Immunology #5 - Complement

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General Features of Complement

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- A family of ~30 activation and regulatory proteins found either bound to the cell membrane or in circulation
- Synthesized mainly by the liver
- Many circulating proteins are pro-enzymes whose activation requires their proteolytic hydrolysis
- 2 pathways: 1. the alternative and 2. the classical
- Both pathways generate “C3b”

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Complement Function

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1. Facilitate the interaction of phagocytes to induce opsonin-mediated phagocytosis
2. Induce osmotic lysis of microbes
3. Enhance vascular permeability by inducing degranulation of mast cells/ basophils
4. Induce chemotaxis of neutrophils
5. Facilitate immune complex elimination

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C3b

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- Bind to immune complexes in circulation
- They can be eliminated by neutrophils (opsonin mediated phagocytosis) or be transported to the liver and/or spleen via red blood cells (erythrocytes)

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Classical Complement Pathway

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- The classical pathway is triggered when IgM or certain subclasses of IgG (IgG1 and IgG3 in humans) bind to antigens (e.g., on a microbial cell surface)
- Fc regions of the antibodies become accessible to complement proteins and 2 or more Fc regions come close together
- When this happens, the C1 complement protein binds to two adjacent Fc regions
- Attached C1 becomes enzymatically active, thus resulting in the binding and cleavage of two other proteins, C4 and C2.
- The resultant C4b2b complex becomes covalently attached to the antibody and to the microbial surface where the antibody is bound
- This complex is the “classical pathway C3 convertase,” which functions to break down C3, and the C3b that is generated again becomes attached to the microbe
- Some of the C3b binds to the C4b2b complex, and the resultant C4b2b3b complex

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C3a and C5a (C4a)

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- C3a and C5a are anaphylatoxins generated by the action of the C3 and C5 convertases
- Bind to cognate receptors on mast cells and basophils inducing degranulation, resulting in the release of histamine and other inflammatory mediators
- Histamine increases vascular permeability
- C5a is also chemotactic for phagocytes, recruiting these cells to the site of infection
- Both the induction of histamine release and chemotaxis of neutrophils contribute to the initiation of the inflammatory response
- The anaphylatoxin C4a is generated only following activation of the classical pathway

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C3b

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- Serves as an opsonin on microbial surfaces
- On immune complexes it facilitates the elimination of immune complexes
- C3b is a component of the alternative pathway C3 convertase (C3bBb)
- Component of the C5 convertases of both the alternative (C3bBbC3b) and the classical (C4b2a3b) complement pathways

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C2b

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- C2b is produced by C1 acting on C2 in the classical pathway
- A weak kinin that increases vascular permeability and contributes to the genesis of inflammation

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C2a and C4b

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- Form the C3 convertase Classical Pathway (C4bC2a)
- They are also components of the C5 convertase classical pathway (C4b2a3b)

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C5b

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- Generated by the action of C5 convertases on the complement protein, C5.
- Formation and deposition of the proteolytically derived C5b proteolytic fragment onto cell surfaces serves as the stimulus for the formation of the membrane attack complex

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C4b2a

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C3 convertase, classical pathway

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C3bBb

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C3 convertase, alternative pathway

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C4b2aC3b

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C5 convertase, classical pathway

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C3bBbC3b

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C5 convertase, alternative pathway

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C5b

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- Activation of either the alternate or the classical complement pathways generates C5b
- Initiator molecule for the formation of the membrane attack complex (MAC) in the terminal complement pathway (MAC= C5b, C6, C7, C8, C9)
- Insertion of the MAC into the target membrane induces osmotic lysis and death of the microbe (or cell)

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Alternative Complement Pathway

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- C3b prompts the mobilization of the alternative pathway
- Derived from circulating C3
- In the absence of microbial infection, the spontaneously generated C3b is rapidly inactivated
- In presence of bacteria, C3b binds to microbial surfaces activates complement
- Factor B hydrolyzed to Ba and Bb by Factor D
- C3bBb complex generated by C3 convertase
- C3 convertase stabilized by a tetrameric protein, properdin, that extends its half-life 6 to 10-fold
- C3b is required for the formation of the alternative pathway C5 convertase (C3bBbC3b), which cleaves C5 to generate C5a and C5b
- Formation of C5b = initiating step for the formation of the membrane attack complex

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C1 INH

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Classical Pathway Regulator:
- C1 INH (C1 esterase inhibitor (C1 INH)) forms a complex with C1, preventing the spontaneous activation of the classical complement pathway
- Inactivates the protease, kallikrein.

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DAF

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Classical & Alternative Pathway Regulator:
Decay accelerating factor (DAF) binds to membrane bound C4b and C3b, blocking the formation of the alternative and classical pathway C3 convertases

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C4bp

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Classical Pathway Regulator:
- C4 binding protein (C4bp) binds to fluid phase C4b, preventing its attachment to cells.
- When the classical C3 convertase has already formed, C4bp competitively binds with C4b, promoting its dissociation

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Factor I

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Classical Pathway Regulator:
Cleaves C4b (bound or soluble) to its inactive form, C4bi, in the presence of one of several cofactors

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AI

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Classical & Alternative Pathway Regulator:
Anaphylatoxin inhibitor (AI) binds to C3a, C4a, and C5a, inhibiting their binding to receptors on mast cells and basophils

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Factor H

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Alternative Pathway Regulator:
- Binds to C3b in the fluid phase thus preventing C3b from binding to the cell surface.
- When the alternative pathway C3 convertase has formed, Factor H competitively binds with C3b, inducing the dissociation of this convertase

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Factor I

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Alternative Pathway Regulator:
Cleaves C3b (soluble or membrane bound) to yield the inactive form, C3bi.

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Terminal Pathway

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MAC inhibitors (MAC INH) include homologous restriction factor (HRF), S-protein, and CD59. These inhibit formation of MAC on autologous cells

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Kallikrein

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- Links the complement and the contact intrinsic coagulation systems
- Enhance the intrinsic coagulation pathway by proteolytically converting Factor XII to Factor XII(a) in the presence of a cofactor, kininogen

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Bradykinin

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potent vasodilator that increases vascular permeability

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Complement Test

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- Tests for (a) the classical pathway (CH50) and (b) the alternative pathway (AH50) measure overall complement activity.
- Tests for C3 and C4 are often used if the CH50 test is abnormal.