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Assoc. Prof. Ma. Jennifer R.

Tiburcio,MSMT
Department of Med Tech
UST Faculty of Pharmacy

• Complement
Group of nonimmunoglobulin plasma proteins More than 25 plasma proteins Sequentially activated by Ag-Ab complexes Directly by microbial constituents Irreversible damage to membranes of the target cells Important functions related to host’s defense mechanisms

• Synthesized in the Liver (except C1 – intestinal epithelial cells) Adipose tissue – factor D Monocytes, macrophages, neutrophils & T cells - properdin & C7

inactive precursors or zymogens numbered sequentially from C1 to C9 activation – horizontal bar over the designated C’ components cleavage products: small cleavage product – a large cleavage product – b letter i – inactive form

additional proteins
Factor B – C3 proactivator Factor D – C3 proactivator convertase C3bBb – C3 activator CVF – cobra venom factor P – properdin factor

Activities of the Complement
1. Cell lysis
the later C’ component complexes disrupt biologic membranes

2. Stimulation of an inflammatory response
the cleavage products of several C’ components regulate the activities of cells & tissues through chemotactic & anaphylatoxic fragments

3. Opsonization
several C’ component fragments coat target cells (opsonization) to make them more palatable to phagocytic cells, which carry receptors for some C’ fragments

4. Removal of Ag-Ab (immune) complexes
C’ fragments reduce tissue-damaging immune complexes to small, soluble aggregates while in the circulation & facilitate clearance of circulating immune complexes

Classical
Immunologic activators IgM or IgG (3, 1, 2) or bound to an antigen Non-immunologic activators Apoptotic cells Staphylococcal protein A, CRP, Gram neg. bacteria DNA Possess a recognition unit Requires presence of calcium & requires C1; C2 & C4 are utilized for its activation

Alternative
Immunologic activators Aggregated IgA in some instances IgG4 & IgE Non-immunologic activators Bacterial & plant polysaccharide LPS, zymosan. Inulin, CVF, viruses & tumor cells No recognition unit Lack of dependence on calcium Activation immediately starts w/ C3 Other components are Factors B, D & properdin

• Routes or Pathways of C’ activation A. Classical
initiation (or recognition/activation phase) C1 specifically C1q amplification (or enzymatic activation) C4, C2 and C3 membrane attack leading to cell destruction (membrane attack complex) C56789

B. Alternate
normal serum proteins initiation of soluble C3 Factor B – analogous to C2 Factor D – similar to C1s Properdin – gamma globulin, when complexed w/ C3b stabilized the alternative C3 convertase

C. Lectin Pathway
activates C’ without Ab being present MBL (mannose-binding lectin) binds to mannose or related sugars such as N-acetylglucosamine to initiate the pathway resembles C1q after MBL is bound to the surface of a cell, two MBL-associated serine proteases (MASPs) become activated MASP-1, homologous to C1r MASP-2, homologous to C1s Cleave C4 and C2

interaction of MBL w/ a carbohydrate on the surface polysaccharide of microbes to the formation of enzymatic complex that binds and activates C4 and C2

Fluid Phase Regulators
1. C1 inhibitor (C1INH) – dissociates C1r & C1s from C1q 2. Factor H – inhibits convertase activity by binding to C3b, thus preventing the binding of factor B 3. Factor I (C3b inactivator) – inactivates C3b and C4b only when they are associated with factor H

4. C4 binding protein (C4BP) – acts as cofactor
for factor I in the inactivation of C4b 5. S protein (vitronectin) – interacts with the C5b67 complex & prevents binding to cell membranes

Cell Bound Regulators
1. CR1 (CD 35) – binds C3b,iC3b & C4b then degraded by Factor I 2. Decay Accelerating Factor (DAF)(CD55 – dissociates both classical & alternative C3 convertases 3. Membrane Cofactor Protein (MCP)(CD46) – most efficient cofactor for Factor I-mediated cleavage of C3b & C4b, binding of C2 to C4b and of factor B to C3b is inhibited 4. Membrane inhibitor of reactive lysis (MRL)(CD59) - blocks the formation of MAC

Additional 1. AI – anaphylatoxin inhibitor binds to C3a, C4a, C5a 2. MAC INH Homologous restriction factor (HRF), S protein CD59

S protein, vitronectin binds to soluble (C5bC6C7) complexes preventing their insertion into autologous membranes CD59 HRF bind to 8, preventing & formation of MAC polymerization of C9

Biologic Manifestations of C’ Activation 1. Anaphylatoxins – increased vascular permeability, contraction of smooth muscle & release of histamine from basophils & mast cells C3a, C4a & C4b, C5a (most potent)

2. Chemotaxins C5a serves as chemotaxins for neutrophils, eosinophils , basophils & monocytes
3. Opsonization C4b, C3b & iC3b bind w/ specific receptors on erythrocytes, neutrophils, monocytes, & macrophages

Congenital deficiencies
C1 esterase inhibitor – hereditary angioedema complement is activated by trauma C1, C4, C2, or C3 - increased susceptibility to infections w/ pyogenic bacteria C6, C7, C8 or C9 – Neisseria C1, C2, or C4 – rheumatic diseases (impaired clearance of immune complexes)

• In Vitro Destruction of Complement 1. Addition of chelating agent 2. Inactivation of serum at 56oC for 30 mins 3. Treatment with zymosan

Assays for the classical pathway
Hemolytic titration (CH50) assay
Because all proteins from C1 to C9 are necessary for this to occur, absence of any one component will result in an abnormal CH50 essentially reducing this number to zero

Biologic properties of C’ components & activation products
C’ component C4, C2 C4a C3a C3b C5a Virus neutralization Some anaphylatoxin activity (induction of histamine release leading to vasodilation & smooth muscle contraction) Anaphylatoxin activity, aggregation of platelets Opsonization; inhibits immune complex formation; promotes clearance of immune complexes Anaphylatoxin activity, chemotaxis of neutrophils; immune adherence to vascular endothelium through specific macrophage receptors Migration inhibition; induction of monocyte & macrophage spreading (nonspecific) Membrane damage (lysis of target cell) Activity

Bb C5b-9