14. Macrophages, their ontogenesis and function.

15. T-lymphocytes, ontogenesis, surface markers. Subpopulations of T-lymphocytes and their functions.

16. The role of thymus. Positive and negative selection of T-lymphocytes.

17. B-lymphocytes - ontogenesis, surface markers, function.

18. Primary immune organs and their role in the immune system.

19. Secondary immune organs - structure and function of lymphatic node and spleen.

20. Mucosal immune system.

Macrophages- ontogenesis

are a tissue- based phagocytic cells, derived from blood monocytes

play important roles in innate and adaptive immune responses

their development courses in the bone marrow an undifferentiated stem cell gives rise to the

myeloid and lymphoid progenitor myeloid progenitor cells differentiate into the

erythrocytic, granulocytic and monocytic cell lines and megakaryocytes

Conversion

of the myeloid precursor cells into monocytes and macrophages is affected by :

GM-CSF: granulocyte-monocyte colony stimulating factor – bone marrow (BM)stromal cells, lymphocytes- production of monocytes from BM

M-CSF: monocyte colony stimulating factor- stromal cells, lymphocytes, endothelial cells, epithelial cells- production and maturation of monocytes

IL-3 : lymphocytes- production of monocytes (other blood cells) from BM

Macrophages- development

Monocytes- in the blood

Macrophages - in tissues

Macrophages - terminology

histiocytes

Macrophages

a monocyte enter damaged tissue through the endothelium of a blood vessel

a monocyte is attracted to damaged site by chemokines, triggered by stimuli including damaged cells, pathogens and cytokines released by macrophages

after migration of monocytes to the tissues they differentiate into different form of macrophages

macrophages survive several months

Macrophage surface molecules

MHC gp class I, II assist in the presentation of epitopes to T lymphocytes

CD 35 - complement receptor 1 (CR 1), binds complement C3b

Receptor for the Fc portion of IgG

Function of macrophages

Phagocytosis Production of monokines Presentation of epitops with MHC class II Presentation of epitops with MHC class I

Phagocytosis

a foreign substances are ingested

a living organisms are killed and digested

follows sparing of antigenic epitopes and their distribution on the cell membrane

Presentation epitopes with MHC gp class II After endocytosis and degradation of the antigen,

preservation of its epitopes follows

epitope is coupled with the MHC gp class II- moved to the cell surface and contact the T-cell receptor

MHC (major histocompatibility complex) = complex of genes that governs the production of the major histocompatibility antigens - in humans are termed HLAs (human leukocyte antigens)

Presentation epitopes with MHC gp class I intracelular parasites are degradeted in proteasomes

of macrophages

their peptides are coupled to TAP (transporters associated with antigen processing molecules 1,2) that carry the epitope and MHC gp class I to the cell surface- protect epitopes from phagocytic destruction

Monokines = cytokines produced by macrophages IL- 1 α, ß- stimulate both T and B cells, IG synthesis,

activation of other macrophages, sensitizing cells to IL-2 and IFN

TNF- α - similar in function to IL-1 IL- 8 - secreted by activated macrophages - chemokine for neutrophils, T cells IL-12 - promotes induction of Th1 cells, inhibits Th2

cells IFN- α- activates host cells to induce enzymes that

inhibit protein synthesis needed for viral replication; increases expression of MHC gp I class on host cells; activates NK cells, T cells, other macrophages

Macrophage - functions

•Macrophages provide line of defense against tumor cells and body cells infected with fungus or parasites.

• a T- cell becomes an activated effector cell after recognition of an antigen on the surface of the APC, releases chemical mediators that stimulate macrophages into a more aggressive form

15. T-lymphocytes, ontogenesis, surface markers. Subpopulations of T-lymphocytes and their functions.

T lymphocytes- ontogenesis

The undifferentiated stem cell in BM gives rise to the lymphoid precursor cell which matures into 3 types of lymphocytes:

T lymphocytes B lymphocytes Natural killer (NK) cell

Pro-thymocytes come to the thymus where continue the maturation into T lymphocytes

Maturation of B lymphocytes continue in BM

Surface markers of T cells

CD (cluster of differentiation) proteins- molecules on the cells membrane, allow the identification of cells

TCR- receptor for antigen

MHC gp I or II class

CD proteins

allow an identification of T-cell subsets CD 2 = adhesion molecule CD 3 = important in intracellular signaling to initiate

an immune response; closely associated with TCR CD 5,7 CD 4,8 = are expresed on subclasses of mature T

cells; CD4 reacts with MHC gp II.class),CD8 reacts with MHC gp I. class on macrophages

CD 28- receptor for costimulator molecules CD80 and 86

Maturation of T lymphocytes

Consist of three types of processes:

Proliferation of immature cells Expression of antigen receptors genes Selection of lymphocytes that express useful

antigen receptor (TCR)

TCR

Antigen receptors are encoded by several gene segments that recombine during lymphocyte maturation

Heterodimer consisting of 2 nonidentical polypeptide chains linked together by disulfide bonds

> 95% T cells express the αß heterodimer, 5% γδ TCR heterodimer is noncovalently associated with the

γ,δ,ε chains of the CD3 molecule COMPLEX TCR- CD3 makes contact with both the Ag

and MHC gp

Subpopulation of T cells

Subpopulation of T cells have been defined according to their particular function and their CD membrane markers

Cytotoxic T lymphocytes = Tc;CD8+ - recognize the foreign epitope in association with class I MHC molecules

Helper T-lymphocytes = Th; CD4+ - recognize the epitopes in association with class II MHC molecules

Cytotoxic T lymphocytes (Tc;CD8+)

cause lysis of target cells; are active against tumors, virus-infected cells, transplanted allogenetic tissue

release TNF- depresses proteosynthesis recognize the foreign epitope in association with

class I MHC molecules destroy their target cells by releasing perforin (create

poresin the cell membrane and cytoplasm escapes) and granzymes (degrading essential macromolecules)

Helper T-lymphocytes(Th; CD4+)

recognize the epitopes in association with II MHC p II.class

help B cells to produce antibodies and help phagocytes to destroy ingested microbes

subsets of Th cells: Th1, Th2 cells

Th1 cells

secrete: INF-γ (gamma interferon) : activates macrophages to become

more effective at killing phagocytosed microbes, supresses the development of Th2 cells

IL- 2 : stimulates survival and proliferation of T cells, called T-cell growth factor

TNF (tumor necrosis factor)- stimulates the recruitment of neutrophils and monocytes to sites of infection, activates these cells to eradicate microbes

IL-3 : promotes expansion of immature marrow progenitors of all blood cells

GM-CSF : acts on progenitors in the bone marrow to increase production of neutrophils and monocytes

Th2 cells

secrete: IL-4 : induces differentiation of Th2 cells from naive

CD4+ precursors, stimulation of IgE production by B cells

IL-5 : activates mast cells IL-6 : stimulates the synthesis of acute phase

proteins by hepatocytes IL-10 : inhibits activated macrophages, supresses

Th1 production IL-3, GM-CSF

Regulatory T cells

Express CD4, CD25, FoxP3 Regulate the activation or effector function of

other T cells Are necessary to maintain tolerance to self

antigens

16. The role of thymus. Positive and negative selection of T lymphocytes.

The role of thymus

In the two thymic lobes, lymphocyte precursors from the bone-marrow become thymocytes, and subsequently mature into T cells

Once mature, T cells emigrate from the thymus and constitute the peripheral T cell repertoire responsible for directing many facets of the specific immune system

Phases of thymocyte maturation

A rare population of hematopoietic progenitors enters the thymus from the blood, and expands by cell division to generate a large population of immature thymocytes

Immature thymocytes each make distinct T cell receptors by a process of gene rearrangement.

This process is error-prone, and some thymocytes fail to make functional T cell receptors, whereas other thymocytes make T cell receptors that are autoreactive

Positive and negative selection

Immature thymocytes undergo a process of selection, based on the specificity of their T cell receptors.

This involves selection of T cells that are functional (positive selection), and elimination of T cells that are autoreactive (negative selection)

Thymus – positive selection of T - cells

1. precursor T cells enter thymus from the blood

2. they are presented with self-antigens complexed with MHC molecules on the surface of cortical epithelial cells

3. only those thymocytes which bind the MHC/antigen complex with adequate affinity will receive a vital "survival signal"

4. the other thymocytes die (>95%)

Thymus – negative selection of T - cells

1. thymocytes that survive positive selection migrate towards the boundary of the thymic cortex and thymic medulla

2. they are again presented with self-antigen in complex with MHC molecules on antigen-presenting cells

3. thymocytes that interact too strongly with the antigen receive an signal for apoptosis

17. B-lymphocytes - ontogenesis, surface markers, function.

B-lymphocytes

are an essential component of the innate immune system

Maturation of B cells course in the BM B cells ordinate from stem cells and need to be in

touch with the stromal cells in the bone marrow Stromal cells produce SCF (stem cell factor) needed

for development at early period, IL-7 needed at later period of maturation

Ig gene rearrangements and the appearance of surface markers identify the stage of B-cell development

Development of B lymphocytes

Lymphoid progenitor gives rise to precursors of B cells = pro- B cells

During maturation from the pro-B cells into the pre-B cells – Ig genes of the heavy chain recombine; pre-B cells express pre-BCR

During maturation from the pre-B cells into the B cells – Ig genes of the light chain recombine Immature B cells express membrane IgM Mature B cells express membrane IgM and IgD = BCR

and are able to respond to antigen in peripheral lymphoid tissues

Negative selection

If an immature B cell binds an antigen in the bone marrow with high affinity- further maturation is stopped and B cell dies by apoptosis

Negative selection eliminates potentially dangerous cells that can recognize and react against self antigens

B cells that survive this selection process leave the bone marrow through efferent blood vessels

B-lymphocytes – surface markers

CD 10 - immature B cells, malignant cells CD 35 - receptor for the C3b of the

complement CD 19 - a characteristic marker of B cells CD 20 - a typical surface antigen of Ig-

positive B lymphocytes IgM, IgD - antigen receptors = BCR MHC class II - antigen-presenting molecules

B-lymphocytes – functions

After stimulation B lymfocytes convert into the plasma cells and produce antibodies against soluble antigens

Other functions are :

antigen presentation

cooperation with complement

18. Primary immune organs and their role in the immune system.

Primary immune organs

Bone marrow Thymus

are places of development, differenciation and maturation of immunocompetent cells and elimination of autoreactive cells

T and B lymphocytes mature and become competent to respond to antigens in PIOs

Bone marrow

is the central cavity of bone that is the site of generation of all circulating blood cells in the adult, including immature lymphocytes, and the site of B-cell maturation.

The pluripotent stem cell gives rise to the progenitor of all immune cells

Production of cells course in the places divided by vascullar sinuses

Endothelial cells of the sinuses produce cytokines

Sinuses are borded by reticular cells

Differentiation in the BM

Differentiation from the stem cell is influenced by:

membrane interaction between the stem

cells and the stromal cells cytokines (CSF, IL-3, trombopoetin,

erytropoetin)

Thymus

is located between the sternum and the major vessel trunks

It consist of two lobes

Each lobe is surrounded by a capsule and is divided into lobules, which are separated from each other by strands of connective tissue = trabeculae

Structure of the thymus

Each lobule is organized into two compartments:

- the cortex (outer compartment) – contains lymphocytes that proliferate

- the medulla (inner compartment)- mature lymphocytes, Hassall´s bodies

Thymus - morphology

stromal cells composed of: thymic epithelial cells – produce thymulin,

thymopoetin, thymosin that influence the maturation of T cells

dendritic cells macrophages

The thymus contain a large number of blood vessels and efferent lymphoid vessels that drain into the mediastinal lymph nodes

19. Secondary immune organs - structure and function of lymphatic node and spleen.

Secondary immune organs

spleenlymphatic nodes tonsils appendix

Peyer´s patchesMALT

• consist of the spleen, the lymph nodes, the mucosal and cutaneous immune system• are organized to optimize interactions of antigens, APCs and lymphocytes• are places of the development of adaptive immune responses

Lymphatic node

are nodular aggregates of lymphoid tissues located along lymphatic channels throughout the body

Lymph comes from tissues and most parenchymal organs to the lymph nodes

Lymph contains a mixture of substances absorbed from epithelia and tissues

as the lymph passes through lymph nodes, APCs in the LN are able to sample the antigens of microbes that may enter through epithelia into tissues

Lymphatic node

• lymph circulates to the lymph node via afferent lymphatic vessels and drains into the node just beneath the capsule in a space called the subcapsular sinus

• the subcapsular sinus drains into trabecular sinuses and finally into medullary sinuses

• the sinus space is criss-crossed by the pseudopods of macrophages which act to trap foreign particles and filter the lymph

• the medullary sinuses converge at the hilum and lymph then leaves the lymph node via the efferent lymphatic vessel

Lymphatic node- medulla

The medullary cords are cords of lymphatic tissue, and include plasma cells and T cells

• The medullary sinuses are vessel-like spaces separating the medullary cords; contain histiocytes (= immobile macrophages) and reticular cells.

• Lymph flows to the medullary sinuses from cortical sinuses, and into efferent lymphatic vessels

Contains lymphoid folicles = acumulation of B-lymphocytes and folicular dentritic cells

When a lymphocyte recognizes an antigen, B cells become activated and migrate to germinal centers = to the secondary nodule

Lymphatic node- cortex

Spleen

is a secondary lymphoid organ positioned high in the left abdominal cavity

is surrounded by a capsule, which sends trabeculae into the interior to form a compartmentalized structure

there are two types of compartments -red pulp and white pulp with a marginal zone in between

is NOT supplied by afferent lymphatics

Spleen

Red pulp : place of mechanical filtration and elimination of senescent red and white blood cells and microbes

White pulp : T lymphocytes CD4+,CD8+ are around arterioles (periarteriolar lymphoid sheaths), B lymphocytes are in the folicles; final maturation of B lymphocytes course in germinal center of secondary folicles

Mucosal immune system

MALT = mucosal-associated lymphoid tissue GALT = gut-associated lymphoid tissue BALT = bronchus-associated lymphoid tissue digestive, respiratory, and urogenital systems are

lined by mucous membranes includes loose clusters of lymphoid cells in lamina

propria of intestinal villi contains a very large population of plasma cells that

synthetize IgA antibodies

M cells

are epithelial cells that are specialized for the transport antigen from the lumen of the respiratory, digestive, and urogenital tracts to the underlying MALT

contain a characteristic pocket filled with B cells, T cells, and macrophages

are found at inductive sites that overlie organized lymphoid follicles in the lamina propria

antigens are endocytosed and transported within vesicles from the luminal membrane to the pocket membrane, where the vesicles fuse and deliver their contents to antigen-presenting cells

DC: dendritic cells, IEC: intestinal epithelial cell (Nu-nucleus), MC: M cell, IEL: intra epithelial lymphocytes, PP: Peyer’s patches, MØ: macrophages

Pv: particulate Ag in pinocytic vesicle of M cell

Secretory IgA

daily production of secretory IgA into mucous secretions exceeds that of any other class of immunoglobulin (5-15 g each day)

is an important line of defense for mucosal surfaces against bacteria

binding of secretory IgA to bacteria and viruses also prevents attachment to mucosal epithelial cells, thereby inhibiting infection and colonization

Cutaneous immune system

Epidermis contains keratin cells that produce IL-1, 6 and TNF during inflamation; and IL-10, TGF-β during healing

Dermis contains fibroblasts that produce collagen, remove apoptotic cells

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