Pharm 41
Introduction
 Inflammation – complex web of responses to tissue injury and infection, characterized by classic signs of rubor
(redness), calor (heat), tumor (swelling), dolor (pain), and functio laesa (loss of function)
 Normal inflammatory response – acute process that resolves after removal of inciting stimulus
o Diseases of inflammation and immunity can occur due to inappropriate inflammation or when normal
inflammatory response regresses to chronic inflammation, either because of long-term inappropriate
response to stimulus (i.e., allergies) or because offending agent not removed (i.e., chronic infection,
transplantation, autoimmunity)
 Pharmacologic strategies used to target pathophysiology of immune diseases
o Modification of signaling mediators of inflammatory process or suppression of components of immune
system; rationale for drugs that affect eicosanoid pathways, histamine, and cells of immune system
 Dependent on understanding molecular events in relevant pathways
o Modification of underlying pathophysiologic stimulus, thus removing impetus for inflammation
 Used to combat peptic ulcer disease, asthma, and gout
o Some drugs use both methods of rationale (line becomes blurred, categories not mutually exclusive)
Overview of the Immune System
 Fundamental role of immune system is to distinguish “self” form “non-self”; terms infection and infectious agent
used to denote inciting stimulus for immune response, regardless of cause of immune response
 Skin and other barrier tissues form first line of defense against infection; once offending agent penetrates these
barriers, immune system mounts response
o Innate responses – stereotyped reactions to stimulus (e.g., release of histamine, phagocytosis of
bacterium); in some cases, sufficient to neutralize the offending agent
 Cells of innate immune system, especially APCs, can also process offending agent into small
fragments; processing necessary for activation of adaptive immune system
o Adaptive responses – neutralizing reactions specific to the offending agent
o In general, innate immune system recognizes non-self and initiates and activates response to offending
non-self agent; adaptive immune system creates response that specifically neutralizes or kills agent
 Cells of immune system derive from 2 types of pluripotent cells in bone marrow: myeloid stem cells and
lymphoid stem cells (common lymphoid stem cells)
o
In general, myeloid stem cells give rise to precursors of innate immune system, whereas lymphoid stem
cells generate precursors of adaptive immune system (some exceptions)
Innate Immunity
 Innate immune cells defend against bacterial and parasitic infections (either by neutralizing infectious agent
with secreted cytotoxic proteins or by phagocytosis)
o Phagocytosis of offending agent initiates proteolytic digestion of microbial macromolecules to
fragments (antigens) that they display, together with major histocompatibility complex (MHC) class II
proteins, on surface of APCs, activating cells of adaptive immune system
o Secrete numerous cytokines that further amplify immune response
 Major cell types include granulocytes (neutrophils, eosinophils, and basophils), mast cells, and antigenpresenting cells (macrophages and dendritic cells)
o Neutrophils envelop invading bacteria in phagocytic vesicles and destroy them using enzymes such as
myeloperoxidase
o Eosinophils attach to parasite’s exterior and secrete cytotoxic substances directly on parasite
o Basophils (circulating) and mast cells (tissue-resident) bind IgE antibody, display the IgE on cell surface,
and maintain histamine-containing granules that are released when exogenous antigen binds to and
cross-links the IgE
 APCs process macromolecules (especially proteins) of invading agent to display processed fragments on surface
of APC; fragments serve as molecular fingerprints used by cells of adaptive immune system to recognize invader
o Important initiators of immune responses because, in addition to displaying non-self antigens to T cells,
they provide co-stimulatory signals necessary for T-cell activation
o Macrophages process and present antigenic fragments of invading pathogen for recognition by T cells;
ability of macrophages to envelop and destroy pathogens enhanced by other components of immune
system, including antibodies and complement that mediate opsonization and cytokines that enhance
killing ability
 Macrophages produce cytokines such as TNF-α that modify immune responses
o Dendritic cells – APCs that (in mature form) found primarily in T-cell areas of lymphoid tissue
 Most important APCs for initiation of adaptive immune responses
 Immature dendritic cells reside in non-lymphoid tissues, ready to engulf and process foreign
antigens; dendritic cells transport antigens to lymphoid tissues and present them to T cells
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Innate immune cells respond to common determinants present on many invading agents (for example, LPS in
outer membrane of Gram-negative bacteria); use pattern recognition to phagocytose class of infectious agents
rather than a specific infectious agent
o Adaptive immune cells mount specific response to 3D conformation of particular antigen (epitope)
o Innate immunity provides broad gating function, attempting to counteract harmful effects of foreign
invaders in rapid manner and determine whether infectious agent should be further attacked by
adaptive immunity
 Pattern recognition function of innate immune cells mediated by Toll-like receptors (TLRs)
o TLRs are transmembrane proteins that bind to shared microbial components such as LPS expressed by
Gram-negative bacteria, mannans expressed by fungi, and double-stranded RNA expressed by viral
pathogens
o 10 TLRs expressed in humans, and each has characteristic immune cell distribution and set of pathogenassociated ligands
 TLR4 expressed by APCs and binds to LPS
o Binding of TLRs to their ligands activates intracellular signaling cascade that converges on expression of
proinflammatory cytokines, leading to further immune cell recruitment and activation of inflammatory
response
o Fundamental role of innate immunity is to sound the alarm that recruits elements of adaptive immunity;
alarm indicates molecular structure associated with pathogen has been detected and serves as early
warning system that adaptive immune system should initiate immune response against pathogenassociated antigens encountered in context of TLR agonist or another innate immune signal
Adaptive Immunity
 Main features of adaptive immune system – specificity to foreign antigens and tolerance to self-antigens; rely on
o Mechanism to generate specific response to foreign antigen; provided by major histocompatibility
complex (MHC) proteins along with somatic gene recombination in T cells and B cells
o Adaptive immune cells must be able to distinguish native cells and soluble factors from foreign cells and
soluble factors; provided by signals from innate immune system, regulated immune cell development,
and co-stimulation
 MHC proteins – transmembrane proteins that bind and display on their surface proteolytically degraded protein
fragments, and in some cases, glycolipid antigens
o MHC class I proteins primarily display fragments of cytosolic proteins; all nucleated cells express MHC
class I proteins
 Repertoire of protein fragments displayed by MHC class I proteins on cell provides fingerprint
for all proteins expressed within that cell
 If cell expressing recognizable pattern of proteins, it will not be attacked by immune system
 If foreign proteins being generated in cell, then proteolytic fragments of those viral proteins will
be displayed on MHC class I proteins at surface of cell, and immune system will recognize cell is
virally infected; antigens presented by MHC class I proteins recognized by CD8+ T-cells
o MCH class II proteins display protein fragments derived from endocytic vesicles; expressed mostly in
APCs, although some other cell types can be induced to express MHC class II proteins
 Endocytic vesicles contain antigenic protein fragments derived from infectious agents after
phagocytosis and proteolytic processing of those agents
 Protein fragments expressed on MHC class II protein generally identify extracellular foreign
agents (e.g., bacteria)
 CD4+ T-cells recognize antigens presented by MHC class II proteins; T-cell stimulate APC to
produce soluble factors (cytokines and chemokines) which in turn aid the T cells in responding
to antigen
o In general, protein fragments bound to MHC class I identify infected cells, whereas fragments bound to
MHC class II identify infections agents
 Because of phenomenon of cross-presentation, some proteins generated in cytosol can be
presented by MHC class II to CD4+ T cells, and some phagocytosed antigens can be presented by
MHC class I to CD8+ T-cells
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Somatic gene recombination and other processes for generating diversity provide mechanism for generating
specific response to infection
o Ig and T-cell receptor genes semirandomly create millions of modular 3D protein structures (variable
regions); recombined variable regions may undergo somatic hypermutation to create additional
diversity that, in the aggregate, can recognize almost any structure
Adaptive immunity divided into humoral immunity and cellular immunity
o Humoral response involves production of antibodies specific for an antigen; Ig’s secreted by plasma cells
and are effective primariliy against extracellular infectious agents
o Cellular response involves activation and clonal expansion of T cells that recognize specific antigen
 Some T cells recognize infected cells and lyse them using cytotoxic proteins (perforins and
granzymes); effective against many intracellular infectious agents
T cells control extent of immune responses; each T cell evolves so it is activated by only one specific MHCantigen complex; all T cells express an MHC-antigen-specific T-cell receptor (TCR)
o Cytotoxic T cells (TC) – mediators of cellular adaptive immunity; express CD8 co-receptor, which
recognizes constant domain on MHC class I proteins
 Co-receptor function allows antigen-specific TCR on TC cells to bind specific class I MHC-antigen
complex with sufficiently high affinity that TC cell activated by cell expressing that complex
 Specific activation of TC cells initiates chain of events, including secretion of perforins (penetrate
membrane) and granzymes (apoptosis-inducing) that result in death of cell displaying antigen
o Helper T cells (TH) – primarily regulators of adaptive immunity; identified by expression of CD4 coreceptor, which recognizes antigen-independent domain on MHC class II proteins
 Co-receptor function allows antigen-specific TCR on TH cells to bind specific class II MHC-antigen
complex with sufficiently high affinity that TH cell activated by APC
 TH cells control type of immune response by producing one or another set of cytokines
 TH1 cells characteristically produce IFN-γ and IL-1, which influence development of cell-mediated
immune responses of both CD8+ TC cells and other CD4+ TH cells
 TH2 cells characteristically produce IL-4, IL-5, and IL-10, and these cytokines enhance antibody
production by B cells; more often associated with autoimmunity
 TH cells can mediate immunity by secreting cytokines that activate phagocytic cells to kill
infecting microbes more efficiently
 Subtypes of TH cells other than TH1 cells and TH2 cells discovered, some of which are
important in disease
o IL-23 stimulates naïve CD4+ cells to differentiate into TH17 cells, and these cells
produce IL-17 isoforms that recruit neutrophils and amplify immune response
Diversity in variable regions of Ig’s and TCRs create potential for some molecules to recognize and attack native
proteins; 2 mechanisms to prevent this
o Clonal deletion – T cells die during development when they express high-affinity receptors that
recognize self-antigen
o Tolerance (anergy) – cells of immune system undergo carefully regulated series of steps during
development to ensure that mature immune cells don’t recognize native proteins
Co-stimulation (requirement for multiple simultaneous signals to initiate immune response) ensures that
stimulation of single immune receptor doesn’t activate a damaging immune reaction
o One signal provides specificity, and the other is permissive, ensuring inflammatory response appropriate
o Regulation of co-stimulatory molecules is mechanism where innate immune system regulates extent of
immune response
o If antigen presented without co-stimulatory signal (i.e., without innate immune activation) then anergy
results, whereby cell becomes unreactive and won’t respond to further antigenic stimuli
o For T cells, the specificity signal is the MHC-antigen-TCR interaction, and permissive signal is mediated
predominantly by interaction of CD28 on T cells with B7-1 (CD80) or B7-2 (CD86) on activated APCs
 Resting T cells present CD28, which can bind either CD80 or CD86 (not normally present on
APCs, but expression increased by innate immune system during immune response to pathogen)
 Lack of expression of B7 molecules in absence of innate immune response helps limit
inappropriate adaptive immune responses
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When T cell receives both signals, expression of IL02, T-cell activation, and clonal expansion of
TH cells specific for that foreign epitope occur
 Activated T cells eventually downregulate CD28 expression and upregulate CTLA-4 expression
 CTLA-4 binds B7-1 and B7-2, but with much higher affinity than CD28
 Interaction of CTLA-4 with B7-1 or B7-2 inhibits T-cell proliferation
o CD40 ligand (CD40L) – mediator of co-stimulation; also called CD154; expressed by activated T cells
 Interaction of TH-cell CD40L with B-cell CD40 promotes B0cell activation, isotype switching, and
affinity maturation
 Interaction of TH-cell CD40L with macrophage CD40 promotes macrophage expression of B7-1
and B7-2, which are crucial for co-stimulation of T cells
 Positive feedback mechanism where activated T cells can promote further expansion of
activated T cells
 Increased expression of B7-1 and B7-2 on macrophages important for promoting CD8+ TC-cell
activation
 Blockade of CD40L with anti-CD40L antibody can produce tolerance and long-term graft survival
in animal models of organ transplantation
o Peripheral tolerance maintained by subset of T cells (regulatory T cells or Treg); best characterized are
CD4+CD25+; elaborate inhibitory cytokines in response to self-antigens and thereby limit immune
responses to antigens
Chemical Mediators of Inflammation
 Histamine – one of initiators of inflammatory response; constitutively synthesized and stored in granules of mast
cells and basophils, which migrate through tissue on continual basis
o Any injury, from physical trauma to microbial invasion, stimulates mast cells to release histamine into
interstitium; histamine is vasoactive, and release stimulates dilation of arterioles and postcapillary
venules, constriction of veins, and contraction of endothelial cells
o Effects responsible for early changes in hemodynamics and vascular permeability
 Complement – system of serine proteases that is one of first innate mechanisms to be activated in response to
injury; can be activated by antigen-antibody interactions (classical pathway), by direct interactions with foreign
surfaces (alternative pathway), or by interactions with certain complex carbs (lectin pathway)
o In each pathway, series of proteolytic reactions converts complement precursor protein to its active
form or forms (i.e., C3 activated to C3a and C3b)
o General scheme analogous to that of coagulation cascade, in which precursor proteins proteolytically
cleaved to active products that contribute to actions of cascade
o After activation, complement triggers inflammatory responses by
 Several cleavage products of complement cascade are potent stimulators of inflammation
 C3b is important opsonin
 C3a and C5a mediate leukocyte chemotaxis
 Final step in complement activation is assembly of membrane attack complex (complex of
complement proteins that produces large pores in outer membrane of Gram-negative bacteria,
leading to lysis of the bacteria)
o Large number of complement regulatory proteins, both soluble and on cell surface, carefully govern and
localize complement activation to site of inflammation
 Eicosanoids – metabolites of arachidonic acid (fatty acid component of phospholipids in inner leaflet of PM of
many cell types)
o Inflammatory mediators such as cytokines and complement stimulate enzymatic release of arachidonic
acid from PM
o Number of biochemical reactions ensue, resulting in formation of prostaglandins, leukotrienes, and
other eicosanoids
o Certain arachidonic acid derivatives are proinflammatory, whereas others serve to limit inflammatory
process; acute inflammation is self-limited process, and process of pathogen destruction intimately tied
to process of tissue repair
 Cytokines – proteins that act in paracrine manner to regulate leukocyte activity; affect proliferation and function
of cells that mediate innate and adaptive immune responses
o
Interleukins – cytokines secreted primarily by cells of hematopoietic lineage; iL-1 and TNF-α are 2
cytokines elaborated in acute inflammatory response
o Chemokines – subset of cytokines that promote immune cell trafficking and localization to sites of
inflammation
 MCP-1 promotes monocyte transmigration and activation
o Hematopoietic growth factors (i.e., GM-CSF and G-CSF) are also cytokines
 Kinins, platelet-activating factor, NO, oxygen radicals, and other leukocyte and bacterial products released
during phagocytosis also used to coordinate inflammatory response
The Inflammatory Response
 Inflammatory response typically occurs in 4 phases: vasculature around site of injury reacts to recruit cells of
immune system; circulating immune cells migrate from vessels into injured tissues, and mechanisms of innate
and adaptive immunity serve to neutralize and remove inciting stimulus; process of repair and tissue healing
ensues; and acute inflammatory process terminated
o If events of inflammation not halted and continue to smolder, chronic inflammation can occur
 Initially, the 5 classic signs of inflammation result from alterations in vascular hemodynamics at site of injury
o Injury to tissue causes release of inflammatory mediators that dilate arterioles and postcapillary venules
o Vasodilation leads to increased blood flow to site of injury, causing redness and warmth
o Inflammatory mediators cause contraction of vascular endothelial cells, leading to increased capillary
permeability and development of exudate; exudate causes swelling
o Pain develops due to both increased tissue pressure and action of various inflammatory mediators
 Increased vascular permeability allows cells from blood to enter interstitium; leukocyte recruitment
orchestrated to optimize clearing of infection and local repair of injured tissue
o At onset of inflammatory response, endothelial cells at site of injury activated to express adhesion
molecules that bind specific receptors expressed by leukocytes
 Intercellular adhesion molecules (ICAMs) expressed by activated endothelial cells bind integrins
expressed on cell surface of leukocytes; interaction causes leukocytes, which normally roll along
surface of endothelium by means of loose, transient binding interactions, to adhere tightly to
activated endothelium at site of injury
 Adherent leukocytes bind other endothelial cell receptors that promote transmigration
(diapedesis) of leukocytes from vasculature into interstitium
o Specificity of immune response achieved according to pattern of adhesion molecules expressed by
activated endothelium and by various types of leukocytes
 Neutrophils dominate early inflammatory response, while monocytes predominate after 24 hrs
 Once cells of immune system cross endothelial barrier, they migrate through interstitium to specific site of injury
or infection; immune cell targeting accomplished by process of chemotaxis
o Inflammatory mediators released at site of injury, such as N-formyl peptides derived from bacterial
proteins or endogenous mediators such as C5a and leukotriene B4 (LTB4), create chemical gradient to
which leukocytes respond, allowing them to move preferentially toward site of inflammatory reaction
 Upon arrival at site of injury, neutrophils, macrophages, and other cells of immune system ready to perform
their duties; cells require opsonins to coat foreign substance before they can be phagocytosed by leukocytes
o Opsonins – molecular adaptors that coat foreign surfaces and signal leukocytes that particle should be
attacked; major opsonins are complement, Ig’s, and collectins (plasma proteins that bind to certain
microbial carbs)
o Interaction of phagocytic cell with opsonized particle initiates engulfment and destruction of offending
agent; crucial point of interaction between innate and adaptive immunity
o APCs process engulfed particles and present their antigens to B cells and T cells, which then react to
antigens
 Tissue repair and reestablishment of homeostasis are final events in acute inflammatory response
o Same mediators that activate inflammation initiate cascade of tissue repair
o Process mediated by release of growth factors and cytokines, including epidermal growth factor (EGF),
platelet-derived growth factor (PDGF), basic fibroblast growth factor-2 (bFGF-2), TGF-β1, IL-1, and TNF-α
o Factors act as mitogens for endothelial cells and fibroblasts and ultimately stimulate healing and scar
formation through angiogenesis and formation of granulation tissue
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Angiogenesis can be pathologic state when associated with abnormal blood vessel growth or
tumor growth, and pharmacologic inhibitors of angiogenesis currently being used to treat agerelated macular degeneration (abnormal blood vessels obscure vision) and as antineoplastic
agents
Chronic Inflammation
 Pathologic state characterized by continued and inappropriate response of immune system to inflammatory
stimulus; accounts for symptoms of many autoimmune diseases and may be important cause of organ
transplant rejection
 One of hallmarks of chronic inflammation is predominance of macrophages
o Activated macrophages secrete collagenases and growth factors in addition to inflammatory mediators
such as proteases and eicosanoids; secreted products initiate and maintain cycle of tissue injury and
repair, leading to tissue remodeling
 Over time, chronic inflammation can cause relentless tissue destruction
 Promising treatments include cytokine inhibitors that neutralize mediators of signaling cascades that perpetuate
chronic inflammation
Conclusion and Future Directions
 Many anti-inflammatory drugs work in whole or in part by depleting populations of innate or adaptive immune
cells
 Chemical mediators of inflammatory response (histamine, complement, eicosanoids, etc.) are major targets of
current pharmacologic therapies
 Macromolecules playing increasingly important role in modulation of chemical mediators
o Number of anticytokine antibodies, including inhibitors of TNF-α, developed for treatment of
rheumatoid arthritis, psoriatic arthritis, and inflammatory bowel disease
 Different approach is targeting intracellular signaling cascades responsible for initiation of immune responses
o Example is cyclosporine