Download Connective tissue mast cells

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 (FcRI)
 Aggregation of several molecules FcRI
 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)
- 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 antagonistic peptides
 Agonist - antigenic peptide, which induce full T cell
response (proliferation, differentiation, TH or TC and
stimulation of effector functions)
 Antagonist - (partial agonist) peptide structurally similar
to antigen-peptide, which induce qualitatively different
response of T lymphocytes (production of only some
cytokines, anergy, ...)
Negative signals induced by antagonist may overcome
positive signals induced by agonist (which is in the body
in excess), it is used by some microorganisms
Regulation by antibodies
 Antibodies competes with the BCR for antigen (negative
regulator of B lymphocyte stimulating)
 IgG immune complexes bind to the BCR and FcR 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 peptide
MHC gp II + antigenic peptide
CD 80, CD 86 - costimulatory ligands
CD 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)
 CD 8+ TS - suppressor T cells has not yet been isolated
as a separate subset (partly identical with TC)
- negatively regulate the activation of other T cells
 Soluble suppressor factors - some CD 8+ T lymphocytes produce a
soluble form of TCR
 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 by 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 so to T lymphocytes (cytotoxic CD8+)
 Present on all nuclear cells of the organism
 3 isotype classical human MHC gp. (HLA - A,-B,-C)
 3 isotype-classical 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
B2mikroglobulin
  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 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

Peptides binding to MHC gp II
Polymorphism of MHC glycoproteins
 HLA complex is located on chromosome 6
 For MHC gp is typical high polymorphism (except the
non-classical MHC gp, and DR  chain)
 Codominant inheritance of alelic forms
(Individual has 3 cell surface isotypes
of HLA molecules (HLA-A,-B,-C) mostly
in 2 different alelic forms)
 Polymorphism has a protective significance at individual
and population level
 Ppolymorphism MHC gp causes complications in
transplantation
HLA typing = determmination of HLA
antigens on the surface of lymphocytes
Carry out during the testing before transplantation and in
determination of paternity
1) Serotyping - Microlymfocytotoxic test
Allospecific serums (obtained from multiple natal to 6 weeks after birth,
obtained by vaccination of volunteers, 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
 For typing are used hypervariable sections in the II. exon
genes coding for HLA class II; to determine HLA class I is
used polymorphism in II. and III. exon coding genes
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 or radiolabeled DNA probes specific
for individual alleles
2c) PCR-SBT
Sequencing 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 alleles
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