From Blood to Host Defense
Gregory J. Bagby, Ph.D.
gbagby@lsuhsc.edu
Office: 310 (CSRB)
From Blood to Host Defense
• Blood
– Components and function
– Hemostasis and clotting
• The host defense system
– Innate immune system
• Pathogen recognition
• Inflammatory response
• Local to systemic responses and integration
– Adaptive immune system
• Humoral immune system and antibodies
• Cell-mediated immune system
The Relationship between Blood
and Host Defense
Liver
Bone marrow
Vascular system
Lymphoid system
Lymph node
The Relationship between Blood
and Host Defense
• Cellular elements of blood and the immune
system produced and/or originate in bone
marrow via hematopoiesis
– Red blood cells (erythrocytes) –important in O2 and
CO2 transport.
– White blood cells (leukocytes) – key roles in host
defense.
– Platelets – perform role in hemostasis and clotting
• Plasma proteins – many produced in liver or by
cells of the host defense system
From Blood to Host Defense
Blood Components and Function
Gregory J. Bagby, Ph.D.
gbagby@lsuhsc.edu
Office: 310 (CSRB)
•
•
•
•
The components of blood
Assessment of Cell Numbers and Types
Regulation of hematopoiesis
Regulation of erythrocyte production
What is blood?
• Blood is a fluid that normally circulates through the
lumen of the cardiovascular system (heart, arteries,
capillaries, and veins)
• Two major components
– Plasma – liquid (a complex solution)
– Formed elements (cells and cell fragments)
What are the main functions of blood?
• Transport water, ions, nutrients and waste products
to and from tissues
– Ions – sodium, chloride, calcium, bicarbonate, etc
– Nutrients – glucose, amino acids, lipids, oxygen
– Waste products – urea, lactic acid, carbon dioxide
• Transport signaling molecules (hormones) from
cells of origin to target cells
• Defending or protecting the blood and
extravascular compartments
– Hemostasis and clotting
– Role in host defense - means by which elements of the
host defense system travel to the thymus, mucosal
tissues, liver, lymphoid tissue, and sites of tissue injury or
infection.
The Components of Blood
• Plasma – liquid component
• Formed elements
1. Erythrocytes
2. Leukocytes
3. Platelets
Regulation of Blood
Cell Production
These components are maintained within a narrow
range (concentration or counts/ml), but new
replace old. Production and/or removal of each
constituent if regulated to maintain homeostasis.
Hematocrit or PCV
Plasma includes
water, ions, proteins,
nutrients, hormones,
wastes, etc.
Buffy coat of leukocytes
separates RBC from
plasma.
The hematocrit is a rapid
assessment of blood
composition.
It is the percent of the blood
volume that is composed of
RBCs (red blood cells).
Normal values
Men = 45%
Women = 42%
How Much Blood Is in the Body?
• Blood volume normally 8% of body weight
– Blood volume = 5.6 L in 70 kg man
• Erythrocyte Volume (45% hematocrit):
0.45 x 5.6 L = 2.5 L
• Plasma Volume: 5.6 L – 2.5 L = 3.1 L
Constituents of Plasma
Constituents
Amount/Concentration
Function
Water
93%
Solvent
Electrolytes
Total < 1% plasma weight
Osmotic balance; buffers;
membrane excitability;
clotting
Na+
145 mM
K+
4 mM
Ca+2
2.5 mM
Mg+2
1.5 mM
H+
0.0004 mM
Cl-
103 mM
HCO3-
25 mM
Phosphate
1 mM
SO4-2
0.5 mM
Proteins
Albumins
(Liver)
Globulins (Immune)
Fibrinogen (Liver)
Total = 7.3% plasma weight
4.2%
2.8%
0.3%
Lumen confined solutes osmotic balance; buffers;
transporters of lipids, etc;
clotting; enzymes,
antibodies; hormones
Constituents of Plasma (continued)
Constituents
Amount/Concentration
Function
CO2
1 mM
Waste product; pH buffer
O2
0.1 mM
Oxidative metabolism
N2
0.5 mM
No function
Glucose
5.6 mM
Energy metabolism
Amino acids
Sum = 2 mM
Back bone of proteins
Lipids
Cholesterol
Fatty acids
Triglycerides
Vitamins
Hormones
500 mg/dl
Energy metabolism;
Hormone precursors;
Involved in metabolic
pathway control and ion
homeostasis
Gases
Nutrients
Bound to albumin or
in lipoproteins
Constituents of Plasma (continued)
Constituents
Amount/Concentration
Function
Vitamins
0.00005-0.1 mM
Co-enzymes, prehormones, other
Trace elements (Cu,
Zn)
0.003-0.018 mM
Co-factors, other
Urea
5.7 mM
Breakdown from proteins
Creatinine
0.09 mM
From creatine
Uric acid
0.3 mM
From nucleic acids
Bilirubin
0.003 – 0.018 mM
From heme
10-12-10-7 M)
Messengers
Waste products
Individual hormones
Serum and Plasma
• Serum is plasma with fibrinogen and other
proteins involved in clotting removed as a result
of clotting.
• Serum is often used for analysis instead of
plasma
–
–Need anticoagulant to obtain plasma
– Fewer interfering substances in serum (less protein)
Suspended Formed Elements
5,000,000/mm3
7,000/mm3
250,000/mm3
The major forms of “cells” in the blood. Among these,
only the leukocytes are true cells with nuclei.
Normal Range of Blood Cell Numbers
(counts/liter)
Cell Type
Adult women
Adult men
Erythrocytes
3.8 to 5.0 x 1012
4.5 to 6.5 x 1012
Leukocytes
4 to 11 x 109
4 to 11 x 109
Granulocytes
(neutrophils)
2.0 to 7.5 x 109
2.0 to 7.5 x 109
Lymphocytes
1.3 to 4.0 x 109
1.3 to 4.0 x 109
Platelets
150 to 440 x 109
150 to 440 x 109
Oxford Handbook of Clin Med and Anatomy & Physiology in
Health and Illness (Ross and Wilson)
Red Blood Cells: Erythrocytes
• Carry O2 from the lungs
/ CO2 to the lungs
• Contain large amounts
of hemoglobin (35% of
mass)
– Men: 16 g / 100 ml
– Women: 14 g / 100 ml
• Biconcaved discs (high
surface:volume ratio to
maximize diffusion
capacity. Aids in flow
through small vessels.)
Leukocytes and Platelets
• Cells of the Immune System
• Small percentage of total blood cells
• Types:
– Neutrophils (50-70%)
– Eosinophils (1-4%)
– Basophils (0.1%)
– Monocytes (2-8%)
– Lymphocytes (20-40%)
Polymorphonuclear
(PMN) leukocytes
(granulocytes)
• Platelets (250,000 per mm3 of blood)
•
•
•
•
The components of blood
Assessment of Cell Numbers and Types
Regulation of hematopoiesis
Regulation of erythrocyte production
Assessment of Cell Numbers and Types
• Manual counts using a microscope
– Morphology/staining
• Auto analyzers such as a Coulter Counter
– Morphology
• Flow cytometry
– Morphology/Specific Antibody binding to
antigens on/in cells (proteins)
Manual Blood Cell Count Determination Using a
Light Microscope, Hemacytometer and Blood Smear
Auto Analyzyer – Coulter Counter
RBC red blood cell (count 10^6
cell/microliter)
Hgb hemaglobin (gm/dl)
Hct hematocrit (%)
Rtc reticulocytes
Mcv mean corpuscular volume (fl)
(femtoliters) -- normal R.m indoor
adult males 72-76 --- normal adult
humans 86-98
Mch mean cell hemaglobin (pg) - normal
R.m. indoor adult males 21.8-24.6 -- normal adult humans 27-32
Mchc mean cell hemaglobin
concentration(%) - normal R.m.
indoor adult males 29.6-31.2 --normal adult humans 32-36
Rdw red cell distibution width --- normal
adult males 11-15
WBC white blood cell (count
10^3/microcliiter)
Sgs segmented neutrophils (%)
Bnd banded neutrophils (%)
Eos eosenophils (%)
Bso basophils (%)
Mno moncytes (%)
Lym lymphocytes (%)
Plt
platelets (count/microliter)
Flow Cytometry of Fluorescence Activated
Cell Sorting (FACS)
Detection
Laser
FALS Sensor
Fluorescence detector
Scattered light detector
-
Charged Plates
+
Fluorescence
labeled antibodies
against specific
proteins
Single cells sorted
into test tubes
Modified from Purdue University Cytometry Laboratories
Forward Angle Light Scatter (Forward Scatter)
When a cell intercepts the laser beam, the light
scattered in the forward direction (along the same axis
that the laser light is traveling) is detected in the
forward scatter channel.
Laser
FALS Sensor
Forward
scatter
Size and shape of cell –
bigger the shadow the
bigger the cell
90 Degree Light Scatter (Side Scatter)
The amount of light scattered to the side (perpendicular to
the axis that the laser light is traveling) is detected in the
side or 90o scatter channel.
Laser
FALS Sensor
Forward
scatter
Reflected light
90LS Sensor
Side scatter
Shape (irregular) and optical
heterogeneity of cells –
granulation (# of organelles
increases side scatter)
Light Scatter Gating
40
30
20
15
8
800
600
Monocytes
400
50
Lymphocytes
200
100
0
200
Forward scatter
1000
Neutrophils
1000
Scale associated with
# of events or cells
0
200
400
600
800
1000
Side Scatter
Modified from Purdue University Cytometry Laboratories
Detection of Fluorescence
The amount of fluorescence is detected in the side or 90o
scatter channel.
Laser
Excitation
FALS Sensor
Forward
scatter
Emission
intensity
Fluorescence detector
Detection of protein on surface or inside cell
by binding of fluorochrome-conjugated
antibody (Phenotype or function).
Light Scatter Gating
40
30
20
15
8
800
600
Monocytes
400
50
Lymphocytes
200
100
0
200
Forward scatter
1000
Neutrophils
1000
Scale associated with
# of events or cells
0
200
400
600
800
1000
Side Scatter
Modified from Purdue University Cytometry Laboratories
Gating on Lymphocytes and Detecting
CD3+ Cells that are either CD4+ or CD8+
CD3+ (T lymphocytes)
100
(CD4)
Log PE Fluorescence
10
1
2
45%
1
2%
27% 3
4
.1
26%
.1
1
10
100
Log FITC Fluorescence (CD8)
1000
Immunophenotyping of Lymphocytes
CD # = cluster designation number
CD4
CD3
CD8
CD3
IFNgamma
IFNgamma
T helper cell
CD20
Cytotoxic T cell
CD3+CD4+
CD3+CD8+
(Th1 cell)
(Antigen specific CTL)
B cell
CD3-CD20+
Stem cells in the bone marrow constitute an important
precursor of many of the formed components in the blood.
Hematopoietic Growth Factors
HGF:receptor
binding activates
intracellular signaling
cascades
Major Hematopoietic Growth Factors:
(Derived from Table 14-4)
Name:
Product:
Erythropoietin
Colony-Stimulating Factors
Interleukins
Thrombopoietin
Stem Cell Factor
Others (TNF, Interferons)
Erythrocytes
Granulocytes and monocytes
Various Leukocytes
Platelets
Many Blood types
Erythrocytes
• Produced in the bone marrow
– Lose nuclei and organelles
• Life-span = 120 days
– 250 billion cells made per day
• Destroyed in liver and spleen
– Bilirubin is the breakdown product (gives plasma its
color)
• Erythrocyte production tightly regulated by
hormones.
Erythropoiesis is
hormonally regulated:
decreased oxygen
delivery to the kidney
causes the secretion of
erythropoietin, which
activates receptors in
bone marrow, leading to
an increase in the rate of
erythropoiesis.
Erythropoietin Used
Clinically:
• Blood loss
• Renal failure
• In conjunction with
chemotherapy
Erythrocytes
• Produced in the bone marrow
– Lose nuclei and organelles
• Life-span = 120 days
– 250 billion cells made per day
• Destroyed in liver and spleen
– Bilirubin is the breakdown product (gives plasma its
color)
• Erythrocyte production tightly regulated by
hormones.
• Erythrocyte production dependent on folic acid,
vitamin B12 and iron
Folic Acid and Vitamin B12
Folic acid
• A vitamin found in leafy plants, yeast, and liver
• Is required for synthesis of the nucleotide base
thymine
• Essential for the formation of DNA and normal
cell division
Vitamin B12
• Is found only in animal products- Strict
vegetarian diets are often deficient in B12
• Absorption in GI tract requires “intrinsic factor”
• Is required for the action of folic acid (DNA
replication)
Iron Metabolism
50%
25%
The availability of dietary
iron can be a limiting
factor in rbc production,
so storage and recycling
mechanisms are highly
developed in humans as
a protection from anemia.
Balance input vs output (95%
recycled).
Transferrin
Maintaining iron balance
important for adequate
hemoglobin/rbc production.
Iron deficiency – anemia
25%
Ferritin
Hemochromatosis –iron
toxicity
Anemia and Polycythemia
• Anemia: Decreased ability of blood to carry
oxygen
- Decreased # erythrocytes
- Decreased concentration or performance of hemoglobin
within erythrocytes
- Combination of both
 Polycythemia: More erythrocytes than
normal (opposite of anemia)
- Increased viscosity of blood
- altitude
Sickle-cell anemia
• Genetic mutation alters one amino acid in
hemoglobin
• Fiber-like structures form during low [O2],
distorting erythrocyte into sickle shape
– Capillary blockage
– Tissue damage
– Destruction of deformed erythrocyte
– Anemia
Regulation of Total Blood Cell
production
• All blood cells are derived from
multipotent/pluripotent hematopoietic stem
cells
• Differentiation and proliferation of stem
cells (the path taken) is stimulated by
hematopoietic growth factors (HGF’s).