The Complement System

Immunology 10: The Complement System

Rowan University
SHREIBER SCHOOL OF VETERINARY MEDICINE
Dr. Rao Dukkipati
Associate Professor
dukkipativ@rowan.edu

Study Resources

  • Lecture PowerPoints

  • Tizard (2025) Veterinary Immunology, Eleventh Edition: Chapter 5

  • Catchpole and HogenEsch (2023) Day’s Veterinary Immunology, Third Edition: Chapter 3

  • Punt et al. (2019) Kuby Immunology, Eighth Edition: Chapter 5 (Electronic copy available via Rowan Library: https://ebookcentral.proquest.com/lib/rowan/detail.action?docID=31360771)

Learning Objectives

  • Describe the nature of complement and the different pathways (classical, lectin, and alternative) leading to activation of the complement cascade.

  • Describe the consequences of complement activation.

  • Provide examples of inherited complement deficiencies in animals.

Components of Innate (Non-specific) Immunity

  1. Physical Barriers

    • Examples: skin, mucous membranes, microbiota, cough, sneeze, vomit, diarrhea

  2. Physiological Barriers - Humoral

    • Examples: temperature, pH, enzymes, antimicrobial proteins, complement factors

  3. Phagocytic Barriers - Cellular

    • Key cell types: Neutrophil, macrophage, dendritic cell

  4. Inflammatory Barriers - Humoral

    • Examples: Pro-inflammatory cytokines, chemokines, acute phase proteins

Complement System

  • Defined as a collective term for a group of proteins that play a key role in host immune responses.

  • Activated by both innate and acquired immune mechanisms.

  • Composed of 25-30 proteins, primarily found in plasma: globulin fraction.

  • Some complement proteins are located on cell membranes.

  • Predominantly produced in the liver, macrophages, and fibroblasts.

  • Comprises about 5% of total serum proteins.

Discovery of the Complement System

  • Discovered in the 1890s by Jules Bordet at the Institut Pasteur, Paris.

    • Experiment:

      • Combination of antiserum and Vibrio cholerae resulted in lysis of bacteria.

      • Heated antiserum with Vibrio cholerae led to survival of the bacteria.

      • Fresh serum without antibodies also caused lysis of bacteria.

    • Conclusion: Bacteriolytic activity required two substances: heat-stable specific antibodies and heat-sensitive components.

  • Paul Ehrlich conducted similar experiments and coined the term complement, defining it as “the activity of blood serum that completes the action of antibody.”

Naming of Complement Proteins

  • Designated with a capital C, followed by a number (e.g., C1, C2, C3).

  • C3 is the most abundant complement factor in serum.

  • Some proteins in the alternative pathway are designated with B, P, and D.

  • The number after C denotes the order of discovery, not their appearance in the pathway (e.g., C4 reacts before C3).

  • Activated proteins are denoted with a bar above the symbol (e.g., C3).

  • Peptide fragments formed during the complement cascade indicate suffixes ‘a’ and ‘b’ (e.g., C3a, C3b).

    • The ‘b’ fragments are larger and continue to participate in the cascade reaction.

    • The ‘a’ fragments are released to induce inflammatory responses.

    • ‘a’ stands for anaphylatoxin, which is a substance produced by complement activation that increases vascular permeability through the release of pharmacologically active mediators from mast cells.

Functions of Complement Proteins

  • Complement proteins circulate in serum in an inactive form.

  • Upon activation, outcomes include:

    • Target cell membrane lysis

    • Chemotaxis

    • Opsonization to enhance phagocytosis

    • Inflammation

Functional Categories of Complement Proteins

  • Convertase Activators:

    • C1r, C1s, C4b, C2a and enzymatic mediators (C3 convertase, C5 convertase)

  • Phagocyte Initiators:

    • C1q, MBL, ficolins

  • Opsonins:

    • C3b

  • Anaphylatoxins:

    • C5a

  • Membrane Attack Complex (MAC)

  • Complement Receptors:

    • CR1, CR3

  • Regulators:

    • Factor I, which degrades complement components and prevents their deposition.

Known Complement Activation Pathways

  1. Classical Pathway

    • Part of the acquired immune system, initiated by antibody binding to antigen (Ab-Ag complex).

    • Trigger: Binding of Ab to Ag, activating C1, which cleaves and activates C4 and C2.

    • This forms C3 convertase (C4bC2b) that splits C3 into C3a (anaphylatoxin) and C3b.

    • C3b combines with C3 convertase to form C5 convertase (C4bC2bC3b) that cleaves C5 into C5a and C5b.

  2. Lectin Pathway

    • Part of the innate immune system, initiated by mannose-binding lectin (MBL) binding to microbial mannose residues.

    • MBL forms a complex with MBL-associated serine proteases (MASP1, MASP2), leading to activation of MASPs.

    • Activated MASP2 acts on C4 and C2 to form C3 convertase (C4bC2b), generating C3a and C3b.

    • C3b then combines with C4bC2b to form C5 convertase.

  3. Alternative Pathway

    • The oldest pathway in evolution, initiated by bacterial/fungal cell wall components (e.g., lipopolysaccharides, teichoic acids).

    • Requires binding of factor B to C3b and cleavage facilitated by factor D, forming C3 convertase (C3bBb).

    • C3 convertase breaks down C3 into C3a and C3b, leading to formation of C5 convertase (C3bBbC3b).

    • Factor H acts as a regulator protein, accelerating decay of C3 convertase.

Terminal Complement Pathway

  • Initiated with either alternative or classical/lectin C5 convertases, cleaving C5 into C5a and C5b.

  • C5b binds to C6 and C7 to form the C5bC6C7 complex, which then binds to the target cell membrane.

  • This complex attaches to C8 and multiple C9 molecules, forming a ‘doughnut-like’ structure known as the membrane attack complex (MAC).

  • MAC punches holes into the invader's membrane, leading to osmotic lysis.

  • The smaller C5a acts as an anaphylatoxin, attracting immune cells (e.g., neutrophils, macrophages).

Biological Consequences of Complement Activation

  • C3a and C5a mediate vasodilation, contributing to tissue edema and the extravasation of leukocytes and plasma proteins into the tissue.

  • These fragments cause mast cell degranulation, further activating recruited leukocytes, thus contributing to inflammation.

  • Complement activation results in cytolysis via MAC, opsonization through C3b, and inflammation via C3a and C5a.

Complement Factor Functions in Phagocytosis

  • C3b enhances opsonization during phagocytosis.

  • Interaction with phagocytic cells enhances binding:

    1. Attachment by nonspecific receptors

    2. Binding via antibodies

    3. Binding of C3b to complement receptors on phagocytes, enhancing phagocytosis

Regulation of Antibody Responses by Complement

  • Complement regulates antibody production through C3d binding to antigens.

  • Antigen binding to B cell receptors (BCR) leads to CD21/CD19 complexes on B cells.

  • Activation of the CD21/CD19 complex stimulates maturation of B cells.

  • Low levels of C3 correlate with decreased primary antibody responses.

  • Coating antigens with C3d enhances binding to dendritic cells.

Complement Genes

  • Genes for complement proteins are located throughout the genome.

  • Two main clusters identified:

    1. MHC class III region (includes genes for C4, C2 and factor B).

    2. Regulation of complement activation (RCA) cluster (includes genes for C4BP, CD55, CD35, CD21, CD46, and factor H).

Examples of Complement Deficiencies

  • C3 Deficiency in Brittany Spaniel Dogs:

    • Autosomal recessive mutation causing lack of C3 protein, leading to reduced antibody production.

    • Clinical signs: recurrent sepsis, pneumonia, pyometra.

    • Heterozygous individuals may exhibit half the normal levels but remain clinically normal.

  • H Factor Deficiency in Yorkshire Pigs:

    • Recessive autosomal mutation, absence of H factor, leads to uncontrolled C3b production.

    • Affected animals appear normal at birth but experience tissue lesions, particularly in kidneys, leading to eventual death.

Complement Fixation Test (CFT)

  • A serological assay used to detect serum antibody levels to a specific antigen.

  • Step 1: Dilutions of serum from the individual (heated to 56ºC to inactivate complement) are mixed with appropriate antigen and known complement (often guinea pig serum).

    • If antibodies are present, they bind to the antigen and fix the complement.

  • Step 2: Measure remaining guinea complement by adding sheep red cells coated with antibodies.

    • If complement was consumed in Step 1, intact sheep red cells will remain cloudy.

    • Positive reaction indicates absence of lysis (complement used up).

    • If complement is unbound, it will lyse the red cells, indicating a negative result for antibody presence.

Summary of CFT Results

  • Positive Reaction: Serum is positive for antibodies → no free complement is available to lyse sheep RBC → no lysis occurs.

  • Negative Reaction: Serum indicates no antibodies → free complement is available → sheep RBC are lysed.