Complement

• Complement system

A multi-component proteolytic cascade

• Critically important to immune defense and immune regulation

• Synthesized by liver macrophages and monocytes, hepatocytes and intestinal epithelia

• C3 is the most abundant (1 g/L of plasma) and most important complement component

Major activities

1. Cell lysis

2. Macrophage and neutrophil activation

3. Optimization and stimulation of phagocytosis

4. Activation of inflammation

5. Plays a major role in innate and adaptive immunity

Interaction with adaptive immune system

• Augments Ab response by immune complexes and allows phagocytosis

• Helps memory generation by immune complexes on follicular DCs

• Helps clear apoptotic cells

• Also can suppress immune responses

  • A fact some pathogens make use of - Clq, Cls, C2 & C4 deficiencies lead to SLE-like disease

  • C3d is a strong suppressor of immune responses (as well as an adjuvant) - elicits IL10 production when soluble

• Tumors, yeast, viruses, parasites and mycobacteria have active mechanisms to coat themselves with C3d to escape the immune system

Activation

• Is a triggered enzyme cascade that involves components of ____, where the product of one reaction is an enzyme that catalyzes the next reaction

Three Pathways

1. Classical

2. Alternative

3. Lectin

Classical pathway

Utilizes antibodies and is the last one activated

1. Is activated primarily by Ag-Ig binding

2. Amplification due to enzymatic activity of the complement components

3. Activation sequence is: C1, C4, C2, C3, C5, C6, C7, C8, C9

Activation

step 1: Activation of C1

1. C1 is actually a complex consisting of C1q, C1r & C1s

   1. Two molecules of each of C1r & C1s are bound to one molecule of C1q

   2. C1 activation occurs when C1q bonds to the CH2 domain of IgG or the CH4 domain of IgM

   3. A single C1q molecule must bind simultaneously to at least two Fc portions of Ig, each Fc portion having a single C1q binding site

   4. One IgM molecule is able to stimulate C1 since it is a pentamer with 5 Fc regions.

      1. At least 2 IgG molecules are required to activate C1

   5. Therefore, IgM is a more efficient complement binding (fixing) antibody than IgG

   6. IgA and IgE lack C1q receptors and cannot activate complement

   7. Only Ag-Ab complexes, and not free of soluble Abs, activate complement

   8. Activated C1q activates C1r which activates C1s

2. Step 2: Activation of C4

   1. Activated C1s cleanses C4 to produce C4a (the small fragment) and C4b (the large fragment)

   2. C4b associates with C2 on cell surface

3. Step 3: Activation of C2

   1. C2 is also cleaved by C1s to form C2a and C2b

   2. C2a associates with C4b to form C4bC2a (called the C3 convertase)

      1. As denoted by the line on top of the complex

4. Step 4: Activation of C3

   1. C3 is activated by C3 convertase to form C3a, C3b

   2. C3b associates with C4bC2a to form C4bC2aC3b (called the C5 convertase)

5. Steps 5-7: Activation of C5, C6, C7

   1. The membrane attack complete (MAC) consisted of activation and assembly of C5-C9 which will induce cell lysis

   2. C5 is cleaved by C5 convertase to form C5a & C5b

   3. C5b associates with C6 and C7 to form C5bC6C7. Binding to C7 exposes a hydrophobic site on C7 which inserts it into the lipid bilayer of the cell membrane

6. Steps 8-9: Activation of C8 and C9

   1. C8 binds C5bC6C7 and insets into the lipid bilayer as well

   2. C9 binds to C5bC6C7C8, polymerizes and forms pores in the cell membrane

   3. Subsequent passage of ions through the pore leads to water influx and osmotic cell lysis

Alternative pathway

Is activated in the absence of antibody

• Is activated by bacterial products (lipopolysaccharide (LPS)), yeast and fungal cell wall (e.g. zymosan), viruses, and parasites (e.g. trypanosomes)

• Depends on the presence of small amounts of C3b normally present in serum due to proteolysis of C3 by plasmid or thrombin (as well as by coagulation factors X1a, Xa, IXa)

Activation

1. C3b binds to Factor B to form C3bB

2. C3bB is cleaved by Factor D to form C3bBb (the C3 convertase) and a byproduct called Ba

3. C3bBb is stabilized by binding to Properdin (Factor P, which can be secreted by PMN, half life extended from 5 min to 30 min)

4. C3bBb generates more C3b molecules from C3 (the feedback amplification step)

5. C3b associated with C3bBb to from C3bBbC3b (the C5 convertase). The subsequent steps are similar to the classical pathway

6. This pathway and its byproducts are positive feedback amplifiers of neutrophil activation

Lectin pathway

Activated by binding of a serum protein, the mannose binding lectin (MBL), to mannose containing proteins or to carbohydrates (e.g. polysaccharide capsules) on bacteria (e.g. Haemophilus spp., Neisseria spp.), fungi (e.g. Candida) or viruses (e.g. HIV, influenza)

1. Activated the classical pathway in an antibody and C1q-independent manner (I.e., does not require antibody for C1q)

2. MBL is structurally similar to C1q. Instead of C1r and C1s, MBL associates with mannose binding lectin associated serum proteases (MASP-1 and MASP-2, 2 molecules of each) to activate C4 and C2

3. Activation by the MBL:MASP complex generates a C3 convertase (C4b2a). Progression is similar to the classical pathway

4. MBL can also activate the alternative complement pathway via direct cleavage of C3 by the MBL:MASP complexes

Mannose binding lectin

MBL stands for

Mannose binding lectin associated serum proteases

MASP stands for

• Regulation of complement

Is necessary to prevent

• formation of MAC on self tissues leading to pathogenesis

• Excessive production of inflammatory mediators, which can result in autoimmunity

Regulation of complement activity

• Natural decay of the individual components

• Inhibitors: Protease inhibitors and specific complement inhibitors

Regulation of the complement activation

• C1 inhibition

  • C11NH, a serine protease inhibitor, binds to and inactivates C1r and C1s

  • Most of the C1 in blood is bound to C11NH to prevent spontaneous complement activation

  • Binding of C1q to Ag-Ab complex releases C1 from C11NH inhibition

• Inhibition of formation of C3 convertase (C4bC2a)

  • C4 binding protein (C4Bp) binds C4b and competitively inhibits the binding of C2a

  • C4b bound to C4Bp is more susceptible to cleavage by factor I

  • Factor I catabolizes C4b to release C4c and C4d. Factor I also catabolizes C3b

  • Membrane Cofactor Protein (MCP) binds C3b and C4b promoting their catabolism by Factor I

  • Decay Accelerating Factor (DAF) binds to C4b and competitively inhibits binding of C2a or facilitates its dissociation from C2a

  • NOTE ~ Membrane MCP and DAF serve to protect host cells from the deleterious effects of complement activation

• Regulation of the alternative pathway

  • Factor H binds to C3b and prevents binding of Factor B and therefor renders it (C3b) susceptible to cleavage by Factor I.

    • Also promotes dissociation of B from C3b

  • DAF and Complement Receptor type 1 (CR1) binds C4b to C3b and inhibit its binding to Factor

• Regulation of membrane attack complex

  • Homologous restriction factor (HRF, also known as C8 binding protein) binds to C8 and prevents binding of C9. Expressed on leukocytes, epithelial cells, RBCs, and platelets

  • Membrane inhibitor of reactive lysis (MIRL, also referred to as CD59 or Protectin) competitively blocks binding of C7 and C8 to C5bC6

  • S protein (vitronectin) is a plasma membrane protein that binds to C5b, 6, 7 and prevents insertion into lipid membrane

Complement receptors

CR1

CR2

CR3

CR4

CR1

Functions

1. Inhibits C3 convertase activity by inhibiting binding of Factor B to C3b or of C4b to C2a

2. Facilitates immune adherence for phagocytosis of C3b or C4b coated microorganisms

3. Clearance of immune complexes consisting of high affinity antibodies bound to soluble bacterial proteins (e.g. toxins)

CR2

Functions

1. B cell activation, EBV mediated activation of B cells

CR3

Function

Phagocytosis of C3bi-coated microorganisms, attachment of monocytes and neutrophils to endothelium

CR4

Function

1. Phagocytosis of complement coated particles

Complement deficiency

Consequence of ____ due to:

• Deficiency in individual complement components

• Deficiency in soluble and membrane bound complement regulatory proteins

Membrane inhibitor of reactive lysis

MIRL stands for

Membrane attack complex

MAC stands for

Membrane Cofactor Protein

MCP stands for

Decay Accelerating Factor

DAF stands for

Decay Accelerating Factor

DAF stands for

Membrane inhibitor of reactive lysis

MIRL stands for

• also referred to as CD59 or Protectin