# Lecture 6: The Complement System

## Immunology

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the three pathways of complement activation (classical, lectin, and alternative)
2. Explain the formation and function of C3 and C5 convertases
3. Identify the major biological effector functions of complement (opsonization, inflammation, lysis)
4. Describe the regulatory mechanisms that prevent inappropriate complement activation on host tissues
5. Discuss clinical consequences of complement deficiencies

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## Lecture Content

### I. Overview of the Complement System

The complement system consists of over 30 plasma proteins and membrane-associated regulatory proteins that constitute a major effector arm of innate immunity. Most complement proteins are produced by hepatocytes in the liver, with additional contributions from macrophages and epithelial cells. These proteins circulate as inactive precursors (zymogens) and become activated through sequential proteolytic cleavage in a cascade fashion. The system carries out three major functions: **opsonization**, in which C3b coats pathogens to enhance phagocytosis; **inflammation**, in which the anaphylatoxins C3a and C5a recruit and activate immune cells; and **lysis**, in which the membrane attack complex (MAC, C5b-C9) directly kills pathogens. Additional functions include clearance of immune complexes, enhancement of antibody responses, and bridging of innate and adaptive immunity.

### II. The Classical Pathway

The classical pathway is initiated by **antibody-antigen complexes**, primarily involving IgM and IgG. The first step is binding of the C1 complex, which consists of C1q, C1r, and C1s. C1q has six globular heads that bind the Fc regions of IgG or IgM bound to antigen, with IgM being most efficient since a single pentameric IgM molecule can activate the pathway (whereas at least two IgG molecules are required). C1q binding causes a conformational change that leads to C1r auto-activation, which in turn cleaves and activates C1s. Activated C1s then cleaves C4 into C4a and C4b, and C2 into C2a and C2b. C4b binds covalently to the pathogen surface, and C2a (the larger fragment) remains associated with C4b, forming **C4b2a, the classical pathway C3 convertase**. This enzyme cleaves C3 into C3a and C3b; C3b deposits on the pathogen surface for opsonization and can join the convertase to form C4b2a3b, the **C5 convertase**. The classical pathway can also be activated by C-reactive protein (CRP) and pentraxins binding to pathogens.

### III. The Lectin Pathway

The lectin pathway is initiated by **pattern recognition** of microbial carbohydrates and does not require antibody. The key recognition molecules are **mannose-binding lectin (MBL)**, which binds terminal mannose, fucose, and GlcNAc on microbial surfaces; **ficolins**, which bind N-acetylglucosamine and other acetylated compounds; and **collectin-11 (CL-11)**, which binds L-fucose and D-mannose. MBL and ficolins associate with **MASPs (MBL-associated serine proteases)**, specifically MASP-1 and MASP-2, which are analogous to C1r and C1s. MASP-2 cleaves C4 and C2 to form the same C3 convertase as the classical pathway (C4b2a), and all downstream events are identical.

### IV. The Alternative Pathway

The alternative pathway is constitutively active at low levels through a "tick-over" mechanism, providing continuous immune surveillance without requiring antibody or specific pattern recognition molecules. The process begins with spontaneous hydrolysis of the internal thioester bond in C3, producing C3(H2O). This molecule binds Factor B, which is then cleaved by Factor D into Bb and Ba. The resulting C3(H2O)Bb acts as a short-lived fluid-phase C3 convertase that cleaves C3 into C3b. When C3b deposits on nearby surfaces, it binds Factor B, which is again cleaved by Factor D to form **C3bBb, the alternative pathway C3 convertase**. **Properdin (Factor P)** stabilizes this convertase, extending its half-life. An amplification loop then ensues: C3bBb generates more C3b, which forms more C3bBb convertases, leading to exponential amplification. The addition of another C3b molecule creates C3bBbC3b, the alternative pathway **C5 convertase**. Importantly, the alternative pathway also amplifies complement activation initiated by the classical or lectin pathways.

<image>A three-panel diagram comparing the three complement activation pathways side by side. Panel A (Classical): C1q binding to antibody-antigen complexes on a bacterial surface, leading through C1r/C1s activation, C4/C2 cleavage, to C4b2a (C3 convertase). Panel B (Lectin): MBL binding to mannose residues on a bacterial surface, with MASP-1/2 activation, same C4/C2 cleavage, same C4b2a convertase. Panel C (Alternative): spontaneous C3 tick-over, Factor B/Factor D involvement, formation of C3bBb convertase stabilized by properdin. All three panels converge at the bottom on the common terminal pathway: C3 cleavage → C3b opsonization and C5 convertase formation → C5 cleavage → MAC assembly (C5b-C6-C7-C8-poly-C9). Key fragments are color-coded: C3a/C5a (red, anaphylatoxins), C3b (green, opsonin), MAC (blue, lysis).</image>

### V. The Terminal Pathway and Membrane Attack Complex (MAC)

The terminal pathway is shared by all three activation pathways and begins when the C5 convertase cleaves C5 into C5a (a potent anaphylatoxin) and C5b. The MAC then assembles sequentially: C5b binds C6 to form C5b6, which binds C7 to form C5b67 (this complex inserts into the lipid bilayer), followed by C8 binding to begin forming a small pore (C5b678), and finally the recruitment of 10-16 C9 molecules that polymerize into a ring. The complete MAC (C5b6789n) forms a transmembrane pore approximately 10 nm in diameter that disrupts membrane integrity, causing osmotic lysis of the target cell. The MAC is most effective against Gram-negative bacteria (which have a thin outer membrane) and enveloped viruses, while Gram-positive bacteria are relatively resistant due to their thick peptidoglycan layer. Nucleated cells can resist MAC through membrane repair mechanisms and the regulatory protein CD59.

### VI. Biological Effector Functions of Complement

**Opsonization and phagocytosis** represent the most important effector function of complement. C3b and its cleavage products (iC3b and C3dg) coat pathogen surfaces. Phagocytes express complement receptors to capture these opsonized targets: CR1 (CD35) binds C3b and C4b to promote phagocytosis, CR3 (CD11b/CD18, also known as Mac-1) binds iC3b to provide a strong phagocytic signal, and CR4 (CD11c/CD18) also binds iC3b.

The **anaphylatoxins** C5a, C3a, and C4a (in decreasing order of potency) mediate inflammation through several mechanisms: they trigger mast cell and basophil degranulation leading to histamine release, vasodilation, and increased vascular permeability; C5a is the most potent chemotactic factor for neutrophils; they upregulate adhesion molecules on endothelium; they cause smooth muscle contraction; and C5a activates the oxidative burst and degranulation of neutrophils and monocytes.

**Direct lysis** through the MAC is primarily effective against Gram-negative bacteria, particularly Neisseria species, and also lyses enveloped viruses and abnormal host cells. **Immune complex clearance** relies on C3b and C4b binding to immune complexes, which are then captured by CR1 on erythrocytes and transported to the liver and spleen for clearance by macrophages, preventing immune complex deposition in tissues (a process relevant to SLE). Finally, complement **enhances adaptive immunity** through C3d, which binds CR2 (CD21) on B cells. CR2 is part of the B cell co-receptor complex (CR2-CD19-CD81), and C3d-antigen complexes lower the threshold for B cell activation by up to 1,000-fold. Complement also enhances antigen retention on follicular dendritic cells in germinal centers.

### VII. Regulation of Complement

Regulation of complement is essential to prevent damage to host tissues. **Fluid-phase regulators** include C1 inhibitor (C1-INH), which inactivates C1r, C1s, and MASPs (deficiency causes hereditary angioedema); Factor I, a serine protease that cleaves and inactivates C3b and C4b with the help of cofactors; Factor H, which serves as a cofactor for Factor I in cleaving C3b and competes with Factor B for C3b binding, making it a key regulator of the alternative pathway on host surfaces; C4b-binding protein (C4BP), which acts as a cofactor for Factor I cleavage of C4b; and clusterin and vitronectin, which prevent MAC insertion.

**Membrane-bound regulators** on host cells include DAF (CD55), which accelerates the decay of C3 and C5 convertases by displacing Bb and C2a; MCP (CD46), which serves as a cofactor for Factor I-mediated cleavage of C3b and C4b; CD59 (protectin), which prevents C9 polymerization and thereby blocks MAC assembly; and CR1 (CD35), which has both decay-accelerating activity and cofactor activity for Factor I. Host cells express these regulators while microbial surfaces do not, enabling selective activation on pathogens.

<image>A diagram of a host cell surrounded by complement regulatory proteins. On the cell membrane: DAF (CD55) shown accelerating the decay of C3 convertase (C4b2a and C3bBb falling apart), MCP (CD46) shown acting as a cofactor for Factor I to cleave C3b into iC3b, and CD59 shown blocking C9 polymerization at the final step of MAC assembly. In the fluid phase around the cell: Factor H binding to C3b on the host cell surface (recognizing sialic acid markers), C1 inhibitor blocking C1r/C1s, Factor I cleaving C3b with Factor H as cofactor. A nearby bacterial surface is shown lacking these regulators, with complement activating freely -- C3b deposition, MAC formation, and C5a release occurring unimpeded.</image>

### VIII. Clinical Significance of Complement Deficiencies

Complement deficiencies cause distinct clinical syndromes depending on which component is affected. **C1q, C2, and C4 deficiency** (classical pathway components) increases the risk of SLE due to impaired immune complex clearance, with C2 deficiency being the most common complement deficiency overall. **C3 deficiency** causes severe recurrent pyogenic infections because of opsonization failure and is also associated with glomerulonephritis. **C5-C9 deficiency** (terminal pathway and MAC components) leads to increased susceptibility to Neisseria infections, including both meningococcal and gonococcal disease. **Factor H or Factor I deficiency** results in uncontrolled alternative pathway activation with consumption of C3, leading to atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy. **MBL deficiency** causes increased infections in early childhood before adaptive immunity fully matures. **C1 inhibitor deficiency** results in hereditary angioedema due to uncontrolled bradykinin production. **CD59/DAF deficiency**, as seen in paroxysmal nocturnal hemoglobinuria (PNH), arises from a somatic mutation in the PIGA gene that eliminates GPI-anchored proteins including CD55 and CD59, leading to complement-mediated lysis of red blood cells, hemolytic anemia, and thrombosis. PNH is treated with eculizumab, an anti-C5 monoclonal antibody.

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