The Complement System

Complements are series of serum proteins involved in the mediation of immune reactions. The complement cascade is triggered classically by the interaction of antibody with specific antigen.

Complement was historically known as alexin. The complement components were designated by the symbol C’ for complement, with a digit denoting the order of their discovery. The early proteins were defined in terms of their activities rather than their biochemical purity. The first 9 components of complement were perceived as individual proteins with designations C₁, C₂, C₃, C₄, C₅, C₆, C₇, C₈, C₉.

Functions of Complement

1. Ability to kill invading bacteria directly or to enhance ingestion and killing by phagocytic cells.

a. All three pathways are involved in initiating bacterial killing and opsonization but the alternative pathway is the most efficient at the latter.

2. Ability to neutralize viruses

a. The binding of complement proteins not only leads to opsonization of the virus & lysis of the virion, but it also interferes with its ability to interact with the membrane of its target cells, and thus blocks its entrance into the cell.

b. While complement clearly plays a role in defense against viral infection, many viruses have evolved to take advantage of complement proteins, both for control of the deposition of complement on their surfaces and for gaining entry to the target cells.

(1)   MCP (Membrane Cofactor Protein) – used by Paramyxoviruses as receptor for entry to host cells.

(2)   DAF (Decay Accelerating Factor) – used to infect epithelial cells.

(3)   CR1, CR2, CR3 (Complement Receptor 123) – to gain entry to mononuclear cells.

3. Protective role in reproduction

a. Functional complement is present in the female reproductive tract, tissues and gametes.

b. Spermatozoa are protected from complement attack by MCP, DAF and CD59.

4. Controls the formation and clearance of immune complexes

a. Complement controls the formation of large insoluble complexes to form because they accumulate in tissues such as skin and kidneys where they cause inflammation and damage to surrounding cells.

b. Clearance of immune complexes is accomplished by one of the following:

(1)   Role of CR1 on erythrocytes – small soluble immune complexes with surface C₃b become bound to CR1 and are carried through the circulation to the liver and spleen. An exchange occurs in which the complex is transferred from the erythrocyte CR1 to macrophage CR3 and Fc receptors taken into phagocytes and destroyed.

5.     As anaphylatoxins

a. This is accomplished through C3a and C5a which induces contraction in ileal, bronchial, uterine and vascular smooth muscle. Interaction of C3a and C5a with mast cells in the area also leads to the release of additional inflammatory mediators including histamine.

6. Enhancement of immune response

Through the binding of CR2 and CR3d,g on the antigen followed by cross linking of the antigen through surface IgM on B cell surface. CR1 is often found with CR2 on B cell and may bind C3b bearing immune complexes that can then be processed to CR3d,g by the action of Factor I and transferred to the CR2. Human CR2 is also found in a complex with CD19, CD81, Leu–B so that transmembrane signals are transmitted via phosphorylation of CD19 when CR2 and mIgM are crosslinked by the C3d,g antigen. Binding of C3d,g to the antigen also appears to alter the enzymatic digestion of the antigen as it processed by antigen–presenting cells. Antigen bound to C3d appears to have an adjuvant effect as well.

7. Aids in the cleanup of debris, dead tissues and foreign substances

Three processes involved in Complement activation

1.     Recognition

2.     Enzyme Activation

3.     Expression of biologic activities

Components of Human Complement System

Component Symbol	Plasma Concentration (µg/ml)	Molecular Weight (Dalton)	Number of chains	Pathway	Enzymatic Activity
C1q	180	462,000	18	CP	No
C1r	100	92,000	1	CP	Yes
C1s	110	86,000	1	CP	Yes
C2	25	117,000	1	CP, MBL	Yes
C4	640	206,000	3	CP, MBL	Cofactor
C3	1200	185,000	2	CP,MBL, AP	Cofactor
C5	80	180,000	2	Terminal	Cofactor
C6	75	120,000	1	Terminal	Yes?
C7	55	110,000	1	Terminal	No
C8	80	163,000	3	Terminal	No
C9	50	71,000	1	Terminal	No
MBL	0.1 – 5	540,000	18	MBL	No
MASP1	ND	94,000	1	MBL	Yes
MASP2	ND	90,000	1	MBL	Yes
C3b	Trace	170,000	2	AP	Cofactor
D (factor D)	2	25,000	1	AP	Yes
B (factor B)	200	93,000	1	AP	Yes
P (properdin)	25	220,000		AP	No
C1–inhibitor	25	110,000	1	CP control	No
Factor I	35	88,000	1	Control, all Pathways	Yes
H (factor H)	500	150,000	1	AP control	Cofactor
C4–binding protein	250	550,000	7	CP control	Cofactor
CFI (CPB–N)	35	310,000	1	Control	Yes
SP40,40	50	80,000	1	Terminal Control	No
S (vitronectin)	500	83,000	1	Terminal Control	No
C4a	1.6	12,000		CP split prod.	No
C4d	8.9	30,000		CP split prod.	No
C3a	0.6	11,000		TP split prod.	No
iC3b	8.5	170,000		TP split prod.	No
C5a	0.01	11,000		TP split prod.	No
Bb	0.4	60,000		AP split prod.	No
SC5b–9 TCC	0.3	>1,000,000		Terminal C	No
CP – classical pathway; MBL – mannose binding lectin pathway; AP – alternative pathway; TP – terminal pathway

Pathways of Complement Activation

1.    The Classical Pathway

a. Initiated when C1 (C1q–r₂s₂) binds to an activating substance such as antigen– antibody complex. C1q–r₂s₂ requires eight Calcium ions to remain complexed. Free C1q can bind to immunoglobulins or other activators but without the C1r₂s₂, no activation occurs.

Substances that activate human complement

Substance	C Pathway activated
Antigen–antibody complexes	Classical
ß Amyloid (Alzheimer’s plaques)	Classical
DNA, polyinosinic acid	Classical
Polyanion–polycation complexes (heparin–protamine)	Classical
C–reactive protein complexes	Classical
Enveloped viruses (some)	Classical
Monosodium urate crystals	Classical
Lipid A of bacterial lipopolysaccharide	Classical
Plicatic acid (from Western red cedar)	Classical
Ant venom polysaccharide	Classical
Mannose–rich bacterial cell walls, etc.	MBL
Inulin	Alternative
Yeast cell walls (zymosan)	Alternative
Sephadex	Alternative
Endotoxin (bacterial lipopolysaccharide)	Alternative
Rabbit erythrocytes	Alternative
Desialylated human erythrocytes	Alternative
Cobra venom cofactor	Alternative
Phosphorothioate backbone oligonucleotides	Alternative
Aggregated immunoglobulins	Classical and alternative
Subcellular membranes	Classical and alternative
Cell– and plasma–derived enzymes Plasmin, kallikrein Activated Hagemen Factor Neutrophil esterase, cathepsins	Classical, alternative, terminal

           

b. Binding of C1q to immunoglobulin is through ionic and hydrophobic bonds between the globular head regions of C1q and the Fc region of immunoglobulin, so the binding is affected by ionic strength: raising the ionic strength above physiologic levels will dissociate the interaction, while decreasing it promotes stronger binding.

c. It requires on IgM molecule bound to antigen or two closely spaced antigen–bound IgG molecule for C1q–r₂s₂ to bind in a stable enough configuration to allow C1 activation.

Immunoglobulin activating C1 (decreasing order):

(1)   IgM

(2)   IgG3

(3)   IgG1

(4)   IgG2

Non–activators of C1

(1)   IgG4

(2)   IgA

(3)   IgE

(4)   IgD

d. During the activation process, approximately 10% of the C4b binds covalently to the surface of the activating substance and provides a Mg²⁺–dependent binding site for C2, which can then be cleared by C1s. The C2a fragment formed by this cleavage remains bound to the C4b and contains a protease domain that is expressed in the C4b2a complex. This enzyme is the classical pathway C3 convertase.

2. Lectin Pathway

a. Initiated when MBL (Mannose–binding lectin) binds to carbohydrate residues on the surface of the activating bacteria or other substance and undergoes a conformational change similar to what happens with C1q.

b. MASP1 (MBL–associated serine proteases) becomes active and cleaves C4 and C2 to form C4b2a, whereas MASP2 appears to be able to cleave C3 directly.

3.    Alternative Pathway

A mechanism of complement activation that does not involve activation of the C1–C4– C2 pathway by Ag–Ab complexes and begins with activation of C3.

a. Initiated by preformed C3b, serum factors B & D and properdin.

b. Preformed C3b which binds B more efficiently is created whenever C3 is cleaved by ongoing complement activation or by proteases derived from the coagulation pathways, from inflammatory cells or bacteria.

c. Factor D can cleave B only when the B is bound to C3b or C3•H₂O and the resulting enzyme, C3bBb (C3•H₂OBb) is the alternative pathway C3 convertase.

d. Control of the alternative pathway occurs at several levels. The short half–life of C3b* forms a limiting mechanism so that most of the C3 that is cleaved becomes inactive C3bi (i for inactive) in the fluid phase. Only 10% of the C3b that is produced becomes bound to the surface of the activator.

e. Control of the enzyme C3bBb is by the following mechanism:

(1)   Downregulation which is accomplished when

(a)   Factor H binds to C3b and displaces Bb.

(b)   H then serves as a cofactor for cleavage of C3b by factor I and the resulting fragments, iC3b, C3c, C3d are unable to participate in the lytic pathway.

(2)   Upregulation which give it the ability to establish a highly efficient amplification loop (C3–feedback loop)

(a)   Properdin can bind to the C3bBb enzyme complex and prevent its dissociation by RCA proteins.

(b)   The long half life of C3bBb P complex allows more C3b generation in a short time to which factor B can bind.

4.    Terminal pathway

a. Cleavage of C5 produces 2 fragments:

(1)   C5a has potent anaphylatoxin properties and is strongly chemotactic for neutrophils and other inflammatory cells.

(2)   C5b forms the nucleus for formation of the membrane attack complex (MAC) consisting of complement components C5b–9. MAC can lead to lysis or to formation of the fluid phase terminal complement complex.

(a)   MAC formation occurs when C6 binds to the C5b–α‘chain.

(b)   C7 appears to bind to the C5b–α‘chain as well.

(c)    C5b67 has affinity for membrane lipid, but the structure at this stage of insertion into the membrane has no lytic capability.

(d)   The C5b portion is available to aqueous phase probes, while C6 and C7 appear to be inserted into the outer membrane leaflet.

(e)   When C8 binds to the C5b67 complex, conformational changes in the C8–α chain allow the complex to penetrate deeper into the lipid bilayer.

(f)    The C8 can be identified by lipid–soluble labeling methods as the primary membrane–perturbing component.

(g)   The C5b–8 complex on a cell membrane serves as a receptor for C9, which binds to the C8–α chain and then unfolds and inserts even more deeply into the membrane.

(h)   Additional C9 molecules interact with the first to form poly–C9 through C9– C9 interactions.

(i)     Changes in the conformations of C9 in the membrane attack complex have been demonstrated through the appearance of C9 neoantigenic epitopes, altered susceptibility to proteases and ultrastructural changes.

b. The surface protein CD59 prevents reactive lysis

(1)   CD59 is attached to the cell membrane by a glycosylphosphatidylinositol anchor (GPI anchor) and binds to C5b–8 on the cell surface, preventing C9 from polymerizing.

(2)   A similar widely distributed membrane protein with MAC inhibitory activity called homologous restriction factor (HRF) acts by binding to C8 and C9 and prevents their insertion into the cell membrane.

(3)   In the fluid phase, S–protein (vitronectin) or SP–40,40 bind to the hydrophobic regions of C5b6, C5b67, C5b–8 and C5b–9 and prevent interaction with membranes. These soluble forms of C5b complexes can be identified in the circulation after complement activation occurs. 

The C3

1. The activation of C3 is the central step in all of the complement pathways and the point at which they converge.

2. The C3 convertase–mediated cleavage of C3–α chain created 2 fragments:

a. C3a – a small fluid phase anaphylatoxin with proinflammatory properties.

b. C3b – a larger fragment which has multiple roles in host defense, inflammation and immune regulation as well as continuation of the complement – activation cascade.

(1)   The binding of C3b to acceptor carbohydrate or protein molecules was thought for a long time to be random but recent experiments have shown that there are a limited number of residues to which the thiolester will bind.

(2)   One result of short–half life of C3b* is that the C3b deposited on surfaces tends to be localized in clusters around the activating enzyme and some will bind to the C4b or C3b of the enzyme.

(3)   The extra C3b converts the specificity of the C3 convertase to that of C5 convertase by providing a site for C5 to bind to the enzyme: C4b2a3b, C3bBb3b.

Diseases of Complement Deficiencies

1. Deficiency in C1, C4, C2 – has an increased risk of developing Systemic Lupus Erythematosus

2. Deficiency in C3

a. Increased susceptibility to recurrent infections with pyogenic organisms (also associated with Factors H and I deficiency).

b. C3–nephritic Factor (C3NeF) – an autoantibody found in patients with SLE or partial lipodystrophy. C3NeF binds to the alternative pathway C3 convertase (C3bBb) in a way that stabilizes it and produces a highly efficient and long–lived fluid phase convertase. If the condition persists, C3 levels can decrease enough to render the patient profoundly C3–deficient.

3. Deficiency in C1 INH

a. Acquired Angioedema (AAE) results when C1 INH levels decrease due to increased utilization. AAE may be a result of abnormal complement activation by lymphoma proteins or cell surface Ig or by other lymphoproliferative or autoimmune disorders.

b. Hereditary Angioedema (HAE) – bradykinin formation is abnormal thus edema is formed.

4. Paroxysmal Nocturnal Hemoglobinuria (PNH) – characterized by chronic intravascular hemolysis that result in hemoloytic anemia and nocturnal hemoglobinuria, venous thrombosis and inability to produce viable erythrocytes.

a. The condition is due to a somatic mutation in the gene that controls the production of the GPI anchor that attaches type I membrane proteins to the cell surface.

b. This anchor is produced in the endoplasmic reticulum by multiple steps that end with the attachment to the appropriate proteins. The anchored protein is then transported from the Golgi apparatus to the cell surface.

c. In PNH, the anchor is not made properly and the unattached proteins are secreted from the cell into the fluid phase.

Proteins that control complement

The regulators of complement activation (RCA) include fluid phase protein factors H and C4bp along with cell surface proteins decay accelerating factor (DAF), membrane cofactor protein (MCP) and complement receptors 1 and 2 (CR1, CR2)

a. CR1 (CD35) is the 220–kDa C3b/C4b receptor found on erythrocytes, monocytes, macrophages, eosinophils, neutrophils, B cells, some T cells, follicular dendritic cells and mast cells. It can also bind iC3b and C3c, though with lower affinity. CR1 has both decay accelerating factor and cofactor activities.

A soluble recombinant form of CR1 (SCR1) has been made by deletion of the transmembrane segment of the protein. The recombinant protein, which has cofactor activity for Factor I, is undergoing clinical testing for efficiency as an inhibitor in a variety of inflammatory disorders.

b. CR2 (CD21) is a 140–kDa membrane glycoprotein expressed on late premature and mature B lymphocytes, some T cells, including thymocytes and follicular dendritic cells. The CR2 molecule binds C3b, iC3b and C3d,g fragments of C3. CR2 does not have cofactor activity for Factor I.

c. Decay Accelerating Factor (DAF / CD55) dissociate bound C3 convertase thus preventing lysis. Without these surface molecules, granulocytes, platelets and particularly red blood cells are susceptible to spontaneous lysis.

d. Membrane cofactor protein

Other Complement Receptors

a. CR3 (CD11b/CD18, Mac–1, Mo1, OKM–1, α–M/ß–2)

·  CR3 is expressed on mononuclear phagocytes, natural killer (NK) cells and granulocytes. It has diverse ligand–binding capacity, including coagulation Factor X, fibrinogen, lipopolysaccharide, zymosan, ICAM–1 and iC3b

·  The number of CR3 molecules per cell is upregulated by inflammatory stimuli, including C3a and C5a and CR3 facilitates binding of phagocytes to endothelial cells so that extravasation can occur when the cells are responding to chemotactic signals.

b. CR4 is expressed on myeloid cells, dendritic cells, activated B cells, NK and some CTLS and platelets. It has a broad ligand repertoire similar to that of CR3 with iC3b the predominant complement ligand. There is still some question whether this receptors serves primarily as a complement receptor, or if it has an alternative role to play in the inflammatory process.

c. C1qRp is associated with phagocytosis of C1q – or mannose–binding lectin–coated particles. It is a type I membrane glycoprotein with the capability of transmitting cellular activation signals.

d.     The C5a receptor (C5aR/CD88) reacts with N–formyl bacterial chemoattractants. It can be found on granulocytes, platelets, mast cells, liver parenchymal cells, lung vascular smooth muscle, endothelium, bronchial and alveolar epithelial cells and astrocytes and microglial cells in the brain.

e. C3a receptor (C3aR) is located on platelets, mast cells, macrophages, neutrophils, basophils, eosinophils, monocytes and endothelial cells.

f.  C3aR messenger RNA has also been identified in cells of the thymus, heart, liver, kidneys, colon, small intestine, placenta, testis, ovaries and several regions of the brain.