1. The complement system, antigens
Martin Liška

2. The complement system
A complex consisting of at least 20 serum proteins, which, once activated, acts like a part of the innate immune defense
The complement components are present in serum in inactive form
The complement is activated in a cascading manner (= each protein activates that following) and it has widespread physiologic and pathophysiologic effects
Complement proteins are synthesized mainly in the liver, but tissue macrophages and fibroblasts can synthesize some complement proteins as well

3. The complement system nomenclature
C1q, C1r, C1s, C2-9
Factor B, Factor D
Properdin
Regulatory proteins (C1-inhibitor, Factor I, C4bBP, Factor H, S protein, anaphylatoxin inactivator)

4. The classical pathway of complement activation
The pathway is activated through antigen-antibody complexes: initially, C1 component binds to a site on the Fc fragment of Ig (IgG (but not IgG4) or IgM); however, native Ig molecules do not interact with C1
C1 component – contains three polypeptides (C1q, C1r, C1s); C1q attaches first to Ig (for initiation of complement activation, C1q has to interact with two or more Ig monomers) → C1q activates proenzyme C1r → C1r cleaves proenzyme C1s → C1s is able to cleave C4 component

6. The classical pathway of complement activation
Activated C1s cleaves C4 to C4a (an anaphylatoxin) + C4b → C4b binds to cell membranes → the next component becomes susceptible to enzymatic attack by activated C1
C4b + C2 + C1s → removal of C2a → enzymatically active molecular complex C4b2b (= C3 convertase of the classical pathway)
Formation of C3 convertase represents the nodal point for all pathways of complement system activation

12. The alternative pathway of complement activation
The alternative pathway is considered to be a primitive „bypass“ mechanism, that does not require C1, C2 and C4
The pathway is activated through reaction of the complement system and some substances of microbial origin (polysaccharides – e.g. lipopolysaccharides of G negative bacteria, teichoic acid of G positive bacteria, zymosan from yeast cell walls, surface components of some animal parasites) or other foreign materials

13. The alternative pathway of complement activation
C3 cleaves into C3a + C3b spontaneously; however, these are inactive under standard conditions
In this case, C3b binds to microbial surface → it reacts with factor B → removal of Ba (it is chemotactic for neutrophils) → C3bBb (= C3 convertase of the alternative pathway) – it is stabilized by properdin (P)

14. The lectin pathway of complement activation
The pathway is activated through binding of MBL (mannose-binding lectin) to microbial surface → C4 → C4a is released + C4b → C4b reacts with C2 → C2b is released → C4b2a complex (= C3 convertase)

15. Further events of complement activation process
C3 convertase causes generation of:
a/ C3a, C4a, C5a fragments
- C3a and C4a act as peptide mediators of inflammation
- C5a is chemotactic for phagocytes
b/ C3b
it binds to complement receptors and causes opsonization of microbes and immunocomplexes

18. Further events of complement activation process
c/ MAC (membrane attack complex)
- formed from terminal complement components (C5b, C6, C7, C8, C9); it attacks membrane of cells (e.g.microbes), causing their osmolysis

23. Complement receptors
Fragments of complement components can bind to complement receptors, which are expressed on the surface of different cells.
CR1 – erythrocytes, granulocytes,
monocytes and B cells
- important for IC clearance
CR2 – B cells and follicular dendritic cells
- immunoregulation
CR3, CR4 – phagocytes
- opsonization

24. Regulatory mechanisms of the complement system
Some serum proteins enzymatically attack complement components, thereby inactivating them (factor I inactivates C3b; anaphylatoxin inactivator inactivates anaphylatoxins (C3a, C4a, C5a))
Some serum proteins bind to, and thus inhibit, complement components (C1-INH inhibits C1; C1-INH deficiency → HAE = recurrent episodes of local edema; factor H acts with factor I the inhibition of C3b; S protein binds to C5b67 → prevention of MAC binding to cell membrane)

25. Regulatory mechanisms of the complement system
Regulatory proteins in cell membranes (DAF (decay-accelerating factor) – it has the same function as factor H → the inactivation of C3b and C4b; membrane cofactor protein – it serves as a cofactor for inactivation of C4b and C3b)

26. Functions of the complement system
C3a, C5a – anaphylatoxins (= they cause release of histamine and other vasoactive compounds from basophils and mast cells, increasing capillary permeability)
C3b, C4b – opsonization (they bind IC to macrophages and neutrophils, enhancing phagocytosis; also binds complexes to erythrocytes, facilitating removal by the liver and spleen)
C5a – chemotaxis (attracts phagocytic cells to sites of inflammation and increases their overall activity)

27. Functions of the complement system
C8, C9 – components of MAC
Ba – neutrophil chemotaxis
Bb – macrophage activation

28. Functions of the complement system - overview
Inflammation (mast cell degranulation, chemotaxis, increases vascular permeability, margination and diapedesis of polymorphonuclears, smooth muscle contraction, activation of polymorphonuclears, NK cells and macrophages)
Clearance of immune complexes
Cell lysis (G negative bacteria, Protozoa, some viruses)
Viral neutralization
Opsonization

29. The complement system - overview
The alternative and lectin pathways are clear components of innate immune system, whereas the classical pathway depends on addaptive immune response (it is triggered through antigen-antibody reaction)
Three functions of the complement system:
1/ C3b coats microbes and promotes the binding of these microbes to phagocytes (by receptors for C3b)

30. The complement system - overview
2/some breakdown products of complement proteins are chemoattractants for neutrophils and monocytes and promote inflammation at the site of complement activation
3/ complement activation results to the formation of a polymeric protein complex (MAC), causing osmolysis or apoptosis of microbes

31. Antigen
A substance, which is recognized by immune system and induces the immune response
It comes from environment (exoantigen), or from individual’s own structures (autoantigen)
Usually proteins or polysaccharides (lipids and nucleic acids can act as antigens when combined with proteins or polysaccharides)

32. Haptens
Small and well-defined chemical structures, which are not immunogenic themselves, but can add a new epitope when combined to an existing antigen
The antibody directed against the new epitope will react with the free hapten as well as the hapten-epitope site in the altered antigen
Typically drugs (e.g.penicillin ATB, hydralazine)

33. Epitope
The portion of antigen, which is recognized by the immune system (lymphocytes, Ig)
Epitopes may be linear (amino acid sequence important), conformational (space conformation important)
Some epitopes are on the antigen’s surface, others are internal
Cross-reactive antigens – share one or more identical or similar epitopes

34. Antigen-antibody reaction
Binding sites of antibodies (paratope) interract with the corresponding sites of the antigen (epitope)
The bonds that hold the antigen-antibody complex are non-covalent (hydrogen, electrostatic and hydrophobic bonds, Van der Waals forces)
Antigen-antibody complex is reversible

35. T cell dependent and independent antigens
1/ T cell dependent antigens
more common, typically contain protein component
a help from T helper cells is necessary for specific humoral immune response generating, otherwise the response is not so effective
the help comes in form of cytokines secreted by the T cell

36. T cell dependent and independent antigens
2/ T cell independent antigens
in some antigens, antibody production can be induced directly, without help from T cells
bacterial lipopolysaccharides and polymeric forms of proteins (e.g. Haemophilus, Str.pneumoniae)

37. Superantigens
Antigenic structures, capable to induce response of T cells by external binding to MHC molecules (i.e. outside of the usual binding site)
The stimulation is polyclonal and extensive
Some bacterial toxins (Staph.aureus, Str.pyogenes, Pseud.aeruginosa)

38. Sequestered antigens
The antigens, which are normally hidden from the immune system at privileged sites, and thus the immune system cannot identify them (e.g. lens, testes)
However, if these allergens are released (injury), the immune system could response to them (the potential mechanism of autoimmunity development)

39. Immunologically privileged tissue
In allogeneic transplantation, some tissues are rejected less frequently (e.g. CNS, cornea, gonades).
The mechanisms of protection from the immune system: separation from the immune system (haematoencephalic barrier); the preference of Th2- and suppression of Th1-reactions; active protection from effector T cells
The privileged status is not absolute (see MS)

40. Molecular size of antigens
Molecules < 5 kDa are not able to induce immune response, the optimal molecular size for immune response induction is approximately 40 kDa

41. Degree of foreignness An antigen must be foreign or alien to the host
The greater the phylogenetic difference, the more foreign something becomes; based on this fact, we can distinguish the following types of antigens:

42. Degree of foreignness
Autologous – are found within the same individual (e.g. a skin graft from an individual’s thigh to his chest); that is, they are not foreign
Syngeneic – are found in genetically identical individuals (e.g. identical twins); that is, they are not foreign
Allogeneic (alloantigens) – are found in genetically dissimilar members of the same species (e.g. a kidney transplant from mother to daughter); it is foreign
Xenogeneic (heterogeneic) – are found in different species (e.g. a transplant of monkey kidneys to human); it is foreign