The Complement System in Immunity

Overview

• Cellular and Molecular Immunology, p. 838-839
• Robbins, Cotran & Kumar Pathologic Basis of Disease, p. 97

Three Activation Pathways

1. Alternative Pathway (Phylogenetically oldest - innate immunity)

• Trigger: C3 spontaneously hydrolyzes in plasma at low levels ("tick-over"), and C3b binds to any nearby surface. On host cells, regulatory proteins (Factor H, DAF) rapidly degrade C3b. On microbial surfaces (LPS, fungal cell walls, complex polysaccharides), these regulators are absent, so C3b is stabilized.
• C3b binds Factor B, which is cleaved by Factor D to generate the alternative pathway C3 convertase: C3bBb.
• A positive feedback loop amplifies activation - more C3b deposits, generating more C3 convertase.
• This pathway distinguishes self from non-self by the presence/absence of host regulatory proteins.

2. Classical Pathway (Adaptive immunity effector; discovered first)

• Trigger: C1q (part of the C1 complex with C1r and C1s) binds to the Fc region of IgM or IgG already bound to antigen on microbial surfaces. Pentameric IgM is particularly potent (one molecule can fix C1). IgG requires two adjacent molecules.
• Pentraxins (e.g., C-reactive protein, CRP) can also bind C1q and activate this pathway.
• C1r and C1s become activated, then cleave C4 (→ C4a + C4b) and C2 (→ C2a + C2b) to form C3 convertase: C4b2a.
• This is the major complement mechanism linking humoral adaptive immunity to complement effector functions.

3. Lectin Pathway (Innate immunity)

• Trigger: Mannose-Binding Lectin (MBL), a collectin with structural similarity to C1q, recognizes terminal mannose residues on microbial glycoproteins/glycolipids. (Mammalian proteins are typically sialylated and lack free mannose termini.)
• MBL associates with MASP1 and MASP2 (functional analogs of C1r/C1s), which activate C4 and C2 in the same manner as the classical pathway.
• C3 convertase formed: C4b2a (identical to classical pathway).
• Cellular and Molecular Immunology, p. 840-842
• Robbins Pathologic Basis of Disease, p. 97-98

The Central Event: C3 Cleavage

• Robbins Pathologic Basis of Disease, p. 98

Three Main Effector Functions

1. Inflammation (C3a, C4a, C5a - "Anaphylatoxins")

• Stimulate histamine release from mast cells and basophils
• Increase vascular permeability and vasodilation
• C5a is the most potent chemotactic factor for neutrophils, monocytes, eosinophils, and basophils
• C5a activates the lipoxygenase pathway in neutrophils/monocytes, amplifying inflammatory mediator release

2. Opsonization and Phagocytosis (C3b, iC3b)

• C3b and its breakdown product iC3b coat microbial surfaces (opsonization)
• Phagocytes express complement receptors: CR1 (recognizes C3b/C4b), CR3/CR4 (recognize iC3b)
• Binding dramatically enhances phagocytic uptake and killing

3. Cell Lysis - Membrane Attack Complex (MAC)

• C5b recruits C6, C7, C8, and multiple copies of C9 to form the MAC (C5b-9)
• C9 polymerizes (~10-16 molecules) to form a transmembrane pore (~10 nm diameter)
• The pore allows free passage of water and ions, causing osmotic lysis
• Particularly important against gram-negative bacteria with thin cell walls (e.g., Neisseria)
• Deficiency of terminal complement components (C5-C9) specifically predisposes to disseminated Neisseria infections
• Robbins Pathologic Basis of Disease, p. 98

Regulation of Complement

• Robbins Pathologic Basis of Disease, p. 98-99

Complement Deficiency States

Role in Adaptive Immunity

1. Enhancing B cell activation: C3d (a breakdown product of C3b) on antigen binds CR2 (CD21) on B cells. CR2 is part of the B cell co-receptor complex (CR2-CD19-CD81), which lowers the B cell activation threshold by ~1000-fold. This is why complement dramatically amplifies antibody responses.
2. Classical pathway as adaptive effector: IgG and IgM antibodies produced by adaptive immunity activate complement, linking humoral immunity to complement-mediated opsonization and lysis.
3. Complement in memory: C3d-coated antigens are trapped by follicular dendritic cells (which express CR1 and CR2), creating antigen depots that sustain long-term B cell memory.
4. Immune complex clearance: CR1 on erythrocytes mediates transport of complement-coated immune complexes to the liver and spleen for disposal. Deficiency (as in early-component complement deficiencies) leads to immune complex disease resembling SLE.

Microbial Evasion of Complement

• Capsules (pneumococcus, meningococcus) physically block C3b deposition
• Sialylation of surface proteins mimics host cells to recruit Factor H
• Staphylococcus aureus secretes SCIN (Staphylococcal Complement Inhibitor) that blocks C3 convertases
• Streptococcus pyogenes expresses M protein, which binds Factor H
• Borrelia burgdorferi outer surface proteins bind complement regulators
• Some viruses encode homologs of host complement regulatory proteins

Summary Table: Three Pathways at a Glance

• Cellular and Molecular Immunology (Abbas, Lichtman, Pillai), pp. 254-255, 838-842
• Robbins, Cotran & Kumar Pathologic Basis of Disease, pp. 97-99

The Complement System in Immunity - Detailed Account

1. Historical Background and Definition

• Cellular and Molecular Immunology, pp. 838-839

2. General Principles of Complement Activation

1. Microbe-surface focus: Activation is triggered by microbes and by antibodies/lectins bound to microbes. Complement attacks are therefore directed specifically at microbial surfaces, not in the plasma.
2. Proteolytic cascade amplification: Sequential zymogen activation (like the coagulation cascade) produces rapid, massive amplification. Millions of C3b molecules deposit within 2-3 minutes.
3. Covalent attachment to targets: Biologically active cleavage products (especially C3b) become covalently attached to microbial surfaces via a reactive thioester bond. In the fluid phase, these bonds hydrolyze within seconds rendering proteins inactive - so full activation only occurs on surfaces.
4. Self vs. non-self discrimination: Regulatory proteins on host cells (DAF, CD59, MCP, Factor H) rapidly inactivate any complement components that land on self-cells. Microbes lack these proteins - so complement activation amplifies on microbes but is extinguished on host cells.

3. The Three Activation Pathways

3A. The Alternative Pathway (Oldest evolutionarily - innate immunity)

• Sialic acid on mammalian cell surfaces selectively favors binding of Factor H (regulatory) over Factor B (activating) - another self/non-self discrimination mechanism.
• LPS, fungal cell walls, cobra venom factor, complex polysaccharides, and IgA aggregates can trigger this pathway.

3B. The Classical Pathway (Adaptive immunity effector - discovered first)

• C1q: 6 globular heads connected by collagen-like stalks, arranged like a bunch of tulips. Each head can bind to the Fc region (CH2 domain) of IgG or the Fc region of IgM.
• C1r: two molecules forming a tetramer with C1s
• C1s: two molecules; the effector serine protease

• IgM (pentameric): extremely efficient because a single molecule provides 5 potential Fc sites in close proximity → 1 IgM molecule suffices
• IgG (monomeric): requires two adjacent IgG molecules bound to antigen → less efficient

• C4 → C4a (released, weak anaphylatoxin) + C4b (covalently binds to nearby surface via thioester bond)
• C2 → C2b (released) + C2a (remains bound to C4b)

3C. The Lectin Pathway (Innate immunity)

• MASP1 (MBL-associated serine protease 1) - functional analog of C1r
• MASP2 (MBL-associated serine protease 2) - functional analog of C1s

4. The Common Terminal Pathway - C5 Cleavage to MAC

• C5a (small, released): most potent anaphylatoxin - ~20-fold more potent than C3a; chemotaxin for neutrophils/monocytes/eosinophils/basophils; acts directly on endothelium
• C5b (large, surface-bound): initiates MAC assembly

• Most bacteria have evolved thick cell walls or capsules preventing MAC access to their membranes
• MAC-mediated killing is critically important only for Neisseria species (very thin cell walls): deficiency of C5-C9 gives specific susceptibility to N. meningitidis and N. gonorrhoeae
• C9 polymerization is structurally homologous to perforin used by cytotoxic T cells, indicating evolutionary conservation of this lytic mechanism

5. Complement Receptors - Detailed

6. Regulatory Proteins - Detailed Mechanisms

6a. Inhibition of C1 Activation

6b. Inhibition of C3/C5 Convertase Assembly - The RCA (Regulators of Complement Activation) Family

6c. Inhibition of MAC Formation

• CD59 (protectin): GPI-anchored membrane protein on virtually all host cells. Binds to C5b-8 complex and prevents binding and polymerization of C9, thereby blocking MAC formation.
• S protein (vitronectin): Plasma glycoprotein. Binds to hydrophobic C5b-7 complex in the fluid phase, blocking its insertion into host cell membranes. Converts C5b-7 into a soluble, non-lytic complex.

7. Effector Functions - Detailed

7a. Opsonization and Phagocytosis

• C3b (native) binds CR1 on neutrophils and macrophages. CR1 alone does not efficiently trigger phagocytosis unless there is co-stimulation (by IFN-γ or C5a). This two-signal requirement prevents inadvertent phagocytosis of host cells that have small amounts of C3b.
• iC3b (generated by Factor I cleavage of C3b) binds CR3 and CR4, which are more efficient phagocytic receptors.
• When microbes are dual-opsonized (both C3b and IgG), CR1 and FcγR synergize dramatically for phagocytosis and killing.

7b. Stimulation of Inflammation (Anaphylatoxins)

• Mast cell and basophil degranulation → histamine release → vasodilation, increased permeability
• Neutrophil chemotaxis (the most potent chemoattractant complement fragment)
• Neutrophil activation: firm adhesion to endothelium, respiratory burst (ROS generation), lysosomal enzyme release, upregulation of CR1/CR3
• Direct action on vascular endothelium: P-selectin upregulation, increased permeability
• Activates the lipoxygenase pathway in neutrophils/monocytes → further inflammatory mediator generation

• Mast cell degranulation
• Chemoattractant for eosinophils specifically

• Binds PAR1/PAR4 receptors; minimal biological effect in humans

7c. Complement-Mediated Cytolysis

• Platelet activation and aggregation
• Endothelial cell activation → procoagulant surface → thrombosis
• Smooth muscle cell activation
• Cellular responses including upregulation of inflammatory mediators

7d. Immune Complex Clearance

1. Sterically block Fc-Fc interactions that promote lattice formation → solubilize immune complexes
2. C3b on immune complexes binds to CR1 on erythrocytes → complexes are transported to liver and spleen → stripped and degraded by phagocytes (Kupffer cells)

7e. Enhancement of Adaptive Humoral Immunity (B Cell Co-receptor)

• Bind antigen through the BCR (Ig receptor)
• Bind C3d on the same antigen through CR2 (CD21)

7f. Apoptotic Cell Clearance

8. Complement Deficiency States - Clinical Correlates

• CH50 (total hemolytic complement): detects deficiency of any classical/terminal pathway component
• AH50: detects alternative pathway deficiency
• C3, C4: consumed in active disease (e.g., lupus nephritis: ↓C3, ↓C4)
• Flow cytometry for CD55/CD59: diagnoses PNH

9. Microbial Evasion of Complement

• Many pathogens express sialic acids (mimicking host) → recruit Factor H → inhibit alternative pathway. N. gonorrhoeae, Haemophilus, schistosomes enzymatically transfer sialic acids from host to their surfaces. E. coli K1 and some meningococci have their own sialic acid biosynthesis.
• HIV-1 gp41 binds Factor H
• Streptococcus pyogenes M protein binds Factor H and C4BP
• Staphylococcus aureus protein Efb binds C3b; SCIN (Staphylococcal Complement Inhibitor) directly blocks C3 convertases

• Thick capsules (gram-positive bacteria, fungi) physically prevent MAC access
• Long polysaccharide chains on gram-negative LPS (smooth LPS) divert MAC away from the outer membrane

• Staphylococcal CHIPS (Chemotaxis Inhibitory Protein of Staphylococcus) blocks C5aR1 and FPR1 → inhibits neutrophil chemotaxis
• Herpesvirus proteins (e.g., HCMV UL18, HSV glycoprotein C) block C3b deposition

10. Therapeutic Targeting of Complement

Summary: Complement System at a Glance

• Cellular and Molecular Immunology (Abbas, Lichtman & Pillai), pp. 838-882
• Robbins, Cotran & Kumar Pathologic Basis of Disease, pp. 97-99
• Janeway's Immunobiology 10e, pp. 1462-1469
• Roitt's Essential Immunology, pp. 850-862
• Henry's Clinical Diagnosis, pp. 1112-1113

The Complement System - Abbas (Cellular and Molecular Immunology)

Historical Background

General Principles of Complement Activation

1. Complement attacks microbial surfaces: The system is activated by microbes and by antibodies/lectins bound to microbes. Complement therefore focuses immune attack specifically on microbial surfaces.
2. Proteolytic cascade amplification: Activation involves sequential proteolysis of proteins to generate enzyme complexes. Proteins that acquire proteolytic activity by the action of other proteases are called zymogens. This is also characteristic of the coagulation and kinin systems. Proteolytic cascades allow tremendous and rapid amplification because each active enzyme molecule at one step can generate multiple activated enzyme molecules at the next step.
3. Surface restriction via covalent attachment: Several biologically active cleavage products become covalently attached to microbial cell surfaces, to antibodies bound to microbes and other antigens, and to apoptotic bodies. In the fluid phase, complement proteins are inactive or only transiently active (for seconds), but they become stably activated after attachment to microbes, antibodies, or dying cells. Thus, full activation and biologic functions of the complement system are limited to microbial cell surfaces or to sites of antibody-antigen binding - they do not occur freely in the blood.
4. Self vs. non-self discrimination via regulatory proteins: Complement activation is inhibited by regulatory proteins present on normal host cells but absent from microbes. These regulatory proteins are an adaptation of normal cells that minimize complement-mediated damage. Because microbes lack these proteins, complement activation can proceed on microbial surfaces.

Three Pathways of Complement Activation

The Alternative Pathway

The Alternative Pathway - Step by Step

The Classical Pathway

Structure of C1

• C1q: 6 identical subunits arranged to form a central core with symmetrically projecting radial arms. The globular heads (H) at the end of each arm are the contact regions for the Fc portions of antibodies (IgG or IgM).
• C1r₂s₂: a tetramer of two C1r and two C1s molecules that wraps around the radial arms of C1q, juxtaposing the catalytic domains of C1r and C1s.

Initiation

• IgM (pentamer): highly efficient - one IgM molecule provides up to 5 potential Fc sites in close proximity. Even a single IgM molecule bound to antigen can activate complement.
• IgG (monomer): requires two adjacent IgG molecules bound to antigen. Soluble IgG does not activate the classical pathway.

Downstream Steps

• C4 → C4a (small, released, weak anaphylatoxin) + C4b (large, covalently attaches to surface via thioester bond)
• C2 → C2b (released) + C2a (remains associated with C4b)

The Lectin Pathway

Late Steps: C5 Cleavage and the Membrane Attack Complex (MAC)

MAC Assembly - Step by Step

• C5a (small, released): potent pro-inflammatory fragment
• C5b (large): released but rapidly binds plasma C6, undergoing a conformational change

• ~20 nm external diameter
• 1-11 nm internal diameter (varies with number of C9 molecules)
• ~15 nm height
• Fully permeable to water and ions

Receptors for Complement Proteins

CR1 (CD35) - Type 1 Complement Receptor

1. Promotes phagocytosis of C3b- and C4b-coated particles. The binding of C3b- or C4b-coated particles to CR1 transduces signals that activate microbicidal mechanisms of phagocytes, especially when the Fcγ receptor is simultaneously engaged by antibody-coated particles (dual opsonization).
2. Immune complex clearance: CR1 on erythrocytes binds circulating immune complexes with attached C3b/C4b and transports them to the liver and spleen. Phagocytes there remove the immune complexes from the erythrocyte surface, and the erythrocytes continue to circulate.
3. Complement regulation: CR1 is also a regulator of complement activation (cofactor for Factor I).

CR2 (CD21) - Type 2 Complement Receptor

1. B cell coreceptor: CR2 is expressed on mature B cells as part of a trimolecular complex with CD19 and CD81 (TAPA1). This complex delivers signals that enhance B cell responses to antigen (detailed below).
2. Antigen trapping on FDCs: CR2 on FDCs traps iC3b-, C3d-, and C3dg-coated antigen-antibody complexes in germinal centers.
3. CR2 is also the receptor for Epstein-Barr virus (EBV), which uses it to infect B cells.

CR3 (MAC-1, CD11b:CD18) - Type 3 Complement Receptor

1. Promotes phagocytosis of microbes opsonized with iC3b.
2. May directly recognize some microbial molecules for phagocytosis.
3. Binds ICAM-1 on endothelial cells and promotes stable attachment of leukocytes to endothelium - leading to recruitment of leukocytes to sites of infection and tissue injury.

CR4 (CD11c:CD18)

Regulation of Complement Activation

1. Low-level complement activation occurs spontaneously, and if allowed to proceed, it can damage normal cells and tissues.
2. Even when appropriately activated on microbial cells, degradation products can diffuse to adjacent host cells and injure them.

Regulatory Proteins Table (from Abbas Table 13.9)

Detailed Regulatory Mechanisms

• Factor H inhibits binding of only Bb to C3b → regulates the alternative pathway only (not the classical pathway)
• MCP, CR1, and DAF are produced by mammalian cells but not microbes → selectively inhibit complement on host cells while permitting activation on microbes
• Sialic acid on mammalian cells (higher than on microbes) further favors binding of Factor H over Factor B

• CD59 (protectin): GPI-linked membrane protein. Binds to C5b-8 complex and prevents binding and polymerization of C9, blocking MAC formation.
• S protein (vitronectin): Plasma protein. Binds to hydrophobic C5b-7 in the fluid phase, blocking its membrane insertion.

Functions of the Complement System

1. Opsonization and Phagocytosis

• Microbes are simultaneously coated with IgG (Fcγ receptor and CR1 co-engagement)
• Macrophages are activated by the cytokine IFN-γ

2. Stimulation of Inflammatory Responses (Anaphylatoxins)

• All three peptides bind mast cells and induce degranulation → release of vasoactive mediators (histamine) → vasodilation and increased vascular permeability
• They are called anaphylatoxins because mast cell reactions they trigger are characteristic of anaphylaxis

• Stimulates neutrophil motility
• Stimulates firm adhesion of neutrophils to endothelium
• At high doses, stimulates the respiratory burst and production of reactive oxygen species
• Acts directly on vascular endothelial cells → increased vascular permeability and expression of P-selectin → promotes neutrophil binding
• These combined actions on mast cells, neutrophils, and endothelial cells contribute to inflammation at sites of complement activation

3. Complement-Mediated Cytolysis

4. Functions in Humoral Immunity - B Cell Co-receptor

• Bind antigen via their membrane Ig (BCR)
• Bind attached C3d via CR2 (CD21)

5. Immune Complex Clearance and Solubilization

1. Sterically block Fc-Fc interactions that promote lattice formation → solubilize immune complexes
2. C3b on immune complexes binds to CR1 on erythrocytes → complexes transported to liver and spleen → phagocytes remove the complexes, erythrocytes continue to circulate

6. Clearance of Apoptotic Cells

Complement Deficiencies

Pathologic Effects of Complement

1. Complement activation by antibodies against vascularized organ transplants and immune complexes in autoimmune diseases may bind to vascular endothelium → inflammation → MAC damage to endothelial surface → favors coagulation and intravascular thrombosis → ischemic injury.
2. Some late complement proteins may activate prothrombinases in the circulation → thrombosis independent of MAC-mediated endothelial damage.
3. Immune complex-mediated diseases: Systemic vasculitis and immune complex glomerulonephritis result from deposition of antigen-antibody complexes in vessel walls and kidney glomeruli. Complement activated by these complexes initiates acute inflammatory responses that destroy vessel walls or glomeruli → thrombosis, ischemic damage, scarring.
4. The function of regulatory proteins may be overwhelmed when large amounts of antibodies deposit on host cells, generating enough active complement proteins that regulatory molecules are unable to keep up.

• Antibodies against C5 that block proteolytic cleavage of C5 → used for PNH, complement-mediated HUS, and neuromyelitis optica
• Recombinant human C1-INH → used for hereditary angioedema

Evasion of Complement by Microbes

Group 1 - Recruiting Host Complement Regulatory Proteins

• Schistosomes, N. gonorrhoeae, and certain Haemophilus species scavenge sialic acids from the host and enzymatically transfer them to their cell surfaces
• E. coli K1 and some meningococci have evolved their own biosynthetic routes for sialic acid generation
• HIV gp41 binds Factor H → virion protection
• HIV also incorporates GPI-anchored host regulatory proteins DAF and CD59 into its envelope when budding from infected cells
• S. pyogenes, B. burgdorferi, N. gonorrhoeae, N. meningitidis, Candida albicans, and Echinococcus granulosus synthesize proteins that recruit Factor H to their cell walls

Group 2 - Producing Proteins That Inhibit Complement Steps

• E. coli makes a C1q-binding protein that inhibits association of C1q, C1r, and C1s
• S. aureus makes Staphylococcal Complement Inhibitor (SCIN) that binds to and stably inhibits both classical and alternative pathway C3 convertases → inhibits all three complement pathways
• Herpes simplex virus glycoprotein C destabilizes the alternative pathway convertase by preventing its C3b from binding to properdin
• Trypanosoma cruzi GP160 binds C3b, prevents C3 convertase formation, and accelerates its decay
• Vaccinia virus complement inhibitory protein structurally resembles human C4BP but binds both C4b and C3b → accelerates decay of both C3 and C5 convertases

Group 3 - Inhibiting Complement-Mediated Inflammation

• S. aureus synthesizes CHIPS (Chemokine Inhibitory Protein of Staphylococci) - an antagonist of the C5a anaphylatoxin, blocking neutrophil chemotaxis

Summary (as stated in Abbas)