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Slide # (2)

By Ala Balawi
Doctor Nasser M. Kaplan Thursday, 27/6/2013

To accomplish GREAT things, We must DREAM As well as ACT

Complement
Some definitions of terms commonly used in discussing complement: C-activation: Alteration of C proteins, enabling them to interact with another component. C-fixation: Utilization of C by antigen-antibody complexes. Hemolytic unit (CH50): dilution of serum lysis of 50% of standard amount of antibody-coated RBCs. C-Inactivation: denaturation (usually by heat) of early complement component loss of hemolytic activity. Convertase/Esterase: altered C protein which acts as proteolytic enzyme for another C component. Some conventions of complement nomenclature: Activated components of complement are over-lined When C is enzymatically cleaved 1. Larger fragment (with letter b usually added to name) binds to activation complex or membrane e.g. C3b. 2. Smaller fragment (with letter a usually added to name) is released into microenvironment e.g. C3a. EXCEPTION: C2 Larger membrane-binding fragment with a designation & smaller fragment with b designation.

Complement system:
Consists of >20 different serum proteins capable of lysing antibody-coated cells. Produced by variety of cells including hepatocytes, macrophages & gut epithelial cells. Some complement proteins bind to Igs or to membrane components of cells. Others are proenzymes which when activated, cleave one or more other complement proteins. Upon cleavage some of complement proteins yield fragments which activate cells, vascular permeability or opsonize bacteria.

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I. Complement Function:
1. Beneficial effects by contributing to host defenses: Opsonize bacteria for enhanced phagocytosis. Recruit & activate various cells including PMNLs & macrophages. Participate in regulation of antibody responses. Aid in clearance of immune complexes & apoptotic cells. 2. Detrimental effects for host: Contributes to inflammation & tissue damage. Trigger anaphylaxis.

II. Pathways of complement activation:

Complement (C) activation can be divided into four pathways: 1. Classical pathway 2. Lectin pathway 3. Alternative pathway 4. Membrane attack (or lytic) pathway. Both classical & alternative pathways activation of C5 convertase production of C5b which is essential for activation of membrane attack (lytic) pathway.
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1. Classical Pathway:
(*) C1 activation: C1 = multi-subunit protein containing 3 different proteins (C1q, C1r & C1s). Binds to Fc region of IgG & IgM antibody molecules which have interacted with antigen. Binding requires Calcium & Magnesium ions. Binding does NOT occur to antibodies which have not complexed with antigen. In some cases C1 can bind to aggregated Ig (e.g. aggregated IgG) or to certain pathogen surfaces in absence of antibody. Binding of C1 to antibody is via C1q which must cross link at least 2 antibody molecules before it is firmly fixed. Binding of C1q activation of C1r activation of C1s activated C1qrs = enzyme (**) C4 & C2 activation (Generation of C3 convertase): 1. C4 cleavage C4b (binds to membrane) & C4a (released into microenvironment) fragments. 2. C2 cleavage C2a (binds to membrane) & C2b (released into microenvironment) fragments. 3. C4bC2a complex = C3 convertase

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Pathways of Complement System

(***) C3 activation (Generation of C5 convertase) C3 cleavage C3b (binds to membrane) & C3a (released into microenvironment) fragments. C4bC2aC3b = C5 convertase END of classical pathway. Generation of C5 convertase in Classical Pathway Activation of C3 by classical pathway:
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Generation of C3 convertase in Classical Pathway

Products of classical pathway: Several have potent biological activities which contribute to host defenses as C3b & C4b. Some may have detrimental effects if produced in unregulated manner as C2b, C3a & C4a there must be some ways to regulate activity of classical pathway to avoid continued production of C2b, C3a, & C4a.

Component: All C2b C3a

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Biological Activity: Prokinin Anaphylotoxin

Regulation: C1-INH x C1r & C1s from C1q C3a-INA

C3b C4a C4b

Opsonin Anaphylotoxin (Weaker) Opsonin

Factor H & I X C3a-INA C4-BP x C4bC2a & Factor I x

Example : C1-INH (C1-inhibitor) deficiency hereditary angioedema.

2. Lectin Pathway:

Very similar to classical pathway. Biological activities & regulatory proteins of lectin pathway are same as those of classical pathway. BEGIN by binding of mannan-binding protein (MBP) or lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides (mannans) binding of two mannan-associated serine proteases (MASP-1 & MASP-2). MBL, MASP-1 & MASP-2 are similar to C1q, C1r & C1s respectively. Formation of MBL/MASP-1/MASP-2 tri-molecular complex activation of MASPs C4 & C2 activation (generation of C3 convertase) 1. C4 cleavage C4b (binds to membrane) & C4a (released into microenvironment) fragments. 2. C2 cleavage C2a (binds to membrane) & C2b (released into microenvironment) fragments. 3. C4bC2a complex = C3 convertase C3 activation (generation of C5 convertase) C3 cleavage C3b (binds to membrane) & C3a (released into microenvironment) fragments. C4bC2aC3b = C5 convertase END of lectin pathway. Lectin-initiated pathway:

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3. Alternative (Alternate) Pathway:

May be the more primitive pathway and both classical & lectin pathways probably developed from it. Provides first line of defense & non-specific resistance against certain pathogens before antibody response (without participation of antibodies). Alternative pathway can be activated by: 1. Many Gram negative bacteria (most significantly, Neisseria meningitidis & N. gonorrhoea), some Gram positive bacteria, certain viruses & parasites lysis of these organisms. C3 deficiency susceptibility to these organisms. 2. Heterologous RBCs, aggregated Igs (esp IgA) & some other proteins (e.g. proteases, clotting pathway products & cobra venom factor (CVF)). BEGIN with C3 activation & requires Factors B & D and Mg++ cation, all present in normal serum. END with generation of C3bBbC3b complex (C5 convertase). (*) Amplification loop of C3b formation: In serum there is low level spontaneous hydrolysis of C3 C3i. Factor B binds to C3i & becomes susceptible to Factor D Factor B cleavage Bb. C3iBb complex acts as C3 convertase C3 cleavage C3a & C3b. Factor B binds to C3b & becomes susceptible to cleavage by Factor D C3bBb complex = C3 convertase continuous generation of more C3b (amplifying C3b production). If this process continues unchecked consumption of all C3 in serum spontaneous C3b production should be tightly controlled. Spontaneous activation of C3 (C3 tick-over):

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(**) Control of amplification loop: Importance: patients with genetic deficiencies of Factor H or I C3 deficiency & susceptibility to certain infections. How? 1. Blocking formation of C3 convertase. 2. Dissociating C3 convertase. 3. Enzymatically digesting C3b. 1) As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF (Decay Accelerating Factor) which: a) Prevents binding of Factor B with C3b preventing production of additional C3 convertase. b) Dissociates Bb from C3b in already formed C3 convertase preventing production of additional C3b. Control of spontaneous C3 activation via DAF:

DAF prevents binding of Factor B with C3b

DAF dissociates Bb from C3b in already formed C3 convertase

2) Some cells possess Complement Receptor 1 (CR1): a) Binding of C3b to CR1 facilitates enzymatic degradation of C3b by Factor I. b) Binding of C3 convertase (C3bBb) to CR1 also dissociates Bb from complex. 3) Finally, Factor H can bind to C3b bound to cell or in fluid phase & facilitate enzymatic degradation of C3b by Factor I.

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Regulation of activated C3 by CR1:

(***) Stabilization of C convertase by activator (protector) surfaces: Activators of alternative pathway are components on surface of pathogens & include: LPS of Gram-negative bacteria and cell walls of some bacteria & yeasts. Binding of C3b to activator surface formation of stable C3 convertase which will continue to generate additional C3a & C3b by C3 cleavage. When bound to appropriate activator of alternative pathway, C3b will bind Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb). However, C3b is resistant to degradation by Factor I & C3 convertase is NOT rapidly degraded, since it is stabilized by activator surface. The complex is further stabilized by properdin binding to C3bBb.

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Stabilization of C3 convertase:

(****) Generation of C5 convertase: Some of C3b generated by stabilized C3 convertase on activator surface associates with C3bBb complex to form C3bBbC3b complex (= C5 convertase) END of alternative pathway. Stabilized C5 convertase of alternative pathway:

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4. Membrane attack (lytic) pathway:

C5 convertase from classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathway cleavage of C5 into: 1) C5a which remains in fluid phase. 2) C5b which rapidly associates with C6 & C7 & inserts into membrane. Subsequently C8 binds, followed by several molecules of C9. C9 molecules form pore in membrane through which cellular contents leak & lysis occurs. Lysis is NOT enzymatic process; it is thought to be due to physical damage to membrane. The complex consisting of C5bC6C7C8C9 is referred to as membrane attack complex (MAC). The lytic pathway:

C5a generated in lytic pathway has several potent biological activities: 1. Most potent anaphylotoxin. 2. Chemotactic factor for neutrophils & stimulates respiratory burst in them. 3. Stimulates inflammatory cytokine production by macrophages. Its activities are controlled by inactivation by carboxypeptidase B (C3-INA). Some of C5b67 complex formed can/may dissociate from membrane & enter fluid phase binding to & lysis of other nearby cells.
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 Protein S (vitronectin) binds to soluble C5b67 & prevents its binding to other bystander cells & prevents their consequent damage.

III. Biologically active products/molecules of complement activation which contribute to resistance, anaphylaxis & inflammation:
1. Kinin: C2b (prokinin) generated by classical pathway of C activation, becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) (serpin) which displaces C1rs from C1qrs complex. Genetic deficiency of C1-INH overproduction of C2b hereditary angioneurotic edema Rx: Danazol which promotes C1-INH production or -amino caproic acid which plasmin activity.

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Regulation of C1rs (C4 convertase) by C1-INH:

2. Anaphylotoxins = C4a, C3a & C5a (in increasing order of activity) basophil/mast cell degranulation & smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA). 3. Chemotactic Factors = C5a & MAC (C5b67). C5a is also a potent activator of neutrophils, basophils & macrophages induction of adhesion molecules on vascular endothelial cells. 4. Opsonins = C3b & C4b in surface of microorganisms attach to C-receptor (CR1) on phagocytic cells & promote phagocytosis. 5. Other Biologically active products of C activation = degradation products of C3 (iC3b, C3d & C3e) which also bind to different cells by distinct receptors & modulate their functions.

Summary:
Complement system takes part in both specific & non-specific resistance & generates number of products of biological & patho-physiological significance.

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 There are known genetic deficiencies of most individual complement components, but C3 deficiency is most serious & fatal. Complement deficiencies also occur in immune complex diseases (e.g., SLE) and acute & chronic bacterial, viral & parasitic infections.

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Thank you

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