Unit 1 Nature of the Immune System
Part 9 Complement
Terry Kotrla, MS, MT(ASCP)BB
Introduction
 Complement 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 Complement
 Primary 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
components
 Cleavage 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 C4b
 C1s cleaves C2 into C2a and C2b
 C4b2a (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 include
 Bacterial cell walls
 Bacterial lipopolysaccharide
 Fungal cell walls
 Virally infected cells
 Rabbit 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 Pathway
 Activation 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 Pathway
 The 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 Blymphocytes (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 Videos
 Go 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
Cascade
 Modulating mechanisms are necessary to regulate complement
activation and control production of biologically active split
products
 First type of control is extreme lability of activated complement
 If 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 proteins
 C1 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 Deficiencies
 Major 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 Deficiencies
 Regulatory 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 going
 Results in increased vascular permeability causing edema.
 Usually spontaneously subsides but can be life threatening – oropharynz
 Inhibitors of factors H and I produces constant turnover of C3
to depletion – recurrent bacterial infections occur.
Tests for Complement
 Can test for individual components to determine if there is a
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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
References
 http://pathmicro.med.sc.edu/ghaffar/complement.htm
 http://student.ccbcmd.edu/courses/bio141/lecguide/unit4/innate/innate.html