Complement

Pro-Inflammatory Components of the Complement System

*C3a, C4a, C5a* have specific receptors on phagocytes, endothelial cells & mast cells Induce LOCALIZED INFLAMMATION: *Contraction* of smooth muscle *Increase* of vascular permeability (→ exudation of plasma) *Degranulation* of mast cells & basophils → release of vasoactive amines (e.g. histamine) & leukotrienes When present at *high levels*, they can induce general circulatory collapse (*anaphylactic shock*)

3 Major Outcomes of Complement Activation

1. *Stimulation of inflammatory reactions* via release proinflammatory mediators, which induce chemotaxis of leukocytes (leading to microbe destruction) & inflammation 2. *Opsonization* of pathogens by depositing fragments of complement proteins on the pathogens, making them more susceptible to *phagocytosis* 3. *Complement-Mediated Cytolysis*, via formation of a Membrane Attack Complex, which can insert itself into bacterial membrane & cause osmotic lysis

C3b on an Activating Surface (process)

1. Amplified C3b deposition occurs on an *activating surface* within seconds (many thousands of C3b) 2. These are slowly converted to iC3b and then C3d by fluid phase Factor I + cofactor 3. This leads to phagocytosis, destruction & immune response.

Alternative Pathway Steps

1. C3 is hydrolyzed to iC3/C3(H₂O) 2. C3 is bound by Factor B 3. Factor D cleaves Factor B into Ba and Bb 4. Ba goes into solution; Bb remains bound to iC3/C3(H₂O), creating the SOLUBLE C3 CONVERTASE OF THE ALTERNATIVE PATHWAY → *Still in Solution* 5. Bb cleaves iC3/C3(H₂O) into C3a and C3b 6. C3a goes into solution, C3b binds to the surface of the cell it is near → *Does not have to be antigen; unspecific* 7. C3b is similar in structure to iC3/C3(H₂O), so Factor B binds 8. Factor D cleaves Factor B into Ba and Bb 9. Bb remains bound to C3b, creating the MEMBRANE-BOUND C3 CONVERTASE OF THE ALTERNATIVE PATHWAY 10. PROPERIDIN binds to the C3 Converstase, stabilizing it so that it may cleave many more C3's 11a. C3b can Opsonize the cell surface 11b. C3b can also bind to the C3 Convertase multiple times, creating the C5 CONVERTASE OF THE ALTERNATIVE PATHWAY

MAC Formation

1. C5 is captured by both types of C5 Convertases and cleaved into C5a and C5b 2. C5b binds C6 & C7, exposing a hydrophobic site on C7 (allows it to bind cell membrane) 3. C8 Can now bind to the complex, which exposes a hydrophobic site and allows it to further insert itself into the membrane 4. C9 binds to C5b to C8 complex, and polymerizes, forming a membrane pore. *This pathway is the same for all three pathways* *All reactions after C5b production are non enzymatic.*

Opsonization

1. C5a increases expression of CR1 on Macrophages 2. CR1 recognizes C3b deposited on Antigen Surfacce 2. Binding induces Endocytosis and Phagocytosis

Classical Pathway (Steps)

1. IgM or IgG Binds to Antigen 2. C1 binds Antibody 3. C1q is activated (through globular heads) → 4. C1r is activated → 5. C1s is activated 6. C1s cleaves C4 into C4a and C4b 7. C4b deposition on surface 8. C1s cleaves C2 into C2a and C2b 9. C2b binds to C4b creating the C3 CONVERTASE OF THE CLASSICAL PATHWAY 10. C3 Convertase cleaves C3 into C3a and C3b 11a. C3b can Opsonize the surface (bind multiple times), marking the antigen for destruction 11b. C3b also binds to the C3 Convertase creating the C5 CONVERTASE OF THE CLASSICAL PATHWAY -Rest of Pathway Covered Later-

Lectin Pathway

1. MBL binds to danger sequences 2. MASP-2 cleaves C4 into C4a and C4b 3. C4b deposition on surface 4. MASP-2 cleaves C2 into C2a and C2b 5. C2b binds to C4b creating the C3 CONVERTASE OF THE LECTIN PATHWAY 6. C3 Convertase cleaves C3 into C3a and C3b 7a. C3b can Opsonize the surface (bind multiple times), marking the antigen for destruction 7b. C3b also binds to the C3 Convertase creating the C5 CONVERTASE OF THE LECTIN PATHWAY

Major Immunological Events at Completion of Complement Cascade (4)

1. Membrane Attack Complex (MAC): lysis of microbes 2. Opsonization of Microbes: Phagocytic uptake 3. Solubilization & removal of immune complexes 4. Proteolytic events produce inflammatory mediators

C1r & C1s

2 Serine Proteases

Properdin

A positive regulatory protein that stabilizes the AP C3 & C5 Convertases, increasing the half-life (>10x) of the enzyme, allowing efficient amplification of C3b deposition → opsonization. This makes the AP a very potent pathway.

Decay Acceleration

A process that accelerates the decay of the enzymatic complexes by removing the member of the Convertase that has Serine Protease Activity. It occurs in all three pathways: CLASSICAL & LECTIN - C2b is removed as it has the Serine Protease activity ALTERNATIVE - Bb is removed as it has the Serine Protease activity.

Lectin Pathway: Recognition Step

Antibody-Independent recognition of carbohydrates on pathogens

C1 Inhibitor

Binds to activated C1r, C1s, removing them from C1q, and binds to activated MASP-2, removing it from MBL.

How Does C1 Recognize IgG or IgM

C1 binds to Fc portions of Antibodies

Waste Disposal

C1q, C3b, C4b or other fragments bind to damaged/dying cells, cellular fragments, sub-cellular membranes, mitochondria, microsomes C5a induces chemotactic response of inflammatory cells & phagocytosis to removed tagged particles & cells

Serine Protease of the C3 Convertase

C2b

REVIEW: C3 and C5 Convertase of AP

C3 CONVERTASE: C3b-Bb C5 CONVERTASE: C3bn-Bb *C3bn = C3b2 or more; multiple C3bs can bind* *Bb contains the Serine Protease activity of these Convertases*

Chemotactic Factors of the Complement System

C5a and C3a; although C5a is more potent Attract Neutrophils, Monocytes, Macrophages & other inflammatory cells → migration of cells to sites of complement activation C4a has no Chemotactic acvitivty

C3a & C5a In Tissues

C5a can induce inflammation in tissues; both are rapidly broken down into C5a des-Arg by cleavage of the N-terminal arginine

CD59 (Protectin)

CD59 is on human cells/tissues. Once complex has formed, CD59 prevents final assembly by blocking polymerization of C9, regulating formation of the MAC

Factor I

CLASSICAL & LECTIN PATHWAYS: Functions to Cleave C4b into iC4b ALTERNATIVE PATHWAY: Function to cleave C3b into iC3b (*inactive* C3b) + C3f (no known function) Follows the action of Factor H/Soluble C4 Binding Protein; but also aids in Opsonization

REVIEW: Regulation of the Three Pathways

CLASSICAL: Membrane Bound Regulators (DAF, MCP, CR1) + Soluble C4 Binding Proteins + C1 Inhibitor + Factor I LECTIN: Membrane Bound Regulators (DAF, MCP, CR1) + Soluble C4 Binding Proteins + C1 Inhibitor + Factor I ALTERNATIVE: Membrane Bound Regulators (DAF, MCP, CR1) + Factor H + Factor I

C-Reactive Protein (CRP) and Classical Pathway

CRP is an acute phase protein and a pentraxin. It recognizes phosphocholine on bacteria and on disrupted cell membranes (apoptotic cells), binds, allowing for binding by C1. *Antibody-Independent Activation* of Complement System

Neisserial Infections Deficiency of Terminal Complement Components

Can be cause by a deficiency of Terminal Complements Components (C5, C6, C7, C8, C9)

Systemic Lupus Erythamatosis

Can be caused by a C1q deficiency, that also leaves patients susceptible to bacterial infection.

iC3b

Can no longer bind Factor B, and can no longer form a convertase. On Non-Activating surfaces, Insignificant amounts of iC3b will remain. On Activating surfaces, iC3b will mark an antigen for Phagocytosis.

Soluble C4 Binding Protein

Carries out Decay Acceleration processes for Classical and Lectin Pathways in the same way as Factor H does for the Alternative Pathway

Activation of a Zymogen

Causes a precursor zymogen to be cleaved & becomes an enzymatically active *serine protease*. This enzyme then cleaves & activates the next zymogen. This begins a *triggered enzyme cascade* where each successive cleavage causes amplification.

Completement System Pathways

Classical Pathway Lectin Pathway Alternative Pathway

Housekeeping Functions of Complement

Clearance of immune complexes Recognition of apoptotic cells Clearance of cellular debris

C1q Composition

Collagenous Tails & Globular Heads

Acute-Phase Response

Complement production increases 3-50x

Structure of C1

Composed of C1q, which is associated with 2 copies of each C1r and C1s. *Do not need to memorize structure description on pic.*

DAF & CR1

DAF = Decay Acceleration Factor CR1 = Complement Receptor 1 Bind to C3b and displace serine protease Bb (or C2b in Classical Pathway), which causes Decay Acceleration.

Erythrocyte Soluble IC Clearance

Erythrocyte CR1 helps facilitate this process Erythrocytes bind C3b on ICs via CR1 on their surface and carries it to the liver or spleen, where: Resident macrophages bind ICs via CR1/CR3/CR4 or Fc Receptors and phagocytose for removal.

Cleavage of C3

Exposes a highly reactive thioester bond in C3b, which allows it to bind covalently to ANY surface that has amino (protein) or hydroxyl (carb) groups, IF it is close enough to the surface If not close enough, thioester bond is spontaneously hydrolyzed and C3b is inactivated very quickly ~90% of C3b or C4b inactivated -C4 is structurally homologous to C3 & has identical thioester group

Complement Receptor 3 (CR3) & Complement Receptor 4 (CR4)

Expressed on mononuclear phagocytes, neutrophils & NK cells. Recognize iC3b

MBL Structure

Has 2 proteins associated with it, MASP-1 & MASP-2

Regulation of Immune Complex (IC) Formation

ICs need to be removed to prevent tissue damage, inflammation, etc. -If ICs are not cleared, can precipitate at basement membrane of small blood vessels (very bad) Complement activation prevents the formation of very large, insoluble ICs AND opsonizes ICs for elimination (phagocytosis of ICs)

Anaphylatoxins and Potency

If Pro-Inflammatory cleavage products are present at high levels, they can induce ANALPHYLACTIC SHOCK POTENCY: C5a > C3a >> C4a

What keeps C1 from binding to antibodies (IgG & IgM) in our blood?

IgG and IgM are circulating in a way that the Fc portions are not accessible to C1q. *IgM*: A pentamer that circulates in planar form (Fc regions inwards) -When IgM recognizes antigen, it changes conformation, & the Fc sites are exposed & C1q can bind. *IgG*: Fc regions must be at a critical difference from each other in order for C1q to bind; they are only in this arrangement after they have bound antigen.

Ability of Ig Isotypes to Activate Complement

IgM > IgG3 > IgG1 > IgG2 IgG4, IgD and IgE DO NOT activate complement 1 molecule of IgM can initiate complement activation

In what form are complement proteins secreted?

Inactive stable *zymogens* (enzyme precursors). These circulate widely through body fluids & tissues.

MBL Deficiency

Increased susceptibility to bacterial infections (especially infants & immunocompromised) Many people have a partial deficiency

ComplementsFunctions Related to Adaptive Immune Response

Influence T cell lineage commitment Influence regulatory T cell development B cell activation B cell memory enhancement

Cell Wall/Capsule of Bacteria

It's presence or absence can make it harder or easier for the MAC to kill a bacteria.

Failure of Housekeeping due to deficiencies of early CP components

Leads to *autoimmune disease* due to increase of dangerous auto antigens and *immune complex disease* (e.g. SLE)

Major Site of Production of Complement Proteins

Liver; 90% produced here

MCP & CR1

MCP = Membrane Cofactor Protein Help Factor I cleave C3b to form inactive iC3b, (or C4b in Classical & Lectin Pathways) which can no longer bind Factor B; *however iC3b still functions as a critical marker for phagocytosis to take place*\

The Complement System: Overview

Major component of Innate Immune System Consists of over 30 proteins, which participate in a tightly regulated *cascade-like activation process*. Protects host from pathogenic microorganims, contributes to immune complex regulation & is an important link between the innate & adaptive immune systems.

MASP-1 & 2

Mannin-binding Associated Serine Protease 1 & 2 Associated with MBL

Polyanions

Molecules that are commonly found on human cells & tissues such as sialic acid & GAGs

How Does the Alternative Pathway Know When to Activate?

NON-ACTIVATING SURFACE (e.g. host): There is *Efficient Regulation* and C3b will be immediately inactivated → No activation or amplification & No complement-mediated damage. ACTIVATING SURFACE: There is *Lack of Regulation* → Complement-mediated opsonization (C3b → iC3b → C3d) and destruction

Evasion Strategies of Pathogens

Non-activating capture of complement initiators (e.g. Igs) Inactivation or depletion of complement components by secreted proteases Recruitment of complement regulators to the pathogen surface or secretion of regulator mimics Molecular inhibition of convertase activity Interference with MAC formation Competitive & antagonistic prevention of immune signaling Enter cells by binding cell-bound complement receptors & regulators via pathogen-expressed surface proteins or via C3b fragments deposited on their surface

C9

Only protein that completely penetrates the cell membrane

CR2

Part of B cell co-receptor complex, which is essential for B cell activation Binds C3d Expressed on B lymphocytes & follicular dendritic cells

Membrane-Bound Regulatory Proteins

Proteins of Host Cells that function in ALL THREE PATHWAYS either carrying out Decay Acceleration or acting as a Cofactor for I: DECAY ACCELERATION: Alternative - Push off Bb Classical & Lectin - Push off C2b FACTOR I COFACTOR: Alternative, Classical & Lectin - Formation of iC3b and iC4b

Alternative Pathway (AP) Activation and Recognition Molecule

RECOGNITION MOLECULE: *None* ACTIVATION: 1. Spontaneous cleavage of C3 (tickover) constantly occurs at a slow rate, creating a constant pool of active C3 called iC3 of C3(H₂O) → called this because it hydrolyzes

Classical Pathway Activation and Recognition Molecule

RECOGNITION MOLECULE: C1 2 Activation Ways: ANTIBODY DEPENDENT: C1 recognizes antigen-bound IgM or IgG ANTIBODY INDEPENDENT: C1 can recognize antigen cell wall components, fragments, proteins, phospholipids or C-Reactive Protein and Serum Amyloid P Protein

Lectin Pathway Activation and Recognition Molecule

RECOGNITION MOLECULE: Mannose-Binding Lecting (MBL) and Ficolins ACTIVATION: Antibody-Independent recognition of "danger" (sugar pattern residues) on pathogens

Factor H

Regulator of the ALTERNATIVE PATHWAY It binds to surfaces where C3b has been deposited and that also contain POLYANIONS. It functions to induce a process called DECAY ACCELERATION.

Human C3 Deficiency

Results in impairments in dendritic cell differentiation, memory B cells, and regulatory T cells. These patients are severely immunocompromised. Demonstrates the major role of complement in bridging innate & adaptive immunity.

REVIEW: C3 and C5 Convertases of the Classical and Lectin Pathways

Same C3 Convertase (C4b,2b) Same C5 Convertase (C4b,2b,3b) *C2b has the Serine Protease activity for both*

Notes About Cleavage Products

Will only bind cell membrane if close enough; otherwise they go into solution. If they do bind, it is covalently to any exposed hydroxyl or amino groups (carbohydrate or protein). The a is typically smaller and will go into solution without an effect. Some, like C3a, however, are Chemotactics and trigger Pro-inflammatory response

iC3b vs. C3d

iC3b, when present in large enough quantities, will trigger Phagocytosis C3d will trigger B cell activation