The Complement System Complement-induced lesions on the membrane of a RBC

Complement system Jules Bordet: Fresh serum(ab) + bacteria (Vibrio cholerae) at 37oC +++ Fresh serum(ab) + bacteria at 56oC ----- Paul Ehrlich: complement: the activity of blood serum that completes the action of antibody Complement: More than 30 serum and cell surface proteins (2 or 3 chains a, B and gamma with s-s bond) Exit as zymogens Two main converging pathways of complement activation co-exist Various biological functions Highly regulated Produced with Mono, MQ, Fibroblast, Epithelial of gut, Parenchyma of liver , Lung, Fatty tissues, genitourinary tracts.

The Complement Components - More than 30 soluble and cell-bound proteins - Participate in both innate and adaptive immunity - - Constitute 15% (by weight) of the serum globulin fraction - Many components are proenzymes (zymogens), which are functionally inactive until proteolytic cleavage, which removes an inhibitory fragment and exposes the active site. - Reaction starts with an enzyme cascade. For defense against microorganisms coming from outside? Is 5% a lot?

The Functions of Complement 1. Lysis of cells, bacteria, and viruses – the major effector of the humoral branch of the immune system 2. Opsonization, which promotes phagocytosis of particulate Ags 3. Binding to specific complement receptors on cells of the immune system, triggering specific cell functions, inflammation, and secretion of immunoregulatory molecules 4. Immune clearance, which removes immune complexes from the circulation and deposits them in the spleen and liver

Complement components C1 (C1q, C1r, C1s ) factor B C2 (C2a, C2b) factor D C3 (C3a, C3b) DAF, CD55 C4 (C4a, C4b) CR1,CD35 C5 (C5a, C5b) factor H C6 factor I C7 C8 C9

Complement Activation 1. Classical Pathway – begins with the formation of Ag-Ab complex 2. Alternative Pathway – is initiated by cell-surface constituents that are foreign to the host – Ab-independent 3. Lection Pathway – is activated by the binding of mannose- binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms

Classical Pathway Begins with Ag-Ab Binding soluble Ag-Ab* or bacteria-Ab* or Ab aggregates, CRP, Pro-A ↓ conformational changes in the Fc portion of Ig expose a binding site on the CH2, IgG & CH3,4 in IgM domain of the Fc portion for the C1 component of the complement system * complement-activating Abs: IgM, IgG1, IgG2, IgG3 (human) C1 molecule C1qr2s2 stabilized by Ca++

Each C1q is able link to 12 IgG and binding of 2 IgG is sufficient for activation of complement.

Classical Pathway – C1q: The Recognition Unit For C1q to initiate the cascade it must attach to at least 2 Fc fragments, requires at least 2 molecules of IgG or one molecule of IgM. C1q binding causes C1r to enzymatically activate C1s as Serine esterase.

Binding of C1q to Fc induces a conformational change in C1r ↓ __ C1q binds to Ag-bound Ab ↓ Binding of C1q to Fc induces a conformational change in C1r ↓ __ C1r converts to an active serine protease enzyme, C1r, __ which cleaves C1s to a similar active enzyme, C1s CH2 domain of the Ab bacterium

C1s has two substrates, C4 and C2 __ ↓ __ ↓ C1s hydrolyzes C4 into C4a and C4b, and hydrolyze C2 into C2b and C2a ↓ ___ C4b and C2a form a C4b2a complex, also called C3 convertase, referring to its role in converting the C3 into an active form. an anaphylatoxin*, or a mediator of inflammation C2a C4b * Anaphylaxis ( )

C4b2a (C3 convertase) hydrolyzes C3 into C3b and C3a ☺ ↓ ____ ______ ↓ ____ ______ C3b binds to C4b2a and form C4b2a3b (C5 convertase) _______ ↓ C4b2a3b cleaves C5 into C5b and C5a (last enzyme!!!!) opsonization inflammatory responses inflammatory responses ©

Membrane-attack Complex (MAC) Formation of C5b6789, Membrane-attack Complex (MAC) C5b attaches to C6, then to C7, and the C5b67 complex inserts into the membrane. ↓ binding of C8 to membrane-bound C5b67 induces a 10 Å pore (leaky patch). binding and polymerization of C9, a perforin-like molecule, to C5b678 The completed membrane-attack complex (MAC) has a tubular form and functional pore size of 70 – 100 Å

B) Alternative or Properdin pathway: C3b: The Recognition Unit This pathway is initiated by: *Immunologic stimuli: IgA, IgG4, IgE (complex with Ag and aggregate form, with Fabʹ2 piece) * Non- immunologic Stimuli: PLS, LPS, Endotoxine of Neg Bac, CVF, C3Nef, Inuline, Zymozane, Parsites, Virus, Cancer cells * It starts at C3 then C5, C6, C7, C8, C9 * The complement component. C1, C4, C2 are by-passed * Antibodies are not required to initiate activation of this pathway * Four serum proteins, C3, factor B, factor D, and properdin, are involved in this pathway.

C1s = factor D C4b2b = C3bBb C4b2b3b = C3bnB3b ___ plasma C3, with an unstable thioester bond, can be hydrolyzed spontaneously into C3a and C3b. C3b attaches to the surface of bacteria, yeasts, viruses (or even host’s own cells). ___ analogous to the C4b2a complex in the classical pathway Mg++ Ba (stabilization of C3bBb) The membranes of most mammalian cells have high levels of sialic acid, which contributes to the rapid inactivation of bound C3b molecules on host cell; consequently this binding rarely leads to further reactions on the host cell membrane. C1s = factor D C4b2b = C3bBb C4b2b3b = C3bnB3b ©

The Lectin Pathway Originates with Host Proteins Binding Microbial Surfaces Lectin: proteins that bind to a carbohydrate MBL (mannose-binding lectin): - an acute phase protein which binds to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms (structurally similar to C1q) MASP-1 & MASP-2: MBL-associated serine protease (structurally similar to C1r and C1s)

MBL is induced during inflammatory responses. After MBL binds to the surface of a microbe, MBL-associated serine proteases, MASP-1 and MASP-2, bind to MBL. The MBL-MASP-1/2 complex mimics the activity of C1r and C1s, and causes cleavage and activation of C4 and C2. Thus, the lectin pathway is Ab-independent. It is an important innate defense mechanism comparable to the alternative pathway, but utilizing the elements of the classical pathway, except for the C1 proteins.

Q2: Why doesn’t mannose-binding lectin (MBL) bind to host carbohydrates? A2: Mammalian cells normally have sialic acid residues covering the sugar groups recognized by MBL and are not a target for binding.

Overview of the main components and effector actions of complement

The Three Complement Pathways Converge at the Membrane-attack Complex the production of an active C5 convertase ↓ C5b6789 membrane-attack complex (MAC)

Formation of Membrane-attack Complexes (MAC) on the cell surface Poly-C9 complex Complement-induced lesions formed in vitro on the membrane of a RBC

Lysis of an E. coli by Complement

Membrane attack complex Innocent bystander lysis, death of un-related cells by complement

The Membrane Attack Complex C5b C5a C5 70-100 Å C9 C9 C9 C9 C9 C9 C9 C9 C9 C9 C9 C7 C8 C6

Classic And Alterenative pathways Classic Pathway Alternative pathway * Specific acquired immunity * Non-specific innate immunity * Initiated by antibody * Bacterial endotoxin, capsule * Interaction of all components * C1, C4, C2 are by-passed * Properdin system not involved * Properdin system is involved

a b c d e f g h i j (Vitronectin)

(B) ____ C4b2a C3bBb C3 convertase

(C) h i j

Biological Effects Mediated by Complement 1. Cell lysis The membrane-attack complex can lyse a broad spectrum of cells: G(-) bacteria parasites viruses erythrocyte nucleated cells (tumor cells) Because the activation of alternative and lectin pathways is Ab-independent, these pathways serve as important innate immune defenses against infectious microorganisms.

2. Inflammatory response - Various peptides generated during activation of complement play a decisive role in the development of an effective inflammatory response. - C3a, C4a, C5a (called anaphylatoxin) bind to complement receptors on mast cells and basophils and induce degranulation with release of histamine and other mediators. - The anaphylatoxins also induce smooth-muscle contraction, increased vascular permeability, extravasation, and chemoattraction (induced by C5a, C3a, and C5b67)

3. Opsonization - C3b is the major opsonin of the complement system, although C4b and iC3b also have opsonizing activity. binds to the surface of microbes C3 convertase

Opsonization by Ab and complement

4. Viral neutralization - Formation of larger viral aggregates reduces the net number of infectious viral particles - The deposits of Ab and complement on viral particle neutralizes viral infectivity by blocking attachment to susceptible host cells and facilitates binding of the viral particle to cells possessing FcR or CR1.

5. Clearance of immune complexes

Relationship between the factors of the alternative, classic and MBL

Functional protein classes in the complement system

Regulation of the Complement Cascade Short half-time of C3b C3bBb C5b C1 inhibitor Inhibits the C1s activity Protein S in Serum Binds to C5b67 Inhibits Formation of the Membrane Attack Complex HRF or CD59 Bind to C8 Inhibits C9 binding Factor H Binds to C3b Facilitates binding of Factor I cleaves C3b to inactive iC3b cleaves C4b to inactive fragments Decay Accelerating Factor Increased dissociation of C3 convertase (both pathways)

CVF is look like C3b and bond to factor B and reacted with factor D and developed C3bBb or C3 convertase in Alternative pathway. Nephrotic factor is IgG3 against factor B in C3bBb or C3 convertase in Alternative pathway. Complement protein deficiency; 1- acquired with malneutrition and 2- hereditary ; C1 Inhibitor, C3 (infection), C5-C9 deficiency (Neisseria, Meningococcal, Gonococci, ), C1,2,4(Rheumatic disease, SLE)

C1 Inhibitor deficiency Acquired or hereditary disease Autosomal dominant C1 Inhibitor , C2, C4 level decreased Lack C4b2b Edema in the hand, legs, lips, skin, face…….. C2 kinin is responsible for edema In some patients Abnormal form of C1 Inhibitor is produced and is not distinguished with natural form of C1 Inhibitor in RIA. Distinguished with low level of C4 protein.

Complement receptor deficiency CR3, CR4 and LFA-1 on results in Neutrophils repetitive infection in skin and mucosal in children with late dissociation of cord blood. Neutrophils lack CR3 couldn’t established respiratory burst.

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