26. Basophils and mast cells- types, function. 27. Immune mechanisms of the inflammation

(local and systemic reaction) 28. Cytokines (overview, disposal according their

function) 29. Physiological mechanisms of regulation of

the immune system. 30. HLA system, 1st class molecules, their

structure and function 31. HLA system, 2nd class molecules, their

structure and function 32. HLA system, genetic background. HLA

typing.

26. Basophils and mast cells - types, function.

Basophils

A type of bone marrow derived circulating granulocyte

Ability to migrate into the tissue Structural and functional similarities to mast

cells = granules are containing inflammatory mediators; express a high- afinity receptor for Fc fragment of IgE on the cell surface

Participate in immediate hypersensitivity reactions

Produce cytokines (IL-4, 13)

Basophils surface markers

FcεRI – high-affinity receptor for Fc fragment of IgE

CD 123 – receptor for IL- 3

CD 63 – activating marker – expressed after degranulation

Mast cells

Derived from bone marrow precursor

Reside in tissues adjacent to blood vessels

Express a high-affinity receptor for Fc fragment of IgE

Contain numerous mediator-filled granules

Mast cells surface markers

FcεRI – high-affinity receptor for IgE

CD 117- membrane gp – mediates interaction necessary for mast cells proliferation

FcεRI

complex consists: - α- chains with two extracellular

immunoglobulin domains - β- chains – four times cross the membrane –

both their ends are inserted to the cytoplasm - γ- chain = homodimer - β and γ chains – contain activating motives

ITAM (mediate transport of activating signal from mast cell surface after binding of IgE) in their cytoplasmic part

Cytoplasmic granules Histamin (dilation of small blood vessels, increase of

vascular permeability, contraction of smooth muscles), proteases (cause a damage to the tissues)

Cytokines: IL- 4 ( stimulates IgE production by B cells), 5 (activates eosinophils), 13 (stimulates mucus secretion by airway epithelial cells), TNF α, IL-1

Growth factors: TGF- β, VEGF (vascular endothelial growth factor)

Synthetize: PAF (platelet activation factor), LTC4- leukotrienes (stimulate prolonged smooth muscle contraction), PGD2- prostaglandins (cause vascular dilation)

Mast cells function

Participate on defence against bacterial and parasites infection

Develop immediate hypersensitivity reactions (production of IgE antibodies in response to an antigen, binding of IgE to Fc receptors of mast cells, cross-linking of the bound IgE by the antigen and release of mast cell mediators)

Take part in reparation of tissues - angiogenesis, production of intercellular substances

27. Immune mechanisms of the inflammation (local and systemic reaction)

Inflammation

= a complex reaction of the innate immune system in vascularized tissues that involves accumulation and activation of leukocytes and plasma proteins at a site of infection, toxin exposure or cell injury

Iniciated by changes in blood vessels that promote leukocyte recruitment and movement of fluid and plasma proteins into tissue

Inflammation serves a protective function in controlling infections and promoting tissue repair

Inflammatory response

Local - signs: redness, swelling, pain, heat

Systemic – sings: fever

Acute or chronic process

Inflammation - iniciation

1. signal: phagocytes and degranulated mast cells, substances released from damaged cells, parts of intercellular substance exposed by injury

Local inflamation

Increase of vessels permeability – plasma fluid passes through the blood vessels- swelling

Increase of endothelial adhesivity (expression of cell adhesion molecules)- phagocytes and lymphocytes adhere to endothelial cells – pass to the tissues

The coagulation, fibrinolytic, kinin and complement systems are activated

Local nerve endings are influenced (pain) changes in temperature regulation

Local inflamation

phagocytes- accumulate in a site of injury, produce cytokines

Ag + APC home to LN (lymph nodes) - activate T cells and induce their terminal differentiation, activation of B cells and their changes into plasma cells with production of antibodies

Mature Th 1 lymphocytes migrate from LN to the site of infection and stimulate macrophages

Systemic response (SR) to inflammation Development of SR depends on: - range of damage - duration of local inflammation SR can arise also without local inflammation: - after massive enter of microorganisms into

the blood = septic shock - after intravascular antigen impulse of

noninfection character = anaphylactic shock Massive release of mediators (cytokines,

complement, histamin)- leads to a massive vasodilatation- hypotensis- vascular collapse

Sings of systemic inflammation

Fever- caused by stimulation of hyppothalamic center of termoregulation (by proinflammatory cytokines –TNF, IL-1, INF-gama)

Caused also by increase of proteosynthesis, increase of Hsp production (hot shock protein)

Mediators of inflammation

Cytokines produced in the site of inflammation- come through the blood into the liver- stimulate production of plasma proteins of acute inflammation phase (CRP, amyloid P, complement C3,C4), transport proteins (ceruloplasmin, transferin) and inhibitors of proteases (alfa 2-macroglobulin)

Proteins of acute phase – function - opsonisation, activation of complement

Cytokines and mediators cause bone marrow to release and produce leukocytes- leukocytosis

Reparation of damaged tissues

Activation of reparation process:- occurs during later phases of inflammation- phagocytes- eliminate damaged cells- activation of fibroplastic mechanisms- activation of angiogenesis- start regeneration and remodelation of tissues Control by cytokines, hormons, enzymes Chronic inflammation – accompane by fibrotic

process

28. Cytokines (overview, disposal according their function)

Cytokines Humoral factors - provide intercellular

communication between immune cells + communication between immune system and other body systems

Proteins secreted by leukocytes (and by other cells of immune system)

Influence different cells of immune system through specific receptors

Forms- secreted or membrane Effects - pleiotropic (1 cytokine has a several

fysiological effects)

Function of cytokines

Activating signals for cells – activate, regulate cell cycle, mitotic activity

Cause changes of the cell membranes – increase of cytokine receptors expression

Participate in reparation of tissues in the terminal phase of inflammation

Regulate immune cells proliferation and differentiation in the immune organs

Influence migration of the immune cells

Classification of cytokines

Pro-inflammatory Anti-inflammatory With growth activity factor Participating in humoral response IS Participating in cell response IS With antiviral effect

Proinflammatory cytokines

IL-1 – produced by macrophages and T cells; activates neutrophils, endothelial cells and T cells, induces synthesis of acute phase proteins in liver, causes fever

IL-6 – produced by T and B cells, monocytes; regulates B cell differentiation and proliferation, synthesis antibodies; stimulates hepatocytes to produce acute phase proteins

IL-8 – produced by monocytes, macrophages, endothelial cells; chemotactic factor for neutrophils

IL-12, IL-18, TNF

TNF TNF α – produced by macrophages (+T a B cells,

NK cells, neutrophiles,..) after activation by lipopolysacharids binded to plasma protein- complex binds to CD14 on the macrophage surface – causes releasing of TNF α

TNF α – participates on early phase of inflammation, induces expression of adhesive molecules on endothelial cells and leukocytes; stimulates proinflammatory protein production; stimulates catabolic processes

TNF β- produced by T and B cells; similar effects

Antiinflammatory cytokines IL-10 – produced by Th2 cells, monocytes, macrophages,

activated B cells; inhibition of cytokine Th1, Tc and NK cells production; inhibition of synthesis proinflammatory cytokines by macrophages

TGF-β – growth activator but also inhibitor of different cell types

Regulates damaged tissues reparation by stimulation of intercellular substances synthesis, modulate expression of tissue metaloproteases and tissue inhibitors of proteolytic enzymes; regulates cell adhesion; chemoatractant of fibroblasts; can inhibite T and B cell proliferation

IL-4

CYTOKINES with activity of growth factors

IL-2, IL-4, IL-5, IL-6, IL-9, IL-14 SCF- stimulates stem cell proliferation, their

release to the peripheral blood IL-3 – influences maturation of all cell lines IL-7 – growth factor of T cells IL-11 – growth factor of megakaryocytes IL-15 – induces proliferation of mast cells, Th

and Tc cells

CYTOKINES with activity of growth factors

G-CSF, M-CSF, GM-CSF – stimulate granulocytes and/or monocytes/macrophages proliferation and differentiation, prolongate their survival and increase their functional capacity during inflammation

EPO = erythropoetin- stimulates red cells differentiation

CYTOKINE participating in humoral response of IS

IL-4 – produced by Th2 cells, mast cells and basophils; stimulates B cell proliferation, production of IgM,G1,E; stimulates T cell proliferation; activates macrophages; growth factor of mast cells (antiinflammatory effect)

IL-13 – see IL-4 + chemotactic factor for monocytes and macrophages

IL-5 – produced by Th2 cells, lymphocytes and mast cells; activates and stimulates B cells and eosinophils proliferation, stimulates Tc cells

IL-9 – produced by Th2 cells; co-stimulating factor - amplifies Th cells and mast cells proliferation

CYTOKINES participating in cell mediated response

IL-2 – produced by Th cells after stimulation by antigen, autocrine effect to Th cells; activating signal for Tc cells and NK cells; 2. signal for activation and differentiation of B cells

IL-12 – produced by monocytes, macrophages; stimulates maturation Th0 into Th1 cells; activates NK cells; stimulates INF- γ production by Th1 cells; inhibites IgE secretion

IFN-γ, GM-CSF

CYTOKINE with antiviral effect Interferons- anti-inflammatory, anti-prolifferative IFN-α, IFN-β - produced by cells with nucleus after

stimulation – viruses - IFN (after binding to the receptor) induces in noninfected cell status of non-permisivity = decomposition of viral nucleus acids and influence into translation of viral proteins

IFN- α = represents 20 cytokines – differ by glycosylation of general protein structure; produced by leukocytes

- in recombinant form – therapy of chronic active hepatitis B, hepatitis C

Receptors for cytokines

Consist of 2-3 subunits 1- binds cytokine Others mediate contact with intracellular

signaling molecules Results of signalisation: stimulation of cell

mitosis, differentiation, initiation of effector functions (degranulation, secretion of cytokines, activation of membrane enzymes, chemotaxis), inhibition of cell mitosis and induction of apoptosis

Signaling process

RECEPTORS of CYTOKINES are associated with:

Protein-kinases- phosphorylate intracellular substances (after activation )- influence transcription of genes, structure of cytoskelet

G-proteins – iniciate phosphorylation (after cytokine binding to the receptor) – catalyze the replacement of GDP (guanyl diphosphate) to GTP and can activate a variety of cytoplasmic enzymes

Calcium ions – open Ca channels (after cytokine binding to the receptor) – increase of Ca ions concentration in the cytoplasma - activation of proteins by ca ions binding

Regulation of local concentration of cytokines Biological effects are neutralized by

binding to nature inhibitors and soluble forms of receptors for cytokines

MMP (matrix metaloproteases) release cytokines from molecules of intercellular substance

Elastases release binded cytokines

29. Physiological mechanisms of regulation of the immune system.

Immune mechanisms of regulation

Immune system is regulated by: Antigen Antagonistic peptid Antibodies Cytokines and intercellular contact T lymphocytes Neuroendocrine regulation

REGULATION BY ANTIGEN

Antigen competition – peptides from different antigens compete for binding-sites on the MHC gp = antigen is able to suppress expression of other antigen

IMPORTANT IS : the binding strenght of peptide to MHC gp II density of peptid-MHC gp II complex on the

surface of APCImmune response finishes after extinction of antigen

– due to a short life-span of effector lymphocytes

ANTAGONISTIC PEPTIDES

Agonists - peptide fragments of antigen with adequate binding to MHC gp, recognition by T cells with sufficient affinity – induce full response of T cells

Partial agonists – induce a qualitative different response of T cells - peptides have a similar structure, bind adequately to MHC gp, but make too weak or too strong interaction with T cells

Antagonists – induce anergy of T cells

REGULATION MEDIATED BY ANTIBODIES

Antibodies have an effector and regulatory functions

Secreted antibodies – compete with BCR for antigen = negative regulators of B cells stimulation

Immune complexes of antibody and antigen – bind to the surface of B cells – interaction of signaling molecules (protein-kinases, protein-phosphatases) = inhibit B cells activation (= antibody feedback)

REGULATION MEDIATED BY CYTOKINES AND INTERCELLULAR CONTACT

Inhibition of cytokine effect by endocytosis of their receptors or by binding of inhibitors to their receptors

Inhibitory receptors – protection against too easy T cells activation

Apoptotic receptor (Fas) mediates a negative regulation after binding to ligand FasL on activated T cells – causes – lysis of cell

NEGATIVE REGULATION mediated by T lymphocytes

Th2 cells produce IL-4, IL-10- suppress immune response of Th1 cells

Suppression of T cells occurs after recognition an antigen on cells different from APC (missing of co-stimulating signals)

CD8+ T cells – secrete soluble forms of TCR - compete with TCR on the surface of other cells- inhibition

Regulatory Tr1 cells (CD4+)- secrete IL-10-anti-inflammatory effect, induce tolerance to autoantigens

NEUROENDOCRINE REGULATION Neurotrasmiters influence leukocytes by

binding to the specific receptors (noradrenalin) CS, growth hormons, thyroxin, endorfins –

influence the leukocytes by binding to the specific receptors

Leukocytes produce endorfins, TSH, growth hormon, vitamin D3, ACTH

Cytokines influence nerve system (IL-1, IL-6) Stress influences immune system-

cortocosteroids-activity of phagocytes, NK cells

OTHER FACTORS

The same antigen can induce active immune response or active tolerance – depends on:

Condition of immune system Character of antigen (size, structure of

molecule) Dose of antigen (too low or too high doses-

induce tolerance) Route of antigen administration (s.c.-

induce immune response, p.o. or i.v.- induce tolerance)

32. HLA system, genetic background. HLA typing.

MHC (major histocompatibility complex) human MHC = HLA (human leukocyte

antigens)

Genes of MHC complex – are codominantly expressed = alleles inherited from both parents are expressed equally; are polymorphic – many different alleles are present

Contain of MHC complex

MHC gp = are encoded by genes localized on short arm of human 6. chromosome

The MHC locus- contains 4 single blocks of loci - HLA Ags I are encoded by loci A, B, C - HLA Ags II – locus D - HLA Ags III – locus between HLA I a II (contains

genes with nondirected participation in histocompatibility- genes for complement, TNF…)

Polymorphism of MHC gp

A lot of different forms of alleles (of each isotype) exist in the population

Forms of alleles differ in 1 or more amino acids in their binding-site for peptide

Importance of polymorphism:

- positive – allows to bind a lot of types of peptides

- negative – complicates transplantation

MHC gp

The MHC was discovered as the genetic locus that is the principal determinant of acceptance or rejection of tissue grafts exchanged between individuals.

Individuals that are identical at their MHC locus will accept grafts from one another

Individuals that differ at their MHC loci will reject such grafts

Function of MHC gp

Physiologic function of MHC molecules is to display peptides derived from protein antigens to antigen- specific T lymphocytes:

bind peptides transport peptides to the cell surface display peptides to T lymphocytes

Classification of MHC gp

MHC gps class I

- display intracellular proteins to CD8+ T lymphocytes

MHC gps class II

- display extracellular proteins to CD4+ T lymphocytes

HLA typing

The combination of class I and class II antigens expressed by a person is called his/her HLA phenotype.

The combination of class I and class II alleles is the HLA genotype. In most cases, the genotype reflects the phenotype.

HLA typing

HLA typing identifies a person's HLA type and, therefore, helps to characterize the immune function of that person.

Different loci and different levels of resolution are required for different clinical applications

HLA typing

HLA typing is routinely used for : matching donors and recipients in solid

organ or hematopoietic stem cell transplantation

helping in the diagnosis of certain autoimmune or anti-inflammatory diseases

determining eligibility of patients to tumor vaccination protocols when the vaccine is HLA-allele dependent

HLA typing

The peripheral blood lymphocytes from the indiviual are incubated with antibodies that are known to react with specific HLA molecules and complement.

If there is lysis of the cells, that individual carries the HLA protein recognized by that specific antibody.

HLA typing

Molecular techniques can now be used to type HLA molecules.

PCR using primers that amplify only certain HLA sequences allow accurate identification of HLA types.

This has the advantage of not requiring the preparation of live cells from the individual to be typed.

30. HLA system, 1st class molecules, their structure andfunction

MHC gps Ist class

Expressed on all nucleated cells Display intracellular proteins

isotypes of clasic HLA Ist class: HLA- A,- B,- C isotypes of nonclasic HLA Ist class: HLA- E,- F,-

G molecules at structure similar to MHC gp Ist class :

CD1

Structure of MHC gp Ist class

2 polypeptide chains (noncovalent association):

- transmembrane chain alfa: subunits alfa -1,-2,-3

- ß2- microglobulin (noncovalent binding)

binding site for peptides: N-terminal domain between subunits alfa 1 and 2

Binding site of MHC molecules Ist class Peptide-binding cleft on the surface : floor- ß-structure,

sides- α-structure binding site is closed - large enough to accommodate

peptides of 8-10 amino acids α-3 subunit contains the binding site for the T cell co-

receptor CD8

Processing of internalized antigens (MHC gp Ist class)

MHC gps Ist class are synthesized in the ER Proteins can be produced in the cytoplasm from viruses

inside infected cells, from mutated or altered host genes, as in tumors- all targeted for destruction by proteolysis

Proteins are covalently tagged with small peptide called ubiquintin in proteasome- proteins are degraded

TAP = transporters associated with antigen processing binds peptides, enters the ER, binds to MHC gps class I

Complex is transported to the Golgi complex- TAP dissociates and complex of MHC gp class I + peptide is transported to the cell surface and recognized by CD8+ T lymphocytes

31. HLA system, 2nd class molecules, their structure and function

MHC gps II class

Expressed on APC (dendritic cells, monocytes, macrophages, B lymphocytes)

Isotypes HLA-DR,-DQ,-DP

Display extracellular peptides to T lymphocytes

Structure of MHC gp IInd class

2 transmembrane subunits: - transmembrane chain alfa consists of

2 subunits (alfa 1 a 2) - transmembrane chain beta consists of

2 subunits (beta 1 a 2) - binding-site for peptides: N- terminal

domain between subunits alfa 1 and beta 1

Binding-site of MHC gp IInd class

Binding-site is opened Cleft is large enough to accommodate peptides of

15-35- amino acids β- 2 domain contains binding-site for the T cell co-

receptor CD4

Processing of internalized antigens (MHC gp IInd class) MHC gps II are synthesized in endoplasmic

reticulum – carry with invariant chain that contains a sequence – CLIP = binds to the peptide binding cleft- transport vesicle

The microbial proteins enter intracellular vesicles (endosomes) – proteins are broken down by enzymes

Endosome fuses with transport vesicle – CLIP is removed – the cleft accepts the peptide, MHC gps IInd class and peptide are transported to the cell surface where are recognized by CD4+ T cells

MHC restriction

T lymphocytes recognize a foreign peptide antigen only when it is bound to MHC gp

Nonclasic MHC gps I class

expressed only on some cells Specialize to binding of specific ligands HLA-G is expressed on the surface of

trophoblast cells complexes HLA-E,-G with peptides are

recognized by inhibitory receptors of NK cells – induce tolerance of fetus in uterus