HUF1-14 Complement

Overview of the Complement System

• The complement system is a key multiple-protein system essential for both innate and adaptive immunity.

• It consists of nine major complement proteins, whose nomenclature was unified in $1968$ by the World Health Organization (WHO) as $C1$, $C4$, $C2$, $C3$, $C5$, $C6$, $C7$, $C8$, and $C9$. This numbering reflects the order of activation within the Classical pathway.

• The historical discovery of these components followed a timeline: the Classical pathway (Adaptive immunity) was established first, followed by the discovery of the Alternative pathway in the $1950$s, and finally the Lectin pathway and regulatory systems in the $1970$s (Innate immunity).

• The system functions through a sequential assembly of activating proteins on the surface of pathogens. This sequence intensifies the activation and generates products harmful to the pathogen.

• The system is self-regulating, containing both activating proteins and inhibitory (regulatory) proteins. Inhibitory proteins prevent the rapid exhaustion of activating proteins and protect host tissues from accidental attack.

Major Immune Actions of Complement Activation

• Activation of the complement system results in three primary immune actions against pathogens:

  • $1$. Initiation of Inflammatory Responses: Complement activation products (e.g., $C3a$, $C5a$) bind to specific receptors ($C3aR$, $C5aR$) on mast cells at the site of infection. This triggers degranulation and the release of vasoactive amines like histamine, which increases blood vessel permeability. This leads to plasma exudation and the massive infiltration of neutrophils (highly phagocytic cells) into the infected tissue, causing localized swelling.

  • $2$. Opsonization: Complement activation products, specifically $C3b$, bind covalently to the surface of the pathogen. These products also bind to complement receptor $1$ ($CR1$) on phagocytic cells (macrophages and neutrophils). This coating process, known as opsonization, makes the pathogen much easier for phagocytic cells to ingest and kill.

  • $3$. Lysis of Pathogens: The system forms a pore-forming structure known as the Membrane Attack Complex ($MAC$), consisting of components $C5b$, $C6$, $C7$, $C8$, and $C9$. This complex creates holes in the pathogen's membrane, causing leakage of intracellular materials and subsequent lysis. This is particularly effective against Gram-negative bacteria, which have relatively thinner and less rigid cell walls.

The Three Pathways of Complement Activation

• The activation process involves four distinct stages: Initiation, Driving Stage, Central Functional Stage, and Late Functional Stage.

• Classical Pathway:

  • Initiation: Dependent on antibodies (adaptive immunity). It starts with the $C1$ complex ($C1q$, $C1r$, and $C1s$) binding to the $Fc$ regions of antigen-bound antibodies ($IgM$ or $IgG$).

  • Driving Stage: Activated $C1s$ cleaves $C4$ and then $C2$ to form the $C4b2a$ complex ($C3$ convertase).

• Lectin Pathway:

  • Initiation: Triggered by the binding of Mannose-Binding Lectin ($MBL$) or Ficolins to microbial carbohydrates (e.g., mannose, fucose, $GlcNAc$, $GalNAc$).

  • Driving Stage: These complexes include $MASP-1$ and $MASP-2$ (homologous to $C1r$ and $C1s$), which cleave $C4$ and $C2$ to form the $C3$ convertase ($C4b2a$).

• Alternative Pathway:

  • Initiation: Occurs spontaneously via the low-level hydrolysis of $C3$ in plasma, producing a small amount of $C3b$. This $C3b$ binds to the pathogen surface.

  • Driving Stage: $C3b$ captures Factor $B$, which is then cleaved by Factor $D$ to form the unstable $C3bBb$ ($C3$ convertase). Properdin ($Factor P$) stabilizes this complex. This pathway acts as an amplification loop, generating more $C3$ convertase to activate more $C3$.

• Central Functional Stage (Common to all):

  • $C3$ convertase cleaves $C3$ into $C3a$ (inflammation) and $C3b$ (opsonization).

  • $C3b$ joins the $C3$ convertase to form $C5$ convertase ($C4b2a3b$ for Classical/Lectin; $C3b2Bb$ for Alternative).

• Late Functional Stage (Common to all):

  • $C5$ convertase cleaves $C5$ into $C5a$ (inflammation) and $C5b$.

  • $C5b$ recruits $C6$, $C7$, $C8$, and multiple $C9$ molecules to assemble the Membrane Attack Complex ($MAC$).

Detailed Operation of the Classical Pathway

• The $C1$ complex consists of one $C1q$ molecule (the sensor) and two molecules each of $C1r$ and $C1s$.

• $C1q$ has $6$ globular heads that bind to the $Fc$ regions of antibodies. Stable binding requires at least $2$ globular heads to be engaged simultaneously to induce a conformational change and activate $C1r$ and $C1s$.

• Relationship with Antibodies:

  • $IgG$: Exist as monomers. One $Fc$ region binds only weakly to one $C1q$ head. For activation, $IgG$ antibodies must be closely spaced on a pathogen surface so that $C1q$ can bind two or more heads simultaneously.

  • $IgM$: Exist as pentamers in a planar conformation in free serum, where $Fc$ regions are hidden. Upon binding to an antigen, $IgM$ adopts a "staple" conformation, exposing its $5$ $Fc$ regions. This makes antigen-bound $IgM$ very efficient at activating the Classical pathway.

  • Free antibodies do not activate the complement system because $C1q$ cannot bind them stably.

Detailed Operation of the Lectin and Alternative Pathways

• Lectin Pathway Recognition:

  • $MBL$ binds sugar residues such as mannose, fucose, and $N\text{-acetylglucosamine (GlcNAc)}$.

  • Ficolins bind specifically to acetylated sugars, including $GlcNAc$ and $N\text{-acetylgalactosamine (GalNAc)}$.

  • These residues are densely present on the surfaces of Gram-positive and Gram-negative bacteria, mycobacteria, fungi, viruses, and parasites.

• Alternative Pathway Mechanism:

  • It operates as a self-amplification cycle. Spontaneously generated $C3b$ binds Factor $B$, which is cleaved by Factor $D$ into $Ba$ (released) and $Bb$ (remains). High levels of $C3b$ then bind more Factor $B$.

  • Nomenclature Note: While the transcript uses $C4b2a$ for $C3$ convertase, some modern texts use $C4b2b$. In this note, the traditional $C4b2a$ is maintained where "a" was traditionally the larger fragment, though "b" is increasingly used to denote the binding portion.

Immune Complex Removal

• Soluble immune complexes ($IC$) are formed when antibodies bind to antigens. If these complexes deposit on tissue surfaces, they can activate complement and cause tissue damage (e.g., vasculitis, glomerulonephritis, or Systemic Lupus Erythematosus - $SLE$).

• The Classical pathway prevents this by coating $IC$s with $C3b$.

• Erythrocytes (red blood cells) express $CR1$, which binds to the $C3b$ on the immune complexes.

• The erythrocytes transport the $IC$s to the liver and spleen. Resident macrophages in these organs, which also possess $CR1$, strip the $IC$s from the erythrocytes and phagocytose them, returning the intact red blood cells to circulation.

Clinical Deficiencies and Diseases

• $C1$, $C4$, or $C2$ Deficiency: Causes severe impairment in immune complex clearance, leading to $IC$ diseases (vasculitis, $SLE$). Susceptibility to infection is partially covered by the Alternative pathway.

• $C3$ Deficiency: The most severe deficiency. It abolishes all three pathways, leading to extreme susceptibility to bacterial infections and immune complex diseases.

• $C5$ to $C9$ Deficiency: Prevents the formation of the $MAC$. Patients are specifically susceptible to Neisseria species infections. Other complement functions (inflammation/opsonization via $C3a/C3b$) remain intact.

• Lectin Pathway Deficiency ($MBL$, Ficolin, or $MASP$s): Increases susceptibility to pyogenic infections and sepsis in neonates (as maternal antibodies decline) and immunosuppressed adults.

Diagnostic Assays: $CH_{50}$ and $AH_{50}$

• $CH_{50}$ (Classical Pathway Hemolytic Assay): Measures the activity of $C1$ through $C9$. It uses sheep red blood cells ($SRBC$) coated with anti-sheep antibodies. Serial dilutions of patient serum are added. The value is the dilution fold required to achieve $504\%$ hemolysis (e.g., a dilution of $256$ yields a $CH_{50}$ of $256$).

• $AH_{50}$ (Alternative Pathway Hemolytic Assay): Measures individual alternative pathway proteins (Factor $B$, $D$, Properdin) and common components ($C3$, $C5$-$C9$). It uses $SRBC$s without antibodies, relying on spontaneous activation.

• Interpretation:

  • Deficiency in $C1$, $C4$, or $C2$: Affects only $CH_{50}$.

  • Deficiency in Factor $B$, $D$, or Properdin: Affects only $AH_{50}$.

  • Deficiency in $C3$ or $C5$-$C9$: Affects both $CH_{50}$ and $AH_{50}$.

Regulation of Complement Activation

• Regulation occurs via three main mechanisms:

  • $1$. Prevention of $C3$ Convertase Formation: Plasma proteins ($C4BP$: $C4$-binding protein, Factor $H$) and cell surface proteins ($CR1$, $MCP$: membrane cofactor protein) bind to $C4b$ and $C3b$. They act as co-factors for Factor $I$, which cleaves $C4b$ and $C3b$, rendering them inactive.

  • $2$. Disruption of Formed $C3$ Convertase: $C4BP$, Factor $H$, $CR1$, and $DAF$ (decay-accelerating factor) bind to the convertase complexes, displacing $C2a$ or $Bb$ and reverting the complex to an inactive state.

  • $3$. Blocking $MAC$ Assembly: The cell surface protein $CD59$ binds to the $C5b678$ complex, preventing the final polymerization of $C9$ and protecting host cells from lysis.

• Specific Regulatory Deficiencies:

  • Paroxysmal Nocturnal Hemoglobinuria ($PNH$): Caused by a mutation in the synthesis of glycosyl phosphatidylinositol ($GPI$), which anchors $DAF$ and $CD59$ to cell surfaces. Without these, erythrocytes are highly susceptible to lysis as only a single $MAC$ is needed to lyse a red blood cell.

  • Factor $I$ Deficiency: Leads to uninhibited $C3$ convertase formation, causing "complement exhaustion." The rate of protein consumption exceeds supply, resulting in low $C3$ levels and repeated pyogenic bacterial infections.