Basophils and mast cells and their importance in

immune responses

Mast cells Mucosal mast cells - in the mucous

membranes of respiratory and gastrointestinal tract, produce histamine, serotonin, heparin, tryptase, leukotriene C4 ..., participate in parasitosis and allergy

Connective tissue mast cells - the connective tissue, producing tryptase, chymase, prostaglandinD2 ..., are multiplicated in fibrosis, in parasitosis and allergy are not participating

Mast cell functions

Defense against parasitic infections

In pathological circumstances, responsible for the early type of hypersensitivity (immunopathological reaction typeI)

Apply during inflammation, in angiogenesis, in tissue remodeling

Mast cell activation

Mast cells can be stimulated to degranulate by:

cross-linking of IgE Fc receptors

by anafylatoxins (C3a, C4a, C5a)

direct injury, alcohol, some antibiotics

Mast cell activation by cross-linking of IgE Fc receptors

Establishing of multivalent antigen (multicellular parasite)

to IgE on highaffinnity Fc receptor for IgE (FcRI)

Aggregation of several molecules FcRI

Initiate mast cell degranulation (cytoplasmic granules mergers with the surface membrane and release their contents)

Activation of arachidonic acid metabolism (leukotriene C4, prostaglandin D2)

Start of production of cytokines (TNF, TGF, IL-4, 5,6 ...)

Mast cell activation scheme

Secretory products of mast cells

Cytoplasmatic granules: hydrolytic enzymes, proteoglycans (heparin, chondroitin sulphate), biogenic amines (histamine, serotonin)

Histamine causes vasodilation, increased vascular permeability, erythema, edema, itching, contraction of bronchial smooth muscle, increases intestinal peristalsis, increased mucus secretion of mucosal glands in the respiratory tract and GIT (helps eliminate the parasite)

Arachidonic acid metabolites (leukotriene C4, prostaglandin D2)

Cytokines (TNF, TGF , IL-4, 5,6 ...)

The role of mast cells in development of allergy

Basophils Differentiate from myeloid precursor

They are considered to be the circulating form of mast

Receptor equipment, containing granules, the mechanisms of stimulation and functions are very similar to mast cells

They are responsible for the emergence of anaphylactic shock

Basophil activation markers: CD 63, (CD 203)

Immune mechanisms of inflammation

(Local and systemic reactions)

Inflammation

* Is a summary of physiological responses to breach the

integrity of the organism, leading to protection against infection of damaged sites, localization of damage and healing.

* The first signals to the development of inflammatory

responses come from mast cells, phagocytes, and the substances released from damaged cells and extracellular components of matter.

Local body's response to inflammation

Manifestations - pain (dolor), fever (calor), redness (rubor), swelling (tumor) and loss of function (funkcio laesa)

Local inflammation- increased permeability of blood vessels (vasoactive amines, complement components C3a, C5a, leukotrienes ..., swelling at site of inflammation)

- increased expression of adhesion molecules on endothelia

- activation of coagulation, fibrinolytic, kinin and complement system - influence of local nerve endings (prostaglandins, pain)

- changes in temperature (IL-1, IL-6, TNF, prostaglandins)

Systemic response to inflammation

- depends on the extent of damage and duration of local inflammation

- fever (proinflammatory cytokines TNF, IL-1, IFN ; stimulate hypothalamic center of thermoregulation)

- mobilization of tissue metabolism

- induction of expression of Hsp (heat-shock-proteins; function as chaperones)

- production of acute phase proteins (CRP, SAP, C3, C4; opsonization and complement activation) by liver after stimulation with cytokines (TNF-α, IL-1, IL-6)

- increased hepatic synthesis of certain serum transport proteins (ceruloplasmin, transferrin)

- increased synthesis of protease inhibitors ( macroglobulin)

- leukocytosis

Septic shock - the massive penetration of microorganisms into the bloodstream (TNF)

Anaphylactic shock - basophil degranulation and complement activation with allergen (histamine)

Repair of damaged tissue

- elimination of damaged cells with phagocytes

- activation of fibroplastic mechanisms

- activation of angiogenesis

- regeneration and tissue remodeling

Physiological mechanisms of

regulation of the immune system

Regulation by antigen

Induce immune responses and extinction

Affinity maturation of B lymphocytes

Maintaining immunological memory

Antigenic competition

Threshold density of the complex MHC II-gp Ag on APC

Regulation by antibodies

Antibodies competes with the BCR for antigen (negative regulator of B lymphocyte stimulating)

IgG immune complexes bind to the BCR and FcR on B cells, resulting in blocking activation of B lymphocytes

Regulation via idiotypic network

Regulation by cytokines and cellular contact

Interaction APC - T lymphocyte Interaction TH1 – macrophages Interaction TH2 - B lymphocytes Mutual regulation of activity TH1 versus TH2 Development of leukocyte subpopulations

Negative regulation of effector cells: CTLA-4 - T cell inhibitory receptor, binds ligands CD80

and CD86 Inhibitory receptors of NK cells Self-destruction interaction of the apoptotic receptor Fas

with ligand FasL on the surface of activated T lymphocytes

Interaction of APC with T lymphocyte

T cell:TCR - antigen-specific receptor (signal 1)CD4 or CD8 - coreceptor (MHCgp binding)CD 28 - costimulatory receptor (signal 2, binds CD 80, CD 86)CTLA-4 - inhibitory receptor (binds CD 80, CD 86)CD-40L

APC:MHC gp I + antigenic peptideMHC gp II + antigenic peptideCD 80, CD 86 - costimulatory ligandsCD 40

Suppression mediated by T lymphocytes

Mutual negative interaction TH1 and TH2 cytokine-mediated (TH2 lymphocytes produce IL-4 and IL-10 that suppress the immune response based on TH1 cells)

Clonal elimination or anergy of T lymphocytes after contact with antigen on the surface of other cells than APC (lacking costimulating signals)

Regulatory T cells (Tr1 CD 4+) help to maintain tolerance to autoantigens

Factors influencing the outcome of the immune response

The same antigen can induce an active immune response or an active state of tolerance, the result of response depends on many factors:

State of the immune system

Properties of antigen

Dose of antigen

Route of antigen administration

Cytokines (Tissue hormones)

Cytokines Regulatory proteins and glycoproteins produced

by leukocytes and other cells

Essential regulators of the immune system

Apply also outside the immune system (angiogenesis, tissue regeneration, carcinogenesis, treatment of many brain functions, embryonic development ...)

Cytokines - secreted      - membrane (CD 80, CD86, CD40L, FasL ..)

Pleiotropic effect

Operates in a cascade

Cytokine Network

Cytokine system is redundant

Effects of cytokines - autocrine - paracrine

- endocrine

Are known as interleukins (exception: TNF, lymphotoxin, TGF, interferons, CSF and growth factors)

B cells communicate via cytokines with other

inflammatory cells, such as T cells and macrophages

Distribution of cytokines according their function

Proinflammatory cytokines (IL-1, IL-6,IL- 8,IL- 12,IL- 18, TNF)

Antiinflammatory cytokines (IL-1Ra, IL-4, IL-10, TGF)

Cytokines with the activity of hematopoietic cells growth factor (IL-2, 3, 4, 5, 6, 7, 9, 11, 14, 15, CSF, SCF, LIF, EPO)

Cytokines applying in TH2 humoral immunity (IL-4, 5, 9, 13)

Cytokines applying in the cell-mediated immunity TH1 (IL-2, 12, IFN, GM-CSF, lymphotoxin)

Cytokines with anti-virus effect (IFN-, IFN-, IFN- )

Cytokine receptors Consisting of 2 or 3 subunits

One subunit binds cytokine, other are associated with cytoplasmic signaling molecules (protein kinases)

Signaling subunit is shared by several different cytokine receptors - called receptor family

Signaling through these receptors may lead to proliferation, differentiation, activation of effector mechanisms or blocking the cell cycle and induction of apoptosis

HLA system (MHC glycoproteins)

MHC glycoproteins class I (Major histocompatibility complex)

The function of MHCgpI is presentation of peptide fragments from inside the cell (which are produced by cell, including viral peptides if are present) on the cell surface to T lymphocytes (cytotoxic CD8+)

Present on all nuclear cells of the organism

3 isotypes of classical human MHC gp. (HLA - A,-B,-C)

3 isotypes of nonclassical MHC gp. (HLA - E,-F,-G; molecule CD1)

Structure of MHC gp I

MHC gp class I consists of transmembrane chain and non-covalently associated 2mikroglobulin

chain has 3 domains, 2 N-terminal (1, 2 - binding site for peptides) and 1 C-terminal domain (3 - anchored in the cytoplasmic membrane, a structure similar to imunoglobulin domain)

Binding of peptide is necessary for a stable conformation of MHCgp and thus ensure its long presentation on the cell surface

Peptides binding to MHCgpI MHC gp I bind peptides with a length of 8 to 10

aminoacides Certain MHC gp molecule binds peptides sharing

common structural features - coupling motif (critical are aminoacides near the end of peptide)

The binding of endogenous peptides occurs in the endoplasmic reticulum during biosynthesis of MHC gp

After a string and 2mikroglobulin create in the ER, folding into the correct conformation and the mutual association and the association of an appropriate peptide, the complex is further processed in the Golgi apparatus and then is presented on the cell surface

Linked peptides derived from proteins degraded proteasome, which cleaves cytoplasmic proteins for destruction (labeled with ubiquitin), peptide fragments are transported into the ER by specific membrane pump

Peptides binding to MHCgpI

Peptides binding to MHC gp I

Peptides binding to MHC gp I

Non-classical MHC gp I

HLA - E,-F,-G; CD1 molecules

Structurally similar to classical MHC gp

Are less polymorphic

There are only on some cells

They specialize in binding of specific ligands

HLA-E and HLA-G - occurs on the trophoblast cells

Complexes of HLA-E and HLA-G with peptides are recognized by inhibiting receptors of NK cells and contribute to the tolerance of the fetus in utero

CD1 molecules - bind glycolipids (recognized by NK-T lymphocytes)

MHC glycoproteins class II The function of MHC gpII is the presentation of

peptide fragments from protein whitch were engulfed by antigen presenting cell on the cell surface to T lymphocytes (auxiliary CD4)

Occur on the APC (dendritic cells, monocytes, macrophages, B lymphocytes)

3 isotypes of MHC gpII (DR, DQ, DP)

Structure of MHC gp II MHC gp II consist of 2 non-covalently associated

transmembrane subunits and

The peptide binding site consists of N-terminal domains 1 and 1

Binding of peptide is necessary for a stable MHC gp conformation and thus ensure its long presentation on the cell surface

Binding of peptides to MHC gp II

MHC gpII bind peptides with a length of 15 to 35 aminoacides (but possibly longer - because the peptide binding site is open at both ends)

Certain MHC gp molecule binds peptides sharing common structural features - coupling motif

After a string and are created in ER, fold into the correct conformation and the mutual associated are connected with another transmembrane chain called invariant chain, which blocks the binding site for the peptide, this complex is further processed in the Golgi apparatus, secretory vesicles isolated from GA merge with endosomes, then split the invariant chain and peptide fragments from cell absorbed proteins bind into binding site of MHC gp and the complex is then presented on cell surface

Binding of peptides to MHC gp II

Peptides binding to MHC gp II

HLA system – genetic background

HLA complex is localized on chromosome 6

Codominant inheritance of HLA( Individual has 3 cell surface isotypes of HLA molecules (HLA-A,-B,-C) mostly in 2 different alelic forms )

For MHC gp is typical high polymorphism (except the non-classical MHC gp)

Polymorphism has a protective significance at individual and population level

Ppolymorphism MHC gp causes complications in transplantation

Polymorphism of MHC glycoproteins

Carry out during the testing before transplantation and in determination of paternity

HLA typing1) Serotyping - Microlymfocytotoxic test Allospecific (typing) serums (obtained from multiple

natal to 6 weeks after birth, or commercially prepared sets of typing serums (monoclonal antibodies))

Principle - the incubation of lymphocytes with typing serums

in the presence of rabbit complement, then is added the vital dye which stained dead cells

- cells carrying a specific HLA are killed by cytotoxic Ab against the Ag

- the percentage of dead cells is a measure of serum toxicity (forces and antileukocyte antibody titre)

Positive reaction is considered more than 10% dead cells (serological typing can be done also by flow cytometry)

2) Molecular genetic methods

2a) PCR-SSP = Polymerase chain reaction with sequential specific primers Extracted DNA is used as a substrate in a set of PCR

reactions Each PCR reaction contains primers pair specific for a

certain allele (or group of alleles) Positive and negative reactions are evaluated by

electrophoresis, each combination of alleles has a specific electrophoretic painting

2b) PCR-SSO PCR reaction with sequence-specific

oligonucleotides Multiplication of hypervariable sections of

genes coding HLA Hybridization with enzyme-labeled or

radiolabeled DNA probes specific for individual alleles

2c) PCR-SBT (Sequence based typing) The most accurate method of HLA typing. We get the exact sequence of nucleotides, which

compares with a database of known sequences of HLA allels

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