1. 180 Morphology and development of blood and immunity cell (Blood and hemopoiesis) Lecture 2013

2. Blood structure and function „connective tissue“ (cells and ECM ) blood cells and plasma erythrocytes (red blood cells) leukocytes (white blood cells) platelets serum – differs from plasma by the absence of factors (oraganic and anorganic), which leave plasma during coagulation (has been involved in clot) hematocrit - the volume of combined formed elements in the blood after centrifugation erythrocytes – 43% leukocytes and platelets (buffy coat) 1% 40-50% male 30-40% female

3. Blood structure and function total volume 5,5L transport : oxygen, CO 2, nutrients, waste products, hormones… maintaining of acid/base and osmotic balance, body temperature plasma: water, proteins (albumin, globulins, clotting proteins, complement, lipoproteins), inorganic salts, ions, nitrogenous compounds, nutrients, gases the interstitial fluid of connective tissues is derived from blood plasma

4. Formed elements transport O 2, protection, clotting

5. Erythrocytes I. red blood cells biconcave-shaped discs without nucleus and typical organells shape provides the cell with larger surface area (140  m 2 an important attribute in gas exchange size: 7,5 x 2,6 μ m (in the middle only 0,8 μ m) number: 3,9 – 5,5 milions in 1 μ L (geographical difference - particular reference values) they survive 120 days in circulation 7,2um

6. Erythrocytes II. the shape of RBC is maintained by membrane proteins integral membrane proteins: glycophorins glycosylated – express specific blood groups + bind to the cytoskeleton, band3 protein – binds hemoglobin peripheral membrane proteins arranged into two- dimensional hexagonal lattice network (spectrin, actin tetramers).. purpose: elasticity and stability – to be able to mechanical deformities, osmotic pressure.. hemoglobin (33%), HbA, HbA2, HbF - fetal – particular types acc. to the polypeptide chain (! myoglobin – oxygen- binding protein in striated muscle) enzymes:- anaerobic glycolysis, hexose- monophosphate shunt

7. Clinical applications anemia – decrease in the concentration of Hb polycythemia – physiological adaptation or haemoblastosis – it increases blood viscosity anisocytosis – higher variability in size macrocytes – greater – impairment of maturation (deficiency of vit. B12) microcytes – smaller (defeiciency of iron) defects in cytoskeleton: hereditary spherocytosis – primary defect in spectrin gene expression sicle cell disease/thallasemia – point mutation in the gene for hemoglobin – HBS shape – abnormal/crescent shape of RBC (red blood cell) clinically manifested as anemia, small vessels/venules obstruction, hemolysis

8. Leukocytes – white blood cells types acc. to presence and absence of the granules granulocytes neutrophils (polymorphonuclear leucocytes) eosinophils basophils agranulocytes/ mononuclear – see mononuclear phagocyte system lymphocytes, B, T monocytes neutrophileosinophil basophilllymphocyte monocytes

9. Leukocytes circulate in blood stream, use it for their transport to connective tissue (by diapedesis), where they perform their function an important property of WBC is their motility non-specific immune response

10. Granulocytes content of two granule types: specific with specific function for particular granulocyte type nonspecific/azurophilic (lysosomes) nuclei are divided in two or more lobes /segments all granulocytes in periferal blood are terminal postmitotic stages of the cell development – cells do not divide. They survive only few days in peripheral blood nuclei are divided in two or more lobes

11. Neutrophil the most common WBC

12. Neutrophilic granulocytes 60-70% of leucocytes size: 12-15 μ m (in smear) nucleus is divides in two or three lobes that are connected by chromatin bridges; (small appendage to the nucleus inactive second X chromosome – Barr body in females´neutrophils good indentified) 2 types granules: specific granules – small, near the limit of resolution in LM (collagenase, phospholipidase, complement activators. Phosphatase, metalloproteinases - migration) azurophilic granules/lysosomes – 0,5 μ m (myeloperoxidase – generates highly reactive bactericidal hypochlorite and chloramines) glycogen – source of energy – anaerobic metabolism phagocytosis, oxidative burst – (H 2 O 2 ) and oxygen radicals, enzymatic degradation

13. Neutrophil – „microphage” the first line cells that enter the area of tissue damage chemoattraction to sites of microorganism invasion phagocytosis of microorganisms (with/without previous opsonisation– coating by antibody - Ab/complement to make bacteria more attractive) antigen-Ag engulfment,internalization - phagosome digestion - release of hydrolytic enzymes and by reactive oxidative compounds in phagosome (superoxide, hydrogen peroxide, hypochlorous acid (oxidative stress) only once perform a phagocytosis and die (pus) production of IL-1 – pyrogen(fever-inducing agent) – induces Pg synthesis – termoregulatory centre in hypothalamus - fever

14. Eosinophilic granulocytes associated with allergic reaction, parasitic infections and chronic inflammation 2-4% of WBC size: 12-15 μ m bilobed nucleus specific granules: crystalline core (internum) – major basic protein and other proteins with cytotoxic activity arylsulfatase, histaminase, collagenase, cathepsin matrix (externum) - lighter azurophilic granules – lysosomes thanks to histaminase moderates deleterious effect of vasoactive substances Ag/AB phagocytosis antihelmintic host defense chronic inflammation - present in lamina propria

15. Basophilic granulocytes acts like mast cell less that 1% size 12-15 μ m nucleus is divided in irregular lobes, but it is not distinct because it is covered by granules (basophilic nucleus/ basophilic granules – heparan-sulfate) specific granules – metachromatic, content of heparan-sulfate, histamine and SRS-A after binding of Ag specific for IgE (binded on the surface through Fc receptors) the release of vasoactive agent is triggered – severe vascular disturbances – hypersensitivity and anafylaxis

16. Lymphocytes the main functional cell of the immune system in the blood they are represent the circulating immunocompetent cells, in transit from one lyphatic tissue to other 30% of WBC on the basis of size: small (6-8 μ m), medium-sized and large (up to 30 μ m) – activated lymphocytes or NK small lymphocytes prevail in blood – memory cells dark, large round nucleus, chromatin is condensed thin rim of cytoplasm, ribosomes, azurophilic granules

17. Lymphocytes types T cells, B cells, NK3 functionally distinct BUT morphologically identical

18. T lymphocytes cell-mediated immunity differentiate in thymus, prevail in peripheral blood: 65% - 75% specific immunity T lymphocytes subtypes cytotoxic CD8+, recognise Ag bound to MHC I (induce apoptosis) supressor CD8+, CD45+ helper CD4+ recognise Ag bound to MHC II B lymphocytes humoral immunity, Ab CD 19+ (B-cell differentiates in plasma cell producing antibody) 20-30% in blood differentiate in bursa Fabricii in birds and bursa equivalent (e.g. bone marrow) in mammals NK (natural killers) CD16+, CD56+, CD94+ medium-sized lymphocytes with granules 10-15% in peripheral blood – nonspecific recognition of tumor and virus infected cells - innate immune response a bit morphological difference – larger and with granules

19. Monocytes mononuclear-phagocytic system size: 12 -20 μ m oval, horseshoe or kidney shaped nucleus, excentrically placed basophilic cytoplasm, azurophilic granules (lysosomes), RER, polyribosomes, mitochondria, Golgi complex monocytes differentiate into macrophages phagocytosis repeatadly antigen presenting cells (express Ag in complex with MHC II)

20. Leukocytes - differential white cell count: the distribution of the different types of white cells present in circulating blood 6 000 -10 000 in 1 μL granulocytes neutrophiles 60 -70%, 3500-7500 eosinophiles 2-4%, 150 - 400 basophiles 0-1% 50-100 agranulocytes lymphocytes 20-30% 1500-2500 monocytes 3-8% 200-800

21. Platelets - thrombocytes nonnucleated, disc-like cell fragments of megakaryocytes size - 2-4 μ m 200 000– 400 000 in uL glycocalyx – reaction surface for fibrin from fibrinogen formation central zone containing granules – granulomera:  granules platelet-specific proteins: fibrinogen, PdGF, FGF, von Willebrandt factor, platelet-specific factor IV, δ granules dense bodies – ATP and serotonin; λ – lysosomes, mitochondria, peroxisomes, glycogen and peripheral lighter zone – hyalomera - open canalicular system – membrane channels – develiopmental remnant, dense tubular system – RER – Ca2+ storage, marginal bundle of microtubules, actin and myosin – contractile hemophilia – inherited deficiency of factor VIII or IX von Willebrand disease – the most frequent bleeding disorder, hereditary disease with abnormal or defect of von Willebrand factor

22. Platelets blood clot formation, surveillance of endotehel continuity, repair of injured tissue

23. Hemopoeisis

24. Chick embryo – blood islands, 2ED mesoderm of yolk sac (3 week) intraembryonic splanchnic mesoderm (3week – 6week) liver spleen (hepato-lineal period) bone marrow (from8week)

25. Development of blood cells regulation microenvironment – cells of stroma, extracellular matrix growth factors: growth factors – stimulation of mitotic activity colony-stimulating factors (CSF) hematopoietins (erythropoietin – synthesis in kidney, thrombopoeitin - synthesis in liver)

26. Red bone marrow stroma: hematopoietic cords and sinusoids stroma – reticular connective tissue (reticular cells and reticular fibres – (collagen type 1 and 3, fibronectin, laminin and proteoglycans) sinusoids – capillaries with discontinuous endothelium stem cells – they can differentiate also in other cellular types that blood cells – appropriate stimulation is necessary

27. Hemopoeisis hemopoiesis is a result of simultaneous proliferation and differentiation hemopoetic stem cells – types and terms: pluripotent stem cell – self-renewal - for life-long reserve – low mitotic activity multipotent - progenitor - stem cell - lymphoid and myeloid stem cells unipotent - - colony forming cells (CFU - self-renewing – high mitotic activity precursor cells (blasts) – not self-renewing (lymphoblast, erythroblast....) mature cells – (cytes) (lymphocyte, erythrocyte)

28. Hematopoiesis

29. Haemopoiesis

30. Erythropoiesis erythropoietin, iron, folic acid, cyancobalamin (vit B12) 3 - 5 division from proerythroblast to erythrocyte approximately 7 days from proerythroblast to reticulocyte decrease of the cell volume condensation of chromatin (pycnotic nucleus) and its expulsion synthesis of hemoglobin and successive loss of basophilia (polyribosomes)

31. Erythroblastic island non-mature red blood cells with still present nucleus, dense chromatin

32. Erythropoiesis Proerythroblast – large cell with loose chromatin, nucleoli and basophilic cytoplasm Basophilic erythroblast – condensed nucleus, basophilic cytoplasm - polyribosomes Polychromatophilic erythroblast – polyribosomes decrease, hemoglobin appears Orthochromatophilic erythroblast – eosinophilic cytoplasm- hemoglobin, maximally condensed nucleus – nucleus expulsion Reticulocyte – rest of ribosomes – substantia reticulo- filamentosa – 1% in peripheral blood Erythrocyte

33. Granulopoiesis in bone marrow more frequent than RBC (short life-time) gradual synthesis of azurophilic and later also specific granules myeloblast – finely dispersed chromatin, no granules promyelocyte – the 2nd largest, basophilic cytoplasm, Golgi complex and azurophilic granules neutrophilic, basophilic and eosinophilic myelocytes – condensation of nucleus, appearance of specific granules neutrophilic metamyelocyte, post-mitotic – band cell – mature granulocyte

34. Bone marrow: promyelocyte, metamyelocyte, lymphocyte Excentrical, nucleus, flat from one side metamyelocyte l ymphocyte

35. Maturation of lymphocytes no specific morphologic differences stem cells - progenitors in bone marrow –– they do not have antigens specific for B and T lymphocytes T – lymphocytes - maturation in thymus (precursors T- lymphoblasts – differentiate in mature lymphocytes) or in bone marrow (B – lymphoblasts, lymphocytes), they may divide in periphery in lymphoid organs (spleen, lymph node, tonsil)

36. Maturation of monocytes monoblast – identical with myeloblast promonocyte – large cell – up to 18 μ m; basophilic cytoplasm, large slightly indented nucleus, large amount of RER, large Golgi complex formation of azurophilic granules – lysosomes in blood, they circulate approximately 8 hours they survive in periphery for several months – macrophages - antigen presenting cells

37. Origin of platelets megakaryoblast – 15 - 50 μ m, large ovoid nucleus, numerous nucleoli. Polyploid cell - up to 30 sets of chromosomes (reflecting its size) megakaryocyte – giant cell - 35 – 150 μ m. irregular nucleus, numerous mitochondria, well- developed RER and extensive Golgi complex. Formation of granules. Invagination of plasmalemma – demarcation membranes – fragmentation of cytoplasm – release of platelets into the blood after desintegration of cytoplasm into the platelets, megakaryocyte die by apoptosis