Complement J. Ochotná

Complement  humoral component of nonspecific immunity  helps remove microorganisms and own altered cells (apoptotic cells)  complement proteins are synthesized in the liver, less by tissue macrophages and fibroblasts

 system of about 30 serum and membrane proteins  complement components are present in serum in inactive form  complement activation has cascade character Complement

 the main complement components : C1-C9 (C3 is the central component)  other complement components : factor B, factor D, factor P  regulatory proteins : C1 - inhibitor, factor I, factor H, C4bp, DAF, MCP, CR1, factor S (vitronectin), CD59 (protektin), anaphylatoxin inactivator Complement

Biological significance  Opsonization (C3b, C4b)  Chemotaxis (C3a, C5a)  Osmotic lysis (MAC C5b-C9)  Anaphylatoxins (C3a, C4a, C5a)

Riedem ann N.C. Pathways of complement activation Classical pathway Alternative pathway Lectin pathway

Classical and alternative pathway

Alternative complement pathway  C3 component of complement rarely spontaneously breaks into C3b and C3a  C3b can covalently bind on the surface of a particle (own cell, microorganism) or reacts with water, thereby inactivates

 to bound C3b joins a factor B, which is cleaved by factor D to Ba and Bb, resulting complex C3bBb is stabilized by factor P (properdin) and functions as an alternative C3 convertase Alternative complement pathway

 C3 convertase cleaves C3 to C3a (chemotaxis) and C3b, which binds to the surface of the particles (opsonization), or gives rise to other C3 convertases Alternative complement pathway

 some C3 convertases form C3bBbC3b that act as an alternative C5 convertase, which cleaves C5 to C5a (chemotaxis) and C5b (starts terminal lytic phase) Alternative complement pathway

 C5b starts terminal lytic phase

Classical complement pathway  can be initiated by antibodies IgG ( not by IgG4) and IgM or by pentraxins (CRP, SAP - acute phase proteins)  after binding of antibodies to the bacteria surface, Ab changes its conformation and than can bind C1 protein

 C1 changes its conformation and acquires proteolytic activity, than cleaves C4 and C2 proteins Classical complement pathway

 fragments C4b and C2a bind to the surface of microorganism and create the classic C3 convertase (C4bC2a), which cleaves C3 to C3a and C3b Classical complement pathway Description of C3 convertase has been modified to C4bC2a

 then creates a classic C5 convertase (C4bC2aC3b) that cleaves C5 to C5a and C5b Classical complement pathway

Lectin complement pathway  is initiated by serum mannose binding lectin (MBL)  MBL binds to mannose residues on the surface of some microbes, after this binding starts cleave C4 and C2  this way is similar to the classical pathway

Lectin complement pathway

Terminal (lytic) phase of the complement cascade  C5b fragments creates a complex with C6, C7 and C8, the complex dive into the lipid membrane of the cell and attache into a circle molecules of C9 => MAC (membrane attack complex), forms a transmembrane channel, which causes osmotic lysis of the target cell (G-bacteria, protozoans, some viruses).  Most microorganisms are resistant to this lytic effect of complement (protection by cell wall).

Terminal phase of the complement cascade

Complement activation and efector functions

Complement regulation and protection of own cells  Activation of complement cascade is controlled by the plasma and membrane inhibitors. MCP DAFProtectin Anaphylatoxin inactivator

Complement regulation  C1 inhibitor (C1-INH) – inhibits C1; if missing→ HAE  factor I with cofactors: MCP (membrane cofactor protein), CR1, factor H – C3b, C4b cleavage  DAF (decay-accelerating protein)-degradation of C3 and C5 convertase

 factor S (vitronectin) – inhibits complex C5bC6  CD 59 (protectin) - prevents the polymerization of C9  anaphylatoxin inactivator (CPN) - inactivates anafylatoxins (C3a, C4a, C5a) Complement regulation

Complement receptors  Bind fragments of complement components  CR1 - on various cells - promotes C3b, C4b decay - stimulate phagocytosis - erythrocyte transport of immunecomplexes  CR2 - on B lymphocytes and FDC - activation of B cells

 CR3, CR4 - on phagocytes - participation in opsonization, adhesion  C3aR, C5aR – receptors for anaphylatoxins - mast cell activation Complement receptors

4 basic complement functions  Opsonization (C3b, C4b)  Chemotaxis (C3a, C5a)  Osmotic lysis (MAC C5b-C9)  Anafylatoxins (C3a, C4a, C5a)

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 – in 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 type I)  Apply during inflammation, in angiogenesis, in tissue remodeling

Mast cell activation Mast cells can be stimulated to degranulate by:  cross-linking of IgE receptors (Fc  RI)  anafylatoxins (C3a, C4a, C5a)  TLR

Mast cell activation by cross-linking of IgE Fc receptors  Binding of IgE to highaffinnity Fc receptor for IgE (Fc  RI)  Binding of multivalent antigen (multicellular parasite) to IgE  Aggregation of several molecules of Fc  RI

Mast cell activation  Mast cell degranulate (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...)

Secretory products of mast cells  Cytoplasmatic granules: hydrolytic enzymes, histamine, heparin, chondroitin sulphate, serotonin Histamine - vasodilation, ↑ vascular permeability (erythema, edema, itching), bronchoconstriction, ↑ intestinal peristalsis, ↑mucus secretion in the respiratory tract and GIT (helps eliminate the parasite)  Arachidonic acid metabolites (leukotriene C4, prostaglandin D2)  Cytokines (TNF, TGF , IL-4, 5,6...)

Basophils  Differentiate from myeloid precursor  Receptor equipment, content of granules, the mechanisms of stimulation and functions are very similar to mast cells  Play a role in parasitic infections and allergies  Basophil activation markers: CD 63, (CD 203)

Immune mechanisms of inflammation (Local and systemic reactions)

Inflammation Is a protective physiological response leading to protection against infection in damaged sites, localization of damage, elimination of necrotic cells and tissue repair.

Local body's response to inflammation Classical signs - pain (dolor), fever (calor), redness (rubor), swelling (tumor)

Inflammation The first signals for the development of inflammatory response come from mast cells, phagocytes, and the substances released from damaged cells and components of extracellular matrix. With longer duration of local inflammation are activated an antigen-specific mechanisms (T and B lymphocytes).

Inflammation

Inflammation – local reaction vasodilation, ↑ vascular permeability (histamine, serotonin, bradykinin, complement components C3a, C5a, leukotrienes, prostaglandins, …) → rednes, swelling ↑expression of adhesion molecules on endothelia ( TNF , IL-1) → leukocyte adhesion to the endothelium influence of local nerve endings via prostaglandins → pain Increased local temperature (IL-1, IL-6, TNF, prostaglandins)

Inflammation - systemic reaction Leukocytosis Fever (TNF, IL-1, IL-6, IFN  ↑ tissue metabolism ↑ mobility of leukocytes ↑ formation of IFN, cytokines, Ig ↑ expression of Hsp Acute phase proteins (IL-6, TNF , IL-1) CRP, SAP - opsonization and complement activation

Inflammation - systemic reaction Septic shock - the massive penetration of microorganisms into the bloodstream (TNF) Anaphylactic shock - basophil and mast cells activation with allergen (histamine)

Tissue repair elimination of damaged cells with phagocytes activation of fibroplastic mechanisms activation of angiogenesis regeneration and tissue remodeling

Antigens

Antigen (immunogen)  substance which provokes specific immune response  usually proteins or polysaccharides (lipids and nucleic acids only in the combination with proteins or polysaccharides)  molecules > 5 kDa (optimal size of the antigen molecules is about 40 kDa)

Hapten  small molecules, that are able to induce specific immune response only after the attachment to the macromolecular carrier  separate haptens are not immunogenic  typically drugs (eg penicillin antibiotics, hydralazin)

Epitope (antigenic determinant)  part of the antigen which is recognized by immune system (lymphocytes- BCR, TCR, Ig)  cross-reactive antigens - shares one or more identical or similar epitopes

Interaction antigen – antibody  Binding site of antibody ( paratop ) form non-covalent complexes with the corresponding part on antigen molecule ( epitope )  participation: the hydrogen bonds, electrostatic and hydrophobic interactions, van der Waals forces  antigen-antibody complex is reversible

Antigen  endogenous antigens - autoantigens (self Ag)  exogenous antigens - foreign substances from the environment  allergen is exoantigen that in the susceptible individuals can cause pathological (allergic) immune response

Properties of antigen  immunogenicity proteins> carbohydrates> macromolecule complexes ( glycoproteins, nucleoproteins, and glycolipids )> lipids  specificity

Factors affecting immunogenicity  Physical: solubility - insoluble Ag more immunogenic molecular weight - ideal 5-40 kDa  Chemical: structure - the number of determinants degradability - "ease" of uncovering the determinants in antigen presenting cells (APC cell)

 Biological: biological heterogeneity genetic and physiological disposition of the body Properties of antigen

Degree of foreignness  Autogeneic - antigens of the same individual  Syngeneic - antigens of genetically identical individuals (eg twins)  Allogeneic (alloantigens) - antigens genetically different individuals of the same species  Xenogeneic (heterologous) - antigens derived from individuals of different species (eg monkey kidney transplant man)

Types of antigens according to antigen presentation  T- dependent antigens  T- independent antigens

Thymus dependent antigens  more frequently, mostly protein Ag  for specific humoral immune response to antigen is necessary assistance of T H lymphocytes (or response isn´t enough effective)  assistance implemented in the form of cytokines produced by T H lymphocytes

T- independent antigens  can stimulate B cells directly  mainly bacterial polysaccharides, lipopolysaccharides and polymer forms of proteins (e.g. Haemophilus, Str.pneumoniae)

T-independent pathway

Superantigens  stimulate T cells polyclonaly and massively (massive cytokine release)  massive activation of T cells can cause shock  e.g. bacterial toxins (Staph.aureus, Str.pyogenes, Pseud.aeruginosa)

Proteins (microbial products) which have 2 binding sites, one interacts with the epitope presented on all MHCgpII, second interacts with other structures presented in many different TCR molecules (connection of T lymphocyte with APC) Superantigens

Differcence between antigen and superantigen binding

Sequestered antigens  autoantigens, that are normally hidden to immune system and therefore unknow (e.g. brain, the lens of the eye, testes)  if they are "uncovered" by demage, exposed to the immune system, are recognized as foreign (one of the theories of autoimmune processes)

Immunologically privileged sites  brain, eye, gonads  are protected from potentially damaging inflammatory immune responses  this tissues are far less rejected in allogeneic transplant (cornea)  this privileged position is not absolute

Immunologically privileged sites  Mechanisms of protection from the immune system :  isolation from the immune system (blood-brain barrier)  preferences of Th2 and suppression of Th1-response  production of immunosuppressive cytokines ( TGFβ )  FasL expression -active protection against effector T-lymphocytes  increased expression of membrane complement inhibitors

Thank you for your attention

Komplement – klasická cesta Komplement – klasická a alternativní cesta Komplement – alternativní cesta Akutní zánět