From Blood to Host Defense Gregory J. Bagby, Ph.D. [email protected] Office: 310 (CSRB)
Dec 22, 2015
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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
Vascular system Lymphoid system
Bone marrow
Liver
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, [email protected]: 310 (CSRB)
Blood Components and Function
• 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.
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.
Plasma includes water, ions, proteins, nutrients, hormones, wastes, etc.
Plasma includes water, ions, proteins, nutrients, hormones, wastes, etc.
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).
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%
Buffy coat of leukocytes separates RBC from plasma.
Buffy coat of leukocytes separates RBC from plasma.
Hematocrit or PCV
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 PlasmaConstituents of PlasmaConstituents 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 Total = 7.3% plasma weight Lumen confined solutes - osmotic balance; buffers; transporters of lipids, etc; clotting; enzymes, antibodies; hormones
Albumins 4.2%
Globulins 2.8%
Fibrinogen 0.3%
(Liver)
(Liver)
(Immune)
Constituents of Plasma (continued)Constituents of Plasma (continued)Constituents Amount/Concentration Function
Gases
CO2 1 mM Waste product; pH buffer
O2 0.1 mM Oxidative metabolism
N2 0.5 mM No function
Nutrients
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
Bound to albumin orin lipoproteins
Constituents of Plasma (continued)Constituents of Plasma (continued)Constituents Amount/Concentration FunctionVitamins 0.00005-0.1 mM Co-enzymes, pre-
hormones, other
Trace elements (Cu, Zn)
0.003-0.018 mM Co-factors, other
Waste products
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
Individual hormones 10-12-10-7 M) Messengers
Serum and PlasmaSerum 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)
The major forms of “cells” in the blood. Among these,only the leukocytes are true cells with nuclei.
5,000,000/mm3
7,000/mm3 250,000/mm3
Suspended Formed ElementsSuspended Formed Elements
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
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 PlateletsLeukocytes 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%)
• Platelets (250,000 per mm3 of blood)
Polymorphonuclear (PMN) leukocytes (granulocytes)
Polymorphonuclear (PMN) leukocytes (granulocytes)
Blood Components and Function
• 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 reticulocytesMcv mean corpuscular volume (fl) (femtoliters) -- normal R.m indoor adult males 72-76
--- normal adult humans 86-98Mch 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 32-36Rdw 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)
Flow Cytometry of Fluorescence Activated Cell Sorting (FACS)
Charged PlatesCharged Plates
Single cells sortedinto test tubes
LaserLaser
+-
Modified from Purdue University Cytometry Laboratories
FALS Sensor
Fluorescence detectorScattered light detector
Detection
Fluorescence labeled antibodies against specific proteins
Forward Angle Light Scatter (Forward Scatter)Forward Angle Light Scatter (Forward Scatter)
FALS Sensor
Laser
Size and shape of cell – bigger the shadow the bigger the cell
Size and shape of cell – bigger the shadow the bigger the cell
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.
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.
Forward scatter
Forward scatter
90 Degree Light Scatter (Side Scatter)90 Degree Light Scatter (Side Scatter)
FALS Sensor
90LS Sensor
Laser
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.
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.
Shape (irregular) and optical heterogeneity of cells – granulation (# of organelles increases side scatter)
Side scatterSide scatter
Forward scatter
Forward scatter
Reflected light
Light Scatter GatingLight Scatter Gating
8
15
20
30
40
50
100
200
1000
Scale associated with # of events or cells
Modified from Purdue University Cytometry Laboratories
Side ScatterSide Scatter0 200 400 600 800 1000
0 2
00 4
00
60
0 8
0010
00
Lymphocytes
Monocytes
Neutrophils
For
war
d sc
atte
r
Detection of FluorescenceDetection of Fluorescence
FALS Sensor
Laser
The amount of fluorescence is detected in the side or 90o scatter channel.The amount of fluorescence is detected in the side or 90o scatter channel.
Forward scatter
Forward scatter
Fluorescence detector
Excitation
Emissionintensity
Detection of protein on surface or inside cell by binding of fluorochrome-conjugated antibody (Phenotype or function).
Detection of protein on surface or inside cell by binding of fluorochrome-conjugated antibody (Phenotype or function).
Light Scatter GatingLight Scatter Gating
8
15
20
30
40
50
100
200
1000
Scale associated with # of events or cells
Side ScatterSide Scatter0 200 400 600 800 1000
0 2
00 4
00
60
0 8
0010
00
Lymphocytes
Monocytes
Neutrophils
For
war
d sc
atte
r
Modified from Purdue University Cytometry Laboratories
.1 1 10 100 1000
.1 1
10
100
1 2
3 4
45% 2%
26%
Gating on Lymphocytes and Detecting CD3+ Cells that are either CD4+ or CD8+Gating on Lymphocytes and Detecting CD3+ Cells that are either CD4+ or CD8+
27%
CD3+ (T lymphocytes)CD3+ (T lymphocytes)Lo
g P
E F
luor
esce
nce
(CD
4)Lo
g P
E F
luor
esce
nce
(CD
4)
Log FITC Fluorescence (CD8)Log FITC Fluorescence (CD8)
Immunophenotyping of Lymphocytes
CD # = cluster designation number
CD3CD4
T helper cell
CD3CD8
Cytotoxic T cellCD20
B cell
IFNgammaIFNgamma
IFNgammaIFNgamma
(Th1 cell)(Th1 cell) (Antigen specific CTL)(Antigen specific CTL)
CD3+CD4+CD3+CD4+ CD3+CD8+CD3+CD8+
CD3-CD20+CD3-CD20+
Stem cells in the bone marrow constitute an important precursor of many of the formed components in the blood.
Hematopoietic Growth FactorsHGF:receptor
binding activates intracellular signaling
cascades
Name: Product: Erythropoietin
Colony-Stimulating Factors
Interleukins
Thrombopoietin
Stem Cell Factor
Others (TNF, Interferons)
Erythrocytes
Granulocytes and monocytes
Various Leukocytes
Platelets
Many Blood types
Major Hematopoietic Growth Factors:(Derived from Table 14-4)
Major Hematopoietic Growth Factors:(Derived from Table 14-4)
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 • 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)
Folic Acid and Vitamin B12
The availability of dietaryiron can be a limitingfactor in rbc production,so storage and recycling mechanisms are highlydeveloped in humans asa protection from anemia.
50%50%
25%25%Ferritin
Transferrin Balance input vs output (95% recycled).
Maintaining iron balance important for adequate hemoglobin/rbc production.
Iron deficiency – anemia
Hemochromatosis –iron toxicity
25%25%
Iron Metabolism
Anemia and PolycythemiaAnemia 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 anemiaSickle-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
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).
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