RED AND WHITE BLOOD CELLS

WBC (leucocytes) : Granulocytes (PMN) neutrophils basophils

eosinophils
Monocytes macrophage Lymphocytes L.B. antibodies L.T. cellular immune mechanism

Neutrophils : - phagocytose bacteria - role in acute inflammation Basophils : - contain histamin & heparin - role in immunologic hypersensitivity reactions Eosinophils : - allergic reactions - parasitic infections Monocytes : - precursor of macrophages - phagocytosis Lymphocytes : role in chronic inflammation

Neutrophils & lymphocytes : quantitatively major classes in normal blood

RBC (Erythrocytes) Function : delivering O2 from lung to the tissue and helping disposal of CO2 & proton from the tissue Structure : - simple, composed of concentrated solution of Hb (95% of intracell protein) surrounded by a membrane - no intracellular organelles

- non nucleated - biconcave shape (discoid) increase surfacevolume ratio fascilitating gas exchange - contains cytoskeletal components determine shape

RBC count: - men : 4.6 - 6.2 million / L ~ 14 18 g/dL Hb - women: 4.2 - 5.4 million / L ~ 12 16 g/dL Hb Total number in circulation : 2.5 x 1013 Hematocrit (packed cell volume / PCV) : - men : 42 - 52% - women : 37 - 47% Life span : 120 days
Reticulocytes ( 1% of total RBC count ) : - contain ribosome & endoplasmic reticulum active in protein

synthesis. - in hemolytic anemias : - life span RBC : shortened - reticulocytes

* Production of RBC (erythropoiesis) is regulated by erythropoietin H erythropoietin : - glycoprotein of 166 A.A (34 kDa) - synthesized by kidney, released in response to hypoxia to the bone marrow : interacts with RBC progenitor : (via specific receptor) 1. Burst -forming unit erythroid (BFU E) proliferate & differentiate 2. Colony-forming unit erythroid (CFU E) proliferate & differentiate Requires the cooperation of IL-3, insulin like growth factor

cDNA encoding erythrop. recombinant erythrop. : treatment of anemia due to renal failure

* Production of WBC is regulated by : hematopoietic growth factors : - glycoprotein - specific cell surface receptor on target cells via intra cell signals gene expression promoting differentiation 1. Granulocyte colony stimulating factor ( G-CSF) : specific, inducing granulocytes 2. Granulocyte-macrophage col. stim. F (GM-CSF) : affects variety of progenitor cells induces granulocytes, macrophages & eosinophils

Neutropenia : - production of neutrophils is depressed - in patients treated with chemotherapeutic / after bone marrow transplantation develop infections G - CSF is useful

METABOLISM OF RBC
Glycolysis Glucose

Pentose-P pathway NADPH

* Glycolysis: - lactate - ATP - 2,3 Bis-P glycerate (BPG/DPG) - NADH

1. ATP: - maintain biconcave shape - regulation of ion transport : - Na+ - K+- ATPase. Ca2+-ATPase - anion exchange protein - water in and out of the cell

2. 2,3 BPG: modulating O2 affinity of Hb Glycolysis

1,3 Bis-P-glycerate
ADP B-P-glycerate mutase

Glucose

2,3 Bis-P-glycerate
ATP Pi 3-P-glycerate 2,3 Bis-P-glycerate phosphatase

Pyruvate

3. NADH: RBC posses a system:

NADH-cytochrome b5 met Hb-reductase system reduced

Fe3+ Fe2+

Hb Fe3+ Met Hb

Cyt.b5 red

NAD Cyt b5 reductase (met Hb reductase)

Hb Fe2+

Cyt.b5 ox

NADH

* PPP NADPH, catalyzed by glucose 6-P dehydrogenase . (G6PD) Function of NADPH : reduced GSSG GSH GSSG GSH glutathion reductase NADPH + H+ NADP+

G-SH: counteract the action of toxic peroxides protect RBC from oxidative damage

* Entry of glucose into RBC : facilitated diffusion by means of glucose transporter (glucose permease) : GLUT I : - 2% of protein RBC membrane - specific for D glucose & related D hexose - affinity for D glucose is highest - inhibited by glucose analog (phloretin) - not insulin dependent - contain 12 transmembrane (hydrophobic) helical segment - function by generating a gated pore in the membrane ( conformationally dependent on the presence of glucose)

During metab. RBC generates oxidants oxidative stress & damage : - superoxide (O2), peroxide (H2O2) - peroxyl radicals : ROO*

- hydroxyl radicals : OH* (reactive), react with

proteins, nucleic acid, lipids & other mol alter structure tissue damage RBC contains cytosolic enzymes to protect from oxidative stress : - superoxide dismutase - catalase - glutathion peroxidase (Se)

* Superoxide (O2) :

- formed by autooxidation of Hb met Hb (3%)

- dismutates spontan H2O + O2 or by superoxide dismutase:

O2 + O2 + 2H+ H2O2 + O2
* H2O2 : - catalase H2O2 H2O + O2

- myeloperoxidase : H2O2 + X + H+ HOX + H2O

(neutrophils) X = Cl,Br, SCN

- glutathion peroxidase : H2O2 + 2G - SH GSSG + 2H2O R-O-O-H + 2GSH GSSG + H2O+ ROH

* OH* & OH : formed from H2O2: - nonenzymatic (Fenton reaction), catalyzed by Fe2+ Fe2+ + H2O2 Fe3+ + OH* + OH - Iron catalyzed (Haber-Weiss reaction)

O2 +H2O2 O2 + OH* + OH
O2 released Fe ions from ferritin production of OH* mechanism of tissue injury due to iron overload

* Pro oxidants : compounds & reactions capable of

generating toxic oxygen species

* Anti-oxidants : compounds & reactions disposing toxic

oxygen species, scavenging, suppressing their formation or

opposing their actions (include : NADPH, GSH, vit C, vit E)

Normal : appropriate pro-oxidant-antioxidant balance

Shifted toward pro-oxidant :

- production of oxygen species

- antioxidants level oxidative stress cell damage

(chemicals, drugs)

(inactivation of enzymes)

* Def. G6PD (mutation: > 300 genetic variants) hemolytic. An. - consumpsion of beans : Vicia faba (contains oxidants) - drugs : sulfonamides, primaquine - chemicals : naphthalene

generation of H2O2 or O2Normal : H2O2 is disposed by catalase & glutathion peroxidase ( GSSG)
* RBC with def.G6PD : cannot regenerate GSH impair the ability to dispose H2O2 & O2 radicals

- oxidation of SH groups in protein - peroxidation of lipid membranes lysis of RBC membr. - oxidation of-SH group of Hb precipitates inside RBC Heinz Bodies

* Fe2+ of Hb is susceptible to oxidation by superoxide met Hb Normal blood: very small amount of met Hb

* Methemoglobinemia : - inherited - acquired : ingestion of certain drugs (sulfonamides), chemicals (aniline) Inherited : - def. Met Hb reductase: - abnormal Hb (Hb M) : mutation changes A.A residue to which heme is attached altering affinity for O2 favoring oxidation * Cyanosis : bluish discoloration of skin & mucous membrane - deoxy Hb in arterial blood - met. Hb (>10%) Treatment : - mild met Hb (def.enz) : ingest methylene blue or ascorbic acid ( reducing agents) - acute massive met Hb (ingest chemicals) methylene blue I.V.

Membrane of RBC

The membrane is a bilayer composed of about 50% lipid and

50% protein. The major lipid classes are phospholipids and cholesterol; the

major phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine(PE), and phosphatidylserine (PS) along with sphingomyelin (Sph).

The choline-containing phospholipids, PC and Sph, predominate in the outer leaflet and the amino-containing phospholipid (PE and PS) in the inner leaflet.

Glycosphingolipids (GSLs) (neutral GSLs, gangliosides, and

complex species, including the ABO blood group substances) constitute about 5 10% of the total lipid.

Membrane of RBC

Analysis by SDS PAGE shows that the membrane contains

about 10 major proteins and more than 100 minor species. The major proteins (which include spectrin, ankyrin, the anion exchange protein, actin, and band 4.1) have been studied intensively, and the principal features of their disposition

(eg, integral or peripheral ), structure

been established.

and function have

Many of the proteins are glycoproteins (eg, the glycophorins)

containing O or N linked (or both) oligosaccharide chains

located on the external surface of the membrane.

Major Proteins of The Membrane of Human RBC

Spectrin Ankyrin and Isoforms
1 2 2.1 2.2 2.3 2.6

Anion exchange protein

3 4.1 4.2

Glycophorins Actin
5 G3PD 6 7

Globin

Membrane

Skeleton

Membrane PAS stain

Coomassie Blue stain

Membrane of RBC * SDS - page analysis : 10 major proteins some are glycoproteins : staining with periodic acid schiff (PAS) * Sensitive staining methods or two-dimensional gel electrophoresis : minor components (glucose transporter) Integral membrane proteins: * Anion exchange protein : - transmembr. glycoprot C term. : on external surface N term. : on cytosolic surface - multipass membrane protein - dimer forms tunnel exchange Cl- for HCO3- N-term. binds many proteins : Hb, Pr.4.1, 4.2, ankyrin & several glycolytic enzymes.

* Glycophorins A, B, & C : - transmembrane glycoprot

- single pass
Glycoph. A : - 131 A.A., 60% is glycosylated

- N-term. contains 16 oligosach. chains, extrudes out from the surface - contains sialic acid (90% of sialic acid of RBC membrane) - transmembrane segment (23 A.A), helical - C-term. extrudes into cytosol, binds Pr.4.1 - spectrin - polymorphism MN blood group system - contains binding sites for influenza virus & plasmodium falciparum

Peripheral membrane proteins - peripheral cytoskeletal protein - attached to the inner membrane - preserving shape & flexibility of membrane * Spectrin : - 2 polypeptides : - spectrin 1 ( -chain) - spectrin 2 ( -chain) - 100 nm length, anti paralel - loosely intertwined dimer - one dimer interact with another head-to-head tetramer - 4 binding sites for : - self association - ankyrin - actin - Pr.4.1

* Ankyrin

: - pyramid-shaped

- binds spectrin & binds tightly band-3 - sensitive to proteolysis bands: 2.2,2.3,2.6 all derived from band 2.1
* Actin : - short, double-helical filaments of F-actin - binds tail end of spectrin dimer & Pr.4.1.

* Protein 4.1: - globular protein - binds to tail end of spectrin, near actin binding site Pr.4.1-spectrin-actin complex - binds to glycophorins A & C attaching the complex to membrane - interact with membr. P.L connecting lipid bilayer to cytoskeleton

* Abnormal spectrin ( amount or structure) hereditary spherocytosis & elliptocytosis Hereditary spherocytosis: - spherocytes in peripheral blood - hemolytic anemia, splenomegaly - def.amount or abnormal structure of spectrin no tightly binds the other proteins weakens the membr. spherocytic shape - subject to destruction in spleen shortening life in circulation , curable by splenectomy

Hereditary elliptocytosis :

- genetic ~ her. spherocytosis

- elliptic, disk.like shape - abnormal spectrin - some cases : abnormal of band 4.1 / glycophorin C

Blood group system

The surface of RBC : - covered with specific antigenic

determinants - 100 blood group determinants 21 H blood group system

The best known : - ABO system important in blood - Rhesus (Rh) system transfusion

ABO system RBC membrane of individuals contain one blood group substance : type A, type B, type AB, or type O

Type A : - do not produce antibodies A,

- posses antibodies B agglutinate type B / type AB blood Type B Type AB recipient Type O : neither A nor B substance posses antibodies to both A & B universal donor : anti A antibodies type AB blood : neither anti A nor anti B antibodies universal agglutinate type A /

ABO substance ABO substance : complex oligosaccharida : glycosphingolipid H. substance : - precursor of A & B substance - O blood group substance - specific oligosach formed by the action of fucosyltransferase which adds fucose to peripheral galactose of its precursor (heterosach.) * Definite correlation between gene activity - specific glycosyl transferase synthesis - oligosaccharida structure H. gene : codes fucosyl transferase ABO gene : - located on long arm of chromosome 9 - 3 alleles : - A & B : codominant -O : recessive 4 phenotypic products : A,B, AB, O substance

A gene : encodes N-acetyl galactosamine (GalNAc)

trasnferase adds terminal Gal Nac to O substance

B gene : encodes galactosyl transferase : adding gal. residue to O substance

O gene : encodes inactive enzyme

Type AB posses both enzyme - 2 oligosach. chains : - 1 type A chain, 1 type B chain

* Specificity of ABO blood types is determined by sugars

GalNAc : immuno determinant of blood type A galactose : immuno determinant of blood type B

Removal of Gal NAc from type A erithrocytes or galactose

from type B erythrocytes convert both type to O erythrocytes

Rhesus (Rh) system

* 3 closely linked genes is involved, located on chromosome 1

the products : C or c, E or e, D or d
* D (RhO) antigen : - major antigen Rh factor - integral membr. protein (30 kDa)

- interact with membr. p -lipids

antigenicity * 15% caucasians lack the antigen Rh - negative

A female (Rh - neg) is transfused with Rh. pos. blood and

become pregnant, if her infant is Rh - pos lysis of infants erythrocytes ( hemolytic disease of the new born)

ANEMIA
- Blood contain fewer erythrocytes / decreased Hb content - Caused by many factors decreased O2 carrying capacity impaired O2 delivery to the tissue symptoms : - fatigue - faintness - headache - lack of concentration The causes are varies 3 broad categories : 1. Decreased number of RBC due to shortened life span - abn. Hb : HbS, HbC, thalassemia - abn. membr. prot. defect cell shape : spherocytosis - abn. enzyme : G6PD def. - infection : malaria - immun. desorders : hemolytic disease of the newborn 2. Decreased production of RBC - impaired Hb synthesis : Fe Def. - defect in stem cell maturation : Vit. B12 def. 3. Abnormal red cell

The body has compensatory mechanism delaying the

onset of anemia

Impaired O2 delivery

* Increasing 2,3 DPG synthesis modulates O2 affinity of

Hb O2 dissociates more readily at low O2 tension in the tissues Hb function more efficient * Releasing erithropoietin accelerates RBC maturation in bone marrow

* In hemolytic anemia bone marrow produces more cells &

sometimes are released prematurely circulation reticulocytes in

Hemolytic anemia

- Increased rate of RBC destruction

- Jaundice - Hb in urine dark colour (black water fever) * Inherited hemolytic anemia 1. RBC membr. defects spherocytosis - spherocytes - defect in spectrin - actin network - membr. is leaky to Na+ Na+- K+- ATP ase ATP ( high glycolytic rate) activity of demand for

2. Abnormal Hb : single A.A. substitution in - chain Hb S sickle cell anemia

Hb C, Hb D, Hb E
decreased solubility in deoxy- form

3. Thalassemia : synthesis of globin chains are depressed - - thal

- - thal

4. Abnormal enzyme
- G6Pd def. G6PD gene : on x - chromosome - all RBC are affected in males & homozygous females - heterozygous females : part of RBC with full enzyme

activity & the remainder with

no G6PD activity - Pyruvate kinase def. - Met Hb reductase def.

* Acquired Hem. An - malaria - bacterium : clostridium welchii - snake venoms, spider releases P lipase lysis cell membr.

- mismatched blood transfusion

- hemolytic disease of the newborn

Neutrophils - active aerobic glycolysis, active PPP - moderate oxidative phosphorylation - rich in lysosomal enzymes - contain : - unique enzyme : - myeloperoxidase (MPO) - NADPH oxidase - defensins (20 33 A.A) kill bacteria - lactoferrin (Fe-binding prot.) inhibit growth of bacteria - CD 11a / CD 18, CD 11b / CD 18, CD 11c / CD18 integrins (adhesion molecules) - receptors Fc fragments of IgG : bind Fc fragments of IgG Neutrophils : motile phagocytic cells defense against bacterial infection role in acute inflammation

Bacteria enter tissues acute inflammatory response A variety of agents are released from cells & plasma proteins vascular permeability tissue edema - mast cells & basophils histamin - platelets serotonin - neutrophils platelet activating factor (PAF), eicosanoids - plasma Pr. - C3a, C4a, C5a, from complement syst. - bradykinin & fibrin split products - neutrophils are recruited from blood stream into the tissue by chemotactic factors (C5a, leukotrienes) - adhesion of neutrophils to endothel cells is mediated by integrins & specific receptor in endothel

- neutrophil is activated turn on metabolic processes involved in phagocytosis & killing of bacteria

Integrins : - surface protein - hetero dimers, contain & subunit subunit contain : extracell, transmembr. & intracell segments: extracell segm. : binds ligands (specific prot. of

extracell matrix / surfaces of other cells

intracell segm. : binds various proteins of cytoskeleton

link outsides of cells to their insides

- involved in adhesion cells to cells or to specific components of extracell matrix

 Activation of neutrophills ~ platelets activation

- specific receptor - interaction with - bacteria, - binding of chemotactic factors - antibody antigen complexes Ca2+ intracell - secretion of granules content - motility ready to destroy bacteria
- rapid increase in O2 consumption (respiratory burst)

rapid utilization of O2 produce large amount of

reactive derivates of O2 : O2 , H2O2, OH* & OCl

- electron transport chain system : NADPH oxidase : - cytochrome b558 : - in the plasma membr. - heterodimer : 2 polypeptides (91 kDa & 22 kDa)

- 2 cytoplasmic polypeptides ( 47 kDa & 67 kDa) :

recruited to plasma membr. - NADPH : generated by PPP

O2 formed is discharged to outside of cell or into phago lysosomes kill bacteria :

combined action of : pH, O2, HOCl - defensins, cathepsin G - certain cationic proteins
O2 H2O2 : spontan or by superoxide dismutase O2 + O2 + 2H+ H2O2 + O2 H2O2 MPO HOCl : powerful oxidant, highly microbicidal disposed of by glut. peroxidase / catalase

 Genetic def. in NADPH oxidase syst. - recurrent infection

- widespread granulomas in skin, lung & lymph nodes

Some patients respond to treatment with - interferon transcription of 91 kDa component

Mutations in genes for the polypeptide components of the NADPH oxidase system Diminished production of superoxide ion and other active derivatives of oxygen
Diminished killing of certain bacteria

Recurrent infections and formation of tissue granulomas in order to wall off surviving bacteria

 Neutrophils contain proteinases (lysosomal enz.) - elastase, collagenase, gelatinase, cathepsin G - hydrolyze elastin. collagen & other protein in extracell matrix tissue damage - inactive in normal neutrophils - small amount are released into normal tissues & increasing during inflammation

- the activities are normally controlled by anti proteinases in plasma & extracell fluid : forming complex inhibition

Antiproteinases : - 1 antitrypsin, - 2 macroglob, - 1 anti-chymotrypsin

Genetic def. of 1 antitrypsin excessive action of elastase digest pulmonary tissue participate in the cause of

emphysema

Hemolytic anemia
Causes :1. Outside membrane - hypersplenisme

- immunologic abnormalities (transfusion reactions)

- toxins - certain bacteria (clostridium) - snakes venoms

2. Within the membrane

- abnormalities of protein (her. spherocytosis & her elliptocytosis) 3. Inside RBC - hemoglobinopathies ( sickle cell anemia) - enzymopathies (def. G6PD, def. pyruvat kinase)