Terry Kotrla, MS, MT(ASCP)BB

Unit 1 Nature of the Immune System

Part 9 Complement

IntroductionComplement refers to a complex set of

14 distinct serum proteins (nine components) that are involved in three separate pathways of activation.Components numbered in order of discovery.Sequence of activation is not in numerical

order.Components circulate in inactive precursor

form, develop activity upon activation.Complement proteins designated by “C”

followed by numbers and letters.

Two major functions:Promote the inflammatory response by

opsonization which enhances susceptibility of coated cells to phagocytosis.

Alter biological membranes to cause direct cell lysis.

General Properties of ComplementPrimary role is cell lysis.Activity of complement destroyed by heating

sera to 56 C for 30 minutes.IgM and IgG are the only immunoglobulin

capable of activating complement (classical pathway).

Complement activation can be initiated by complex polysaccharides or enzymes (alternative or properdin pathway).

Portions of the complement system contribute to chemotaxis, opsonization, immune adherence, anaphylatoxin formation, virus neutralization, and other physiologic functions

Sequential interaction of complement componentsCleavage of components generates a small

fragment which is released, and a larger molecule which attaches to cell surface and continues in reaction sequence.

Sequence of activation referred to as a cascade reaction.

Three Pathways

Classical Pathway

C1: The Recognition Unit C1 consists of 3 subunits: C1q, C1r, and C1s.C1q molecule consists of a collagenous region

with six globular head groups globe end serves as recognition unit

When antibody binds to antigen, binding sites for the globular head groups of C1q are exposed on the Fc region of the antibody.

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.

Classical Pathway – C1: The Recognition Unit

The Activation Unit (C4b2a3b)C1s cleaves C4 into C4a and C4bC1s cleaves C2 into C2a and C2bC4b2a (C4b2b in some texts) is

enzymatically active and can cleave many molecules of C3 into C3a and C3b.

Membrane Attack Unit (C5,6,7,8,9)In the presence of C5b, molecules of C6,

C7, C8 and a variable number of C9 molecules assemble themselves into aggregates.

This molecular complex causes a change in membrane permeability.

Exact cause of lysis unknown, one theory is change in lipid membrane causes exchange of ions and water molecules across membrane.

Cells can lyse without C9 but it’s slower.

Membrane Attack Unit

Order of Activation in Classical Pathway C1, C4, C2, C3, C5, C6, C7, C8, C9

Classical Pathway

Alternative Pathway (Properdin Pathway)Cleavage of C3 and activation of the

remainder of the complement cascade occurs independently of antibody.

Triggers for the alternative pathway includeBacterial cell wallsBacterial lipopolysaccharideFungal cell wallsVirally infected cellsRabbit erythrocytes

Alternative Pathway (Properdin Pathway)Molecules of C3 undergo cleavage at

continuous low level in normal plasma.At least 4 serum proteins (factor B, factor

D, properdin (P), and initiating factor (IF) function in this pathway.

C3b attaches to appropriate site (activating surface) which is actually a protective surface

Alternative Pathway (Properdin Pathway)Action by the 4 serum proteins on C3b

proceeds to the C3 activator stage without participation of C1, C4 or C2.

Activation sequence: C3, C5, C6, C7, C8, C9.

Lectin PathwayActivation of the lectin pathway begins when

mannan-binding protein (MBP) binds to the mannose groups of microbial carbohydrates.

Two more lectin pathway proteins called MASP1 and MASP2 (equivalent to C1r and C1s of the classical pathway) bind to the MBP.

This forms an enzyme similar to C1 of the classical complement pathway that is able to cleave C4 and C2 to form C44bC2a, the C3 convertase capable of enzymatically splitting hundreds of molecules of C3 into C3a and C3b

Lectin PathwayThe beneficial results are the same as in the

classical complement pathway:Trigger inflammation (C5a>C3a>C4a);Chemotactically attract phagocytes to the

infection site (C5a);Promote the attachment of antigens to

phagocytes via enhanced attachment or opsonization (C3b>C4b;

Serves as a second signal for the activation of naive B-lymphocytes (C3d);

Cause lysis of gram-negative bacteria and human cells displaying foreign epitopes (MAC).

And remove harmful immune complexes from the body (C3b>C4b).

View VideosGo to

http://www.youtube.com/watch?v=gNvHLStz-VA or do your own search for complement activation videos which illustrate the 3 pathways.

Regulation of the Complement CascadeModulating mechanisms are necessary to regulate

complement activation and control production of biologically active split products

First type of control is extreme lability of activated complementIf activated complement does not combine within

milliseconds the activity is lost or decreased.Active fragments rapidly cleared from the body.

Second type of control involves specific control proteinsC1 inhibitor blocks activity of C1r and C1s.Factor I in activator in the presence of certain cofactors

inactivates C3b and C4b.A number of proteins act to control membrane attack

unit

Complement DeficienciesMajor Pathway components

Hereditary deficiency of any component except C9 results in increased susceptibility to infection and delayed clearance of complexes.

Deficiency of MBL important during infant transition to making their own antibody, associated with pneumonia, sepsis and meningococcal disease.

C3 deficiency most serious, key mediator in ALL pathways. Prone to serious life threatening infections and immune complex disease.

Deficiency of terminal components causes increased susceptibility to Neisseria infections.

Complement DeficienciesRegulatory Factor components

PNH – RBCs deficient in DAF, results in hemolysis due to “bystander” affect.

Hereditary angiodema results in excess cleavage of C4 and C2,keeps classical pathway goingResults in increased vascular permeability causing

edema.Usually spontaneously subsides but can be life

threatening – oropharynzInhibitors of factors H and I produces constant

turnover of C3 to depletion – recurrent bacterial infections occur.

Tests for ComplementCan test for individual components to determine

if there is a deficiency.Can perform assays to test the classical pathway

using the hemolytic titration (CH50) assay, lytic activity, and ELISA.

Can perform assays to test the alternative pathway using the AH50 or an ELISA test.

Test all three pathways using test strips coated with reagents specific for each pathway.

Use complement fixation test to detect viral, fungal and rickettsial antibodies, hemolysis of RBCs is a positive result.

Summary of Complement Function

Referenceshttp://pathmicro.med.sc.edu/ghaffar/compleme

nt.htm http://student.ccbcmd.edu/courses/bio141/lecguide/unit4/innat

e/innate.html