Chapter 5: The Complement System — Comprehensive Study Notes

The Complement System

• Describes a group of serum proteins that circulate in inactive form.
• Upon activation, outcomes include:
  • target cell membrane lysis,
  • chemotaxis,
  • opsonization to enhance phagocytosis,
  • inflammation.
• Activation pathways converge on common effector steps, notably the formation of C3b and C5 convertases, leading to inflammation, opsonization, and MAC formation.
• Key terms:
  • C3 convertases: enzyme complexes that cleave C3 to C3a and C3b.
  • C5 convertases: formed when C3 convertases acquire additional components, cleaving C5 to C5a and C5b.
  • MAC: membrane attack complex that forms a pore in target membranes.
• Major outcomes: lysis, chemotaxis, opsonization, inflammation.

Activation Pathways

• Three initiation routes exist:
  • Classical pathway
  • Lectin pathway
  • Alternative pathway
• All three converge to generate C3b, and ultimately drive MAC formation via C5 convertases.
• Core convertases:
  • Classical and Lectin pathways use the C3 convertase $C4b2a$.
  • Alternative pathway uses $C3bBb$ as its C3 convertase.
• C5 convertases:
  • Classical/Lectin pathway: $C4b2a3b$.
  • Alternative pathway: $C3bBbC3b$ (also called the C5 convertase).
• C5a is a potent inflammatory mediator; C5b initiates MAC assembly.
• C4b’s thioester bond is exposed upon C4 cleavage; the bond is reactive and binds to target surfaces; if no surface present, the bond is hydrolyzed and inactivated. $ext{C4 has an exposed thioester bond that can react with surface amines or hydrolyze if no surface is available.}$
• Figures referenced in slides: Fig. 5-2 (activation pathways), Fig. 5-3, Fig. 5-5, Fig. 5-6, Fig. 5-7, Fig. 5-8, Fig. 5-10, Fig. 5-11, Fig. 5-12, Fig. 5-13, Fig. 5-15, Fig. 5-16, Fig. 5-17, etc.

Classical Pathway Overview

• Requires binding of either:
  • IgM or IgG to multivalent antigen (soluble immune complexes or microbial membrane-associated antigens).
• Initiation:
  • C1 complex (C1qr2s2) binds to Fc regions on adjacent antibodies or via IgM bound to antigen.
  • C1q binding induces conformational changes activating C1r, which then activates C1s.
  • Result: cleavage of C4 and C2 by C1s, generating C4b and C2a that form the C3 convertase $C4b2a$ on the membrane near the site of activation.
  • C4 cleavage: C4a (an anaphylatoxin) and C4b (proximal to membrane) produced.
• Downstream steps:
  • C3 convertase hydrolyzes many C3 molecules to generate C3b and C3a.
  • Some C3b combines with the C3 convertase to form C5 convertase $C4b2a3b$.
  • C5 convertase cleaves C5 into C5a (anaphylatoxin) and C5b which initiates MAC assembly.
• Key details:
  • C1 binds Fc regions on IgG or IgM bound to antigen (epitopes).
  • C4 is cleaved first; C4b binds membrane near C1; C4b binds C2 and makes C4b2a (the C3 convertase).
  • C3b participates in opsonization and in forming C5 convertase.
• Illustrative sequence (in order):
  • C1q binds to antibody–antigen complex → conformational changes → activation of C1r and C1s → cleavage of C4 and C2 by C1s → formation of $C4b2a$ (C3 convertase) → hydrolysis of C3 to C3b and C3a → formation of $C4b2a3b$ (C5 convertase) → cleavage of C5 → generation of MAC components.
• Thioester bond exposure in C4 allows surface attachment; if surface absent, bond hydrolyzed and C4b is inactivated.

Lectin Pathway Overview

• Initiation occurs when soluble lectins recognize microbial carbohydrates on surfaces, e.g., mannose-binding lectin (MBL) or ficolins.
• Lectins serve as docking sites for MASPs (MBL-associated serine proteases).
• MASPs cleave C4 and C2 to form the C3 convertase, identical to downstream steps of the classical pathway: $C4b2a$, followed by formation of $C4b2a3b$, cleavage of C3 to C3b and C3a, formation of C5 convertase, C5 cleavage, and MAC assembly.
• Figure reference: Fig. 5-7.

Initiating and Amplifying Proteins of Classical and Lectin Pathways

• Table 5-1 highlights biologically active fragments and functions:
  • IgM, IgG: bind pathogen surface; initiate complement cascade (Classical pathway).
  • MBL and ficolins: bind carbohydrates on microbial surfaces; initiate classical-like cascade (Lectin pathway).
  • C1 complex: initiates classical pathway by binding Ig; C1r and C1s are serine proteases that cleave C4 and C2.
  • MASP-1 and MASP-2: MASP-2 cleaves C4 and C2; MASP-1 supports activation.
  • C2 and C4: form C3 convertase when bound with the appropriate partner; C2a is a serine protease fragment; alternative nomenclature notes that some sources call C2a the larger fragment and C2b the smaller one.
  • C3: cleavage products include C3a and C3b; C3b participates in opsonization and forms C5 convertases in both classical and lectin pathways.

The Alternative Pathway

• Initiation can occur in three ways:
  • Alternative tickover pathway
  • Properdin-initiated pathway
  • Protease-activated pathway
• The Alternative Tickover Pathway:
  • Small, constitutive cleavage of C3 occurs (tickover), generating C3b in the absence of pathogens.
  • Cleaved C3b binds to target cell membranes.
  • Factor B binds to surface-bound C3b.
  • Factor D cleaves Factor B into Bb and Ba; the complex C3bBb forms the C3 convertase on the surface.
  • Properdin stabilizes C3bBb, increasing convertase half-life and activity; this allows cleavage of many more C3 molecules.
  • Newly activated C3b binds C3bBb to form C3bBbC3b, i.e., the C5 convertase of the alternative pathway.
  • C5 convertase then cleaves C5 to C5a and C5b, continuing the cascade toward MAC.
• The Alternative Properdin-Activated Pathway:
  • Properdin can directly bind to a surface; recruits C3b and Factor B; Factor D cleaves B; forms C3bBb (active C3 convertase).
  • Downstream steps identical to the alternative tickover pathway.
• The Alternative Protease-Activated Pathway:
  • Coagulation/clotting cascades can stimulate complement activation.
  • Thrombin has been shown to cleave C3 and C5 in vitro.
  • Platelet activation releases ATP and Ca^{2+} and kinases that could stabilize C3b in fluid phase, potentially promoting inflammatory amplification.
• Figures referenced: Fig. 5-8 (tickover), Fig. 5-9 (properdin-activated), Fig. 5-9 (protease-activated).

C5 Convertase and the Membrane Attack Complex (MAC)

• All three pathways converge at the formation of the C5 convertase.
• C5 initiates MAC formation.
• MAC results from deposition of components C5b, C6, C7, C8, and C9 into target membranes, forming a pore that disrupts osmotic integrity and leads to cell death.
• MAC assembly sequence (illustrative):
  • C5b binds C6 → C7 → C8 → multiple C9 molecules; polymerization of C9 creates a transmembrane pore.
  • Figure reference: Fig. 5-10.
• Summary of MAC components: ext{MAC} = C5b
  ightarrow C6
  ightarrow C7
  ightarrow C8
  ightarrow C9_{ ext{polymer}} (pores formed by multiple C9 units).

The Proteins of the Complement Membrane Attack Complex (MAC)

• Key components and roles:
  • C5: C5a (anaphylatoxin) and C5b (MAC initiator); $C5a$ promotes inflammation; $C5b$ initiates MAC.
  • C6, C7, C8: contribute to MAC assembly and membrane insertion.
  • C9: polymerizes to form the pore.
• Table 5-3 provides a detailed breakdown of each fragment’s role within the MAC.
• The MAC disrupts cellular membranes leading to lysis of susceptible target cells.

C4b and C4d; C4b Binding to the Membrane

• A thioester bond in C4 becomes exposed upon cleavage; C4b can form covalent bonds with target membranes via the reactive thioester, anchoring the C3 convertase complex to the surface.
• If no surface is present, the thioester bond is hydrolyzed, and C4b is neutralized.
• Figure reference: Fig. 5-6.

Receptor-Mediated Functions and the Interfaces with Innate and Adaptive Immunity

• Receptors that bind complement components and their breakdown products:
  • CR1 (CD35): binds C3b, iC3b, C3d, C3dg, C4b, C1q; functions include clearance of immune complexes, enhanced phagocytosis, and regulation of C3 breakdown.
  • CR2 (CD21): binds C3d, C3dg, iC3b; on B cells and FDCs; enhances B-cell activation and serves as B-cell co-receptor; retention of C3d-tagged immune complexes.
  • CR3 (Mac-1, CD11b/CD18) and CR4 (CD11c/CD18): bind iC3b; facilitate leukocyte adhesion, extravasation, and opsonization.
  • CRIg (VSIG4): binds iC3b; on fixed tissue macrophages; promotes phagocytosis of immune complexes by macrophages and inhibits alternative pathway on the surface.
• C1q receptors and lectin pathway receptors:
  • C1qR (CD93) and SIGN-R1 (CD209) participate in recognition and opsonization processes.
• C3aR and C5aR are receptors on granulocytes that mediate inflammatory responses:
  • C3aR and C5aR binding C3a and C5a stimulates degranulation and release of inflammatory mediators from mast cells, basophils, and granulocytes.
  • C5L2 (C5a receptor-like 2) may downregulate proinflammatory effects of C5a.
• Table references: Tables 5-5 and 5-6 summarize receptors, ligands, and regulatory proteins.
• Also important: C3aR, C5aR expression on leukocytes; roles in chemotaxis and inflammation.

Opsonization and Inflammation

• Opsonization: covalently bound C3b and C4b tag pathogens and immune complexes for enhanced phagocytosis.
• Complement contributes to host defense via three main activities:
  • Innate defense through MAC-mediated lysis of susceptible cells.
  • Opsonization for efficient phagocytosis of pathogens and immune complexes.
  • Induction of inflammation via anaphylatoxins C3a and C5a and their receptors on leukocytes.
• Diagram reference: Fig. 5-13 and Fig. 5-15 illustrate opsonization and inflammatory roles.

Complement Interfaces with Innate and Adaptive Immunity

• Complement enhances antigen uptake via binding to receptors on APCs (MBL, C1q, C3b, C4b).
• Increases B-cell responses through higher avidity of B-cell binding to complement-tagged antigens; supports antigen presentation.
• Lyses immature T cells with low sialic acid content, which can change during maturation.
• C3a, C5a, and C3b receptors on mature T cells support growth, differentiation, and survival.

Complement in the Contraction Phase of the Immune Response

• Complement aids in the contraction phase by:
  • disposal of apoptotic cells and bodies as lymphocytes are no longer required,
  • removal/disposal of immune complexes formed during responses,
  • helping to prevent unnecessary inflammation after antigen clearance.
• This regulatory role helps avoid collateral tissue damage once an infection is cleared.

Which Statement Is Not True of Complement?

• From practice item (Page 23): false statement is often that all three pathways lead to MAC formation or that all three pathways form the same convertases.
• Specifically, while all three pathways form C3 convertases and C5 convertases, the exact composition differs; e.g., the C3 convertase for classical/lectin is $C4b2a$, while the alternate pathway uses $C3bBb$. Additionally, not all pathways use the same initiating components.

Regulation of Complement Activity

• Complement activity is tightly regulated to prevent damage to host tissues.
• Key points:
  • C3 convertases have short half-lives unless stabilized by properdin.
  • Self-cells employ different surface carbohydrates that recruit soluble proteases to inactivate C3b rapidly.
  • Numerous regulatory proteins prevent unintended complement activation on host cells.
• Major regulatory proteins include: C1 inhibitor (C1INH), Decay-accelerating factors (DAF/CD55, CR1/CD35), C4BP, Factor H, Factor I, MCP (CD46), S protein (vitronectin), CD59 (Protectin), and carboxypeptidases N, B, and R.
• Figure 5-16 illustrates regulatory networks including dissociation and decay of convertases, cofactor activities, MAC inhibition, and des-Arg formation of anaphylatoxins.

C1 Inhibitor (C1INH)

• C1INH promotes the dissociation of the C1 components.
• It binds in the active site of serine proteases and causes C1r2s2 to dissociate from C1q, preventing further cleavage of C4 and C2.
• Inhibits initiation of classical and lectin complement pathways.
• Figure reference: Fig. 5-16(a).

Regulation of C3 Convertases

• Decay-accelerating factors promote decay of C3 convertases; examples:
  • DAF (CD55)
  • CR1 (CD35)
  • C4BP (C4-binding protein)
• Factor H binds negatively charged cell surface sialic acid and heparin (host-specific markers) to facilitate decay of the C3 convertases on host cells.
• Function: accelerate decay of both classical/lectin C4b2a and alternative C3bBb convertases.
• Figure reference: Fig. 5-16(b).

Factor I and Cofactors

• Factor I is a soluble serine protease that cleaves C3b and C4b into inactive fragments.
• Requires cofactors such as MCP (CD46) and CR1 to function on host-cell surfaces.
• Additional cofactors include C4BP, Factor H, and CR1 for degradation of C3b/C4b.
• Figure reference: Fig. 5-16(c).

Inhibition of MAC-Mediated Lysis

• Protectin (CD59) inhibits MAC by binding C5b-678 complexes deposited on host cells and preventing their insertion into the plasma membrane and C9 recruitment.
• Soluble S-protein (vitronectin) binds fluid-phase C5b67 and prevents MAC insertion into host cell membranes.
• Figure reference: Fig. 5-16(d).

Cleavage and Inactivation of Anaphylatoxins

• Carboxypeptidases N, B, and R cleave C3a and C5a by removing C-terminal arginine residues to form des-Arg forms, inactivating chemotactic and inflammatory functions.
• Figure reference: Fig. 5-16(e).

Proteins Involved in Regulation of Complement Activity (Table 5-6)

• Summary of regulators, their form (soluble or membrane-bound), pathways affected, and function:
  • C1INH: soluble; classical and lectin; initiates dissociation and inhibition of C1r2s2 from C1q.
  • DAF (CD55): membrane-bound; acts on classical, alternative, and lectin pathways; accelerates dissociation of C4b2a and C3bBb.
  • CR1 (CD35): membrane-bound; broad activity; acts as regulator and cofactor for factor I; binds C4b and C3b.
  • C4BP: soluble; classical and lectin; inhibits formation or accelerates dissociation of C4b2a; cofactor for factor I.
  • Factor H: soluble; alternative; inhibits C3 convertase formation; cofactor for factor I in degradation of C3b.
  • Factor I: soluble; all pathways; cleaves C3b and C4b; requires cofactors.
  • S protein (vitronectin): soluble; all pathways; binds soluble C5b67 and prevents insertion into membranes.
  • CD59 ( Protectin): membrane-bound; all pathways; prevents MAC formation by blocking C9 insertion.
  • Carboxypeptidases N, B, and R: soluble; all pathways; inactivate C3a and C5a.

Complement Deficiencies and Disease Relevance

• Genetic deficiencies exist for most complement components with varied outcomes.
• C1q, C1r, C1s, C4, or C2 deficiencies are often associated with immune complex disorders due to poor clearance.
• MBL deficiency may lead to higher susceptibility to infections by encapsulated bacteria due to reduced opsonization.
• Animal models exist for most complement deficiencies, aiding study of function and disease associations.

Microbial Evasion of Complement (Table 5-7)

• Microbes employ diverse strategies to evade complement:
  • Interference with antibody–complement interaction:
  • Staphylococcal protein A depletes IgG.
  • Staphylokinase removes IgG.
  • Binding and inactivation of complement proteins:
  • S. aureus SCIN binds C3bBb (C3 convertase) and inactivates it.
  • Parasitic C2 receptors disrupt C2–C4 interaction.
  • Elastase and alkaline phosphatase from Pseudomonas degrades C1q, C3, and C3b.
  • ScpA and ScpB from Streptococcus degrade C5a.
  • Microbial mimicry of complement regulatory proteins:
  • Streptococcus pyogenes M proteins bind C4BP and factor H to the surface, promoting decay of C3 convertases bound to the bacterial surface.
  • Variola and Vaccinia viruses express cofactors for Factor I to degrade C3b and C4b.
  • Disruption of interactions between complement proteins:
  • C2 receptor trispanning proteins from Schistosoma and Trypanosoma disrupt C2a–C4b interaction.

Evolutionary Origins and Comparative Aspects

• Evolutionarily, genes encoding complement components fall into five families.
• The alternative pathway genes appear earliest in evolution; terminal MAC components appeared last.
• Complement originally aided phagocytosis prior to the evolution of adaptive immunity.
• Figure reference: Fig. 5-17.

Pathways Across Major Groups of Deuterostome Animals

• Table 5-8 summarizes presence/absence of complement components across animal groups (on/off for alternative, classical, lectin pathways, MAC, and antibodies present):
  • Mammals: all five features present (A/P MAC and antibodies).
  • Birds, Reptiles, Amphibians, Teleost fish, Cartilaginous fish, Agnathan fish, Tunicates, Echinoderms: varying patterns; generally, the classical and lectin pathways and MAC are present in many vertebrates, with some groups lacking antibodies.

Summary Points

• The complement system links innate and adaptive immunity and is tightly regulated to prevent host damage.
• Activation proceeds via classical, lectin, or alternative pathways, all converging on C3 activation and MAC formation.
• The system contributes to host defense through lysis, opsonization, and inflammation, while regulating contraction and clearance of immune complexes and apoptotic cells.
• Regulatory proteins ensure self-tolerance and prevent excessive inflammation.
• Microbial evasion strategies demonstrate co-evolution with host immunity, including mimicry, proteolysis, and binding of regulatory proteins.
• Evolutionary analyses show that complement predates adaptive immunity and offers insights into the evolution of immune defense.

Concept Map and Practice Activities (Study Aids)

• Concept map exercises help relate core terms: C3 convertase, C5 convertase, PAMP-bound lectins, Properdin, C3, C3(H2O), Proteases, Classical pathway, Lectin pathway, Alternative pathway, Immune complexes, MAC.
• Suggested activity: draft a concept map and compare with a partner to explore alternative connections and pathways.

Math and Key Notation Used in This Chapter

• Classical and Lectin pathway C3 convertase: $C4b2a$
• Alternative pathway C3 convertase: $C3bBb$
• Classical/lectin C5 convertase: $C4b2a3b$
• Alternative C5 convertase: $C3bBbC3b$
• MAC composition: ext{MAC} = C5b
  ightarrow C6
  ightarrow C7
  ightarrow C8
  ightarrow C9_{ ext{polymer}}
• Opsonization factors: $C3b,\, C4b$
• Receptors: CR1/CR2/CR3/CR4/CRIg (CD35, CD21, CD11b/CD18, CD11c/CD18, VSIG4 respectively)
• Anaphylatoxins and receptors: $C3a, C5a$ with receptors $C3aR, C5aR$

Practice Question Highlights (from slides)

• Place the events of classical pathway activation in order from earliest to latest. Sequence key steps including C1 binding, C4/C2 cleavage, formation of $C4b2a$, C3 activation, and C5 convertase formation.
• Identify which statements about complement activation pathways are true/false, e.g., whether all three pathways form identical convertases or all generate MAC, etc.

Connections to Foundational Principles and Real-World Relevance

• The complement system exemplifies innate-immunity signaling that bridges to adaptive responses via antigen tagging and enhanced B-cell activation.
• Therapeutic targeting of complement components is clinically relevant in inflammatory diseases, autoimmune conditions, and transplant biology.
• The balance between activation and regulation is critical; dysregulation can underlie tissue injury and autoimmune phenomena.
• Understanding complement evolution provides insights into how immune systems diversified to defend against pathogens prior to adaptive immunity.

Ethical, Philosophical, and Practical Implications

• Therapeutic interventions targeting complement must preserve essential host defense while reducing pathological inflammation.
• Balancing immune activation with tolerance highlights principles applicable to autoimmune disease management and vaccination strategies.
• Studying microbial evasion informs public health approaches and the design of anti-infective therapies.

Connections to Previous Lectures (Foundational Principles)

• Innate immunity concepts: pattern recognition, opsonization, phagocytosis, and inflammation.
• Adaptive-immunity synergy: antibody involvement in classical pathway activation and B-cell receptor co-stimulation via CR2.
• Regulatory biology: how host cells protect themselves using surface molecules (e.g., sialic acid) and plasma cofactors to distinguish self from non-self.

Key Tables and Figures to Review

• Table 5-1: Initiating and amplifying proteins; biologically active fragments and pathways.
• Table 5-2: Reactions involving Factor D, Factor B, C3(H2O), and properdin in alternate pathways.
• Table 5-3: MAC components and their roles.
• Table 5-4: The three main classes of complement activity in host defense.
• Table 5-5: Receptors that bind complement components and breakdown products.
• Table 5-6: Regulators of complement activity (DAF, CD59, Factor H, MCP, etc.).
• Table 5-7: Microbial evasion strategies.
• Table 5-8: Complement system pathways across major deuterostome animals.
• Fig. 5-2, 5-3, 5-5, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-15, 5-16, 5-17 for pathway visuals and regulatory networks.

End of Chapter 5 Notes