The Complement System

Introduction

Complements the Antibacterial Activity of Antibody by:

Recruiting inflammatory cells (chemotaxis via C4a, C3a, C5a)

Increasing vascular permeability (also via C4a, C3a, C5a)

Opsonzing pathogens (altering the surface of a pathogen so that it can be ingested by phagocytes)

Killing pathogens (via Membrane-attack complexes (MAC))

Terminology:

*1. PATHWAYS*:	Classical, Alternative, Terminal
2. PROTEINS:	Classical/Terminal Components: C1, C4, C2, C3, C5, C6, C7, C8, C9 Alternative Factors: B, D, P
3. ACTIVATED PROTEIN (i.e. enzyme)	Anything that has a bar over it is an enzyme (not shown here)
4. *ACTIVATION PRODUCTS*:	Lower case letters indicate the fragment’s status, a = released, b = bound. Examples: C3b, C5a, Bb. EXCEPTION: C2 – C2a is bound form, C2b is released form
5. UPPER CASE	An upper case letter after a component indicates it is a factor, inhibitor, or gene product. Examples: C4A and C4B are gene products.

Overview

Classical Pathway

C1 is a molecule made by endothelial cells and liver cells, and it floats around in the plasma

first molecule in the complement pathway
C1 starts the pathway when it binds either two IgG antibodies or one IgM antibody
The reason for this is that C1q (the "vessel" part of the molecule) has five globular heads that must bind at least two Fc regions in order to activate
IgG only has one Fc region per Ig, while IgM has six Fc regions per Ig… so in this case C1q can bind all of them at once!

After C1q binds to either IgM or two IgG (IgG 1,2,or 3, but not 4) antibodies (all of which are already on the surface of the pathogen) it activates an internal structure called C1r which cleaves and activates the serine protease C1s.

C1q binds antibody Þ C1q Þ C1r Þ C1s

C1s proceeds to do two things

(1) cleaves C4, which is also floating around in the plasma

C4 is cleaved into C4a (floats away) and C4b (binds to either an -NH₂ or -OH on the surface of the pathogen)

(2) cleaves C2 into C2b (floats away) and C2a (joins the C4b already there)

this forms the important enzyme called C4b2a which is also called C3 Convertase for the Classical Pathway

C1s + C4 Þ C4a + C4b

C1s + C2 Þ C2b + C2a

C4b and C2a combine to form C4b2a, the C3 convertase

C4b (from A gene) Þ binds -NH₂ groups on the surface of the pathogen, while C4b (from B gene)Þ binds -OH

C3, the most abundant complement component in the serum, is cleaved by the C3 convertase (C4b2a) into C3a (floats away) and C3b (binds in massive quantities to the surface of the pathogen)

C3b binding takes place by means of a thioester bond between the a chain (between cys and glu) of the C3b (or C4b) and the surface of the pathogen

not to be confused by internal thioester bonds of the C3b or C4b, which hold these molecules together

the presence of huge quantities of C3b on the surface of a cell signals phagocytes and membrane attack complexes into action

Alternative Pathway

C3 deposits on the surface of the pathogen on its own (unlike the classical pathway, no antibody is involved)

The first C3 that binds to the surface is termed C3^*

C3^* is activated by factors B and D and P, to form C3bBb, the C3 Convertase for the Alternative Pathway

C3^* + B Þ C3bBb + C3 + P Þ C3bBbP Þ reacts with many more C3 molecules, thus creating a lot of C3b

factor P (properidin) plays an important function in stabilizing the C3bBb (the C3 convertase)

Terminal Pathway

pathogen is now completely covered with C3b – two possibilities at this point:

(1) activation of phagocytes

(2) Terminal Pathway

If either of the C3 Convertases (classical or alternative) randomly binds an additional C3b molecule it becomes a C5 convertase (C4b2aC3b or C3bBbC3b)

C5 convertase starts a chain reaction which cleaves C5 into C5a (floats away) and C5b

C5b activates C6, which activates C7, which activates C8, which activates one to twenty C9s to insert into the membrane and form a pore that interferes with membrane functions and destroys the cell

takes one C9 to mess up an RBC, it takes twenty C9s to insert and mess up a nucleated cell

C3 Convertase + C3b Þ C5 convertase + C5 Þ C5b Þ C6 Þ C7 Þ C8 Þ insertion of C9 and creation of pore

Complement Activation – serves three functions in inflammation:

(1) alter target surfaces and render them susceptible to destruction and/or disposal
(2) liberate soluble peptides which focus inflammation to target sites
(3) upregulate cells from resting to activated states that can mediate their effector functions

Complement Activation Pathways and Regulation

Regulation of Complement Activation – several mechanisms:

(1) C1INH – regulates the enzymatic activity of C1s by covalently attaching to C1r and C1s thus dissociating them from the C1 complex

(2) carbyoxypeptidase B – regulates the activity of anaphylatoxins (C4a, C3a, C5a)

these molecules (which floated away during complement activation) normally recruit fluid and inflammatory cells to sites of antigen deposition
carboxypeptidase B which cleaves their C-terminal arginine (producing C4a des arginine, etc) which abolishes all biological activities of anaphylatoxins except recruitment of inflammatory cells

(3) factor I, factor H, and C4bp – accellerates delay of C3 and C5 convertases

C4bp (C4 binding protein), factor H and factor I, dissociate C3 and C5 convertases, and inhibit further convertase formation
Factor I additionally irreversibly cleaves C4b and C3b forming iC4b and iC3b

(4) factor S – binds to C7 and blocks its ability to interact with cell membranes or activate C8

Prevention of Attack by Complement on Native Cells:

(1) Decay Accelerating Factor (DAF) – 70 Kda glycoprotein expressed by host cells which come in contact with serum complement proteins

prevents assembly of C3 convertases if C3b is deposited on native cells

C3b + DAF Þ dislodges Bb

C4b + DAF Þ dislodges C2a

(2) CD59 – expressed on all blood cell surfaces

interferes with binding of C9 to C8 and formation of MACs

Pathological States

(1) Hereditary Angioneurotic Edema (HAE) – autosomal dominant deficiency of C1INH

Characterized by recurrent edema of the skin and upper respiratory and GI tracts.

Two forms of C1INH deficiency:

(1) severely decrased levels of C1INH
(2) nonfunctional C1INH

results in too much activated C1s Þ Ý cleavage of C2 Þ Ý [C2b] Þ subfragment of C2b Ý vascular permeability and edema formation seen in disease

C1INH production can be stimulated by androgen therapy, this will reverse all clinical and biochemical abnormalities in patients with HAE

(2) Cobra venom factor (CoVF)

CoVF is an analog of C3b which cannot be regulated by factors H and I

causes complement depletion leading to vascular collapse (excessive C3b Þ vascular permiability)

(3) Idiopathic Membranoproliferative Nephritis and C3Nef

C3Nef is an autoantibody to C3bBb that cannot be broken down by Factor H
causes alternative pathway complement activation and C3b deposition in kidneys

(4) Adult Respiratory Distress Syndrome (ARDS) – alternative pathway has been implicated as a mediator of tissue injury in ARDS

Complement activation Þ PMN accumulation in lung Þ injury secondary to release of enzymes from PMNs and the generation of O₂ radicals

(5) Paroxymal nocturnal hemoglobinuria (PNH) – deficiency of decay accelleration factor (DAF, see above)

acquired anemia characterized by bouts of hemoglobinuria frequently occurring at night
PNH cells exhibit heightened sensitivity to complement
disease arises from mutation of a progenitor cell in the marrow which results in deficiencies of both DAF and CD59 (both anchored by a common glycoinositol phospholipid structure)
critical mutation underlying PNH is in the 1^st step of the pathway for glycolipid anchor assembly.

C3 Receptors – Extrinsic Regulation – regulates complement on target cells

(1) CR1 – on RBCs and lymphocytes except T cells (only on 20% of T cells)

IC Transport – after opsonization, C3b attaches to CR1 on RBCs which then flow to the liver where hepatic macrophages grab opsonized pathogen and release RBCs back to circulation
opsonization

(2) CR2 – on B-cells

IgM to IgG switching

(3) CR3 – on phagocytes (macrophages, neutrophils, monocytes)

ingestion

Complement Genes

MHC gene – C4, C2 factor B

C4A gene – C4b product that binds -NH₂ groups on surface of the pathogen

C4B gene – C4b product that binds -OH groups on surface of the pathogen

Measurement of Complement

Total Hemolytic Complement – measures whole pathway functionally

suspension containing erythrocyte:antibody complexes (EAs) proceed through the complement pathway ending up with MACs
hemoglobin pours out of the cell and is measured to functionally quantify the complement proteins

Radial Immunodiffusion – measures antigen levels, usually C3, C4, and factor B

filling wells in agarose plates with antiserum specific for one complement component
after a 12 hr incubation measure the diameter of the precipitate ring formed around the well, and compare to diameter of precipitate formed around wells with standard solution

Complement and Disease

Complement Deficiency

Deficiencies of all complement components (except factors B and D) have been described

C1-C4 Þ lupus-like syndrome

C3 Þ infections

C5-C8 Þ neisserial infections or rheumatic disorders

C9 Þ none

C1INH Þ hereditary angioedema

Complement component deficiencies follow autosomal recessive inheritance (Exceptions: C9 and C1INH deficiencies are autosomal dominant traits)

Complement components are synthesized primarily by hepatocytes, epithelial cells of the genito-urinary tract, epithelial cells of the intestinal tract, and reticuloendothelial system (monocytes/macrophages) cells

Diseases associated with complement deficiency:

(1) Systemic Lupus Erythematosus and other immune diseases

associated with deficiencies of C2, C4, C1r, C1s, and C1INH
may be related to abnormal clearance of immune complexes
Impaired viral neutralization
Close genetic linkage (on chromosome 6 between the genes for C2 and C4 and certain genes, e.g., DR4 and B8 of the MHC)

(2) C3 Deficiency – increased susceptibility to severe, life-threatening infections with pyogenic organisms

(3) Disseminated Neisserial Disease – Meningococcal Meningitis, Gonococcal arthritis, etc.

associated with deficiency in C5, C6, C7, C8 or C9.

(4) "Rheumatic" Disorders

possibly associated with deficiencies in C5, C6, C7, C8 or C9