Lecture 5 PPT Notes: The Complement System: Detailed Architecture, Activation, and Regulation

The Discovery and Nature of the Complement System

• Historical Discovery: The system was discovered through the observation of anti-sera against Vibrio cholera, which caused bacterial lysis.

  • Heat Sensitivity: Investigators found that heating the anti-sera destroyed its lytic activity, although the antibodies themselves remained present and active.

  • Restoration of Activity: The killing/lytic activity could be restored by adding normal sera that lacked specific antibodies.

  • Postulated Substances: These findings led researchers to conclude there were two substances involved in the immune response:

    • Antibodies: Specific for the bacteria but unable to kill alone without the heat-sensitive factor.

    • Complement: A heat-sensitive "mystery substance" described as the "activity of blood that completes the activity of the antibody."

• General Characteristics: The complement system is a complex network involving dozens of different proteins.

Synthesis and Biochemical Properties of Components

• Production Sites: The majority of complement components are synthesized by liver hepatocytes. Significant amounts are also produced by:

  • Monocytes and macrophages (MF).

  • Epithelial cells located in the gastrointestinal and genitourinary tracts.

• Serum Abundance: Complement proteins constitute approximately $15\%$ of the total serum protein.

• Zymogen State: Most components circulate in an inactive form known as a Zymogen.

  • Activation: Conversion to the active form occurs via proteolytic cleavage.

  • Mechanism: Cleavage removes an inhibitory fragment and exposes the active site of the protein.

  • Enzyme Cascade: The cleavage of one component initiates a sequential enzyme cascade, where one active enzyme activates the next precursor in the line.

Immunological Functions Mediated by Complement

• Lysis: Destruction of targets including cells, bacteria, and viruses via the Membrane Attack Complex (MAC).

• Opsonization: The process of "coating" a target to promote ingestion by phagocytic cells.

• Receptor Activation: Complement fragments bind to surface molecules on effector cells to trigger responses such as inflammation, generalized cell activation, and the secretion of cytokines.

• Immune Clearance: Removal of aggregated immune complexes. These are deposited in the spleen and liver for final removal.

• Regulatory Protection: Specific components protect host cells from accidental complement-mediated damage:

  • Factor I: Degrades $C3b$.

  • CD59: Inhibits the formation of the MAC.

Mechanisms of Complement Activation

• Classical Pathway (Antibody Dependent): Requires the attachment of an antibody to the target, which subsequently activates $C1$ to trigger the cascade.

• Alternative Pathway (Antibody Independent): Direct activation of $C3$. This requires accessory factors including Factor B and Factor D.

• Lectin Pathway (Carbohydrate Dependent): Triggered by specific sugar/carbohydrate molecules on the surface of pathogens.

The Classical Pathway: Initiation and Components

• C1 Complex: Initiates the pathway and consists of three components:

  • C1q: Composed of 18 polypeptide chains organized into two domains:

    • Collagen-like structural domain: Mediates the assembly of the $C1$ complex.

    • Globular head domain: Mediates interaction with antibodies.

  • C1r and C1s: Two molecules of each protein associate with the fully assembled $C1$ complex to mediate enzymatic activity.

• Activation Requirements:

  • Antibody Interaction: The globular head of $C1q$ must interact with at least two Immunoglobulin ($Ig$) molecules at the $C_{H}2$ domain.

  • Isotype Differences:

    • IgM: Pentameric structure ensures $5$ molecules are in close proximity, allowing a single antibody to activate complement.

    • IgG: Monomeric; many $IgG$ molecules must be attached to the target in close enough proximity to facilitate activation.

• C-Reactive Protein (CRP): Can also activate $C1$. It binds to Damage-Associated Molecular Patterns (DAMPs) on damaged surfaces (DNA, histones, annexins A2 and A5 on apoptotic cells) and serves as an opsonin.

The Classical Pathway: Cascade and Convertases

• C1 Activation Sequence:

  • Antibody interaction with $C1q$ causes a conformational change.

  • This change induces $C1r$ to cleave itself (autocatalytic activation).

  • Active $C1r$ then cleaves the second associated $C1r$.

  • Both active $C1r$ subunits cleave the two associated $C1s$ subunits.

• Creation of the C3 Convertase:

  • $C1s$ cleaves $C4$ into $C4a$ and $C4b$. $C4b$ associates with the target membrane.

  • $C2$ associates with the membrane-bound $C4b$.

  • $C1s$ cleaves $C2$ into $C2a$ and $C2b$.

  • $C2a$ remains with $C4b$ to form the complex $C4bC2a$, known as the C3 convertase.

• Creation of the C5 Convertase:

  • $C4bC2a$ cleaves $C3$ into $C3a$ and $C3b$.

  • $C3b$ joins the existing complex to form $C4bC2aC3b$, known as the C5 convertase.

  • The $C3b$ portion specifically binds $C5$ to alter its conformation for cleavage.

The Membrane Attack Complex (MAC) and Cell Death

• Assembly Sequence:

  • The $C5$ convertase cleaves $C5$ into $C5a$ and $C5b$.

  • $C5b$ remains on the membrane but is unstable; it is stabilized by binding $C6$.

  • $C5b6$ then binds $C7$. This induces a hydrophilic to amphipathic shift, allowing the complex to insert into the lipid bilayer.

  • $C5b67$ recruits $C8$. The $C8 \alpha \gamma$ component undergoes a similar hydrophilic-amphiphilic transition and inserts into the membrane.

  • The $C5b678$ complex recruits multiple $C9$ subunits ($10$ to $19$ molecules). These insert into the membrane to create a pore.

• Pore Size and Death Mechanisms:

  • Large Pore ($17$ to $19$ C9 molecules): Causes direct cell lysis.

  • Small Pore ($10$ or fewer C9 molecules): Does not cause immediate lysis but still results in cell death.

  • Apoptotic Necrosis: An influx of $Ca^{++}$ through smaller pores activates signaling pathways leading to nuclear fragmentation. This is distinct from true apoptosis and is termed apoptotic necrosis.

• Eukaryotic Resistance: Eukaryotic cells are difficult to lyse because they contain inhibitory factors on the membrane and can endocytose and remove MAC complexes.

The Lectin Pathway

• Mannose Binding Lectin (MBL): Constitutively produced by the liver. It is structurally homologous to $C1q$.

  • Sugar Binding: Recognizes mannose, N-acetylglucosamine, D-glucose, and L-fucose polymers on cell surfaces.

• MASP Proteins: MBL associates with MBL Associated Serine Proteases (MASP-1, -2, and -3).

  • MASP-2: The critical protease for the Lectin pathway.

  • MASP-1 and -3: Primarily important for the Alternative pathway.

• Pathway Execution: Binding of MBL to sugars activates MASP-2, which then cleaves $C4$ and $C2$. From this point, the pathway is identical to the Classical pathway.

The Alternative Pathways

• Alternative Tickover Pathway:

  • Relies on the spontaneous, autocatalytic cleavage of $C3$, creating $C3(H_{2}O)$.

  • $C3(H_{2}O)$ binds Factor B (requiring $Mg^{++}$, which exposes the cleavage site for Factor D.

  • Factor D cleaves Factor B into $Ba$ and $Bb$. The complex $C3(H_{2}O)Bb$ is the fluid-phase C3 convertase.

  • In the absence of a challenge, these molecules are unstable and the pathway stops.

  • If a challenge exists, soluble $C3b$ binds the target, associates with Factor B, and is cleaved by Factor D to form a membrane-bound $C3$ convertase ($C3bBb$).

• The Role of Properdin:

  • Factor P (Properdin): Binds to and stabilizes the unstable $C3bBb$ membrane-bound convertase.

  • Amplification: This stabilized complex cleaves more $C3$ into $C3a$ and $C3b$, creating a repeating cycle that duplicates convertases.

  • C5 Convertase: When a second $C3b$ remains associated with the stabilized complex, it forms $C3bBbC3b$, the Alternative C5 convertase.

• Alternative Properdin Activated Pathway: Properdin can bind directly to membranes of bacteria or damaged host cells, recruiting $C3b$ produced by Tickover and initiating the process with higher specificity.

• Alternative Protease Activated Pathway:

  • Thrombin: A clotting factor that can directly cleave $C3$ and $C5$, generating anaphylatoxins ($C3a$, $C5a$).

  • Platelets: During clotting, platelets activate protein kinases that phosphorylate $C3b$, making it more stable and enhancing all complement pathways.

Complement Receptors and Effector Physiology

• CR1 (CD35):

  • Expression: Erythrocytes (RBC), MF, PMN, eosinophils, follicular dendritic cells, and B cells.

  • Functions: Promotes clearance of immune complexes (via RBCs to spleen/liver), promotes phagocytosis (opsonization), and assists B cell differentiation on follicular dendritic cells.

• CR2 (CD21):

  • Expression: B cells and follicular dendritic cells.

  • Function: Component of the trimeric B cell co-receptor ($CR2/CD81/CD19$). Binds C3d (a cleavage product of $C3b$).

  • Impact: Binding of $C3d$ to $CR2$/$CD19$ amplifies the B cell activation signal via ITAMs, decreasing the antigen concentration required for activation by 100-fold.

• CR3 (CD11b/CD18) and CR4 (CD11c/CD18):

  • Binds iC3b (a product of $C3b$ degradation by Factor I).

  • Acts as an opsonin for phagocytes and a chemotactic factor promoting extravasation for non-phagocytic cells.

• CRIg: Expressed on tissue-resident macrophages; binds $C3b$-opsonized antigens.

• Anaphylatoxin Receptors (C3aR, C5aR, C5L2):

  • Expressed on MF, PMN, basophils, eosinophils, and mast cells.

  • Triggers chemotaxis, oxidative burst, and degranulation (initiating inflammation).

  • C5L2: Acts as a down-regulator for $C5a$ signaling.

• C1qRp and SIGN-R1:

  • C1qRp: Expressed on monocytes, neutrophils, endothelial cells, platelets, and T cells; binds $C1q$ and $MBL$.

  • SIGN-R1: Expressed on macrophages in the marginal zone of the spleen and lymph nodes; binds $C1q$.

Impact on Adaptive Immunity

• Antigen Presentation: $MBL$, $C1q$, $C3b$, and $C4b$ facilitate antigen uptake and processing by dendritic cells ($pAPC$).

• Cytokine Commitment: $C5a$ binding to $C5aR$ on dendritic cells can induce $IL-12$ expression (committing to a $T_{H}1$ response) or suppress it (committing to a $T_{H}2$ response) depending on the timing of binding.

• T-Cell Maturation and Selection:

  • Sialic Acid: Mature T cells increase sialic acid on their membrane, which recruits inhibitors (Factor H) and prevents antibody binding.

  • Immature T Cells: Lack sialic acid and the inhibitor CD55, making them susceptible to complement-mediated lysis, ensuring only mature cells survive thymus exit.

• T-Cell Survival and Shutdown:

  • Activated T cells secrete small amounts of $C3a$ and $C3b$.

  • $C3b$ binds CD46 on the T cell surface, which is essential for survival.

  • Later, complement proteins induce IL-10 production to trigger apoptosis and shut down the immune response.

The Contraction Phase and Immune Clearance

• Clearing Apoptotic Cells: Dying cells express DAMPs, including phosphatidylserine (normally on the cytosolic layer) and DNA/histones on the outer membrane.

  • Annexin 1 binds phosphatidylserine; $C1q$ then binds Annexin 1 or directly binds DNA.

  • $C1qRp$ on phagocytes recognizes the bound $C1q$, promoting clearance.

• Clearing Immune Complexes:

  • Antigen/Antibody complexes are coated with $C3b$.

  • $C3b$ attaches to CR1 on erythrocytes.

  • Erythrocytes transport the complexes to the liver or spleen, where they are engulfed by phagocytic cells.

Regulation of the Complement System

• Passive Regulation: Short half-lives of components (e.g., $C3bBbC3b$ convertase degrades in $5$ minutes without properdin). High sialic acid content on host cells binds proteases to degrade $C3b$.

• Specific Inhibitors:

  • C1Inh: A Serine Protease Inhibitor (SERPIN) that binds $C1r_{2}s_{2}$ and MASP2, causing them to dissociate. Inhibits Classical and Lectin pathways.

  • CD55 (Decay Activating Factor): Associates with glycosaminoglycans like heparin sulfate on host cells. It dissociates $C4bC2a$ and, with Factor H, dissociates $C3bBb$.

  • Factor I: A protease that degrades membrane-associated $C3b$ and $C4b$ to prevent reinitiation of the cascade. Requires cofactors like MCP, CR1, Factor H, and C4BP.

  • CD59 (Protectin): Binds $C5b678$ to prevent MAC insertion into host cell membranes.

  • S protein (Vitronectin): Fluid-phase protein that binds released $C5b67$ complexes to prevent accidental membrane insertion.

  • Carboxypeptidases (N, B, and R): Inactivate anaphylatoxins $C3a$ and $C5a$ by cleaving arginine from their carboxy termini.

Clinical Complement Deficiencies

• Early Classical Pathway Deficiencies ($C1q, C1r, C1s, C2, C4$): Correlate with immune complex diseases (SLE/Lupus, glomerulonephritis, vasculitis) due to poor clearance of complexes and apoptotic cells.

• MBL Deficiencies: Lead to pyogenic bacterial infections in children and upper respiratory tract infections.

• C3 Deficiencies: Severe; characterized by recurrent bacterial infections and immune complex disease.

• Factor B, D, and Properdin Deficiencies: Specifically associated with Neisseria infections.

• MAC Component Deficiencies ($C5$ through $C9$): Confer susceptibility to N. meningitidis.

• C4 Alleles: Human populations have between $2$ and $6$ copies of the $C4$ allele. Low copy number increases SLE risk.

Microbial Evasion Tactics

• Antibody Modulation: Many viruses produce Fc-binding proteins to block complement interaction or promote the endocytosis of immune complexes.

• Direct Interference:

  • Schistosoma and Trypanosoma produce a C2 receptor trispanning protein to disrupt the $C3$ convertase.

  • P. aeruginosa produces elastase to cleave $C1q$ and $C3/C3b$.

  • Streptococcal species produce ScpA/ScpB (degrade $C5a$) and pyrogenic exotoxin B (degrades properdin).

  • S. pyogenes M protein recruits Factor H or C4BP to inhibit pathways.

• Regulatory Mimicry: H. pylori recruits CD59 to its surface. Enveloped viruses incorporate high sialic acid levels to recruit Factor I cofactors.

• Proteolytic Destruction: Aspergillus fumigatus (mold) produces Alp1 protease, which degrades $C1q$, $C3$, $C4$, $C5$, and $IgG$ molecules.