Proliferation of remnant cells (to replace injured cells) and Endothelial Cells (ECs; to form new vessels), both driven by growth factors (GFs) • Integrity of the extracellular matrix (ECM) (creates a framework for cell and tissue repair) • Activation of stem cells within the tissue and differentiation into mature cells Based on their intrinsic proliferative capacity, there are: a. Labile Tissues b. Stable Tissues c. Permanent Tissues Tissue Regeneration a.Labile Tissues Injured cells of these tissues are continuously being lost and replaced by rapidly proliferating residual immature progenitor cells or by stem cells . Examples: - Surface epithelia (e.g., basal layers of stratified squamous epithelia of skin, oral cavity, vagina, and cervix) - Cuboidal epithelia of ducts in draining exocrine organs (e.g., salivary glands, pancreas, biliary tract) - Columnar epithelium of the GI tract, uterus, and fallopian tubes - Transitional epithelium of urinary tract - Hematopoietic cells in bone marrow Tissue Regeneration b. Stable (Quiescent) Tissues/Cells Cells of these tissues are normally quiescent (arrested in G0 stage of cell cycle). However, some cells can go into mitosis in response to injury or loss of tissue mass to compensate the loss of parenchymal cells by equivalent newly formed cells. Examples: - Parenchymal cells of most solid organs (e.g., liver*, kidney, and pancreas) - Endothelial cells, fibroblasts, and smooth muscle cells * With the exception of liver, stable tissues have a limited capacity to regenerate after injury. Tissue Regeneration c. Permanent Tissues Terminally differentiated and nonproliferative in postnatal life. An injury in these tissues is usually repaired by replacement by cells that cannot perform the specific function of the original cells (scar formation). Examples: - Neurons - Cardiac muscle cells - Skeletal muscle cells, though satellite cells attached to endomysial sheath can provide some regenerative capacity (does not fully compensate) Tissue Regeneration Growth Factors and Tissue Repair Proliferation of cells involved in tissue regeneration is driven by growth factors (GFs) that are produced close to the site of injury by different cells (mainly MØs but also others like epithelial and stromal cells). Some GFs are cell-specific, whereas others act on multiple cell types. GFs bind to specific receptors on the cells, stimulating signaling pathways that: a) induce changes in gene expression that drive the cell through the cell cycle b) support the synthesis of molecules and organelles that are needed for mitosis The ECM can bind to and sequester GFs (protecting them from degradation) that are displayed at high concentrations at the site of injury. Tissue Regeneration Liver as a Classical Example of Repair by Regeneration Extraordinary capacity for regeneration after resection of up to 90% of the liver. Liver regeneration may occur through two main mechanisms: a. If partial hepatectomy (PHx): proliferation of residual “quiescent adult hepatocytes” (normally in G0) of the remnant liver. Three phases: • Priming phase: cytokines (mainly IL-6) secreted by Kupffer cells prepare (prime) hepatocytes to respond to GFs. >100 genes are activated few minutes after PHx. • Proliferative phase: activation of epidermal growth factor receptor (EGFR) and c-Met (receptor for hepatocyte growth factor, HGF) → from G0 to G1 and then S. • Termination phase: inhibition of proliferation (return to quiescence) by TGF-β. b. If chronic liver injury or inflammation: repopulation from progenitor cells in the liver that proliferate and differentiate into mature hepatocytes (in rodents, progenitor cells are called oval cells, not clear in humans). The integrity of the Extracellular Matrix (ECM) is Crucial for Tissue regeneration The integrity of the stroma (made of ECM containing connective tissue cells) of the parenchymal cells is critical for the organized regeneration of tissues. In all organs, maintenance of normal tissue structure and function require a stromal scaffold. In liver (as well as in other organs) “regeneration” only takes place if the ECM is not damaged. If the injury also damaged the ECM, there is deposition of connective tissue and collagen → “repair by scar formation”. Kumar, V, Abbas AK, Fausto N, Aster JC. Robbins and Cotran Pathologic Basis of Disease, 8th ed. Copyright © 2010 by Saunders, an imprint of Elsevier Inc. Tissue Regeneration Liver as a Classical Example of Repair by Regeneration Tissue Repair 1. Tissue Regeneration 2. Repair by Scarring 2. Repair by Scarring: When tissue injury cannot be repaired by regeneration alone, the injured parenchyma is replaced by connective tissue, leading to the formation of a scar. Repair by scar formation only happens when the injury to parenchymal cells and the connective tissue framework is severe or chronic. If some regeneration is still possible, there is a combination of tissue regeneration and tissue scarring. Repair by scarring “patches” the tissue but does not restore the original architecture or function. The term scar is usually linked to skin wound healing but is also used to describe replacement of parenchymal cells by collagen in any tissue (e.g., in heart after myocardial infarction). Repair by scar formation occurs through sequential steps (next slide). Repair by Scarring Steps in Scar Formation A. Hemostasis. In minutes, platelets aggregate, deposit fibrin, and form a hemostatic plug that stops bleeding and creates a scaffold for migrating inflammatory cells. Scar formation in a large wound in skin (healing by second intention) B. Inflammation (classical acute and chronic). • During the next 6 to 48 hours, neutrophils and monocytes are recruited to the site of injury. • Activated leukocytes followed by macrophages eliminate the offending agents and clear dead cells and debris. • The inflammation resolves. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Repair by Scarring Steps in Scar Formation (cont.) C. Cell Proliferation. ~ 10 days after the injury, several cell types proliferate and migrate to close the now-clean wound: • Epithelial cells respond to GFs and migrate over the wound to cover it, depositing basement membrane components. • Endothelial and other vascular cells form new blood vessels (angiogenesis). • Fibroblasts proliferate and migrate to lay down collagen fibers that form the scar. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Steps in Scar Formation (cont.) Repair by Scarring • Granulation tissue: combination of proliferating fibroblasts, loose connective tissue, new blood vessels and scattered chronic inflammatory cells, beneath the scab of a skin wound. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. D. Remodeling. The connective tissue deposited by fibroblasts is reorganized to produce a stable fibrous scar. This occurs 2 to 3 weeks after injury and may continue for months or years. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Healing of Skin Wounds May involve epithelial regeneration (mainly in healing by first intention), or a combination of regeneration and scarring (in healing by second intention), depending on the amount of tissue damage: Incised wounds with good apposition of the edges (clean and uninfected): Healing by first intention Open wounds with separated edges, large defects, extensive loss of cells: Healing by second intention Wound edges are joined by a small fibrin plug. The wound is filled by a large fibrin clot, with necrotic debris and exudate. Regeneration by cells of basal layer of epidermis, as the fibrin clot begins to be lysed. Little granulation tissue. Much larger amounts of granulation tissue are formed (many proliferating fibroblasts, new blood vessels and inflammatory cells). New ECM produced by fibroblasts, with more abundant collagen fibers to bridge the incision. Provisional ECM with fibronectin, type III collagen, then replaced by an ECM primarily of type I collagen. At the end, myofibroblasts participate in wound contraction. Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. Process by which new blood vessels are formed. Radial convergence of new blood vessels sprouting from larger vessels towards a wound in the center (mouse skin flap). It is critical to develop collateral circulation to make up for blood vessels lost at the site of injury, necessary to provide nutrients and oxygen. In normal circumstances, the proliferation of vascular endothelial cells (ECs) occurs once every 1000 days and is maintained physiologically through the balanced effects of angiogenic (stimulatory) factors and anti-angiogenic (inhibitory) molecules. In tissue injuries, many angiogenic factors are released by platelets, inflammatory cells, and ruptured cells. These factors activate ECs in nearby pre-existing blood vessels. Li WW, et al. Advances in Skin & Wound Care 18(9): 491-500, 2005. Repair by Scarring • Angiogenesis in Tissue Repair by Scaring 1. Angiogenic factors (mainly vascular endothelial growth factor, VEGF) activate a specialized EC called "tip cell” (aka leading cell) from a pre-existing intact blood vessel. 2. The “tip cell”, which has VEGFR on its filopodia, degrades the subendothelial basement membrane (BM) through proteases (mainly, matrix metalloproteinases - MMPs). 3. Mural vascular contractile cells (pericytes in small capillaries or vascular smooth muscle cells [vSMCs] in larger vessels) separate. 4. The “tip cell” migrates towards the VEGF coming from the wound bed, followed by proliferative “stalk cells” that support the elongation of the angiogenic bud (aka vascular stalk). 5. Two tip cells meet, buds merge and a vascular lumen is created. The new vessel is then stabilized by the synthesis of a new BM and interaction of ECs with pericytes (proteases inhibitors, such as TIMPs, increase). Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Repair by Scarring • Angiogenesis in Tissue Repair by Scaring (cont.) Repair by Scarring • Angiogenesis in Tissue Repair by Scaring (cont.) • The most important inducer of VEGF is hypoxia-inducible factor 1 (HIF-1) secreted by hypoxic injured cells. • Other GFs that participate in the angiogenesis process include: Fibroblast Growth Factor (FGF-2), by stimulating the proliferation of ECs (it also promotes migration of MØs and fibroblast to the damaged area and stimulate epithelial cells migration to cover epidermal wounds). Platelet-derived Growth Factor (PDGF), by recruiting pericytes and vSMCs to stabilize newly generated blood vessels generated by sprouting angiogenesis. Angiopoietins (Angs) – In conjunction with VEGF, Ang-2 promotes neovascularization, whereas Ang-1 plays an important role in vessel stabilization (aka vessel maturation). Transforming growth factor-β (TGF-β) – inhibits EC proliferation and migration, and stimulates synthesis of ECM proteins as part of the stabilization process. • Except for TGF-β that binds to the single pass serine/threonine kinase receptor TGFβR, all the GFs mentioned above bind to receptor tyrosine kinases (RTKs) (VEGFR for VEGF, FGFR for FGFs, PDGFR for PDGF, and Tie-2 for Ang-1 and -2). Repair by Scarring • Granulation Tissue • Combination of migrating and proliferative fibroblasts, new thin-walled delicate capillaries (from angiogenesis), and some inflammatory cells (mainly MØs) in a loose ECM. • Microscopically seen beneath the scab of a skin wound as a characteristic pink, soft, granular gross appearance with Hematoxylin and eosin (H&E) stain. Granulation tissue Inflammatory cells Blood vessels Loose ECM (minimal collagen at this point) H&E stain Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. • The amount of granulation tissue depends on the size of the tissue deficit created by the wound and the intensity of the inflammation. Repair by Scarring • Deposition of Connective Tissue The laying down of connective tissue that leads to scar formation occurs through two main steps: 1) Migration and proliferation of fibroblasts into the site of injury, and 2) Deposition of ECM proteins produced by fibroblasts. 1) Migration and proliferation of fibroblasts into the site of injury • Inflammatory cells (mainly alternatively activated [M2] MØs) that infiltrate the site of injury release TGF-β, PDGF and FGF-2, which induce the migration of fibroblasts toward the center of the wound and their proliferation. • Some fibroblasts differentiate into myofibroblasts (contain smooth muscle actin and have contractile activity) that help closing the wound by pulling their margins toward the center (scar contraction). Nucleus Fibroblast Mechanical tension Actin Myofibroblast Proto-myofibroblast Adapted from: Strayer DS, Saffitz JE, Rubin E. Rubin's Pathology: Mechanisms of Human Disease, 8th ed., Copyright © 2020 Wolters Kluwer. 2) Deposition of ECM proteins produced by fibroblasts • As healing progresses, the number of proliferating fibroblasts and new vessels diminishes, with fibroblasts starting ECM production (mostly synthesis of collagen). Ultimately, the granulation tissue transforms into a less vascular, and then avascular scar. • TGF-β is the most important cytokine mediating collagen synthesis by fibroblasts, critical to the development of strength in a healing wound site. • Levels of TFG-β depend on its rate of secretion in its active form, as well as of activation of latent TGF-β sequestered in the ECM (usually mediated by integrins). • Net collagen accumulation depends not only on its increased synthesis, but also on its diminished degradation. Mature scar with dense ECM caused by deposition of collagen (blue, due to Trichrome stain) and scattered blood vessels. Kumar, V, Abbas AK, Aster JC. Robbins Basic Pathology, 10th ed. Copyright © 2018 by Elsevier Inc. Repair by Scarring • Deposition of Connective Tissue (cont.) Repair by Scarring • Remodeling of Connective Tissue ▪ After collagen deposition, the ECM in the scar continues to be modified and remodeled. Different components of the ECM are mainly cleaved by Matrix metalloproteinases (MMPs), proteolytic enzymes dependent on metal ions (mainly Zn+). ▪ MMPs are produced by fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells. Their expression and synthesis mainly regulated by GFs and cytokines. ▪ Almost all MMPs (>25) are secreted as latent enzymes (zymogens) that are activated when needed to degrade selective substrates. Examples: interstitial collagenases (MMP-1, -2, and -3) cleave fibrillar collagen, gelatinases (MMP2 and -9) degrade amorphous collagen and fibronectin, and stromelysins degrade proteoglycans, laminin, fibronectin, and amorphous collagen. ▪ The activity of MMPs is controlled by different Tissue Inhibitors of Metalloproteinases (TIMPs), produced by most mesenchymal cells. Factors that Influence Tissue Repair Factors that Influence Tissue Repair Tissue repair may be affected by many different extrinsic or intrinsic conditions, such as: • Infection prolongs inflammation, may increase local tissue injury, and delays healing. • Nutritional status: protein deficiency, and vitamin C deficiency (needed for collagen synthesis) may delay healing. • Diabetes is an important systemic cause of abnormal wound healing. • Glucocorticoids (steroids) have anti-inflammatory effects that may result in weakness of the scar because of inhibition of TGF-β production and diminished fibrosis. However, in corneal infections, along with antibiotics, they reduce the corneal opacity that may result from collagen deposition. • Foreign bodies (e.g., fragments of glass or steel) that impede healing. Factors that Influence Tissue Repair (cont.) • Mechanical factors (e.g., increased local pressure or torsion) may cause wounds to pull apart or dehisce. • Poor perfusion (e.g., caused by arteriosclerosis, diabetes, or varicose veins) also impairs healing. • Extension and type of tissue injury: even in tissues with cells capable of proliferating, incomplete tissue regeneration and loss of function may occur when the injury is very large. In tissues with nondividing cells, injury results in scarring (e.g., healing of a myocardial infarct). • Location of injury: inflammation in tissue spaces (e.g., pleural, synovial cavities) may develop small exudates that can be digested by proteolytic enzymes and resorbed, resulting in resolution of the inflammation and restoration of normal tissue architecture. If the exudate is too large and cannot be resorbed, granulation tissue may grow into the exudate and a fibrous scar ultimately forms. Selected Clinical Examples of Abnormal Wound Healing and Scarring a. Defects in Healing b. Excessive Scarring c. Fibrosis in Parenchymal Organs Abnormal Wound Healing & Scarring a. Defects in Healing: Chronic Wounds Some examples: Venous leg ulcers • Common in elderly people • Result of chronic venous hypertension (caused by varicose veins or congestive heart failure) • Ulcers fail to heal due to poor delivery of O2 to the area Arterial ulcers • Mostly in individuals with atherosclerosis of peripheral arteries (often associated with diabetes) → ↓ blood supply • Ischemia causes atrophy and necrosis of the skin and underlying tissues. Pressure sores • Mostly in bedridden and immobile elderly people • Caused by prolonged soft tissue compression against a bone, resulting in local ischemia, ulceration and necrosis Adapted from: Kumar V, Abbas Ak, Aster JC. Robbins & Cotran Pathologic Basis of Disease, 10th ed, Copyright © 2021 by Elsevier, Inc. Another example: Diabetic ulcers • Usually affecting the lower extremities (mostly feet) of diabetic patients • Tissue necrosis and failure to heal due to small vessel disease, neuropathy, systemic metabolic abnormalities, and secondary infections •Characteristic epithelial ulceration •Extensive granulation tissue formation in the underlying dermis Adapted from: Kumar, V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease,9th ed. Copyright © 2015 by Saunders, an imprint of Elsevier Inc. Abnormal Wound Healing & Scarring a. Defects in Healing (cont.) Exaggerated formation of the components of the repair process may result in the accumulation of excessive amounts of collagen, resulting in: • Hypertrophic scars ▪ Raised scars that often grow rapidly (often with abundant myofibroblasts) but tend to regress after several months ▪ Usually after thermal of traumatic injury that affects deep layers of the dermis • Keloids ▪ Bulging scars that grow beyond the boundaries of the wound and do not regress. ▪ Higher predisposition in African American individuals ▪ Very thick connective deposition in the dermis Adapted from: Kumar V, Abbas Ak, Aster JC. Robbins & Cotran Pathologic Basis of Disease, 10th ed, Copyright © 2021 by Elsevier, Inc. Abnormal Wound Healing & Scarring b. Excessive Scarring Abnormal Wound Healing & Scarring b. Excessive Scarring (cont.) Another deviation in wound healing resulting from an exaggerated formation of the components of the repair is: Exuberant Granulation ▪ Excessive amounts of granulation tissue that projects above the level of the surrounding skin and inhibits reepithelialization ▪ Also known as “proud flesh” ▪ Whenever possible, it must be removed by cautery or surgical excision to permit restoration of the continuity of the epithelium. Abnormal Wound Healing & Scarring b. Excessive Scarring (cont.) Exaggerated contraction in the size of a wound sometimes results in: Contractures • Deformities of the surrounding tissues Severe contracture of a wound after deep burn injury wound and • Particularly prone to develop on the palms, the soles, and the anterior aspect of the thorax. • Most common as a result of serious burns and can compromise the movement of joints Aarabi S et al. PLOS Med 4:e234, 2007. Abnormal Wound Healing & Scarring c. Fibrosis in Parenchymal Organs Excessive deposition of collagen and other ECM components that, most often, refers to abnormal deposition of collagen in chronic diseases of internal organs. • Due to persistent injurious stimuli such as chronic infections and immunologic reactions that leads to chronic inflammation and loss of tissue architecture. • Usually results in organ dysfunction or organ failure. • The main cytokine involved in fibrosis is TGF-β, which stimulates migration and proliferation of myofibroblasts and deposition of collagen and other ECM proteins. Activation of TGF-β would be caused by reactive oxygen species (ROS) derived from necrotic or apoptotic cells. • Fibrotic disorders may affect many organs: liver cirrhosis, chronic pancreatitis, fibrosing diseases of the lung (idiopathic pulmonary fibrosis, pneumoconiosis, etc.), end-stage kidney disease, and constrictive pericarditis. Inflammation and Repair I - III CDM 1125 – 02/24/21 and 02/25/21– Lecturer: R. Daniel Bonfil, Ph.D. Primary Reference for this lecture: ▪ Kumar, V., Abbas, A.K., & Aster, J.C. (eds). Robbins Basic Pathology (10th ed.), Elsevier, 2018. Chapter 3: Inflammation and Repair, pp 57-95. http://novacat.nova.edu/record=b3313497~S13 Secondary Reference for this lecture: ▪ Kumar, V., Abbas, A.K., & Aster, J.C. (eds). Robbins and Cotran Pathologic Basis of Disease (10th ed.), Elsevier, 2021 Chapter 3: Inflammation and Repair, pp 71-112. ▪ Strayer, D.S., Saffitz JE, & Rubin, E. (eds). Rubin’s Pathology: Mechanisms of Human Disease, 8th ed. Wolters Kluwer, 2020. Chapter 2: Inflammation, and Chapter 3: Repair, Regeneration and Fibrosis. Note: This handout comprises information about topics on “Inflammation I to III” that you may require for future clinical rotations and board examinations. Questions in your upcoming exam will address the specific topics selected and covered in these lectures. I. Overview of Inflammation A. Definition. Inflammation is a protective response involving host cells, blood vessels, and proteins and other mediators that is intended to eliminate the initial cause of cell injury, as well as the necrotic cells and tissues resulting from the original insult, and to initiate the process of repair. Without inflammation, infections would go unchecked and wounds would never heal. B. General features of inflammation include: 1. The components of the inflammatory reaction that destroy and eliminate microbes and dead tissues are also capable of injuring normal tissues. 2. The main components of inflammation are a vascular reaction and a cellular response; both are activated by cell-derived mediators and plasma protein-derived mediators which are shared with the innate immune system. 3. Inflammation’s relationship to Innate Immunity and Adaptive Immunity. Inflammation initially and predominantly relies on the components of the Innate Immunity System to address an infectious or non-infectious threat/injury; however, if inflammation’s protective response is insufficient to address the infectious or noninfectious threat/injury, the Adaptive Immunity System will then play a role in the response. 4. The steps of the inflammatory response can be remembered as the five Rs: a. Recognition of the offending agent b. Recruitment of leukocytes c. Removal of the agent d. Regulation (control & termination) of the inflammation reaction e. Resolution (repair of damaged tissue). 5. Inflammation is normally controlled and self-limited. a. Inflammation is induced by cell-derived mediators and plasma protein-derived mediators and b. Inflammation subsides due to several mechanisms that counteract inflammatory mediators and function to limit or terminate the inflammatory response. 6. The external/clinical manifestations of inflammation, often called its cardinal signs, are heat (calor), redness (rubor), swelling (tumor), pain (dolor), and loss of function (functio laesa). 7. Inflammation can be acute or chronic. When acute inflammation achieves its desired goal of eliminating the threat, the reaction subsides, and the residual damage is repaired. But if the initial response fails to resolve the threat, the inflammatory response progresses to a protracted course that is called chronic inflammation. 1 General Features of Acute and Chronic Inflammation Feature Onset Cellular Infiltrate Acute Fast: Minutes or hours (to days) Mainly Neutrophils Chronic Slow: Days (to years) Monocytes/macrophages and lymphocytes Tissue Injury, Fibrosis Usually mild and self-limited Often severe and progressive Local & Systemic Signs Prominent Less prominent; may be subtle Clinical Examples Acute Bronchitis, Skin Tuberculosis, Asbestosis, Infection, 1st deg Sunburn Rheumatoid Arthritis 8. The outcome of acute inflammation is either elimination of the noxious stimulus followed by decline of the reaction and repair of the damaged tissue, or persistent injury resulting in chronic inflammation, or scarring with loss of function, or in some instances it can result in sudden death. II. Acute Inflammation A. Overview of Acute Inflammation. The acute inflammatory response rapidly delivers leukocytes and plasma proteins to sites of injury. At the injury site, leukocytes clear the invaders and begin the process of digesting and getting rid of necrotic tissues and the plasma proteins serve to stimulate several of the inflammatory processes. Acute Inflammation has two major components: 1. Vascular changes. a. Vasodilation - alteration in vessel caliber resulting in increased blood flow. b. Increased vascular permeability - changes in the vessel wall which permits plasma proteins to leave the circulation. c. Endothelial cell activation to facilitate leukocyte adhesion and migration through the vessel wall. 2. Cellular events. a. Leukocyte recruitment which involves emigration of leukocytes from the circulation & accumulation at focus of injury. b. Leukocyte activation to eliminate the offending agent. Neutrophils (polymorphonuclear leukocytes) are the principal leukocytes in acute inflammation. B. Stimuli for Inflammation include the following: 1. Infections and microbial toxins (bacterial, viral, fungal, parasitic): the most common and medically important causes of inflammation. 2. Tissue necrosis results in molecules being released from necrotic cells which trigger inflammation; e.g. Ischemic injury (reduced blood flow – the cause of myocardial infarction and stroke), trauma (crush injury, vascular disruption), physical and chemical injury (thermal injury – burns or frostbite; irradiation; or exposure to environmental chemicals). 3. Foreign bodies (splinters, dirt, sutures, crystal deposits). May elicit inflammation by themselves or because they cause traumatic injury or carry microbes. May be endogenous, such as urate crystals in gout and cholesterol crystals in atherosclerosis. 4. Immune reactions. Reactions in which the normally protective immune system damages the individual’s tissues: a. Adaptive Immune System reactions. Hypersensitivity reactions directed against different types of antigens may result from various causes; e.g., autoimmunity (reactions against self-antigens), reactions against microbes (immune complex deposition or cross-reactivity), and reactions against environmental antigens such as allergies (pollen, animal dander, and dust mites). b. Innate Immune System reactions. Autoinflammatory Diseases characterized by seemingly unprovoked episodes of inflammation, without high-titer autoantibodies or antigen specific T cells caused by a dysregulation of the innate immune response and subsequent cytokine excess; e.g. Familial Mediterranean Fever (FMF) due to defective pyrin which leads to uncontrolled inflammation. c. Genetically complex disorders. These are caused by multiple lesions of both branches of the immune system with potentially self-amplifying loops; e.g., gout, pseudogout, and fibrosing disorders (asbestosis and silicosis). C. Recognition of Microbes, Necrotic Cells, and Foreign Substances 1. Pattern recognition receptors (PRRs). 2 a. Phagocytes, dendritic cells (immune cells in connective tissue and organs that capture microbes & initiate responses to them), & many other cells, such as epithelial cells, express receptors that are designed to sense the presence of infectious pathogens & of substances released from dead cells. b. These receptors recognize structures (i.e., molecular patterns, damage signals) that are common to many microbes or to dead cells. (1) Pathogen-Associated Molecular Patterns (PAMPs). Gram-positive and gram-negative bacteria, viruses, parasites, and fungi all possess a limited number of unique cellular constituents not found in vertebrate animals which are detected by the PRRs. (2) Danger-Associated Molecular Patterns (DAMPs). Damage signals from the release of endogenous peptides and glycosaminoglycans from apoptotic or necrotic host cells which are detected by PRRs. Heat shock proteins, fibrinogen, fibronectin, hyaluronan, and high mobility group box-1 (HMGB-1) have been identified as DAMPs. 2. Two important families of these Pattern Recognition Receptors (PRRs) include; a. Toll-like Receptors (TLRs). TLRs are located in plasma membranes and cytoplasmic endosomes, so they are able to detect extracellular & intracellular microbes. Functions of TLRs: (1) Recognize products of different types of microbes and thus provide defense against essentially all classes of infectious pathogens. (2) Recognition of microbes causes activation of transcription factors that stimulate the production of a number of secreted and membrane proteins. These proteins include mediators of inflammation, antiviral cytokines (interferons), and proteins that promote lymphocyte activation and even more potent immune responses. b. The Inflammasome A multi-protein cytoplasmic complex that recognizes products of dead cells, such as uric acid and extracellular ATP, as well as crystals and some microbial products. (1) The inflammasome consists of a sensor protein (a leucine-rich protein called NLRP3), an adaptor, and the enzyme caspase 1, which is converted from an inactive to an active form. (2) Activation of the enzyme called caspase-1 cleaves precursor forms of the inflammatory cytokine interleukin-1β (IL-1β) into its biologically active form. NOTE: IL-1 is an important (cytokine) mediator of leukocyte recruitment in the acute inflammatory response, and when the leukocytes phagocytose and destroy dead cells. CLINICAL CORRELATION: The joint disease, gout, is caused by deposition of urate crystals, which are ingested by phagocytes and activate the inflammasome, resulting in IL-1 production and acute inflammation. IL-1 inhibitors are effective treatments in cases of gout that are resistant to conventional anti-inflammatory therapy (i.e., colchicine, NSAIDs, steroids, & allopurinol). The IL-1 inhibitors used in gout are anakinra, canakinumab, and rilonacept. D. Vascular Changes in acute inflammation. While the initial encounter of an injurious stimulus, such as a microbe, is with macrophages and other cells in the connective tissue, the vascular reactions triggered by these interactions soon follow & dominate the early phase of the response. The main vascular reactions of acute inflammation (triggered by cell generated chemical mediators) are: 1. Changes in Vascular Caliber and Flow. These changes are characterized by: a. Initial transient vasoconstriction (lasting only for seconds), then arteriolar vasodilation follows, resulting in locally increased blood flow and engorgement of the down-stream capillary beds. Vasodilation is the cause of the redness (rubor) and heat (calor) – the first two of the cardinal signs of inflammation. b. The microvasculature becomes more permeable, and protein-rich fluid moves into the extravascular tissues (exudate). This causes the red cells in the flowing blood to become more concentrated, thereby increasing blood viscosity and slowing the circulation. These changes are reflected microscopically by numerous dilated small vessels packed with red blood cells and are called stasis. c. As stasis develops, leukocytes (principally neutrophils) begin to accumulate along the vascular endothelial surface—a process called margination. This is the first step in leukocyte migration from the blood vessel to the site of injury. 2. Increased Vascular Permeability. a. Increasing vascular permeability leads to the movement of protein-rich fluid and even blood cells into the extravascular tissues. b. This in turn increases the osmotic pressure of the interstitial fluid, leading to more outflow of water from the blood into the tissues. The resulting protein-rich fluid accumulation is called an exudate. 3 Exudate vs Transudate Type of Interstitial Fluid Exudate Transudate Cause Inflammatory Status Composition Specific Gravity Increased vascular permeability due to an increase in interendothelial spaces Increased hydrostatic pressure or decreased intravascular colloid Inflammation Protein-rich May contain cellular debris S.G.>1.020 NonInflammatory conditions Protein poor Little or no cellular debris S.G.<1.012 Fluid accumulation in extravascular spaces, whether due to an exudate or a transudate, produces edema (tumor - the third cardinal sign of inflammation). c. Mechanisms which contribute to increased vascular permeability in acute inflammatory reactions: (1) Endothelial retraction leading to gaps between endothelial cells in postcapillary venules. • The most common cause of increased vascular permeability. • Chemical mediator induced (histamine, bradykinin, leukotrienes & cytokines). • Rapid and short-lived response (15-30 minutes): histamine, bradykinin, leukotrienes (2) Direct Endothelial Injury causing endothelial cell necrosis and detachment results in vascular leakage. • Most cases: leakage begins immediately after injury (trauma) & persists for several hours (or days) until the damaged vessels are thrombosed or repaired. Venules, capillaries, & arterioles can all be affected, depending on the injury site. • Delayed, prolonged leakage that begins after a delay of 2-12 hrs, lasts for several hours or even days, and involves venules and capillaries. Examples: mild to moderate thermal injury, certain bacterial toxins, and x-ray or ultraviolet irradiation. • Injury secondary to Leukocyte accumulation along the vessel wall. Activated leukocytes release many toxic mediators that may cause endothelial injury or detachment. (3) Transcytosis that appears to be caused by opening of intracellular channels that respond to certain mediators such as vascular endothelial growth factor (VEGF). Observed in animals, though not fully confirmed in humans. Although these three mechanisms of vascular permeability are described separately, all of them may be involved in the response to a particular stimulus. For example, in a thermal burn, leakage results from chemically mediated endothelial cell contraction, as well as from direct injury and leukocyte-mediated endothelial damage. E. Cellular Events: Leukocyte Recruitment and Activation An important function of the inflammatory response is to deliver leukocytes to the site of injury and to activate them. Leukocytes ingest offending agents, kill bacteria and other microbes, and eliminate necrotic tissue and foreign substances. 1. Leukocyte Recruitment. The sequence of events in the recruitment of leukocytes from the vascular lumen to the extravascular space consists of 4 phases: • Margination and rolling along the vessel wall. • Firm adhesion to the endothelium. • Transmigration between endothelial cells. • Migration in interstitial tissues toward a chemotactic stimulus. a. MARGINATION AND ROLLING ALONG THE VESSEL WALL. (1) Margination. The laminar flow of blood from capillaries into postcapillary venules causes the larger white cells to be pushed out of the central axial column - occupied by smaller cells (e.g., red blood cells) against the venular wall where they have a better opportunity to interact with the vessel endothelium, especially in the face of the stasis which develops as a result of increased vascular permeability. Leukocyte accumulation at the periphery of blood flow within vessels is called margination. (2) Rolling along the vessel wall. Endothelial cell activation by cytokines and other mediators 4 cause the expression of adhesion molecules on the surface of the endothelium to which the leukocytes attach loosely. A process of rolling then occurs as the leukocytes tumble on the endothelial surface, binding and detaching to these adhesion molecules. The Selectin Family of adhesion molecules mediate the weak and transient interactions involved in rolling. b. FIRM ADHESION TO THE ENDOTHELIUM. (1) Integrins mediate the firm adhesion of Leukocytes to Endothelial Cells. This adhesion is mediated by integrins expressed on leukocyte cell surfaces interacting with their ligands on endothelial cells. Integrins are normally expressed on leukocyte plasma membranes in a low-affinity form and do not adhere to their specific ligands until the leukocytes are activated by chemokines released during an inflammatory response. (2) The role of cytokines and chemokines in adhesion. • Cytokines: Polypeptide products produced by many cell types as mediators of inflammation and immune responses. • Chemokines: Chemoattractant cytokines secreted by cells at sites of inflammation and are displayed on the endothelial surface. (a) When the adherent leukocytes encounter the displayed chemokines on endothelial cells, the adherent leukocytes are activated, and their integrins undergo conformational changes and cluster together converting to a high-affinity form. (b) Simultaneously, other cytokines, notably TNF and IL-1 (also secreted at sites of infection and injury) activate endothelial cells to increase their expression of ligands for integrins. c. TRANSMIGRATION BETWEEN ENDOTHELIAL CELLS. (1) After stable attachment on the endothelial surface, Leukocytes then migrate through the blood vessel wall by squeezing between cells at the intercellular junctions (diapedesis). Diapedesis occurs mainly in the venules of the systemic vasculature. (2) PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1 AKA CD31), a cellular adhesion molecule expressed on leukocytes and endothelial cells, mediates the homotypic binding events needed for leukocytes to traverse the endothelium. (homotypic = similar structure). (3) After passing through the endothelium, leukocytes secrete collagenases that enable them to pass through the vascular basement membrane. d. MIGRATION IN INTERSTITIAL TISSUES TOWARD A CHEMOTACTIC STIMULUS. After migrating from the blood vessels, Leukocytes move toward the site of infection or injury along a chemical gradient by a process called chemotaxis. Both exogenous and endogenous substances can be chemotactic for leukocytes, including the following: • Bacterial products, particularly peptides with N-formylmethionine termini. • Cytokines, especially those of the chemokine family. • Components of the complement system, particularly C5. • Products of the lipoxygenase pathway of arachidonic acid (AA) metabolism, particularly leukotriene B4 (LTB4). Chemotactic molecules bind to specific cell surface receptors, which triggers the assembly of cytoskeletal contractile elements necessary for movement. Leukocytes move by extending pseudopods that anchor to the Extracellular Matrix (ECM) and then pull the cell in the direction of the extension. e. The type of Leukocyte recruited during inflammation. The type of emigrating leukocyte varies with the age of the inflammatory response and with the type of stimulus. (1) In most forms of acute inflammation, neutrophils predominate in the inflammatory infiltrate during the first 6 to 24 hours and are replaced by monocytes in 24 to 48 hours. Several factors account for this early abundance of neutrophils: These cells are the most numerous leukocytes in the blood, they respond more rapidly to chemokines, and they may attach more firmly to the adhesion molecules that are rapidly induced on endothelial cells, such as P- and E-selectins. In addition, after entering tissues, neutrophils are short-lived—they die by apoptosis and disappear within 24 to 48 hours—while monocytes (macrophages) survive longer. (2) There are exceptions to this pattern of cellular infiltration. In certain infections (e.g., those caused by Pseudomonas organisms), the cellular infiltrate is dominated by continuously recruited neutrophils for several days; in viral infections, lymphocytes may be the first cells to arrive; and in some hypersensitivity reactions, eosinophils may be the main cell type. 5 2. Leukocyte Activation. Once Leukocytes have been recruited to the site of infection or tissue necrosis, they must be activated to perform their function in inflammation. When leukocytes are activated due to inflammation, they then promote specific Leukocyte defensive responses. a. Leukocyte defensive responses as a result of activation. The major defensive functions of leukocyte activation include: (1) Phagocytosis of particles. Phagocytosis consists of: (a) Recognition and attachment of the particle to the ingesting leukocyte. Some of the leukocyte receptors recognize components of the microbes and dead cells and other receptors recognize host proteins (called opsonins) that coat microbes and target them for phagocytosis (the process called opsonization). These opsonins either are present in the blood ready to coat microbes or are produced in response to the microbes. (b) Engulfment, with subsequent formation of a phagocytic vacuole. (c) Formation of the phagolysosome due to fusion of the phagocytic vacuole with a cytoplasmic lysosome that contains microbiocidal and degradation substances. (2) Intracellular destruction of phagocytosed microbes and dead cells by substances produced in phagolysosomes, including reactive oxygen species (ROS), reactive nitrogen species, and lysosomal enzymes. (3) Extracellular destruction via liberation of substances that destroy extracellular microbes and dead tissues, which are largely the same as the substances produced within phagocytic vesicles. (a) Direct extracellular release of the lysosomal granules due to regurgitation during phagocytosis, frustrated phagocytosis of materials that cannot be easily digested, and damage of phagolysosomal membranes by injurious substances (e.g., silica). (b) Neutrophil Extracellular Traps (NETs). These “traps” are extracellular fibrillar networks that are produced by neutrophils in response to infectious pathogens (mainly bacteria and fungi) and inflammatory mediators (such as chemokines, cytokines, complement proteins, and ROS). NETs contain a framework of nuclear chromatin with embedded granule proteins, such as antimicrobial peptides and enzymes. The traps provide a high concentration of antimicrobial substances at sites of infection and prevent the spread of the microbes by trapping them in the fibrils. In the process, the nuclei of the neutrophils are lost, leading to death of the leukocytes referred to as NETosis. F. Leukocyte-Induced Tissue Injury. Because leukocytes are capable of secreting potentially harmful substances such as ROS and Lysosomes, they are important causes of injury to normal cells. The mechanisms by which leukocytes damage normal tissues are the same as the mechanisms involved in the clearance of microbes and dead tissues, because once the leukocytes are activated, their effector mechanisms do not distinguish between offender and host. In fact, if unchecked or inappropriately directed against host tissues, leukocytes themselves become the main offenders and may then cause the evolution of a chronic disease. G. Defects in Leukocyte Function. Since leukocytes play a central role in host defense, defects in leukocyte function, both acquired and inherited, lead to increased susceptibility to infections, which may be recurrent and life-threatening. 1. The most common causes of defective inflammation are acquired: a. Bone marrow suppression caused by tumors or treatment with chemotherapy or radiation (resulting in decreased leukocyte numbers), and b. Metabolic diseases such as diabetes which impairs leukocyte adhesion, chemotaxis, phagocytosis, and microbiocidal activity. c. Disease impairment of Leukocyte functions: (1) Adhesion & chemotaxis may be impaired in malignancies, sepsis, and chronic dialysis. (2) Phagocytosis and microbiocidal activity may be impaired in leukemia, anemia, sepsis, and malnutrition. 2. The genetic disorders are rare and can cause defects in leukocyte adhesion, defects in microbiocidal activity, defects in phagolysosome and mutations in TLR signaling pathways. H. Outcomes of Acute Inflammation. Acute inflammation generally has one of four outcomes: 1. Resolution: Regeneration and repair. The conditions which are required for resolution: a. When the injury is limited or short-lived, b. When there has been no or minimal tissue damage, and c. When the injured tissue is capable of regenerating, the usual outcome is restoration to structural and functional normalcy. 6 2. Chronic inflammation may follow acute inflammation if the offending agent is not removed, or it may be present from the onset of injury (e.g., in viral infections or immune responses to self-antigens). Depending on the extent of the initial and continuing tissue injury, as well as the capacity of the affected tissues to regrow, chronic inflammation may be followed by restoration of normal structure and function or may lead to scarring...or may result in persistent unresolving chronic inflammation & chronic disease. 3. Scarring is a type of repair after substantial tissue destruction (as in abscess formation), or when inflammation occurs in tissues that do not regenerate, in which the injured tissue is filled in by connective tissue. In organs in which extensive connective tissue deposition occurs in attempts to heal the damage or because of chronic inflammation, the outcome is fibrosis, a process that can significantly compromise organ function. 4. Rapid deterioration/Death. In some instances acute inflammation may result in rapid deterioration leading to multiorgan failure and possible death due to cytokine storm/cytokine release syndrome or a severe hypersensitivity reaction. III. Morphologic Patterns of Inflammation A. The vascular and cellular reactions that characterize inflammation are reflected in the morphologic appearance of the reaction. The importance of recognizing these morphologic patterns is that they are often provide valuable clues about the underlying cause. B. Morphologic Patterns: 1. Serous inflammation is characterized by a collection of a watery, protein-poor fluid into spaces created by injury to surface epithelia or into body cavities (peritoneal, pleural, or pericardial cavities). Typically, the fluid in serous inflammation is not infected by destructive organisms and does not contain large numbers of leukocytes (cell poor). In body cavities the fluid may be derived from the plasma (as a result of increased vascular permeability) or from the secretions of mesothelial cells (as a result of local irritation); fluid in a serous cavity is called an effusion. The skin blister resulting from a burn or viral infection is a good example of the accumulation of a serous effusion either within or immediately beneath the epidermis of the skin. 2. Fibrinous inflammation occurs as a consequence of more severe injuries, resulting in greater vascular permeability that allows large molecules (such as fibrinogen) to pass the endothelial barrier. Histologically, the accumulated extravascular fibrin appears as an eosinophilic meshwork of threads or sometimes as an amorphous coagulum. A fibrinous exudate is characteristic of inflammation in the lining of body cavities, such as the meninges, pericardium, and pleura. Such exudates may be degraded by fibrinolysis, and the accumulated debris may be removed by macrophages, resulting in restoration of the normal tissue structure (resolution). However, extensive fibrin-rich exudates may not be completely removed and are replaced by an ingrowth of fibroblasts and blood vessels (organization), leading ultimately to scarring that may have significant clinical consequences. For example, organization of a fibrinous pericardial exudate forms dense fibrous scar tissue that bridges or obliterates the pericardial space and restricts myocardial function. 3. Purulent (suppurative) inflammation and abscess formation. These are manifested by the collection of large amounts of purulent exudate (pus) consisting of neutrophils, necrotic cells, and edema fluid. Certain organisms (e.g., staphylococci) are more likely to induce such localized suppuration and are therefore referred to as pyogenic (pus-forming). Abscesses are focal collections of pus that may be caused by seeding of pyogenic organisms into a tissue or by secondary infections of necrotic foci. Abscesses typically have a central, largely necrotic region rimmed by a layer of preserved neutrophils, with a surrounding zone of dilated vessels and fibroblast proliferation indicative of attempted repair. As time passes, the abscess may become completely walled off and eventually be replaced by connective tissue. Because of the underlying tissue destruction, the usual outcome with abscess formation is scarring. 4. An ulcer is a local defect, or excavation, of the surface of an organ or tissue that is produced by necrosis of cells and sloughing (shedding) of necrotic and inflammatory tissue. Ulceration can occur only when tissue necrosis and resultant inflammation exist on or near a surface. Ulcers are most commonly encountered in (1) the mucosa of the mouth, stomach, intestines, or genitourinary tract and (2) in the subcutaneous tissues of the lower extremities in older persons who have circulatory disturbances predisposing affected tissue to extensive necrosis. Ulcerations are best exemplified by peptic ulcer of the stomach or duodenum, in which acute and chronic inflammation coexist. IV. Chemical Mediators and Regulators of Inflammation Leukocyte production of mediators, including arachidonic acid metabolites and cytokines, which amplify the inflammatory reaction, by recruiting and activating more leukocytes. 7 PRINCIPAL MEDIATORS OF INFLAMMATION MEDIATOR CELL DERIVED PRINCIPAL SOURCES Histamine Mast cells, basophils, platelets Serotonin Platelets Prostaglandins Mast cells, leukocytes, Platelets, Endothelial Cells Leukotrienes Mast cells, leukocytes, Platelets, Endothelial Cells Platelet-activating factor Leukocytes, mast cells Cytokines (TNF, IL-1, IL-6) Macrophages, dendritic cells, activated lymphocytes, endothelial cells, mast cells. (NOTE: with the exception of RBCs, every cell can produce and respond to cytokines) Reactive Oxygen Species Leukocytes, activated macrophages Leukocytes Nitric Oxide Endothelium, macrophages Lysosomal Enzymes of Leukocytes Neutrophils, monocytes Neuropeptides Leukocytes, nerve fibers Chemokines PRINCIPAL ACTIONS (Selected) Vasodilation, increased vascular permeability, endothelial activation Increased vascular permeability, Vasoconstriction during clotting Vasodilation, pain, fever Increased vascular permeability, chemotaxis, leukocyte adhesion, and activation Vasodilation, increased vascular permeability, leukocyte adhesion, chemotaxis, degranulation, oxidative burst Local: Leukocyte recruitment, endothelial activation (expression of adhesion molecules, contraction). Systemic: fever, metabolic abnormalities, hypotension (shock). Rarely cytokine storm Chemotaxis, leukocyte activation Microbiocidal, tissue damage Microbiocidal, Vascular smooth muscle relaxation Enzymes that destroy phagocytosed substances, generate C3a and C5a, generate bradykinin-like peptides Transmit pain signals, regulate vessel tone, modulate vascular permeability PLASMA PROTEIN-DERIVED Complement (C5a, C3a, C4a) Plasma (produced in liver) Kinins Plasma (produced in liver) Proteases activated during coagulation Plasma (produced in liver) Fibrinolytic agents Endothelium, leukocytes, and other tissues Leukocyte chemotaxis and activation, direct target killing (Membrane Attack Complex), vasodilation (mast cell stimulation) Increased vascular permeability, smooth muscle contraction, vasodilation, pain Promote clotting, Endothelial activation, leukocyte recruitment Serves to limit clotting, increase vascular permeability, activate Hageman factor 8 General characteristics of all chemical mediators and regulators of inflammation: 1. Cell-derived mediators vs Plasma-derived mediators. Mediators may be produced locally by cells at the site of inflammation or may be derived from circulating inactive precursors (typically synthesized by the liver) that are activated at the site of inflammation. a. Cell-derived mediators are normally sequestered in intracellular granules and are rapidly secreted upon cellular activation (e.g., histamine in mast cells) or are synthesized de novo in response to a stimulus (e.g., prostaglandins and cytokines produced by leukocytes and other cells). b. Plasma protein–derived mediators (coagulation & fibrinolytic factors, complement proteins, & kinins) circulate in an inactive form and typically undergo proteolytic cleavage to acquire their biologic activities. 2. Receptor based vs Direct acting mediators. Most mediators act by binding to specific receptors on different target cells. Other mediators (e.g., lysosomal proteases, ROS) have direct enzymatic and/or toxic activities that do not require binding to specific receptors. 3. Finite Activity. The actions of most mediators are tightly regulated and short-lived. Once activated and released from the cell, mediators quickly decay (e.g., arachidonic acid metabolites), are inactivated by enzymes (e.g., kininase inactivates bradykinin), are eliminated (e.g., antioxidants scavenge toxic oxygen metabolites), or are inhibited (e.g., complement regulatory proteins block complement activation). A. Cell-Derived Mediators. Tissue macrophages, mast cells, and endothelial cells at the site of inflammation, as well as leukocytes that are recruited to the site from the blood, are all capable of producing different mediators of inflammation. Selected Cell-Derived Mediators include: 1. Arachidonic Acid (AA) Metabolites: Prostaglandins, Leukotrienes, and Lipoxins. AA metabolites can mediate virtually every step of inflammation; their synthesis is increased at sites of inflammatory response, and agents that inhibit their synthesis also diminish inflammation. Leukocytes, mast cells, endothelial cells, and platelets are the major sources of AA metabolites in inflammation. These AA-derived mediators act locally at the site of generation and then decay spontaneously or are enzymatically destroyed. AA is a 20-carbon polyunsaturated fatty acid (with four double bonds) produced primarily from dietary linoleic acid and present in the body mainly in its esterified form as a component of cell membrane phospholipids. It is released from these phospholipids through the action of cellular phospholipases that have been activated by mechanical, chemical, or physical stimuli, or by inflammatory mediators such as C5a. AA metabolism proceeds along one of two major enzymatic pathways: (1) Cyclooxygenase stimulates the synthesis of prostaglandins and thromboxanes, and (2) Lipoxygenase is responsible for production of leukotrienes and lipoxins. a. Prostaglandins and thromboxanes. Products of the cyclooxygenase pathway include prostaglandin E2 (PGE2), PGD2, PGF2α, PGI2 (prostacyclin), and thromboxane A2 (TXA2), each derived by the action of a specific enzyme on an intermediate. Some of these enzymes have a restricted tissue distribution. For example, platelets contain the enzyme thromboxane synthase, and hence TXA2, a potent plateletaggregating agent and vasoconstrictor, is the major prostaglandin produced by these cells. Endothelial cells, on the other hand, lack thromboxane synthase but contain prostacyclin synthase, which is responsible for the formation of PGI 2, a vasodilator and a potent inhibitor of platelet aggregation. TXA2 and PGI2 play opposing roles in hemostasis. PGD2 is the major metabolite of the cyclooxygenase pathway in mast cells; along with PGE2 and PGF2α (which are more widely distributed), it causes vasodilation and potentiates edema formation. The prostaglandins also contribute to the pain and fever that accompany inflammation; PGE2 augments pain sensitivity to a variety of other stimuli and interacts with cytokines to cause fever. b. Leukotrienes. Leukotrienes are produced by the action of 5-lipoxygenase, the major AAmetabolizing enzyme in neutrophils. The synthesis of leukotrienes involves multiple steps. The first step generates leukotriene A4 (LTA4), which in turn gives rise to LTB4 or LTC4. LTB4 is produced by neutrophils and some macrophages and is a potent chemotactic agent for neutrophils. LTC4 and its subsequent metabolites, LTD4 and LTE4, are produced mainly in mast cells and cause bronchoconstriction and increased vascular permeability. c. Lipoxins. Once leukocytes enter tissues, they gradually change their major lipoxygenasederived AA products from leukotrienes to anti-inflammatory mediators called lipoxins, which inhibit neutrophil chemotaxis and adhesion to endothelium and thus serve as endogenous antagonists of leukotrienes. Platelets that are activated and adherent to leukocytes also are important sources of lipoxins. Platelets alone cannot synthesize lipoxins A4 and B4 (LXA4 and LXB4), but they can form these 10 mediators from an intermediate derived from adjacent neutrophils, by a transcellular biosynthetic pathway. By this mechanism, AA products can pass from one cell type to another. d. Anti-inflammatory Drugs That Block Prostaglandin Production. The central role of eicosanoids in inflammatory processes is emphasized by the clinical utility of agents that block eicosanoid synthesis. Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, inhibit cyclooxygenase activity, thereby blocking all prostaglandin synthesis (hence their efficacy in treating pain and fever). Glucocorticoids, which are powerful anti-inflammatory agents, act in part by inhibiting the activity of phospholipase A2 and thus the release of AA from membrane lipids. 2. Cytokines a. Cytokines are polypeptide products of many cell types that function as mediators of inflammation and immune responses. With the exception of RBCs, every cell can produce and respond to cytokines. The types of cytokines include: (1) Monokines - Cytokines produced by mononuclear phagocytes. (2) Lymphokines - Cytokines produced by lymphocytes. (3) Interleukins - Represent a broad family of cytokines that act primarily on leukocytes. (4) Chemokines - Cytokines that share the ability to stimulate leukocyte movement (chemokinesis) and directed movement (chemotaxis). b. The major cytokines in acute inflammation are Tumor Necrosis Factor (TNF), IL-1, IL-6, and a group of chemoattractant cytokines called chemokines. (1) Tumor Necrosis Factor (TNF) and Interleukin-1 (IL-1) (a) Produced mainly by activated macrophages, as well as by mast cells, endothelial cells and other cell types. Their secretion is stimulated by endotoxin and other microbial products, immune complexes, physical injury, and a variety of inflammatory stimuli. (b) The actions of TNF, IL-1 and IL-6 contribute to the local and systemic reactions of inflammation. The most important roles of these cytokines in inflammation are the following: ➢ Endothelial activation. Both TNF and IL-1 act on endothelium to induce a spectrum of changes referred to as endothelial activation. These changes include increased expression of endothelial adhesion molecules, mostly E- and P-selectins and ligands for leukocyte integrins; increased production of various mediators, including other cytokines and chemokines, and eicosanoids; and increased procoagulant activity of the endothelium. ➢ Activation of leukocytes and other cells. TNF augments responses of neutrophils to other stimuli such as bacterial endotoxin and stimulates the microbicidal activity of macrophages. IL-1 activates fibroblasts to synthesize collagen and stimulates proliferation of synovial cells and other mesenchymal cells. IL-1 and IL-6 also stimulate the generation of a subset of CD4+ helper T cells called T H 17 cells. ➢ Systemic acute-phase response. IL-1 and TNF (as well as IL-6) induce the systemic acute-phase responses associated with infection or injury which is manifested by fever, lethargy, hepatic synthesis of various acute phase proteins (also stimulated by IL-6), metabolic wasting (cachechia), and hypotension. They also are implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS), resulting from disseminated bacterial infection (sepsis) and other serious conditions. (2) Chemokines. The chemokines are structurally related proteins that act primarily as chemoattractants for different subsets of leukocytes. The two main functions of chemokines are to recruit leukocytes to the site of inflammation and to control the normal anatomic organization of cells in lymphoid and other tissues. Combinations of chemokines that are produced transiently in response to inflammatory stimuli recruit particular cell populations (e.g., neutrophils, lymphocytes or eosinophils) to sites of inflammation. Chemokines also activate leukocytes. Chemokines mediate their activities by binding to specific G protein–coupled receptors on target cells. c. The major cytokines in chronic inflammation include interferon-γ (IFN-γ), IL-4, IL-13, IL-12, and IL-17. Their roles are discussed in Section VI: Chronic Inflammation, p 18-19) d. Cytokine Dual Roles in acute and chronic inflammation. There is considerable overlap between cytokines involved in acute and chronic inflammation; many cytokines associated with acute inflammation may also contribute to chronic inflammation. A cytokine called IL-17 - produced by T lymphocytes and other cells - plays an important role in recruiting neutrophils and monocytes in both acute and chronic inflammation. 11 B. Plasma Protein-Derived Mediators Circulating proteins of four interrelated systems - the complement, kinin, coagulation and fibrinolytic systems - are involved in several aspects of the inflammatory reaction. A description of each of these four systems and their interrelationship is as follows: 1. The Complement System. The complement system consists of plasma proteins that play an important role in host defense (immunity) and inflammation. The role of the complement system in inflammation is summarized as follows: a. Complement components, numbered C1 to C9, are present in plasma in inactive forms, and many of them are activated by proteolysis to acquire their own proteolytic activity, thus setting up an enzymatic cascade b. The critical step in the generation of biologically active complement products is the activation of the third component, C3. C3 cleavage occurs by three pathways: (1) The classical pathway, triggered by fixation of the first complement component C1 to antigenantibody complexes formed on the surface of bacteria or apoptotic cells. (2) The alternative pathway, triggered by bacterial polysaccharides (e.g., endotoxin) and other microbial cell wall components, and involving a distinct set of plasma proteins including factors B and D. (3) The lectin pathway, in which a plasma lectin (carbohydrate binding protein) binds to mannose residues on microbes and activates an early component of the classical pathway (but in the absence of antibodies). c. All three pathways lead to the formation of a C3 convertase that cleaves C3 to C3a and C3b, and then the cascade continues to the formation of C9. d. Effects of Complement System in acute inflammation. (1) Vascular effects. C3a and C5a increase vascular permeability and cause vasodilation by inducing mast cells to release histamine. These complement products are also called anaphylatoxins because their actions mimic those of mast cells, which are the main cellular effectors of the severe allergic reaction called anaphylaxis. C5a also activates the lipoxygenase pathway of AA metabolism in neutrophils and macrophages, causing release of more inflammatory mediators. (2) Leukocyte activation, adhesion, and chemotaxis. C5a, and to lesser extent, C3a and C4a, activate leukocytes, increasing their adhesion to endothelium, and is a potent chemotactic agent for neutrophils, monocytes, eosinophils, and basophils. (3) Phagocytosis. When fixed to a microbial surface, C3b and its inactive proteolytic product iC3b act as opsonins, augmenting phagocytosis by neutrophils and macrophages, which express receptors for these complement products. (4) Membrane Attack Complex (MAC) formation. The MAC, which is made up of multiple copies of the final component C9, kills some bacteria (especially thin-walled Neisseria) by creating pores that disrupt osmotic balance. e. Complement System Regulation (inhibition). The activation of complement is tightly controlled by cell-associated and circulating regulatory proteins. The presence of these inhibitors in host cell membranes protects normal cells from inappropriate damage during protective reactions against microbes. Inherited deficiencies of these regulatory proteins lead to spontaneous complement activation: (1) A protein called C1 inhibitor blocks activation of C1, and its inherited deficiency causes a disease called hereditary angioedema, in which excessive production of kinins secondary to complement activation results in edema in multiple tissues, including the larynx. (2) Another protein called Decay-Accelerating Factor (DAF) normally limits the formation of C3 and C5 convertases. In a disease called paroxysmal nocturnal hemoglobinuria, there is an acquired deficiency of DAF that results in complement-mediated lysis of red cells (which are more sensitive to lysis than most nucleated cells). (3) Factor H is a plasma protein that also limits convertase formation; its deficiency is associated with a kidney disease called the hemolytic uremic syndrome, as well as spontaneous vascular permeability in macular degeneration of the eye. Even in the presence of regulatory proteins, inappropriate or excessive complement activation (e.g., in antibodymediated diseases) can overwhelm the regulatory mechanisms; this is why complement activation is responsible for serious tissue injury in a variety of immunologic disorders. 2. Kinin system activation leads ultimately to the formation of bradykinin from its circulating precursor, high-molecular-weight kininogen (HMWK). Like histamine, bradykinin causes increased vascular permeability, arteriolar dilation, and bronchial smooth muscle contraction. It also causes pain when injected into the skin. The actions of bradykinin are short-lived because it is rapidly degraded by 12 kininases present in plasma and tissues. Of note, kallikrein, an intermediate in the kinin cascade with chemotactic activity, also is a potent activator of Hageman factor and thus constitutes another link between the kinin and clotting systems. 3. Coagulation system activation initiates the proteolytic cascade which leads to the activation of thrombin, which then cleaves circulating soluble fibrinogen to generate an insoluble fibrin clot. Factor Xa, an intermediate in the clotting cascade, causes increased vascular permeability and leukocyte emigration. Thrombin participates in inflammation by binding to protease-activated receptors that are expressed on platelets, endothelial cells, and many other cell types. Binding of thrombin to these receptors on endothelial cells leads to their activation and enhanced leukocyte adhesion. In addition, thrombin generates fibrinopeptides (during fibrinogen cleavage) that increase vascular permeability and are chemotactic for leukocytes. Thrombin also cleaves C5 to generate C5a, which provides another link between coagulation with complement activation. 4. Fibrinolytic System activation occurs concurrently whenever clotting is initiated (e.g., by activated Hageman factor). This mechanism serves to limit clotting by cleaving fibrin, thereby solubilizing the fibrin clot. Plasminogen activator (released from endothelium, leukocytes, and other tissues) and kallikrein cleave plasminogen, a plasma protein bound up in the evolving fibrin clot. The resulting product, plasmin, is a multifunctional protease that cleaves fibrin and is therefore important in lysing clots. However, fibrinolysis also participates in multiple steps in the vascular phenomena of inflammation. For example, fibrin degradation products (FDP) increase vascular permeability, and plasmin cleaves the C3 complement protein, resulting in production of C3a and vasodilation and increased vascular permeability. FDPs also interfere with thrombin influence on fibrinogen. Plasmin can also activate Hageman factor, thereby amplifying the entire set of responses. 5. Interrelationships between the four plasma mediator systems. Some of the molecules activated during blood clotting are capable of triggering multiple aspects of the inflammatory response. Hageman factor (also known as factor XII of the intrinsic coagulation cascade) is a protein synthesized by the liver that circulates in an inactive form until it encounters collagen, basement membrane, or activated platelets (e.g., at a site of endothelial injury). Activated Hageman factor (factor XIIa) has the ability to initiate the four plasma mediator systems in the inflammatory response. C. Anti-inflammatory Mechanisms Inflammatory reactions subside because many of the mediators are short-lived and are destroyed by degradative enzymes. In addition, there are several mechanisms that counteract inflammatory mediators and function to limit or terminate the inflammatory response. 1. Some of these, such as lipoxins, and complement regulatory proteins, have been previously described. 2. Activated macrophages and other cells secrete a cytokine, IL-10, whose major function is to down-regulate the responses of activated macrophages, thus providing a negative feedback loop. 3. Other anti-inflammatory cytokines include Transforming Growth Factor-beta (TGF-β), which is also a mediator of fibrosis in tissue repair after inflammation. 4. Cells also express many intracellular proteins, such as tyrosine phosphatases, that inhibit pro-inflammatory signals triggered by receptors that recognize microbes and cytokines. 13 V. Chronic Inflammation A. Description. Chronic inflammation is inflammation of prolonged duration (weeks to years) in which continuing inflammation, tissue injury, and healing, often by fibrosis, proceed simultaneously. The dominant characteristics of chronic inflammation include: • Infiltration with mononuclear cells, including macrophages, lymphocytes, and plasma cells. • Tissue destruction largely induced by the products of the inflammatory cells. • Repair, involving new vessel proliferation (angiogenesis) and fibrosis • Less prominent local and systemic signs. B. Pathogenetic Mechanisms. Chronic inflammation may arise in the following settings: 1. Persistent infections by microbes that are difficult to eradicate. These include Mycobacterium tuberculosis, Treponema pallidum (the causative organism of syphilis), and certain viruses and fungi, all of which tend to establish persistent infections and elicit a T lymphocyte–mediated immune response called delayed-type hypersensitivity. 2. Immune-mediated inflammatory diseases (hypersensitivity diseases). a. Immune reactions develop against the affected person’s own tissues, leading to autoimmune diseases. In such diseases, autoantigens evoke a self-perpetuating immune reaction that results in tissue damage and persistent inflammation. Examples of several common and debilitating chronic inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, and psoriasis. b. Immune responses against common environmental substances are the cause of allergic diseases, such as bronchial asthma. Immune-mediated diseases may show morphologic patterns of mixed acute and chronic inflammation because they are characterized by repeated bouts of inflammation. Since, in most cases, the eliciting antigens cannot be eliminated, these disorders tend to be chronic and intractable. 3. Prolonged exposure to potentially toxic agents. Examples are nondegradable exogenous materials such as inhaled particulate silica, which can induce a chronic inflammatory response in the lungs (silicosis), and endogenous agents such as cholesterol crystals, which may contribute to atherosclerosis. 4. Mild forms of chronic inflammation may be important in the pathogenesis of many diseases that are not conventionally thought of as inflammatory disorders. Such diseases include neurodegenerative disorders such as Alzheimer disease, atherosclerosis, gout, metabolic syndrome and the associated type 2 diabetes, and some forms of cancer in which inflammatory reactions promote tumor development. In many of these diseases the inflammation may be triggered by recognition of the initiating stimuli by the inflammasome. C. Chronic Inflammatory Cells and Mediators In chronic inflammation there are several cell populations which are active in this response to cell injury. Understanding the pathogenesis of chronic inflammatory reactions requires an appreciation of these cells and their biologic responses and functions. 1. Macrophages. a. The Mononuclear Phagocyte System (AKA Reticuloendothelial System). The Mononuclear Phagocyte System is composed of macrophages which originate from two sources: (1) from progenitors in the embryonic yolk sac and fetal liver during early development and (2) the hematopoietic stem cells in the bone marrow. (1) The macrophages that reside in tissues in the steady state (in the absence of tissue injury or inflammation), such as microglia (central nervous system), Kupffer cells (liver), alveolar macrophages (lung), and sinus histiocytes (spleen and lymph nodes) arise from the yolk sac or fetal liver very early in embryogenesis, populate the tissues, stay for long periods, and are replenished mainly by the proliferation of resident cells. (2) In inflammatory reactions, progenitors in the bone marrow give rise to monocytes, which enter the blood, migrate into various tissues, and differentiate into macrophages. Entry of blood monocytes into tissues is governed by the same factors that are involved in neutrophil emigration, such as adhesion molecules and chemokines. When monocytes reach the extravascular tissue, they undergo transformation into macrophages, which are somewhat larger and have a longer lifespan and a greater capacity for phagocytosis than do blood monocytes. The half-life of blood monocytes is about 1 day, whereas the life span of tissue macrophages is several months or years. Thus, macrophages often become the dominant cell population in inflammatory reactions within 48 hours of onset. (3) In all tissues, macrophages act as filters for particulate matter, microbes, and senescent cells, as well as the effector cells that eliminate microbes in cellular and humoral immune responses. b. Macrophage Activation. There are two major pathways of macrophage activation, called classical and alternative. Which of these two pathways is taken by a given macrophage depends on the 14 nature of the activating signals. (1) Classical macrophage activation may be induced by microbial products such as endotoxin, which engage TLRs and other sensors, and by T cell–derived signals, importantly the cytokine IFN-γ, in immune responses. Classically activated (also called M1) macrophages produce NO and ROS and upregulate lysosomal enzymes, all of which enhance their ability to kill ingested organisms, and secrete cytokines that stimulate inflammation. These macrophages are important in host defense against microbes and in many inflammatory reactions. (2) Alternative macrophage activation is induced by cytokines other than IFN-γ, such as IL-4 and IL-13, produced by T lymphocytes and other cells. These macrophages are not actively microbicidal; instead, the principal function of alternatively activated (M2) macrophages is in tissue repair. They secrete growth factors that promote angiogenesis, activate fibroblasts, and stimulate collagen synthesis. c. The role of macrophages in inflammation. The products of activated macrophages eliminate injurious agents such as microbes and initiate the process of repair, but are also responsible for much of the tissue injury in chronic inflammation. Several functions of macrophages are central to the development and persistence of chronic inflammation and the accompanying tissue injury. (1) Macrophages secrete mediators of inflammation, such as cytokines (TNF, IL-1, chemokines, and others) and eicosanoids. Macrophages are central to the initiation and propagation of inflammatory reactions. (2) Macrophages display antigens to T lymphocytes and respond to signals from T cells, thus setting up a feedback loop that is essential for defense against many microbes by cell-mediated immune responses. (3) Their impressive arsenal of mediators makes macrophages powerful allies in the body's defense against unwanted invaders, but the same weaponry can also induce considerable tissue destruction when macrophages are inappropriately or excessively activated. It is because of these activities of macrophages that tissue destruction is one of the hallmarks of chronic inflammation. d. The Macrophage final role in acute and chronic inflammation. (1) After the initiating stimulus is eliminated and the inflammatory reaction abates, macrophages eventually die or wander off into the lymphatics. (2) In chronic inflammatory sites, however, macrophage accumulation persists, because of continued recruitment from the blood and local proliferation. IFN-γ can also induce macrophages to fuse into large, multinucleate giant cells. 2. Lymphocytes. a. Lymphocyte Recruitment. Lymphocytes are mobilized in the setting of any specific immune stimulus (i.e., infections) as well as non–immune-mediated inflammation (e.g., due to ischemic necrosis or trauma), and are the major drivers of inflammation in many autoimmune and other chronic inflammatory diseases. b. Lymphocyte Activation. The activation of T and B lymphocytes is part of the adaptive immune response in infections and immunologic diseases. Both classes of lymphocytes migrate into inflammatory sites using some of the same adhesion molecule pairs and chemokines that recruit other leukocytes. In the tissues, B lymphocytes may develop into plasma cells, which secrete antibodies, and CD4+ T lymphocytes are activated to secrete cytokines. c. The role of Lymphocytes in inflammation. By virtue of cytokine secretion, CD4+ T lymphocytes promote inflammation and influence the nature of the inflammatory reaction. There are three subsets of CD4+ helper T cells (T H Cells) that secrete different sets of cytokines and elicit different types of inflammation: (1) T H1 cells produce the cytokine IFN-γ, which activates macrophages in the classical pathway. This promotes an pro-inflammatory environment. (2) T H2 cells secrete IL-4, IL-5, and IL-13, which recruit and activate eosinophils and are responsible for the alternative pathway of macrophage activation. Promotes anti-inflammatory/repair. (3) T H17 cells secrete IL-17 and other cytokines that induce the secretion of chemokines responsible for recruiting neutrophils and monocytes into the reaction. Sustains inflammatory response. 3. Other Chronic Inflammation Cells. a. Eosinophils are characteristically found in inflammatory sites around parasitic infections and as part of immune reactions mediated by IgE, typically associated with allergies. Their recruitment is driven by adhesion molecules similar to those used by neutrophils, and by specific chemokines (e.g., eotaxin) derived from leukocytes and epithelial cells. Eosinophil granules contain major basic protein, a highly charged cationic protein that is toxic to parasites but also causes epithelial cell necrosis. b. Mast cells are sentinel cells widely distributed in connective tissues throughout the body, and they can participate in both acute and chronic inflammatory responses. In atopic persons (those prone to allergic reactions), mast cells are “armed” with IgE antibody specific for certain environmental antigens. 15 When these antigens are subsequently encountered, the IgE-coated mast cells are triggered to release histamines and AA metabolites that elicit the early vascular changes of acute inflammation. IgE-armed mast cells are central players in allergic reactions, including anaphylactic shock. Mast cells can also elaborate cytokines such as TNF and chemokines and may play a beneficial role in combating some infections. 4. Neutrophils in Chronic Inflammation. Although the presence of neutrophils is the hallmark of acute inflammation, many forms of chronic inflammation may continue to show extensive neutrophilic infiltrates, as a result of either persistent microbes or necrotic cells, or mediators elaborated by macrophages. Such inflammatory lesions are sometimes called “acute on chronic”—for example, in inflammation of bones (osteomyelitis). D. Granulomatous Inflammation 1. Description. Granulomatous inflammation is a distinctive pattern of chronic inflammation characterized by aggregates of activated macrophages with scattered lymphocytes. Granulomas are characteristic of certain specific pathologic states; consequently, recognition of the granulomatous pattern is important because of the limited number of conditions (some life-threatening) that cause it. 2. Pathogenesis. Granulomas can form under three settings: a. With persistent T-cell responses to certain microbes (such as Mycobacterium tuberculosis, T. pallidum, or fungi), in which T cell–derived cytokines are responsible for chronic macrophage activation. Tuberculosis is the prototype of a granulomatous disease caused by infection and should always be excluded as the cause when granulomas are identified. b. Granulomas may also develop in some immune-mediated inflammatory diseases, notably Crohn disease, which is one type of inflammatory bowel disease and an important cause of granulomatous inflammation in the United States. c. Chronic disease of unknown etiology & inert foreign bodies. They are also seen in a disease of unknown etiology called sarcoidosis, and they develop in response to relatively inert foreign bodies (e.g., suture or splinter), forming so-called foreign body granulomas. 3. Pathology. a. Pathologic significance of granulomas. The formation of a granuloma effectively “walls off” the offending agent and is therefore a useful defense mechanism. However, granuloma formation does not always lead to eradication of the causal agent, which is frequently resistant to killing or degradation, and granulomatous inflammation with subsequent fibrosis may even be the major cause of organ dysfunction in some diseases, such as tuberculosis. b. Microscopic appearance. (1) In the usual H&E preparations, some of the activated macrophages in granulomas have pink, granular cytoplasm with indistinct cell boundaries; these are called epithelioid cells because of their resemblance to epithelia. Typically, the aggregates of epithelioid macrophages are surrounded by a collar of lymphocytes. Older granulomas may have a rim of fibroblasts and connective tissue. Frequently, but not invariably, multinucleate giant cells 40 to 50 μm in diameter are found in granulomas. Such cells consist of a large mass of cytoplasm and many nuclei, and they derive from the fusion of multiple activated macrophages. (2) Caseating vs Noncaseating granulomas. In granulomas associated with certain infectious organisms (most classically the tubercle bacillus), a combination of hypoxia and free radical injury leads to a central zone of necrosis. On gross examination, this has a granular, cheesy appearance and is therefore called caseous necrosis. On microscopic examination, this necrotic material appears as eosinophilic amorphous, structureless, granular debris, with complete loss of cellular details. The granulomas associated with Crohn disease, sarcoidosis, and foreign body reactions tend to not have necrotic centers and are said to be “noncaseating.” Healing of granulomas is accompanied by fibrosis that may be quite extensive. VI. Systemic Effects of Inflammation - The Acute-Phase Response A. Definition. Inflammation, even if it is localized, is associated with cytokine induced systemic reactions that are collectively called the Acute-Phase Response. Anyone who has suffered a severe bout of viral illness (such as influenza) has experienced the systemic effects of inflammation. B. Pathogenesis. The cytokines TNF, IL-1, and IL-6 are the most important mediators of the acutephase reaction. These cytokines are produced by leukocytes (and other cell types) in response to infection or in immune reactions and are released systemically. TNF and IL-1 have similar biologic actions, although these may differ in subtle ways. IL-6 stimulates the hepatic synthesis of a number of plasma proteins (acute phase proteins), which are described in Para C2 below. C. Pathologic & Clinical Features. The Acute-Phase Response consists of several clinical and 16 pathologic changes: 1. Fever, characterized by an elevation of body temperature, is one of the most prominent manifestations of the acute-phase response. Fever is produced in response to substances called pyrogens that act by stimulating prostaglandin synthesis in the vascular and perivascular cells of the hypothalamus. a. Exogenous pyrogens: Bacterial products, such as lipopolysaccharide (LPS) stimulate leukocytes to release cytokines such as IL-1 and TNF (called endogenous pyrogens). b. Endogenous pyrogens (IL-1 and TNF): increase the levels of cyclooxygenases that convert AA into prostaglandins. In the hypothalamus the prostaglandins, especially PGE2, stimulate the production of neurotransmitters, which function to reset the temperature set point at a higher level. NSAIDs, including aspirin, reduce fever by inhibiting cyclooxygenase and thus blocking prostaglandin synthesis. 2. Elevated plasma levels of acute-phase proteins. These plasma proteins are mostly synthesized in the liver, and in the setting of acute inflammation, their concentrations may increase several hundred-fold. Three of the best known of these proteins are C-reactive protein (CRP), Fibrinogen, and Serum Amyloid A (SAA) protein. Synthesis of these molecules by hepatocytes is stimulated by cytokines, especially IL-6. a. Many acute-phase proteins, such as CRP and SAA, bind to microbial cell walls, and they may act as opsonins and fix complement, thus promoting the elimination of the microbes. Elevated serum levels of CRP are now used as a marker for increased risk of myocardial infarction or stroke in patients with atherosclerotic vascular disease. It is believed that inflammation is involved in the development of atherosclerosis, and increased CRP is a measure of inflammation. b. Fibrinogen binds to erythrocytes and causes them to form stacks (rouleaux) that sediment more rapidly at unit gravity than individual erythrocytes. This is the basis for measuring the erythrocyte sedimentation rate (ESR) as a simple test for the systemic inflammatory response, caused by any number of stimuli, including LPS. Serial measurements of ESR and CRP are used to assess therapeutic responses in patients with inflammatory disorders such as rheumatoid arthritis. 3. White blood cell abnormalities in inflammation/an acute phase response . Initially caused by TNF & IL-1 from bone marrow post mitotic reserve; in prolonged infection, CSF induces proliferation of bone marrow precursors. a. Normal white blood cell count (WBC): 4,000 to 11,000 cells/mL. b. Leukocytosis: WBC usually climbs to 15,000 to 20,000 cells/mL Leukocytosis (an increase in the White Blood Cells [WBC] count) occurs initially because of accelerated release of cells (under the influence of cytokines, including TNF and IL-1) from the bone marrow postmitotic reserve pool. c. Leukemoid reactions (similar to leukemia): Leukocytosis of 40,000 to 100,000 cells/mL d. Left Shift: Both mature and immature neutrophils may be seen in the blood. e. Neutrophilia: An increase in the blood neutrophil (bacterial infection). f. Lymphocytosis: An increased numbers of lymphocytes which may be associated with viral infections (infectious mononucleosis, mumps,& German measles). g. Eosinophilia: An increase in the absolute number of eosinophils (bronchial asthma, hay fever, & parasite infestations). h. Leukopenia: A decreased number of circulating white cells (typhoid fever and infections caused by some viruses, rickettsiae, and certain protozoa) likely because of cytokine-induced sequestration of lymphocytes in lymph nodes. Prolonged infection also stimulates production of colony-stimulating factors (CSFs), which increase the bone marrow output of leukocytes, thus compensating for the consumption of these cells in the inflammatory reaction. 4. Other manifestations of the acute-phase response include increased heart rate and blood pressure; decreased sweating, mainly as a result of redirection of blood flow from cutaneous to deep vascular beds, to minimize heat loss through the skin; and rigors (shivering), chills (perception of being cold as the hypothalamus resets the body temperature), anorexia, somnolence, and malaise, probably secondary to the actions of cytokines on brain cells. 5. In severe bacterial infections (sepsis), the large amounts of bacterial products in the blood or extravascular tissue stimulate the production of enormous quantities of several cytokines, notably TNF and IL-1. The high blood levels of cytokines (notably TNF) cause widespread clinical and pathological abnormalities such as (1) disseminated intravascular coagulation, (2) hypotension, & (3) metabolic disturbances including insulin resistance and hyperglycemia; this clinical triad is known as septic shock. 6. Systemic inflammatory Response Syndrome (SIRS). SIRS is defined as a clinical syndrome that is a form of dysregulated inflammation which has routinely been associated with both infectious processes (to include sepsis) and noninfectious insults (autoimmune disorder, vasculitis, thromboembolism, burns, trauma or surgery). SIRS is defined as having 2 or more of the following: 17 ➢ ➢ ➢ ➢ Fever of more than 38oC(100.4oF) or less than 36oC (96.8oF) Heart rate more than 90 beats per minute Respiratory rate > 29 breaths/minute or arterial carbon dioxide tension (PaCO 2) < 32 m Hg Abnormal white blood cell count (>12000/μL or >10% immature (band) forms a. Systemic Inflammatory Syndrome (SIRS). Another form of dysregulated inflammation caused large amounts of cytokines (especially TNF), but may occur as a complication of noninfectious disorders, such as pancreatitis, vasculitis, severe burns, trauma, surgery. Two or more of the following defines SIRS: higher than 100.4oF (38oC) or lower of 96.8oF (36oC) (normal: 98.7oF or 37oC);Heart rate >90 beats per minute; Respiratory Rate>20 breaths per minute (normal: 12-15 per minute) or PaCO 2 (partial pressure of CO2<32 (normally 40); WBC count >12,000 or <4,000 (normal 4,000 to 10,000). Sepsis is often defined as SIRS in the setting of infection. Multiple organ dysfunction syndrome (MODS) is a progressive physiologic dysfunction in ≥2 organs or organ systems, that can occur at the severe end of SIRS or sepsis. VII. Overview of Tissue Repair A. Tissue repair (AKA healing) is the restoration of tissue architecture and function after injury. It begins even before the inflammatory process ends and it involves two types of reactions: (1) Regeneration of the injured tissue, and (2) Scar formation by the deposition of connective tissue. 1. Regeneration: The replacement of damaged cells and return to a normal state. Regeneration occurs by proliferation of residual (uninjured) cells that retain the capacity to divide, and by replacement from tissue stem cells. It is the typical response to injury by the rapidly dividing epithelia of the skin and intestines, and some parenchymal organs, notably the liver. 2. Scar Formation. If the injured tissues are incapable of regeneration, or if the supporting structures of the tissue are severely damaged, repair occurs by the laying down of connective (fibrous) tissue, a process that results in scar formation. a. Fibrous scar cannot perform the function of lost parenchymal cells, but it serves to provide structural stability so that the injured tissue is usually able to function. b. Fibrosis is most often used to describe the extensive deposition of collagen that occurs in the lungs, liver, kidney, and other organs as a consequence of chronic inflammation, or in the myocardium after extensive ischemic necrosis (infarction). c. Organization describes fibrosis which develops in a tissue space occupied by an inflammatory exudate, (as in organizing pneumonia affecting the lung). B. After many common types of injury, both regeneration and scar formation contribute in varying degrees to the ultimate repair. Both processes involve the proliferation of various cells and close interactions between cells and the ECM. VIII. Cell and Tissue Regeneration The regeneration of injured cells and tissues involves cell proliferation, which is driven by growth factors and is critically dependent on the integrity of the Extracellular Matrix (ECM). ► Several cell types proliferate during tissue repair: a. The remnants of the injured tissue (which attempt to restore normal structure). b. Vascular endothelial cells (to create new vessels that provide the nutrients needed for the repair process). c. Fibroblasts (the source of the fibrous tissue that forms the scar to fill defects that cannot be corrected by regeneration). ► The proliferation of these cell types is driven by proteins called growth factors. The production of polypeptide growth factors and the ability of cells to divide in response to these factors are important determinants of the adequacy of the repair process. ► Cell and tissue regeneration occurs within the framework of the Extracellular Matrix. A. Proliferative Capabilities of Tissues 1. The intrinsic proliferative capability of tissues critically influences their repair capability. The tissues of the body are divided into three groups based on proliferative capability: a. Labile (continuously dividing) tissues. Cells of these tissues are continuously being lost and replaced by maturation from stem cells and by proliferation of residual immature cells. Labile cells include hematopoietic cells in the bone marrow and the majority of surface epithelia, such as the stratified squamous surfaces of the skin, oral cavity, vagina, and cervix; the cuboidal epithelia of the ducts draining exocrine organs (e.g., salivary glands, pancreas, biliary tract); the columnar epithelium of the gastrointestinal 18 tract, uterus, and fallopian tubes; and the transitional epithelium of the urinary tract. These tissues can readily regenerate after injury as long as the pool of stem cells is preserved. b. Stable tissues. Cells of these tissues are quiescent and have only minimal replicative activity in their normal state; however, these cells are capable of proliferating in response to injury or loss of tissue mass. Stable cells constitute the parenchyma of most solid tissues, such as liver, kidney, and pancreas. They also include endothelial cells, fibroblasts, and smooth muscle cells; the proliferation of these cells is particularly important in wound healing. With the exception of liver, stable tissues have a limited capacity to regenerate after injury. c. Permanent tissues. The cells of these tissues are considered to be terminally differentiated and nonproliferative in postnatal life. Most neurons and cardiac muscle cells belong to this category. Thus, injury to brain or heart is irreversible and results in a scar, because neurons and cardiac myocytes cannot regenerate. Skeletal muscle is usually classified as a permanent tissue, but satellite cells attached to the endomysial sheath provide some regenerative capacity for this tissue. In permanent tissues, repair is typically dominated by scar formation. 2. With the exception of tissues composed primarily of nondividing permanent cells (e.g., cardiac muscle, nerve), most mature tissues contain variable proportions of three cell types: continuously dividing cells, quiescent cells that can return to the cell cycle, and cells that have lost replicative ability. B. Growth Factors 1. Most growth factors are proteins that stimulate the survival and proliferation of particular cells, and may also promote migration, differentiation, and other cellular responses. a. Growth Factor effects. Growth factors induce cell proliferation by binding to specific receptors and effecting the expression of genes whose products typically have several functions: (1) They promote entry of cells into the cell cycle, (2) They relieve blocks on cell cycle progression (thus promoting replication), (3) They prevent apoptosis, and (4) They enhance the synthesis of cellular proteins in preparation for mitosis. (5) A major activity of growth factors is to stimulate the function of growth control genes, many of which are called proto-oncogenes because mutations in them lead to unrestrained cell proliferation characteristic of cancer (oncogenesis). 2. Growth Factors that contribute to Regeneration and Repair. a. Cells that secrete Growth Factors. Many of the growth factors that are involved in regeneration and repair are produced by macrophages and other cells that are recruited to the site of injury or are activated at this site, as part of the inflammatory process. Other growth factors are produced by parenchymal cells or stromal (connective tissue) cells in response to cell injury. b. The Main Growth Factors involved in Regeneration and Repair. GROWTH FACTOR SOURCES GENERAL FUNCTIONS Mesenchymal cells Vascular endothelial Stimulates proliferation of endothelial growth factor (VEGF) cells; increases vascular permeability. Platelet-derived growth Platelets, macrophages, Chemotactic for neutrophils, factor (PDGF) endothelial cells, smooth macrophages, fibroblasts, and smooth muscle cells, keratinocytes muscle cells; activates and stimulates proliferation of fibroblasts, endothelial, and other cells; stimulates ECM protein synthesis. Fibroblast growth factors Macrophages, mast cells, Chemotactic and mitogenic for (FGFs), including acidic endothelial cells, many other fibroblasts; stimulates angiogenesis and (FGF-1) and basic (FGF-2) cell types ECM protein synthesis. Transforming growth Platelets, T lymphocytes, Chemotactic for leukocytes and factor-β (TGF-β) macrophages, endothelial fibroblasts; stimulates ECM protein cells, keratinocytes, smooth synthesis; suppresses acute muscle cells, fibroblasts inflammation. C. Role of the Extracellular Matrix (ECM) in Tissue Repair. Tissue repair depends not only on growth factor activity but also on interactions between cells and ECM components. 1. ECM Composition and Function. a. Composition. The ECM is a complex of several proteins that assembles into a network that surrounds cells and constitutes a significant proportion of any tissue. ECM sequesters water, providing turgor to soft tissues, and minerals, giving rigidity to bone. 19 b. Function. It regulates the proliferation, movement, and differentiation of the cells living within it, by supplying a substrate for cell adhesion and migration and serving as a reservoir for growth factors. The ECM is constantly being remodeled; its synthesis and degradation accompany morphogenesis, wound healing, chronic fibrosis, and tumor invasion and metastasis. 2. Two types of ECM. ECM occurs in two basic forms: a. Interstitial Matrix. This form of ECM is present in the spaces between cells in connective tissue, and between epithelium and supportive vascular and smooth muscle structures. It is synthesized by mesenchymal cells (e.g., fibroblasts) and tends to form a three-dimensional, amorphous gel. Its major constituents are fibrillar (type I,II,III,V) and nonfibrillar (type IV, VII, IX) collagens, as well as fibronectin, elastin, proteoglycans, hyaluronate, and other elements (described later). b. Basement Membrane. The seemingly random array of interstitial matrix in connective tissues becomes highly organized around epithelial cells, endothelial cells, and smooth muscle cells, forming the specialized basement membrane. The basement membrane lies beneath the epithelium and is synthesized by overlying epithelium and underlying mesenchymal cells; it tends to form a platelike “chicken wire” mesh. Its major constituents are amorphous nonfibrillar type IV collagen and laminin. 3. Components of Extracellular Matrix (ECM). There are three basic components of ECM: a. Fibrous structural proteins which confer tensile strength and recoil (e.g., collagens and elastins). (1) Collagen. The collagens are composed of three separate polypeptide chains braided into a ropelike triple helix. Approximately 30 collagen types have been identified, some of which are unique to specific cells and tissues. (a) Fibrillar Collagens. Some collagen types (e.g., types I, II, III, and V) form fibrils (fibrillar collagens) by virtue of lateral cross-linking of the triple helices. The fibrillar collagens form a major proportion of the connective tissue in healing wounds and particularly in scars. The tensile strength of the fibrillar collagens derives from their cross-linking, which is the result of covalent bonds catalyzed by the enzyme lysyl-oxidase. This process is dependent on vitamin C; therefore, individuals with vitamin C deficiency have skeletal deformities, bleed easily because of weak vascular wall basement membrane, and suffer from poor wound healing (scurvy). Genetic defects in these collagens cause diseases such as osteogenesis imperfecta and Ehlers-Danlos syndrome. (b) Nonfibrillar Collagens. Other collagens are nonfibrillar and may form basement membrane (type IV) or be components of other structures such as intervertebral disks (type IX) or dermal–epidermal junctions (type VII). (2) Elastin. The ability of tissues to recoil and return to a baseline structure after physical stress is conferred by elastic tissue. This is especially important in the walls of large vessels (which must accommodate recurrent pulsatile flow), as well as in the uterus, skin, and ligaments. Morphologically, elastic fibers consist of a central core of elastin surrounded by a meshlike network of fibrillin glycoprotein. Defects in fibrillin synthesis lead to skeletal abnormalities and weakened aortic walls (as in Marfan syndrome). b. Water-hydrated gels which permit resilience and lubrication (e.g., proteoglycans and hyaluronan). (1) Proteoglycans. Proteoglycans form highly hydrated compressible gels conferring resilience and lubrication (such as in the cartilage in joints). Besides providing compressibility to tissues, proteoglycans also serve as reservoirs for growth factors secreted into the ECM (e.g., fibroblast growth factor [FGF], Hepatocyte Growth Factor [HGF]). (2) Hyaluronan (also called hyaluronic acid), a huge mucopolysaccharide without a protein core, binds water, and forms a viscous, gelatin-like matrix. c. Adhesive glycoproteins and Adhesion Receptors, They connect the matrix elements to one another and to cells. Adhesive glycoproteins and adhesion receptors are structurally diverse molecules involved in cell-to-cell adhesion, the linkage of cells to the ECM, and binding between ECM components. The adhesive glycoproteins include fibronectin and laminin. The adhesion receptors, also known as Cell Adhesion Molecules (CAMs), include immunoglobulins, cadherins, selectins, and integrins. 4. Functions of Extracellular Matrix (ECM). The ECM is much more than a space filler around cells. Its various functions include: a. Mechanical support for cell anchorage and cell migration, and maintenance of cell polarity. b. Control of cell proliferation by binding and displaying growth factors and by signaling through cellular receptors of the integrin family. This type of ECM proteins can affect the degree of differentiation of the cells in the tissue, again acting largely through cell surface integrins. c. Scaffolding for tissue renewal. Because maintenance of normal tissue structure requires a basement membrane or stromal scaffold, the integrity of the basement membrane or the stroma of parenchymal cells is critical for the organized regeneration of tissues. Thus, although labile and stable 20 cells are capable of regeneration, disruption of the ECM results in a failure of the tissues to regenerate and repair can be accomplished only by scar formation. d. Establishment of tissue microenvironments. Basement membrane acts as a boundary between epithelium and underlying connective tissue and also forms part of the filtration apparatus in the kidney. D. Role of Regeneration in Tissue Repair. The importance of regeneration in the replacement of injured tissues varies in different types of tissues and with the severity of injury. 1. In labile tissues, such as the epithelia of the intestinal tract and skin, injured cells are rapidly replaced by proliferation of residual cells and differentiation of tissue stem cells provided the underlying basement membrane is intact. 2. Tissue regeneration can occur in parenchymal organs with stable cell populations, but with the exception of the liver, this is usually a limited process. Pancreas, adrenal, thyroid, and lung have some regenerative capacity. 3. The regenerative response of the liver that occurs after surgical removal of hepatic tissue is remarkable and unique among all organs. As much as 40% to 60% of the liver may be removed in a procedure called living-donor transplantation, in which a portion of the liver is resected from a normal person and transplanted into a recipient with end-stage liver disease, or after partial hepatectomy performed for tumor removal. In both situations, the removal of tissue triggers a proliferative response of the remaining hepatocytes (which are normally quiescent), and the subsequent replication of hepatic nonparenchymal cells. 4. The importance of having residual connective tissue framework that is structurally intact. Extensive regeneration or compensatory hyperplasia can occur only if the residual Extracellular Matrix is structurally intact, as after partial surgical resection. By contrast, if the entire tissue is damaged by infection or inflammation, regeneration is incomplete and is accompanied by scarring. For example, extensive destruction of the liver with collapse of the reticulin framework, as occurs in a liver abscess, leads to scar formation even though the remaining liver cells have the capacity to regenerate. IX. Scar Formation If tissue injury is severe or chronic and results in damage to parenchymal cells and epithelia as well as the connective tissue, or if nondividing cells are injured, repair cannot be accomplished by regeneration alone. Under these conditions, repair occurs by replacement of the non regenerated cells with connective tissue, leading to the formation of a scar, or by a combination of regeneration of some cells and scar formation. A. Steps in Scar Formation. 1. Hemostasis. Within minutes after injury, a hemostatic plug comprised of platelets is formed, which stops bleeding and provides a scaffold for infiltrating inflammatory cells. 2. Inflammation. This step is comprised of the typical acute and chronic inflammatory responses. a. Neutrophils and monocytes are recruited to the injury sited during the 6-48 hours after injury. b. Activated leukocytes eliminate the offending agents and clear the debris. c. Macrophages are the central cellular players in the repair process —M1 macrophages clear microbes and necrotic tissue and promote inflammation in a positive feedback loop, and M2 macrophages produce growth factors that stimulate the proliferation of many cell types in the next stage of repair. d. As the injurious agents and necrotic cells are cleared, the inflammation resolves; how this inflammatory flame is extinguished in most situations of injury is still not well defined. 3. Cell proliferation. In the next stage, which takes up to 10 days, several cell types, including epithelial cells, endothelial and other vascular cells, and fibroblasts, proliferate and migrate to close the now-clean wound. Each cell type serves unique functions: a. Epithelial cells respond to locally produced growth factors and migrate over the wound to cover it. b. Endothelial and other vascular cells proliferate to form new blood vessels, a process known as angiogenesis c. Fibroblasts proliferate and migrate into the site of injury and lay down collagen fibers that form the scar. d. Formation of granulation tissue. The combination of proliferating fibroblasts, loose connective 21 tissue, new blood vessels and scattered chronic inflammatory cells, forms a type of tissue that is unique to healing wounds and is called granulation tissue. This term derives from its pink, soft, granular gross appearance, such as that seen beneath the scab of a skin wound. 4. Remodeling. The connective tissue that has been deposited by fibroblasts is reorganized to produce the stable fibrous scar. This process begins 2 to 3 weeks after injury and may continue for months or years. B. Angiogenesis. Angiogenesis is the process of new blood vessel development from existing vessels. It is critical in healing at sites of injury, in the development of collateral circulations at sites of ischemia, and in allowing tumors to increase in size beyond the constraints of their original blood supply. 1. Angiogenesis involves sprouting of new vessels from existing ones, and consists of the following steps: a. Vasodilation in response to NO and increased permeability induced by VEGF. b. Vessel sprout formation due to separation of pericytes from the abluminal surface and breakdown of the basement membrane. c. Migration of endothelial cells toward the area of tissue injury d. Proliferation of endothelial cells just behind the leading front (“tip”) of migrating cells e. Remodeling into capillary tubes f. Mature vessel formation by recruitment of periendothelial cells (pericytes for small capillaries and smooth muscle cells for larger vessels). g. Cessation of angiogenesis by suppression of endothelial proliferation and migration and deposition of the basement membrane. 2. Growth Factors involved in Angiogenesis. Several growth factors contribute to angiogenesis; the most important are Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (FGF2). Other key growth factors include angiopoietins, Platelet-derived Growth Factor (PDGF), and Transforming Growth Factor-Beta (TGF-β). a. Vascular Endothelial Growth Factor (VEGF). Characteristics of VEGFs include: (1) VEGF-A is generally referred to as VEGF and is the major inducer of angiogenesis after injury and in tumors. (2) Of the many inducers of VEGF, hypoxia is the most important; others are platelet-derived growth factor (PDGF), TGF-α, and TGF-β. (3) VEGF stimulates both migration and proliferation of endothelial cells, thus initiating the process of capillary sprouting in angiogenesis. It promotes vasodilation by stimulating the production of NO, and contributes to the formation of the vascular lumen. b. Fibroblast Growth Factor (FGF-2). The FGF family of growth factors has more than 20 members; the best characterized are FGF-1 (acidic FGF) and FGF-2 (basic FGF). (1) FGF-1 functions as a modifier of endothelial cell migration and proliferation, as well as an angiogenic factor. (2) FGF-2 participates in angiogenesis mostly by stimulating the proliferation of endothelial cells. It also promotes the migration of macrophages and fibroblasts to the damaged area, and stimulates epithelial cell migration to cover epidermal wounds. c. Other key angiogenesis growth factors: (1) Angiopoietins Ang1 and Ang2 are growth factors that play a role in angiogenesis and the structural maturation of new vessels. Newly formed vessels need to be stabilized by the recruitment of pericytes and smooth muscle cells and by the deposition of connective tissue. (2) Platelet-derived Growth Factor (PDGF) recruits smooth muscle cells as part of the stabilization process. (3) Transforming Growth Factor-Beta (TGF-β) suppresses endothelial proliferation and migration, and enhances the production of ECM proteins as part of the stabilization process. 3. ECM role in Angiogenesis. ECM proteins participate in the process of vessel sprouting in angiogenesis, largely through interactions with integrin receptors in endothelial cells and by providing the scaffold for vessel growth. Enzymes in the ECM, notably the matrix metalloproteinases (MMPs), degrade the ECM to permit remodeling and extension of the vascular tube. 4. Angiogenesis and edema. Newly formed vessels are leaky because of incomplete interendothelial junctions and because VEGF increases vascular permeability. This leakiness explains why granulation tissue is often edematous and accounts in part for the edema that may persist in healing wounds long after the acute inflammatory response has resolved. C. Activation of Fibroblasts and Deposition of Connective Tissue. 1. The laying down of connective tissue in the scar occurs in two steps: a. Migration and proliferation of fibroblasts into the site of injury. The recruitment and activation 22 of fibroblasts to synthesize connective tissue proteins are driven by many growth factors, including PDGF, FGF-2, and TGF-β. The major source of these factors is inflammatory cells, particularly macrophages, which are present at sites of injury and in granulation tissue. Sites of inflammation are also rich in mast cells, and in the appropriate chemotactic milieu, lymphocytes may be present as well. Each of these cell types can secrete cytokines and growth factors that contribute to fibroblast proliferation and activation. b. Deposition of ECM proteins. As healing progresses, the number of proliferating fibroblasts and new vessels decreases; however, the fibroblasts progressively assume a more synthetic phenotype, so there is increased deposition of ECM. Collagen synthesis, in particular, is critical to the development of strength in a healing wound site. Collagen synthesis by fibroblasts begins early in wound healing (days 3 to 5) and continues for several weeks, depending on the size of the wound. Net collagen accumulation, however, depends not only on increased synthesis but also on diminished collagen degradation. Ultimately, the granulation tissue evolves into a scar composed of largely inactive, spindle-shaped fibroblasts, dense collagen, fragments of elastic tissue, and other ECM components. As the scar matures, there is progressive vascular regression, which eventually transforms the highly vascularized granulation tissue into a pale, largely avascular scar. 2. Growth Factors Involved in ECM Deposition and Scar Formation. Many growth factors (including TGF-β, PDGF, and FGF), and some cytokines are involved in these processes. The major properties of TGFβ, PDGF, and cytokines in ECM deposition and scar formation are: a. Transforming growth factor-β (TGF-β). TGF-β has two main functions: (1) TGF-β stimulates the production of collagen, fibronectin, and proteoglycans, and it inhibits collagen degradation by both decreasing proteinase activity and increasing the activity of tissue inhibitors of proteinases known as TIMPs (discussed later on). (2) TGF-β is an anti-inflammatory cytokine that serves to limit and terminate inflammatory responses. b. Platelet-derived growth factor (PDGF). PDGF is stored in platelets and released on platelet activation and is also produced by endothelial cells, activated macrophages, smooth muscle cells, and many tumor cells. PDGF causes migration and proliferation of fibroblasts and smooth muscle cells and may contribute to the migration of macrophages. c. Cytokines may also function as growth factors and participate in ECM deposition and scar formation. IL-1 and IL-13, for example, act on fibroblasts to stimulate collagen synthesis, and can also enhance the proliferation and migration of fibroblasts D. Remodeling of Connective Tissue. After its synthesis and deposition, the connective tissue in the scar continues to be modified and remodeled. 1. The outcome of the repair process is a balance between synthesis and degradation of ECM proteins. The degradation of collagens and other ECM components is accomplished by a family of Matrix Metalloproteinases (MMPs), which are dependent on zinc ions for their activity. 2. Sources of MMPs. MMPs are produced by a variety of cell types (fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells). 3. Secretion & Regulation of MMPs. MMP synthesis and secretion are regulated by growth factors, cytokines, and other agents. The activity of the MMPs is tightly controlled. They are produced as inactive precursors (zymogens) that must be first activated; this is accomplished by proteases (e.g., plasmin) likely to be present only at sites of injury. In addition, activated MMPs can be rapidly inhibited by specific Tissue Inhibitors of metalloproteinases (TIMPs), produced by most mesenchymal cells. Thus, during scarring, MMPs are activated to remodel the deposited ECM, and then their activity is shut down by the TIMPs. X. Factors That Influence Tissue Repair Tissue repair may be altered by a variety of influences, frequently reducing the quality or adequacy of the reparative process. Variables that modify healing may be extrinsic (e.g., infection) or intrinsic to the injured tissue. Particularly important are infections and diabetes. A. Infection is clinically the most important cause of delay in healing; it prolongs inflammation and potentially increases the local tissue injury. B. Nutrition has profound effects on repair; protein deficiency, for example, and especially vitamin C deficiency inhibit collagen synthesis and retard healing C. Glucocorticoids (steroids) have well-documented anti-inflammatory effects, and their administration may result in weakness of the scar because of inhibition of TGF-β production and diminished fibrosis. In some instances, however, the anti-inflammatory effects of glucocorticoids are desirable. For example, in corneal infections, glucocorticoids are sometimes prescribed (along with antibiotics) to reduce the likelihood of opacity 23 that may result from collagen deposition. D. Mechanical variables such as increased local pressure or torsion may cause wounds to pull apart, or dehisce. E. Poor perfusion, due either to arteriosclerosis and diabetes or to obstructed venous drainage (e.g., in varicose veins), also impairs healing. F. Foreign bodies such as fragments of steel, glass, or even bone impede healing. G. The type and extent of tissue injury affects the subsequent repair. Complete restoration can occur only in tissues composed of stable and labile cells; injury to tissues composed of permanent cells must inevitably result in scarring, as in healing of a myocardial infarct. H. The location of the injury and the character of the tissue in which the injury occurs are also important. For example, inflammation arising in tissue spaces (e.g., pleural, peritoneal, or synovial cavities) develops extensive exudates. Subsequent repair may occur by digestion of the exudate, initiated by the proteolytic enzymes of leukocytes and resorption of the liquefied exudate. This is called resolution, and generally, in the absence of cellular necrosis, normal tissue architecture is restored. In the setting of larger accumulations, however, the exudate undergoes organization: Granulation tissue grows into the exudate, and a fibrous scar ultimately forms. XI. Selected Clinical Examples of Tissue Repair and Fibrosis Thus far we have discussed the general principles and mechanisms of repair by regeneration and scarring. In this section we describe three clinically significant types of repair: (A) The Healing of Skin Wounds (cutaneous wound healing), (B) Fibrosis in Parenchymal Organs, and (C) Tissue Repair Pathology. A. Healing of Skin Wounds Cutaneous wound healing is a process that involves both epithelial regeneration and the formation of connective tissue scar and is thus illustrative of the general principles that apply to healing in all tissues. Depending on the nature and size of the wound, the healing of skin wounds is said to occur by first or second intention. 1. Healing by First Intention. One of the simplest examples of wound repair is the healing of a clean, uninfected surgical incision approximated by surgical sutures. This is referred to as primary union, or healing by first intention. The incision causes only focal disruption of epithelial basement membrane continuity and death of relatively few epithelial and connective tissue cells. Key characteristics of healing by first intention: o Epithelial regeneration is the principal mechanism of repair. o A small scar is formed. o There is minimal wound contraction. The narrow incisional space first fills with fibrin-clotted blood, which then is rapidly invaded by granulation tissue and covered by new epithelium. 2. Healing by Second Intention. When cell or tissue loss is more extensive, such as in large wounds, at sites of abscess formation, ulceration, and ischemic necrosis (infarction) in parenchymal organs, the repair process is more complex and involves a combination of regeneration and scarring. In second intention healing of skin wounds, also known as healing by secondary union, the inflammatory reaction is more intense, and there is development of abundant granulation tissue, with accumulation of ECM and formation of a large scar, followed by wound contraction mediated by the action of myofibroblasts. Secondary healing differs from primary healing in several respects: • Larger clot or scab rich in fibrin and fibronectin forms at the surface of the wound. • Inflammation is more intense because large tissue defects have a greater volume of necrotic debris, exudate, and fibrin that must be removed. Consequently, large defects have a greater potential for secondary, inflammation-mediated, injury. • Larger defects require a greater volume of granulation tissue to fill in the gaps and provide the underlying framework for the regrowth of tissue epithelium. A greater volume of granulation tissue generally results in a greater mass of scar tissue. • Secondary healing involves significant wound contraction. Within 6 weeks, for example, large skin defects may be reduced to 5% to 10% of their original size, largely by contraction. This process has been ascribed to the presence of myofibroblasts, which are modified fibroblasts exhibiting many of the ultrastructural and functional features of contractile smooth muscle cells. 3. Wound Strength. Initially, carefully sutured wounds have approximately 70% of the strength of normal skin, largely because of the placement of sutures. When nonabsorbable sutures are removed, usually at 7-10 days, wound strength is approximately 10% of that of unwounded skin, but this increases rapidly over the next 4 weeks. The recovery of tensile strength results from collagen synthesis exceeding degradation during the 24 first 2 months, and from structural modifications of collagen (e.g., cross-linking, increased fiber size) when synthesis declines at later times. Wound strength reaches approximately 70% to 80% of normal by 3 months and usually does not improve substantially beyond that point. B. Tissue Repair Pathology. Complications in tissue repair can arise from abnormalities in any of the basic components of the process; the resultant pathology can be grouped into three general categories: 1. Deficient scar formation. Inadequate formation of granulation tissue or formation of a scar can lead to two types of complications: wound dehiscence and ulceration. a. Dehiscence or rupture of a wound, although not common, occurs most frequently after abdominal surgery and is due to increased abdominal pressure. Vomiting, coughing, or ileus can generate mechanical stress on the abdominal wound. b. Wounds can ulcerate because of inadequate vascularization during healing. For example, lower extremity wounds in individuals with atherosclerotic peripheral vascular disease typically ulcerate. c. Nonhealing wounds (neuropathic ulcers) also form in areas devoid of sensation. These neuropathic ulcers are occasionally seen in patients with diabetic peripheral neuropathy. 2. Excessive formation of the repair components. a. Excessive formation of the components of the repair process can give rise to hypertrophic scars and keloids. The accumulation of excessive amounts of collagen may give rise to a raised scar known as a hypertrophic scar; if the scar tissue grows beyond the boundaries of the original wound and does not regress, it is called a keloid. Keloid formation seems to be an individual predisposition, and it is more common in African Americans for unknown reasons. Hypertrophic scars generally develop after thermal or traumatic injury that involves the deep layers of the dermis. b. Exuberant granulation is the formation of excessive amounts of granulation tissue, which protrudes above the level of the surrounding skin and blocks reepithelialization (AKA Proud Flesh). Excessive granulation must be removed by cautery or surgical excision to permit restoration of the continuity of the epithelium. Rarely, incisional scars or traumatic injuries may be followed by exuberant proliferation of fibroblasts and other connective tissue elements which form desmoids or aggressive fibromatoses which may recur after excision. These neoplasms may lie in the interface between benign and malignant (though low-grade) tumors. 3. Formation of contractures. Contraction in the size of a wound is an important part of the normal healing process. An exaggeration of this process gives rise to contracture and results in deformities of the wound and the surrounding tissues. Contractures are particularly prone to develop on the palms, the soles, and the anterior aspect of the thorax. Contractures are commonly seen after serious burns and can compromise the movement of joints. C. Fibrosis in Parenchymal Organs 1. Scar vs Fibrosis in Parenchymal Organs. Deposition of collagen is part of normal wound healing and scar formation. The term fibrosis is used to denote the excessive deposition of collagen and other ECM components in a tissue. Even though the terms scar and fibrosis are used interchangeably, fibrosis most often refers to the extensive abnormal deposition of collagen in internal organs in chronic diseases while scar refers to a more localized deposition of collagen. 2. Fibrosis may result in organ dysfunction/failure. The basic mechanisms of fibrosis are the same as those of scar formation during tissue repair. However, tissue repair typically occurs after a short-lived injurious stimulus and follows an orderly sequence of steps, whereas fibrosis is induced by persistent injurious stimuli such as infections, immunologic reactions, and other types of tissue injury. The fibrosis seen in chronic diseases such as pulmonary fibrosis is often responsible for organ dysfunction and even organ failure. XII. AA AKA AMD Ang cAMP CAMs COX CR CRP CSC CSF DAF Abbreviations Arachidonic Acid Also Known As Age-related Macular Degeneration Angiopoietins (e.g., Ang1 and Ang2) cyclic Adenosine Monophosphate Cell Adhesion Molecules Cyclooxygenase enzyme (e.g., COX-1 and COX-2) Complement Receptor C-Reactive Protein Central Serous Chorioretinopathy Colony Stimulating Factor Decay Accelerating Factor 25 DAMP Danger-Associated Molecular Pattern DIC EC ECM EGF ESR FDP FGF G-CSF GDP GTP H2O2 H&E HETE HGF HLA HMWK HPETE HOCl• Hrs ICAM-1 IFN-γ IgG IL KGF JAKs LAD-1 LAD-2 LFA-1 LPS LT LX MAC Mac-1 MAP Min MIP-1α MMP MPO NADPH NETs NO NOS NSAIDs O●2 PAF PAMP PDGF PECAM-1 PG PMN PRR RBCs ROS SAA SIRS Disseminated Intravascular Coagulation Endothelial Cell Extracellular Matrix Epidermal Growth Factor Erythrocyte Sedimentation Rate Fibrin Degradation Products Fibroblast Growth Factor Granulocyte Colony-Stimulating Factor Guanosine Diphosphate Guanosine Triphosphate Hydrogen Peroxide Hematoxylin and Eosin (Stain) Hydroxyeicosatetraenoic acid Hepatocyte Growth Factor Human Leukocyte Antigen High-Molecular-Weight Kininogen Hydroperoxyeicosatetraenoic acid Hypochlorous radical Hours Intercellular Adhesion Molecule-1 Interferon-γ or Interferon gamma Immunoglobulin G Interleukin Keratinocyte Growth Factor Janus kinases Leukocyte Adhesion Deficiency type 1 Leukocyte Adhesion Deficiency type 2 Leukocyte Function Associated Antigen-I Lipopolysaccharide Leukotriene Lipoxins [e.g., Lipoxin A4 (LXA4) and Lipoxin B4 (LXB4)] Membrane Attack Complex Macrophage-I antigen Mitogen-activated Protein (kinase) Minutes Macrophage Inflammatory Protein-1α Matrix Metalloproteinases Myeloperoxidase Nicotinamide Adenine Dinucleotide Phosphate Neutrophil Extracellular Traps Nitric Oxide Nitric Oxide Synthase [e.g., inducible NOS (iNOS); endothelial NOS (eNOA)] Nonsteroidal anti-inflammatory drugs Superoxide ion Platelet-Activating Factor Pathogen-Associated Molecular Pattern Platelet-Derived Growth Factor Platelet Endothelial Cell Adhesion Molecule-1 Prostaglandin [e.g., prostaglandin E2 (PGE2), PGD2, PGF2α, PGI2 (prostacyclin)] Polymorphonuclear (Leukocyte) Pattern Recognition Receptors Red Blood Cells Reactive Oxygen Species Serum Amyloid Protein Systemic Inflammatory Response Syndrome 26 TGF T H Cells TIMPs TLR TNF TXA2 VCAM-1 VEGF VEGFR Transforming Growth Factor CD4+ helper T cells Tissue Inhibitors of Metalloproteinases Toll-Like Receptor Tumor Necrosis Factor Thromboxane A2 Vascular Cell Adhesion Molecule-1 Vascular Endothelial Growth Factor Vascular Endothelial Growth Factor Receptor (e.g., VEGFR-1, -2, and -3) 27 CDM 1125 – Pathology-I Environmental Pathology Ghaith Al-Eyd MBChB (MD), MSc, PhD Associate Professor of Pathology & Medical Education e mail: galeyd@nova.edu Copyright Notice This session includes images, diagrams, and texts from the references listed in the “References Slide” of this session presentation. The faculty used these materials purely for the purpose of teaching, and in order to protect the copyrights of the publishers of the references, no part of this session may be reproduced or transmitted in any form or by any means for purposes other than teaching/learning. SESSION SESSIONLEARNING LEARNING OBJECTIVES: OUTCOMES: 1. Describe the health effects of climate change. 2. Explain the pathophysiology of toxicities induced by environmental chemical and physical agents. 3. Discuss the health effects of outdoor and indoor air pollution and explain the pathophysiology of toxicities induced by some example pollutants including tobacco, alcohol, lead, mercury, and arsenic. 4. Discuss the adverse reactions of therapeutic and non-therapeutic drugs and describe the effects of selected example drugs including exogenous estrogen, oral contraceptive pills, aspirin, and cocaine. 5. Describe injuries induced by physical agents including those of mechanical trauma, thermal injury, electrical injury and radiation. Environmental Disease: ❖ Many diseases are caused or influenced by environmental factors. Broadly defined, the term ambient environment encompasses the various outdoor, indoor, and occupational settings in which humans live and work. ❖ In each of these settings, the air people breathe, the food and water they consume, and the toxic agents they are exposed to are major determinants of health. ❖ Other environmental factors pertain to the individual (“personal environment”) and include tobacco use, alcohol ingestion, therapeutic and “recreational” drug consumption, diet, and the like. ❖ It is generally believed that factors in the personal environment have a larger effect on human health than that of the ambient environment, but new threats related to global warming may change this equation. Health Effects of Climate Change: ❖ Global temperature measurements show that the earth has warmed significantly since the early 20th century, and especially since the mid-1960s. ❖ Record-breaking global temperatures have become common, with 2005, 2010, 2014, and 2015 each setting successive high- temperature records. ❖ The rising atmospheric and ocean temperatures have led to a large number of effects that include changes in storm frequency, drought, and flood, as well as large-scale ice losses in Greenland, Antarctica, and the vast majority of the other glaciated regions. ❖ Among scientists there is a general acceptance that climate change is, at least in part, man-made. The culprit is the rising atmospheric level of greenhouse gases, particularly carbon dioxide (CO 2 ) released through the burning of fossil fuels, as well as ozone, and methane. Health Effects of Climate Change: ❖ Climate change is expected to have a serious negative impact on human health by increasing the incidence of a number of diseases, including the following: ➢ Cardiovascular, cerebrovascular, and respiratory diseases, all of which will be exacerbated by heat waves and air pollution. ➢ Gastroenteritis, cholera, and other food- and waterborne infectious diseases, caused by contamination as a consequence of floods and disruption of clean water supplies and sewage treatment, after heavy rains and other environmental disasters. ➢ Vector-borne infectious diseases, such as malaria and dengue fever , resulting from changes in vector number and geographic distribution related to increased temperatures, crop failures, and more extreme weather variation. ➢ Malnutrition, caused by changes in local climate that disrupt crop production. Such changes are anticipated to be most severe in tropical locations, in which average temperatures may already be near or above crop tolerance levels. Toxicity of Chemical and Physical Agents: ❖ Xenobiotics are exogenous chemicals in the environment that may be absorbed by the body through inhalation, ingestion, or skin contact ROBBINS BASIC PATHOLOGY, Tenth Edition, Figures 8.2 & 8.3 Outdoor Air Pollution: ❖ Air pollution is a serious problem in many industrialized countries like USA. ❖ The major sources of ambient air pollutants are: ➢ Combustion of fossil fuels: Vehicles, factories, barbeques & fireplaces. ➢ Photochemical reactions: Oxides of nitrogen and volatile hydrocarbons interact in the atmosphere to produce ozone (O3) as a secondary pollutant. ➢ Power plants: Theses release sulfur oxide (SO2) & particulates into the atmosphere. ➢ Waste incinerators, industry, smelters: These point sources release acid aerosols, metals, mercury vapor, and organic compounds that may be hazardous for human health. ❖ Lungs are the major target of common outdoor air pollutants, especially vulnerable are children, asthmatics, and people with chronic lung or heart diseases (decreased lung function, increased airway reactivity , respiratory infections, altered mucociliary clearance). Outdoor Air Pollution: Health Effects of Outdoor Air Pollutants ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.1 Outdoor Air Pollution – Examples of Some Pollutants: ❖ Ozone: It is a major component of smog that accompanies summer heat waves. Exposure of exercising children & adults to as little as 0.08 ppm produces cough, chest discomfort, and inflammation in the lungs. It is a highly reactive agent that oxidizes polyunsaturated lipids to hydrogen peroxide & lipid aldehydes which are irritants that cause inflammatory reactions. ❖ Sulfur Dioxide, particles, and acid aerosols are emitted by coal- and oil-fired power plants and industrial processes burning these fuels. Of these, particles appear to be the main cause of morbidity and death. Particles less than 10 µm in diameter are particularly harmful, because when inhaled they are carried by the airstream all the way to the alveoli . These small particles are phagocytosed by macrophages and neutrophils, causing the release of mediators (possibly by activating inflammasomes), and inciting an inflammatory reaction. Outdoor Air Pollution – Examples of Some Pollutants: ❖ Carbon monoxide (CO): It is a nonirritating, colorless, tasteless, odorless gas produced by the incomplete oxidation of carbonaceous materials. Its sources include automotive engines, industries using fossil fuels, home oil burners, and cigarette smoke: ➢ Low levels (found in ambient air) may contribute to impaired respiratory function but are not life threatening. Chronic exposure in confined environment (e.g. underground garages/tunnels) can cause chronic poisoning. The slowly developing hypoxia can evoke widespread ischemic changes in the brain, particularly in the basal ganglia and lenticular nuclei. ➢ CO is an important cause of accidental & suicidal death (in a small closed garage, exhaust from a running engine can induce a lethal coma within 5 min). CO is a systemic asphyxiant that kills by binding to hemoglobin & preventing oxygen transport. Clinically it is marked by a generalized cherry-red color of the skin and mucous membranes, a color imparted by carboxyhemoglobin. Indoor Air Pollution – Examples of Some Pollutants: ❖ Smoke from burning of organic materials: It contains various oxides of nitrogen and carbon particulates, is an irritant that predisposes exposed persons to lung infections and may contain carcinogenic polycyclic hydrocarbons . It is estimated that one-third of the world, mainly in developing areas, burn carbon-containing material such as wood, dung, or charcoal in their homes for cooking, heating, and light. ❖ Radon: It is a radioactive gas derived from uranium, is widely present in soil and in homes. Although radon exposure can cause lung cancer in uranium miners (particularly in those who smoke), it does not appear that low-level chronic exposures in the home increase lung cancer risk, at least for nonsmokers. ❖ Bioaerosols: They may contain pathogenic microbiologic agents, such as those that can cause Legionnaires' disease, viral pneumonia, and the common cold, as well as allergens derived from pet dander, dust mites, and fungi and molds, which can cause rhinitis, eye irritation, and even asthma. Metals as Environmental Pollutants – Lead: ❖ Lead (Pb)-mining and manufacturing industry: More than 4 million tons of lead are produced each year for use in batteries, alloys, and exterior red lead paint. ❖ Compared with adults, the absorption is greater in children & infants and hence they are particularly vulnerable to lead toxicity. Children can absorb more than 50% from diet in food/water while adults absorb about 15%. A more permeable blood–brain barrier in children creates a high susceptibility to brain damage ❖ Clinically, overt lead poisoning has disappeared due to the reduction of lead from many sources, particularly from gasoline and paints. Environmental sources include: ➢ ➢ ➢ ➢ ➢ Urban air is better with unleaded gasoline. Soil contaminated with lead paint. Batteries. Paint chips. Water supply- plumbing. Metals as Environmental Pollutants – Lead: ❖ Pathophysiology of Lead Toxicity: Lead is a readily absorbed metal that binds to sulfhydryl groups in proteins and interferes with calcium metabolism, leading to hematologic, skeletal, neurologic, GI, and renal toxicities. ❖ A dramatic case of lead contamination of drinking water occurred in the U.S. city of Flint, Michigan, in 2014–2016: ➢ The so-called “Flint water crisis” occurred when the source of water supply to the city was changed from Lake Huron to the Flint River. ➢ Because water from the Flint River had a higher chloride concentration than the lake waters, it leached lead from century-old lead pipes. ➢ This caused an increase in lead levels in tap water above the acceptable limit of 15 parts per billion (ppb) in about 25% of the homes and in some cases as high as 13,200 ppb. ➢ As a result 6000 to 12,000 residents developed very high lead levels in their blood. Metals as Environmental Pollutants – Lead: Lead (Pb) Accumulation: ❖ Most absorbed lead (80% to 85%) is taken up into developing teeth and into bone, where it competes with calcium, binds phosphates, and has a half-life of 20 to 30 years. ❖ About 5% to 10% of the absorbed lead remains in the blood, and the remainder is distributed throughout soft tissues. ❖ Free Erythrocyte Protoporphyrin levels (FEP); FEP is a screening method for Lead. (FEP is increased in lead poisoning). ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.4 Pathologic & Clinical Features of Lead Poisoning Metals as Environmental Pollutants – Lead: Lead poisoning: Impaired remodeling of calcified cartilage in the epiphyses (arrows) of the wrist has caused a marked increase in their radiodensity, so that they are as radiopaque as the cortical bone. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.5 Metals as Environmental Pollutants – Lead: Basophilic Stippling The nucleated RBCs contain basophilic stippling of the cytoplasm. This suggests a toxic injury to the bone marrow, such as lead poisoning. Such stippling may also appear with severe anemia, such as a megaloblastic anemia. Metals as Environmental Pollutants – Lead: ❖ Chronic lead poisoning causes gray lines on the gums (Burton’sQ lines) due to lead accumulation : and reaction with oral bacteria metabolites. Answer: ❖ Patients usually have history of chronic chewing of opium into which lead is added to increase its wight when sold. ❖ Treatment includes chelating agents counseling to stop chewing opium. and Burton’s Line from Chronic Lead Intoxication https://www.nejm.org Metals as Environmental Pollutants – Mercury: ❖ Mercury has had many uses throughout history such as a pigment in cave paintings, a cosmetic, a remedy for syphilis, and a component of diuretics. ❖ Poisoning from inhalation of mercury vapors has long been recognized and is associated with tremor, gingivitis, and bizarre behavior, such as that displayed by the Mad Hatter in Alice in Wonderland. ❖ There are three forms of mercury: metallic mercury (also referred to as elemental mercury), inorganic mercury compounds (mostly mercuric chloride), and organic mercury (mostly methyl mercury). ❖ Today, the main sources of exposure to mercury are contaminated fish (methyl mercury) and mercury vapors released from metallic mercury in dental amalgams, a possible occupational hazard for dental workers. In some areas of the world, mercury used in gold mining has contaminated rivers and streams. ❖ Inorganic mercury from the natural degassing of the earth's crust or from industrial contamination is converted to organic compounds such as methyl mercury by bacteria. Metals as Environmental Pollutants – Mercury: ❖ Pathophysiology of Mercury Toxicity: Mercury, like lead, binds to sulfhydryl groups in certain proteins with high affinity, leading to damage in the CNS and several other organs such as the GI tract and the kidneys. ❖ Ingested mercury can injure the gut and cause ulcerations and bloody diarrhea. In the kidneys, mercury can cause acute tubular necrosis and renal failure. ❖ To protect against potential fetal brain damage, the Centers for Disease Control and Prevention has recommended that pregnant women reduce their consumption of fish known to contain mercury to a minimum . Metals as Environmental Pollutants – Mercury: ❖ Disasters caused by the consumption of fish contaminated by the release of methyl from industrial sources, in Minamata Bay and the Agano River in Japan in 1956, caused widespread mortality and morbidity. ❖ Acute exposure through consumption of bread made from grain treated with a methyl mercury–based fungicide in Iraq in 1971 resulted in hundreds of deaths and thousands of hospitalizations. ❖ The medical disorders associated with the Minamata episode became known as “Minamata disease” and include cerebral palsy, deafness, blindness, mental retardation, and major CNS defects in children exposed in utero. ❖ For unclear reasons, the developing brain is extremely sensitive to methyl mercury . The lipid solubility of methyl mercury and metallic mercury facilitate their accumulation in the brain, disturbing neuromotor, cognitive, and behavioral functions. Metals as Environmental Pollutants – Arsenic: ❖ Pathophysiology of Arsenic Toxicity: Arsenic salts interfere with several aspects of cellular metabolism, leading to toxicities that are most prominent in the GI tract, nervous system, skin, and heart. ❖ Arsenic is found naturally in soil and water and is used in wood preservatives, herbicides, and other agricultural products . It may be released into the environment by the mining and smelting industries. Arsenic is present in Chinese and Indian herbal medicine, and arsenic trioxide is a frontline treatment for acute promyelocytic leukemia. ❖ If ingested in large quantities, arsenic causes acute toxicity manifesting as severe abdominal pain, diarrhea; cardiac arrhythmias, shock and respiratory distress syndrome; and acute encephalopathy. GI, cardiovascular and CNS toxicity may be severe enough to cause death. ❖ These effects may be attributed to the interference with mitochondrial oxidative phosphorylation. Chronic exposure to arsenic causes hyperpigmentation and hyperkeratosis of the skin, which may be followed by the development of basal and squamous cell carcinomas (but not melanomas). A symmetrical sensorimotor polyneuropathy can also develop. Effects of Tobacco: ❖ Tobacco is the most common exogenous cause of human cancers, being responsible for 90% of lung cancers. ❖ The main culprit is cigarette smoking, but smokeless tobacco in its various forms (snuff, chewing tobacco) also is harmful to health and is an important cause of oral cancer. ❖ Not only does the use of tobacco products create personal risk, but also passive tobacco inhalation from the environment (“second-hand smoke”) can cause lung cancer in nonsmokers. ❖ Smoking is the most important cause of preventable human death. It reduces overall survival in a dose-dependent fashion. Whereas 80% of nonsmokers are alive at age 70, only about 50% of smokers survive to this age. ❖ It is a risk factor for development of atherosclerosis and myocardial infarction, peripheral vascular disease, and cerebrovascular disease. In the lungs, in addition to cancer, it predisposes to emphysema, chronic bronchitis, and chronic obstructive disease. Maternal smoking increases the risk of abortion, premature birth, and intrauterine growth retardation. Effects of Tobacco: ROBBINS BASIC PATHOLOGY, Tenth Edition, Tables 8.3 & 8.4; Figure 8.7 Adverse effects of smoking Effects of Alcohol: ❖ Acute alcohol abuse causes drowsiness at blood levels of approximately 200 mg/dL. Stupor and coma develop at higher levels. ❖ Alcohol is oxidized to acetaldehyde in the liver primarily by alcohol dehydrogenase, and to a lesser extent by the cytochrome P-450 system, and by catalase. Acetaldehyde is converted to acetate in mitochondria and is used in the respiratory chain. Alcohol oxidation by alcohol dehydrogenase depletes NAD, leading to accumulation of fat in the liver and to metabolic acidosis. ❖ The main effects of chronic alcoholism are fatty liver, alcoholic hepatitis, and cirrhosis, which leads to portal hypertension and increases the risk for development of hepatocellular carcinoma. ❖ Chronic alcoholism can cause bleeding from gastritis and gastric ulcers, peripheral neuropathy associated with thiamine deficiency, and alcoholic cardiomyopathy, and it increases the risk for development of acute and chronic pancreatitis. ❖ Chronic alcoholism is a major risk factor for cancers of the oral cavity, larynx, and esophagus . The risk is greatly increased by concurrent smoking or the use of smokeless tobacco. Injury by Therapeutic Drugs: Adverse Drug Reactions: ❖ Adverse drug reactions (ADRs) are untoward effects of drugs that are administered in conventional therapeutic settings. Some Common Adverse Drug Reactions and Their Agents ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.5 Exogenous Estrogens and Oral Contraceptives: ❖ The most common type of Menopausal Hormone Therapy (MHT) (previously referred to as hormone replacement therapy, or HRT) consists of the administration of estrogens together with a progestogen . Because of the risk of uterine cancer, estrogen therapy alone is used only in hysterectomized women. ❖ It is reported that MHT increases the risk of breast cancer, stroke, and venous thromboembolism. It may have a protective effect on the development of atherosclerosis and coronary disease in women younger than 60 years of age, but there is no protection in women who started MHT at an older age. ❖ MHT effects depend on the type of hormone therapy regimen used (combination estrogen-progestin versus estrogen alone), the age and risk factor status of the woman at the start of treatment, the duration of the treatment, and possibly the hormone dose, formulation, and route of administration. ❖ Oral Contraceptives (OCs) may increase the risk of cervical carcinomas in women infected with human papillomavirus . They are associated with a threefold to sixfold increased risk of venous thrombosis and pulmonary thromboembolism resulting from increased hepatic synthesis of coagulation factors. They are also associated with development of hepatic adenoma. ❖ OCs do not cause an increase in breast cancer risk and have a protective effect against endometrial & ovarian cancers. They do not increase the risk of coronary artery disease in women younger than 30 years or in older women who are nonsmokers, but the risk approximately doubles in women older than 35 years who smoke. Aspirin & Reye Syndrome: ❖ Reye syndrome is a potentially fatal rare diseases that affects young children with viral infections (varicella or influenza) who are treated with aspirin. ❖ The pathogenesis is unknown; however injury of mitochondria and its dysfunction play a key role (Aspirin metabolites suppress β-oxidation by reversible inhibition of mitochondrial enzymes). ❖ Reye syndrome causes massive diffuse fatty change of the liver (hepatic microvesicular steatosis) & cerebral edema/encephalopathy. Presents with hypoglycemia, elevated liver enzymes, nausea, vomiting, irritability, lethargy, and may progress to coma & death. ❖ 75% of patient may recover completely. Patients who do not recover may have coma, permanent neurological deficits, and death. Treatment is supportive. Injury by Nontherapeutic Agents (Drug Abuse): ❖ Drug abuse generally involves the use of mind-altering substances beyond therapeutic or social norms. Drug addiction and overdose are serious public health problems. ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.6 Injury by Nontherapeutic Agents (Drug Abuse) – Cocaine: ❖ Cocaine produces a sense of intense euphoria and mental alertness, making it one of the most addictive of all drugs. The manifestations of cocaine toxicity include: ➢ Cardiovascular effects: ✓ Cocaine is a sympathomimetic agent that has a net effect of the accumulation of these neurotransmitters in synapses and excessive stimulation, manifested by tachycardia, hypertension, and peripheral vasoconstriction. ✓ Cocaine also induces myocardial ischemia, the basis for which is multifactorial. It causes coronary artery vasoconstriction and promotes thrombus formation by facilitating platelet aggregation. ✓ Cigarette smoking potentiates cocaine-induced coronary vasospasm. ➢ Effects on the fetus: In pregnant women, cocaine may cause decreased blood flow to the placenta, resulting in fetal hypoxia and spontaneous abortion. Neurologic development may be impaired in the fetuses of pregnant women who are chronic drug users. ➢ Chronic cocaine use: may cause perforation of the nasal septum in snorters; decrease in lung diffusing capacity in users who inhale the smoke; and the development of dilated cardiomyopathy. ➢ CNS effects: hyperpyrexia (thought to be caused by aberrations of the dopaminergic pathways that control body temperature) and seizures. Injury by Nontherapeutic Agents (Drug Abuse) – Cocaine: The effect of cocaine on neurotransmission. The drug inhibits reuptake of the neurotransmitters dopamine and norepinephrine in the central and peripheral nervous systems. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.12 Injury by Physical Agents: ❖ Injury induced by physical agents is divided into the following categories: mechanical trauma, thermal injury, electrical injury, and injury produced by ionizing radiation. ❖ Mechanical force may inflict soft tissue injuries, bone injuries & head injuries. ❖ Soft tissue injuries can be superficial involving mainly the skin, or deep, associated with visceral damage: ➢ Abrasions: A scrape, in which the superficial epidermis is torn off by friction or force. Regeneration without scarring usually occurs. ➢ Laceration versus incision: Laceration is an irregular tear in the skin produced by overstretching and it can be linear or stellate depending on the tearing force. Typical of laceration are the bridging strands of fibrous tissue or blood vessels across the wound which are not seen in incision. Incision in contrast, is made by a sharp cutting object, e.g. knife or a piece of glass and has a clean margins. ➢ Contusion: This is an injury caused by a blunt force that damages small blood vessels and causes interstitial bleeding, usually without disruption of the continuity of the tissue. ➢Gunshot wounds: This can be entry or exit gunshot wounds. ➢Vehicular accident: It results from hitting interior parts of the vehicle; being thrown from the vehicle; and being trapped in a burring vehicle. Injury by Physical Agents: (A) Laceration of the scalp: The bridging strands of fibrous tissues are evident. (B) Contusion resulting from blunt trauma: The skin is intact, but hemorrhage of subcutaneous vessels has produced extensive discoloration. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.13 Thermal Injury - Burns: ❖ Both excess heat & excess cold are important cause of injury. Thermal burns are too common where many victims are children scalded by hot liquids. ❖ Burns: The clinical significance of burns depends on depth of the burn, percentage of body surface involved, possible presence of internal injuries from inhalation of hot & toxic fumes, and promptness & efficacy of therapy, especially fluid and electrolyte management and prevention or control of wound infections (pseudomonas aeruginosa, S. aures, Candida). ❖ Full thickness burn involves total destruction of epidermis, dermis, with loss of the dermal appendages that would provide cells for epithelial regeneration (3rd & 4th degree burns). ❖ Partial thickness burns (the deeper portions of the dermal appendages are spared) include 1st degree buns (epidermis involvement only) and 2nd degree burn (both epidermis & dermis involvement). ❖ Morphology: Grossly, full-thickness burns are white or charred, dry, and anesthetic (as a result of the destruction of nerve endings), whereas partial-thickness burns, depending on the depth, are pink or mottled, blistered, and painful . Histologic examination of devitalized tissue shows coagulative necrosis adjacent to vital tissue, which quickly accumulates inflammatory cells and marked exudation. Thermal Injury - Burns: https://monarchmedtech.com Thermal Injury – Hyperthermia: ❖ Prolonged exposure to elevated ambient temperatures can result in: ➢ Heat crumps: Loss of electrolytes through sweating. Cramping of voluntary muscles, usually in association with vigorous exercise, is the hallmark sign. Heat-dissipating mechanisms are able to maintain normal core body temperature . ➢ Heat exhaustion: It is the most common heat syndrome, it is of sudden onset with prostration and collapse resulted from a failure of the CVS to compensate for hypovolemia, secondary to water depletion . ➢ Heat stroke: This is associated with high ambient temperatures and high humidity. Thermoregulatory mechanisms fail, sweating ceases, and core body temperature rises (necrosis of muscles & myocardium may occur). ➢ Malignant hyperthermia: a genetic condition resulting from mutations in genes such as RYR1 that control calcium levels in skeletal muscle cells. In affected individuals, exposure to certain anesthetics during surgery may trigger a rapid rise in calcium levels in skeletal muscle, which in turn leads to muscle rigidity and increased heat production . The resulting hyperthermia has a mortality rate of approximately 80% if untreated, but this falls to less than 5% if the condition is recognized and muscle relaxants are administered promptly. Thermal Injury - Hypothermia: ❖ Prolonged exposure to low ambient temperature leads to hypothermia, a condition seen frequently in homeless persons. ➢ At a body temperature of bout 90o F, loss of consciousness occurs, followed by bradycardia & atrial fibrillation at lower core temperature. ➢ Local reactions include chilling or freezing of cells & tissues (direct physical disruption of organelles within cells or indirect through circulatory changes). Frost bite is an example. ➢ Slowly developing, prolonged chilling may induce vasoconstriction and increased permeability, leading to edema & hypoxia . Such changes are typical of “trench foot.” This condition developed in soldiers who spent long periods of time in waterlogged trenches during the First World War (1914–1918), frequently causing gangrene that necessitated amputation. ➢ Alternatively, with sudden sharp drops in temperature, the vasoconstriction and increased viscosity of the blood in the local area may cause ischemic injury and degenerative changes in peripheral nerves. Electrical Injury: ❖ The passage of electric current through the body may be without effect; may cause sudden death by disruption of neural regulatory impulses, producing e.g. cardiac arrest; or may cause thermal injury to organs interposed in the pathway of the current. ❖ The resistance of tissue & the intensity of current will affect the severity of injury , the greater resistance, the greater the heat generated. e.g. dry skin will be more resistant than wet skin. Thus, the electric current may cause only a surface burn of dry skin. The electric current will be transmitted through the wet skin and may produce ventricular fibrillation or respiratory paralysis. ❖ The thermal effects of the passage of electric current depend on its intensity , e.g. high intensity current as lightening coursing along the skin, produces linear arborizing burns known as lightening marks and when the current is conducted around the victim (flashover) it causes blasting & disruption of clothing but with little injury . When lightening is transmitted internally it will cause steaming & explosion of solid organs. Injury Produced by Radiation: ❖ Radiation is energy that travels in the form of waves or high-speed particles. It has a wide range of energies that span the electromagnetic spectrum; it can be divided into nonionizing and ionizing radiation. ❖ The energy of nonionizing radiation, such as ultraviolet (UV) and infrared light, microwaves, and sound waves, can move atoms in a molecule or cause them to vibrate but is not sufficient to displace electrons from atoms. ❖ By contrast, ionizing radiation has sufficient energy to remove tightly bound electrons. Collision of these free electrons with other atoms releases additional electrons, in a reaction cascade referred to as ionization . ❖ The main sources of ionizing radiation are (1) x-rays and gamma rays, which are electromagnetic waves of very high frequencies, and (2) high-energy neutrons, alpha particles (composed of two protons and two neutrons), and beta particles, which are essentially electrons. Injury Produced by Radiation: ❖ The dose of ionizing radiation is measured in several units; e.g. Roentgen, Rad, Gray, …etc. ❖ In addition to the physical properties of the radioactive material & the dose , the biologic effects of ionizing radiation depend on several factors: ➢ Dose rate: a single dose can cause greater injury than divided dose. ➢ Cell proliferation: since DNA is the most important subcellular target, rapidly dividing cells (e.g. Hematopoietic cells, germ cells, GIT epithelium, ..etc) are more radiosensitive than quiescent cells. Cells in G2 & mitotic phases of the cell cycle are most sensitive. ➢ Field size: smaller doses delivered to larger fields may be lethal. A single dose of external radiation administered to the whole body is more lethal than regional doses with shielding. ➢ Vascular damage: damage to endothelial cells, which are moderately sensitive to radiation, may cause narrowing or occlusion of blood vessels, leading to impaired healing, fibrosis, and chronic ischemic atrophy. Different cells subtypes differ in the extent of their adaptive and reparative responses. ➢ Hypoxia: may reduce the extent of damage and the effectiveness of radiotherapy directed against tumors. Since ionizing radiation produces oxygen-derived radicals from the radiolytic cleavage of water, cell injury induced by x-rays & gamma rays is enhanced by hyperbaric oxygen. Injury Produced by Radiation - Acute Injury & Delayed Complications: ❖ The acute effect of ionizing radiation ranges from overt necrosis at high doses (>10 Gy), killing of proliferating cells at intermediate doses (1-2 Gy), and no histopathologic effect at doses less than 0.5 Gy. ❖ Cells usually shows adaptive & reparative responses to low doses of ionizing radiation while extensive radiation induced DNA damage will lead to apoptosis. ❖ Cells which survive DNA damage, may show delayed effects as mutations, chromosomal aberrations, and genetic instability, these cells will become malignant. ❖ Total body irradiation associated with three syndromes: ➢ Hematopoietic (200-500 rad): nausea, vomiting, lymphopnia, thrombocytopnia, neutropnia, and later anemia. ➢ GIT (500-1000 rad): sever GIT symptoms including diarrhea, hemorrhage, emaciation, and at higher doses ; death within days. ➢ Cerebral (>5000 rad): listlessness and drowsiness followed by convulsions, coma, and death within hours. Injury Produced by Radiation: Effects of ionizing radiation on DNA and their consequences. The effects on DNA can be direct or, most important, indirect, through free radical formation. Ionizing radiation can cause many types of damage in DNA, including single-base damage, single- and double-strand breaks, and crosslinks between DNA and protein. In surviving cells, simple defects may be reparable by various enzyme repair systems. However, double-strand breaks may persist without repair, or the repair of lesions may be imprecise (error prone), creating mutations. If cell-cycle checkpoints are not functioning (for instance, because of mutations in TP53 ), cells with abnormal and unstable genomes survive and may expand as abnormal clones to form tumors eventually. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.14 Injury Produced by Radiation: Overview of the major morphologic consequences of radiation injury . Early changes occur in hours to weeks; late changes occur in months to years. ARDS, Acute respiratory distress syndrome. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.16 Injury Produced by Radiation: Estimated Threshold Doses for Acute Radiation Effects on Specific Organs ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.7 Injury Produced by Radiation – Morphology: Vascular changes and fibrosis of salivary glands produced by radiation therapy of the neck region. (A) Normal salivary gland. (B) fibrosis caused by radiation (C) fibrosis and vascular changes consisting of fibrointimal thickening and arteriolar sclerosis. V, Vessel lumen; I, thickened intima. ❖ At the light microscopic level, vascular changes and interstitial fibrosis are prominent in irradiated tissues. During the immediate postirradiation period, vessels may show only dilation. Later, or with higher doses, a variety of degenerative changes appear, including endothelial cell swelling and vacuolation, or even necrosis of the walls of small vessels such as capillaries and venules. ❖ Affected vessels may rupture or undergo thrombosis. Still later, endothelial cell proliferation and collagenous hyalinization with thickening of the media layer are seen in irradiated vessels, resulting in marked narrowing or obliteration of the vascular lumina. ROBBINS BASIC PATHOLOGY, Tenth Edition, Figure 8.15 References & Recommended Readings ❖ References: ➢ Robbins Basic Pathology, Tenth Edition, 2018, ISBN: 978-0-323-48054-C3 ➢ The Internet Pathology Laboratory for Medical Education Hosted By The University of Utah Eccles Health Sciences Library. ❖ Recommended Readings: ➢ Robbins Basic Pathology, Tenth Edition, 2018 ✓ Chapter 8: Environmental Disease Section CDM 1125 – Pathology-I Environmental Pathology Session’s Text Excerpt Handout Copyright Notice This document may include texts, images, tables, and diagrams from the references listed below. The faculty used these materials purely for the purpose of teaching, and in order to protect the copyrights of the publishers of the references, no part of this session may be reproduced or transmitted in any form or by any means for purposes other than teaching/learning. Reference Textbook: ROBINS BASIC PATHOLOGY, Tenth Edition, 2018, ISBN: 978-0-323-48054-C3 ATTENTION! The following material is intended to facilitate watching the PowerPoint presentation in class and on Shark-Media. To study for the exam, students are required to learn from the session presentation as well as the required textbook listed in the syllabus and found on vital source. Please do not limit yourself to learning only from handouts. Listening to the lecture and studying its images/diagrams/tables is required. Environmental Diseases: Many diseases are caused or influenced by environmental factors. Broadly defined, the term ambient environment encompasses the various outdoor, indoor, and occupational settings in which humans live and work. In each of these settings, the air people breathe, the food and water they consume, and the toxic agents they are exposed to are major determinants of health. Other environmental factors pertain to the individual (“personal environment”) and include tobacco use, alcohol ingestion, therapeutic and “recreational” drug consumption, diet, and the like. It is generally believed that factors in the personal environment have a larger effect on human health than that of the ambient environment, but new threats related to global warming may change this equation. Health Effects of Climate Change: ❖ Global temperature measurements show that the earth has warmed significantly since the early 20th century, and especially since the mid-1960s. ❖ Record-breaking global temperatures have become common, with 2005, 2010, 2014, and 2015 each setting successive high-temperature records. ❖ The rising atmospheric and ocean temperatures have led to a large number of effects that include changes in storm frequency, drought, and flood, as well as large-scale ice losses in Greenland, Antarctica, and the vast majority of the other glaciated regions. ❖ Among scientists there is a general acceptance that climate change is, at least in part, manmade. The culprit is the rising atmospheric level of greenhouse gases, particularly carbon dioxide (CO 2 ) released through the burning of fossil fuels, as well as ozone, and methane. ❖ Climate change is expected to have a serious negative impact on human health by increasing the incidence of a number of diseases, including the following: ❖ Cardiovascular, cerebrovascular, and respiratory diseases, all of which will be exacerbated by heat waves and air pollution. ❖ Gastroenteritis, cholera, and other food- and waterborne infectious diseases, caused by contamination as a consequence of floods and disruption of clean water supplies and sewage treatment, after heavy rains and other environmental disasters. ❖ Vector-borne infectious diseases, such as malaria and dengue fever, resulting from changes in vector number and geographic distribution related to increased temperatures, crop failures, and more extreme weather variation. ❖ Malnutrition, caused by changes in local climate that disrupt crop production. Such changes are anticipated to be most severe in tropical locations, in which average temperatures may already be near or above crop tolerance levels. It is estimated that by 2080, agricultural productivity may decline by 10% to 25% in some developing countries as a consequence of climate change. Toxicity of Chemical and Physical Agents: Toxicology is defined as the science of poisons. It studies the distribution, effects, and mechanisms of action of toxic agents . More broadly, it also includes the study of the effects of physical agents such as radiation and heat. Approximately 4 billion pounds of toxic chemicals, including 72 million pounds of known carcinogens, are produced each year in the United States. In general, however, little is known about the potential health effects of chemicals. Of the approximately 100,000 chemicals in use in the United States, less than 1% have been tested experimentally for health effects. Xenobiotics are exogenous chemicals in the environment that may be absorbed by the body through inhalation, ingestion, or skin contact. Pollutants contained in air, water, and soil are absorbed through the lungs, gastrointestinal (GI) tract, and skin. In the body, they may act at the site of absorption, but they generally are transported through the bloodstream to various organs, where they are stored or metabolized. Metabolism of xenobiotics may result in the formation of water-soluble compounds, which are excreted, or in activation of the agent, creating a toxic metabolite. Xenobiotics can be metabolized to nontoxic metabolites and eliminated from the body (detoxification). However, their metabolism also may result in formation of a reactive metabolite that is toxic to cellular components. If repair is not effective, short- and long-term effects develop. Outdoor Air Pollution: ❖ Air pollution is a serious problem in many industrialized countries like USA. ❖ The major sources of ambient air pollutants are: ➢ Combustion of fossil fuels: Vehicles, factories, barbeques & fireplaces. ➢ Photochemical reactions: Oxides of nitrogen and volatile hydrocarbons interact in the atmosphere to produce ozone (O3) as a secondary pollutant. ➢ Power plants: Theses release sulfur oxide (SO2) & particulates into the atmosphere. ➢ Waste incinerators, industry, smelters: These point sources release acid aerosols, metals, mercury vapor, and organic compounds that may be hazardous for human health. ❖ Lungs are the major target of common outdoor air pollutants, especially vulnerable are children, asthmatics, and people with chronic lung or heart diseases (decreased lung function, increased airway reactivity, respiratory infections, altered mucociliary clearance). Health Effects of Outdoor Air Pollutants ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.1 Outdoor Air Pollution – Examples of Some Pollutants: ❖ Ozone: It is a major component of smog that accompanies summer heat waves. Exposure of exercising children & adults to as little as 0.08 ppm produces cough, chest discomfort, and inflammation in the lungs. It is a highly reactive agent that oxidizes polyunsaturated lipids to hydrogen peroxide & lipid aldehydes which are irritants that cause inflammatory reactions. ❖ Sulfur Dioxide, particles, and acid aerosols are emitted by coal- and oil-fired power plants and industrial processes burning these fuels. Of these, particles appear to be the main cause of morbidity and death. Particles less than 10 µm in diameter are particularly harmful, because when inhaled they are carried by the airstream all the way to the alveoli. These small particles are phagocytosed by macrophages and neutrophils, causing the release of mediators (possibly by activating inflammasomes), and inciting an inflammatory reaction. ❖ Carbon monoxide (CO): It is a nonirritating, colorless, tasteless, odorless gas produced by the incomplete oxidation of carbonaceous materials. Its sources include automotive engines, industries using fossil fuels, home oil burners, and cigarette smoke: ➢ Low levels (found in ambient air) may contribute to impaired respiratory function but are not life threatening. Chronic exposure in confined environment (e.g. underground garages/tunnels) can cause chronic poisoning. The slowly developing hypoxia can evoke widespread ischemic changes in the brain, particularly in the basal ganglia and lenticular nuclei. ➢ CO is an important cause of accidental & suicidal death (in a small closed garage, exhaust from a running engine can induce a lethal coma within 5 min). CO is a systemic asphyxiant that kills by binding to hemoglobin & preventing oxygen transport. Clinically it is marked by a generalized cherry-red color of the skin and mucous membranes, a color imparted by carboxyhemoglobin. Indoor Air Pollution – Examples of Some Pollutants: ❖ Smoke from burning of organic materials: It contains various oxides of nitrogen and carbon particulates, is an irritant that predisposes exposed persons to lung infections and may contain carcinogenic polycyclic hydrocarbons. It is estimated that one-third of the world, mainly in developing areas, burn carbon-containing material such as wood, dung, or charcoal in their homes for cooking, heating, and light. ❖ Radon: It is a radioactive gas derived from uranium, is widely present in soil and in homes. Although radon exposure can cause lung cancer in uranium miners (particularly in those who smoke), it does not appear that low-level chronic exposures in the home increase lung cancer risk, at least for nonsmokers. ❖ Bioaerosols: They may contain pathogenic microbiologic agents, such as those that can cause Legionnaires' disease, viral pneumonia, and the common cold, as well as allergens derived from pet dander, dust mites, and fungi and molds, which can cause rhinitis, eye irritation, and even asthma. ❖ Smoke from burning of organic materials: It contains various oxides of nitrogen and carbon particulates, is an irritant that predisposes exposed persons to lung infections and may contain carcinogenic polycyclic hydrocarbons. It is estimated that one-third of the world, mainly in developing areas, burn carbon-containing material such as wood, dung, or charcoal in their homes for cooking, heating, and light. ❖ Radon: It is a radioactive gas derived from uranium, is widely present in soil and in homes. Although radon exposure can cause lung cancer in uranium miners (particularly in those who smoke), it does not appear that low-level chronic exposures in the home increase lung cancer risk, at least for nonsmokers. ❖ Bioaerosols: They may contain pathogenic microbiologic agents, such as those that can cause Legionnaires' disease, viral pneumonia, and the common cold, as well as allergens derived from pet dander, dust mites, and fungi and molds, which can cause rhinitis, eye irritation, and even asthma. Metals as Environmental Pollutants – Lead: ❖ Lead (Pb)-mining and manufacturing industry: More than 4 million tons of lead are produced each year for use in batteries, alloys, and exterior red lead paint. ❖ Compared with adults, the absorption is greater in children & infants and hence they are particularly vulnerable to lead toxicity. Children can absorb more than 50% from diet in food/water while adults absorb about 15%. A more permeable blood–brain barrier in children creates a high susceptibility to brain damage ❖ Clinically, overt lead poisoning has disappeared due to the reduction of lead from many sources, particularly from gasoline and paints. Environmental sources include: ➢ Urban air is better with unleaded gasoline. ➢ Soil contaminated with lead paint. ➢ Batteries. ➢ Paint chips. ➢ Water supply- plumbing. ❖ Pathophysiology of Lead Toxicity: Lead is a readily absorbed metal that binds to sulfhydryl groups in proteins and interferes with calcium metabolism, leading to hematologic, skeletal, neurologic, GI, and renal toxicities. ❖ A dramatic case of lead contamination of drinking water occurred in the U.S. city of Flint, Michigan, in 2014–2016: ➢ The so-called “Flint water crisis” occurred when the source of water supply to the city was changed from Lake Huron to the Flint River. ➢ Because water from the Flint River had a higher chloride concentration than the lake waters, it leached lead from century-old lead pipes. ➢ This caused an increase in lead levels in tap water above the acceptable limit of 15 parts per billion (ppb) in about 25% of the homes and in some cases as high as 13,200 ppb. ➢ As a result 6000 to 12,000 residents developed very high lead levels in their blood. Lead (Pb) Accumulation: ❖ Most absorbed lead (80% to 85%) is taken up into developing teeth and into bone, where it competes with calcium, binds phosphates, and has a half-life of 20 to 30 years. ❖ About 5% to 10% of the absorbed lead remains in the blood, and the remainder is distributed throughout soft tissues. ❖ Free Erythrocyte Protoporphyrin levels (FEP); FEP is a screening method for Lead. (FEP is increased in lead poisoning). ❖ Excess lead is toxic to nervous tissues in adults and children; peripheral neuropathies predominate in adults, whereas central effects are more common in children. The effects of chronic lead exposure in children may be subtle, producing mild dysfunction, or they may be massive and lethal. In young children, sensory, motor, intellectual, and psychologic impairments have been described, including reduced IQ, learning disabilities, retarded psychomotor development, and, in more severe cases, blindness, psychoses, seizures, and coma. Lead-induced peripheral neuropathies in adults generally remit with the elimination of exposure, but both peripheral and CNS abnormalities in children usually are irreversible. ❖ Excess lead interferes with the normal remodeling of calcified cartilage and primary bone trabeculae in the epiphyses in children, causing increased bone density detected as radiodense “lead lines”. Lead lines of a different sort also may occur in the gums, where excess lead stimulates hyperpigmentation. Lead inhibits the healing of fractures by increasing chondrogenesis and delaying cartilage mineralization. Excretion of lead occurs by way of the kidneys, and acute exposures may cause damage to proximal. ❖ Lead has a high affinity for sulfhydryl groups and interferes with two enzymes involved in heme synthesis: aminolevulinic acid dehydratase and delta ferrochelatase. Iron incorporation into heme is impaired, leading to anemia. Lead also inhibits sodium- and potassium-dependent ATPases in cell membranes, an effect that may increase the fragility of red cells, causing hemolysis. The diagnosis of lead poisoning requires constant vigilance. It may be suspected on the basis of neurologic changes in children or unexplained anemia with basophilic stippling in red cells in adults and children. Elevated blood lead and red cell free protoporphyrin levels (greater than 50 µg/dL) or, alternatively, zinc-protoporphyrin levels, are required for definitive diagnosis. In milder cases of lead exposure, anemia may be the only obvious abnormality. ❖ Chronic lead poisoning causes gray lines on the gums (Burton’s lines) due to lead accumulation and reaction with oral bacteria metabolites. Such patients usually have history of chronic chewing of opium into which lead is added to increase its wight when sold. Metals as Environmental Pollutants – Mercury: ❖ Mercury has had many uses throughout history such as a pigment in cave paintings, a cosmetic, a remedy for syphilis, and a component of diuretics. ❖ Poisoning from inhalation of mercury vapors has long been recognized and is associated with tremor, gingivitis, and bizarre behavior, such as that displayed by the Mad Hatter in Alice in Wonderland. ❖ There are three forms of mercury: metallic mercury (also referred to as elemental mercury), inorganic mercury compounds (mostly mercuric chloride), and organic mercury (mostly methyl mercury). ❖ Today, the main sources of exposure to mercury are contaminated fish (methyl mercury) and mercury vapors released from metallic mercury in dental amalgams, a possible occupational hazard for dental workers. In some areas of the world, mercury used in gold mining has contaminated rivers and streams. ❖ Inorganic mercury from the natural degassing of the earth's crust or from industrial contamination is converted to organic compounds such as methyl mercury by bacteria. ❖ Pathophysiology of Mercury Toxicity: Mercury, like lead, binds to sulfhydryl groups in certain proteins with high affinity, leading to damage in the CNS and several other organs such as the GI tract and the kidneys. ❖ Ingested mercury can injure the gut and cause ulcerations and bloody diarrhea. In the kidneys, mercury can cause acute tubular necrosis and renal failure. ❖ To protect against potential fetal brain damage, the Centers for Disease Control and Prevention has recommended that pregnant women reduce their consumption of fish known to contain mercury to a minimum. ❖ Disasters caused by the consumption of fish contaminated by the release of methyl from industrial sources, in Minamata Bay and the Agano River in Japan in 1956, caused widespread mortality and morbidity. ❖ Acute exposure through consumption of bread made from grain treated with a methyl mercury–based fungicide in Iraq in 1971 resulted in hundreds of deaths and thousands of hospitalizations. ❖ The medical disorders associated with the Minamata episode became known as “Minamata disease” and include cerebral palsy, deafness, blindness, mental retardation, and major CNS defects in children exposed in utero. ❖ For unclear reasons, the developing brain is extremely sensitive to methyl mercury. The lipid solubility of methyl mercury and metallic mercury facilitate their accumulation in the brain, disturbing neuromotor, cognitive, and behavioral functions. Metals as Environmental Pollutants – Arsenic: ❖ Pathophysiology of Arsenic Toxicity: Arsenic salts interfere with several aspects of cellular metabolism, leading to toxicities that are most prominent in the GI tract, nervous system, skin, and heart. ❖ Arsenic is found naturally in soil and water and is used in wood preservatives, herbicides, and other agricultural products. It may be released into the environment by the mining and smelting industries. Arsenic is present in Chinese and Indian herbal medicine, and arsenic trioxide is a frontline treatment for acute promyelocytic leukemia. ❖ If ingested in large quantities, arsenic causes acute toxicity manifesting as severe abdominal pain, diarrhea; cardiac arrhythmias, shock and respiratory distress syndrome; and acute encephalopathy. GI, cardiovascular and CNS toxicity may be severe enough to cause death. ❖ These effects may be attributed to the interference with mitochondrial oxidative phosphorylation. Chronic exposure to arsenic causes hyperpigmentation and hyperkeratosis of the skin, which may be followed by the development of basal and squamous cell carcinomas (but not melanomas). A symmetrical sensorimotor polyneuropathy can also develop. Effects of Tobacco: ❖ Tobacco is the most common exogenous cause of human cancers, being responsible for 90% of lung cancers. ❖ The main culprit is cigarette smoking, but smokeless tobacco in its various forms (snuff, chewing tobacco) also is harmful to health and is an important cause of oral cancer. ❖ Not only does the use of tobacco products create personal risk, but also passive tobacco inhalation from the environment (“second-hand smoke”) can cause lung cancer in nonsmokers. ❖ Smoking is the most important cause of preventable human death. It reduces overall survival in a dose-dependent fashion. Whereas 80% of nonsmokers are alive at age 70, only about 50% of smokers survive to this age. ❖ It is a risk factor for development of atherosclerosis and myocardial infarction, peripheral vascular disease, and cerebrovascular disease. In the lungs, in addition to cancer, it predisposes to emphysema, chronic bronchitis, and chronic obstructive disease. Maternal smoking increases the risk of abortion, premature birth, and intrauterine growth retardation. ROBBINS BASIC PATHOLOGY, Tenth Edition, Tables 8.3 & 8.4; Figure 8.7 Adverse effects of smoking: ❖ ❖ ❖ ❖ Cancers: Oral cavity, larynx, esophagus, lung, pancreas, and bladder Chronic bronchitis, emphysema Systemic atherosclerosis; Myocardial infarction Peptic ulcer Effects of Alcohol: ❖ Acute alcohol abuse causes drowsiness at blood levels of approximately 200 mg/dL. Stupor and coma develop at higher levels. ❖ Alcohol is oxidized to acetaldehyde in the liver primarily by alcohol dehydrogenase, and to a lesser extent by the cytochrome P-450 system, and by catalase. Acetaldehyde is converted to acetate in mitochondria and is used in the respiratory chain. Alcohol oxidation by alcohol dehydrogenase depletes NAD, leading to accumulation of fat in the liver and to metabolic acidosis. ❖ The main effects of chronic alcoholism are fatty liver, alcoholic hepatitis, and cirrhosis, which leads to portal hypertension and increases the risk for development of hepatocellular carcinoma. ❖ Chronic alcoholism can cause bleeding from gastritis and gastric ulcers, peripheral neuropathy associated with thiamine deficiency, and alcoholic cardiomyopathy, and it increases the risk for development of acute and chronic pancreatitis. ❖ Chronic alcoholism is a major risk factor for cancers of the oral cavity, larynx, and esophagus. The risk is greatly increased by concurrent smoking or the use of smokeless tobacco. Injury by Therapeutic Drugs: Adverse Drug Reactions: ❖ Adverse drug reactions (ADRs) are untoward effects of drugs that are administered in conventional therapeutic settings. Some Common Adverse Drug Reactions and Their Agents ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.5 Exogenous Estrogens and Oral Contraceptives: ❖ The most common type of Menopausal Hormone Therapy (MHT) (previously referred to as hormone replacement therapy, or HRT) consists of the administration of estrogens together with a progestogen. Because of the risk of uterine cancer, estrogen therapy alone is used only in hysterectomized women. ❖ It is reported that MHT increases the risk of breast cancer, stroke, and venous thromboembolism. It may have a protective effect on the development of atherosclerosis and coronary disease in women younger than 60 years of age, but there is no protection in women who started MHT at an older age. ❖ MHT effects depend on the type of hormone therapy regimen used (combination estrogen-progestin versus estrogen alone), the age and risk factor status of the woman at the start of treatment, the duration of the treatment, and possibly the hormone dose, formulation, and route of administration. ❖ Oral Contraceptives (OCs) may increase the risk of cervical carcinomas in women infected with human papillomavirus. They are associated with a threefold to sixfold increased risk of venous thrombosis and pulmonary thromboembolism resulting from increased hepatic synthesis of coagulation factors. They are also associated with development of hepatic adenoma. ❖ OCs do not cause an increase in breast cancer risk and have a protective effect against endometrial & ovarian cancers. They do not increase the risk of coronary artery disease in women younger than 30 years or in older women who are nonsmokers, but the risk approximately doubles in women older than 35 years who smoke. Aspirin & Reye Syndrome: ❖ Reye syndrome is a potentially fatal rare diseases that affects young children with viral infections (varicella or influenza) who are treated with aspirin. ❖ The pathogenesis is unknown; however injury of mitochondria and its dysfunction play a key role (Aspirin metabolites suppress β-oxidation by reversible inhibition of mitochondrial enzymes). ❖ Reye syndrome causes massive diffuse fatty change of the liver (hepatic microvesicular steatosis) & cerebral edema/encephalopathy. Presents with hypoglycemia, elevated liver enzymes, nausea, vomiting, irritability, lethargy, and may progress to coma & death. ❖ 75% of patient may recover completely. Patients who do not recover may have coma, permanent neurological deficits, and death. Treatment is supportive. Injury by Nontherapeutic Agents (Drug Abuse): ❖ Drug abuse generally involves the use of mind-altering substances beyond therapeutic or social norms. Drug addiction and overdose are serious public health problems. ROBBINS BASIC PATHOLOGY, Tenth Edition, Table 8.6 Injury by Nontherapeutic Agents (Drug Abuse) – Cocaine: ❖ In 2014, it was estimated that there were 1.5 million users (highest among adults 18-25 Yr. of age) of cocaine in the United States, of which approximately 15% to 20% were users of “crack” cocaine. ❖ Cocaine produces a sense of intense euphoria and mental alertness, making it one of the most addictive of all drugs. The manifestations of cocaine toxicity include: ➢ Cardiovascular effects: ✓ Cocaine is a sympathomimetic agent that has a net effect of the accumulation of these neurotransmitters in synapses and excessive stimulation, manifested by tachycardia, hypertension, and peripheral vasoconstriction. ✓ Cocaine also induces myocardial ischemia, the basis for which is multifactorial. It causes coronary artery vasoconstriction and promotes thrombus formation by facilitating platelet aggregation. ✓ Cigarette smoking potentiates cocaine-induced coronary vasospasm. ➢ Effects on the fetus: In pregnant women, cocaine may cause decreased blood flow to the placenta, resulting in fetal hypoxia and spontaneous abortion. Neurologic development may be impaired in the fetuses of pregnant women who are chronic drug users. ➢ Chronic cocaine use may cause: perforation of the nasal septum in snorters; decrease in lung diffusing capacity in users who inhale the smoke; and the development of dilated cardiomyopathy. ➢ CNS effects: hyperpyrexia (thought to be caused by aberrations of the dopaminergic pathways that control body temperature) and seizures. ➢ The effect of cocaine on neurotransmission. The drug inhibits reuptake of the neurotransmitters dopamine and norepinephrine in the central and peripheral nervous systems. Injury by Physical Agents: ❖ Injury induced by physical agents is divided into the following categories: mechanical trauma, thermal injury, electrical injury, and injury produced by ionizing radiation. ❖ Mechanical force may inflict soft tissue injuries, bone injuries & head injuries. ❖ Soft tissue injuries can be superficial involving mainly the skin, or deep, associated with visceral damage: ➢ Abrasions: A scrape, in which the superficial epidermis is torn off by friction or force. Regeneration without scarring usually occurs. ➢ Laceration versus incision: Laceration is an irregular tear in the skin produced by overstretching and it can be linear or stellate depending on the tearing force. Typical of laceration are the bridging strands of fibrous tissue or blood vessels across the wound which are not seen in incision. Incision in contrast, is made by a sharp cutting object, e.g. knife or a piece of glass and has a clean margins. ➢ Contusion: This is an injury caused by a blunt force that damages small blood vessels and causes interstitial bleeding, usually without disruption of the continuity of the tissue. ➢ Gunshot wounds: This can be entry or exit gunshot wounds. ➢ Vehicular accident: It results from hitting interior parts of the vehicle; being thrown from the vehicle; and being trapped in a burring vehicle. Thermal Injury - Burns: ❖ Both excess heat & excess cold are important cause of injury. Thermal burns are too common where many victims are children scalded by hot liquids. ❖ Burns: The clinical significance of burns depends on depth of the burn, percentage of body surface involved, possible presence of internal injuries from inhalation of hot & toxic fumes, and promptness & efficacy of therapy, especially fluid and electrolyte management and prevention or control of wound infections (pseudomonas aeruginosa, S. aures, Candida). ❖ Full thickness burn involves total destruction of epidermis, dermis, with loss of the dermal appendages that would provide cells for epithelial regeneration (3rd & 4th degree burns). ❖ Partial thickness burns (the deeper portions of the dermal appendages are spared) include 1st degree buns (epidermis involvement only) and 2nd degree burn (both epidermis & dermis involvement). ❖ Morphology: Grossly, full-thickness burns are white or charred, dry, and anesthetic (as a result of the destruction of nerve endings), whereas partial-thickness burns, depending on the depth, are pink or mottled, blistered, and painful. Histologic examination of devitalized tissue shows coagulative necrosis adjacent to vital tissue, which quickly accumulates inflammatory cells and marked exudation. Thermal Injury – Hyperthermia: ❖ Prolonged exposure to elevated ambient temperatures can result in: ➢ Heat crumps: Loss of electrolytes through sweating. Cramping of voluntary muscles, usually in association with vigorous exercise, is the hallmark sign. Heatdissipating mechanisms are able to maintain normal core body temperature. ➢ Heat exhaustion: It is the most common heat syndrome, it is of sudden onset with prostration and collapse resulted from a failure of the CVS to compensate for hypovolemia, secondary to water depletion. ➢ Heat stroke: This is associated with high ambient temperatures and high humidity. Thermoregulatory mechanisms fail, sweating ceases, and core body temperature rises (necrosis of muscles & myocardium may occur). ➢ Malignant hyperthermia: a genetic condition resulting from mutations in genes such as RYR1 that control calcium levels in skeletal muscle cells. In affected individuals, exposure to certain anesthetics during surgery may trigger a rapid rise in calcium levels in skeletal muscle, which in turn leads to muscle rigidity and increased heat production. The resulting hyperthermia has a mortality rate of approximately 80% if untreated, but this falls to less than 5% if the condition is recognized and muscle relaxants are administered promptly. Thermal Injury - Hypothermia: ❖ Prolonged exposure to low ambient temperature leads to hypothermia, a condition seen frequently in homeless persons. ➢ At a body temperature of about 90o F, loss of consciousness occurs, followed by bradycardia & atrial fibrillation at lower core temperature. ➢ Local reactions include chilling or freezing of cells & tissues (direct physical disruption of organelles within cells or indirect through circulatory changes). Frost bite is an example. ➢ Slowly developing, prolonged chilling may induce vasoconstriction and increased permeability, leading to edema and hypoxia. Such changes are typical of “trench foot.” This condition developed in soldiers who spent long periods of time in waterlogged trenches during the First World War (1914–1918), frequently causing gangrene that necessitated amputation. ➢ Alternatively, with sudden sharp drops in temperature, the vasoconstriction and increased viscosity of the blood in the local area may cause ischemic injury and degenerative changes in peripheral nerves. Electrical Injury: ❖ The passage of electric current through the body may be without effect; may cause sudden death by disruption of neural regulatory impulses, producing e.g. cardiac arrest; or may cause thermal injury to organs interposed in the pathway of the current. ❖ The resistance of tissue & the intensity of current will affect the severity of injury, the greater resistance, the greater the heat generated. e.g. dry skin will be more resistant than wet skin. Thus, the electric current may cause only a surface burn of dry skin. The electric current will be transmitted through the wet skin and may produce ventricular fibrillation or respiratory paralysis. ❖ The thermal effects of the passage of electric current depend on its intensity, e.g. high intensity current as lightening coursing along the skin, produces linear arborizing burns known as lightening marks and when the current is conducted around the victim (flashover) it causes blasting & disruption of clothing but with little injury. When lightening is transmitted internally it will cause steaming & explosion of solid organs. Injury Produced by Radiation: ❖ Radiation is energy that travels in the form of waves or high-speed particles. It has a wide range of energies that span the electromagnetic spectrum; it can be divided into nonionizing and ionizing radiation. ❖ The energy of nonionizing radiation, such as ultraviolet (UV) and infrared light, microwaves, and sound waves, can move atoms in a molecule or cause them to vibrate but is not sufficient to displace electrons from atoms. ❖ By contrast, ionizing radiation has sufficient energy to remove tightly bound electrons. Collision of these free electrons with other atoms releases additional electrons, in a reaction cascade referred to as ionization. ❖ The main sources of ionizing radiation are (1) x-rays and gamma rays, which are electromagnetic waves of very high frequencies, and (2) high-energy neutrons, alpha particles (composed of two protons and two neutrons), and beta particles, which are essentially electrons. ❖ The dose of ionizing radiation is measured in several units; e.g. Roentgen, Rad, Gray, …etc. ❖ In addition to the physical properties of the radioactive material & the dose, the biologic effects of ionizing radiation depend on several factors: ➢ Dose rate: a single dose can cause greater injury than divided dose. ➢ Cell proliferation: since DNA is the most important subcellular target, rapidly dividing cells (e.g. Hematopoietic cells, germ cells, GIT epithelium, ..etc) are more radiosensitive than quiescent cells. Cells in G2 & mitotic phases of the cell cycle are most sensitive. ➢ Field size: smaller doses delivered to larger fields may be lethal. A single dose of external radiation administered to the whole body is more lethal than regional doses with shielding. ➢ Vascular damage: damage to endothelial cells, which are moderately sensitive to radiation, may cause narrowing or occlusion of blood vessels, leading to impaired ble 8.7 healing, fibrosis, and chronic ischemic atrophy. Different cells subtypes differ in the extent of their adaptive and reparative responses. ➢ Hypoxia: may reduce the extent of damage and the effectiveness of radiotherapy directed against tumors. Since ionizing radiation produces oxygen-derived radicals from the radiolytic cleavage of water, cell injury induced by x-rays & gamma rays is enhanced by hyperbaric oxygen. Injury Produced by Radiation - Acute Injury & Delayed Complications: ❖ The acute effect of ionizing radiation ranges from overt necrosis at high doses (>10 Gy), killing of proliferating cells at intermediate doses (1-2 Gy), and no histopathologic effect at doses less than 0.5 Gy. ❖ Cells usually shows adaptive & reparative responses to low doses of ionizing radiation while extensive radiation induced DNA damage will lead to apoptosis. ❖ Cells which survive DNA damage, may show delayed effects as mutations, chromosomal aberrations, and genetic instability, these cells will become malignant. ❖ Total body irradiation associated with three syndromes: ➢ Hematopoietic (200-500 rad): nausea, vomiting, lymphopnia,thrombocytopnia, neutropnia, and later anemia. ➢ GIT (500-1000 rad): sever GIT symptoms including diarrhea, hemorrhage, emaciation, and at higher doses ; death within days. ➢ Cerebral (>5000 rad): listlessness and drowsiness followed by convulsions, coma, and death within hours. Effects of ionizing radiation on DNA and their consequences: ❖ The effects on DNA can be direct or, most important, indirect, through free radical formation. ❖ Ionizing radiation can cause many types of damage in DNA, including single-base damage, single- and double-strand breaks, and crosslinks between DNA and protein. ❖ In surviving cells, simple defects may be reparable by various enzyme repair systems. However, double-strand breaks may persist without repair, or the repair of lesions may be imprecise (error prone), creating mutations. If cell-cycle checkpoints are not functioning (for instance, because of mutations in TP53 ), cells with abnormal and unstable genomes survive and may expand as abnormal clones to form tumors eventually. Estimated Threshold Doses for Acute Radiation Effects on Specific Organs Injury Produced by Radiation – Morphology: ❖ At the light microscopic level, vascular changes and interstitial fibrosis are prominent in irradiated tissues. During the immediate postirradiation period, vessels may show only dilation. Later, or with higher doses, a variety of degenerative changes appear, including endothelial cell swelling and vacuolation, or even necrosis of the walls of small vessels such as capillaries and venules. ❖ Affected vessels may rupture or undergo thrombosis. Still later, endothelial cell proliferation and collagenous hyalinization with thickening of the media layer are seen in irradiated vessels, resulting in marked narrowing or obliteration of the vascular lumina.
