Complement

Exam 2

Complement is part of which immune response?

Innate

Most complement proteins are synthesizes (made) where? What are the 2 that aren’t made here and where are they made instead?

What are these proteins called before they are converted to active enzymes?

• Most: Liver

  • C1 components: Intestinal epithelial cells

  • Factor D: adipose

• Inactive: zymogens

What can WBCs act as in complement?

C1, C2, C3, & C4

What is the main thing complement activation causes in the innate response?

OIL

• Opsonization (make tasty)

• Inflammation (recruit WBCs)

• Lysis (kill pathogen)

What are the three pathways of complement? What is each dependent on that causes activation?

• Classical pathway: Ag-Ab interaction

• Alternative pathway: bacterial & fungal cell walls, yeast, virus, tumor cells, & some parasites

• Lectin pathway: carbs/sugars on microbe cell wall

Is only one complement pathway active or activated at a time?

No, usually one starts but then one or both of the other pathways join in.

• They can also all be activated at once

What initiates the classical pathway? (6 things)

1. Antibodies bound with antigen

2. some viruses

3. some gram- bact

4. C-reactive protein w/ ligand

5. some protozoa

6. mycoplasmas (tiny bacteria that lack cell wall)

What two antibodies (and how many of them) specifically trigger the classical pathway? Include the specific sub-type if there is one.

• 2 IgG (due to small size, need 2) → IgG3 is best

• 1 IgM (due to large size)

C1qrs is the starting protein in complement activation. Where does it bind on the antibody to trigger activation of the classical pathway?

C1qrs binds on the Fc region of the Ab.

What are all the proteins/enzymes involved in the classical pathway? Try to list them in order of activation.

→ (cleaves)

= (creates)

C1qrs → C4 = C4b = C4b2a (C3 convertase)

C1qrs → C2 = C2a = ^

C4b2a binds C3b = C4b2a3b (C5 convertase) → C5 = C5a + C5b

C5b binds cell membrane to form MAC (C5b6789)

What does MAC (membrane attack complex) do to a cell?

Causes perforins in cell which leads to lysis (cell death)

What is the C3 convertase of the classical pathway? C5 convertase?

C3: C4b2a

C5: C4b2a3b

What does the lectin pathway use to identify/bind the carbohydrates (sugars) on microbe cell walls? What kind of protein is this?

Mannose-binding lectin (MBL) → acute phase reactant

What binds to MBL to put it into its active form? (Acts similar to C1qrs).

MBL binds with MASP1-3 to become activated and bind to mannose (or other sugars) on microbe cell walls

What is the order of cleavage for the lectin pathway? What cleaves the first two proteins?

MASP-2 cleaves both C4 and C2.

Pathway order: MBL/MASP-1-3 → C4 → C2 → C3

What triggers the alternative pathway (3 things)?

• Lipopolysaccharides (LPS) on bact. cell walls & toxins

• Complexes of IgA

• Spontaneous hydrolysis of C3

What are all the proteins/enzymes/Factors involved in the alternative pathway? Try to list them in order of activation.

[→] hydrolyzes

{stabilizes}

→ cleaves

= creates

H2O [→] C3 = C3a & C3b

C3b attaches to cell srfc & binds with factor B = C3bB (inactive)

Factor D → C3bB = C3bBb (C3 convertase) & Ba (floats away into serum)

Factor P {C3bBb} = C3bBbP (stable C3 convertase)

C3bBbP → C3 = C3a & C3b (both float away - C3b helps make C5 convertase in classical and alt.)

C3bBbP + C3b = C3bBbP3b (C5 convertase) → C5 = C5a + C5b6789 (MAC)

What is the C3 convertase of the alternative pathway? C5 convertase?

C3: C3bBbP

C5: C3bBbP3b

What is the main purpose of the alternative function?

Acts as an amplifier for the classical and lectin response.

What protein takes part (converges) in each pathway?

C3

What protein converges the Classical and Lectin pathway?

C4 & C2

What can happen to the body if complement activation goes uncontrolled?

Tissue damage, loss of function, or death (due to septic shock)

What are system controls for complement? What does Factor H effect and how? What does C1-INH and Factor I do?

System controls: prevent formation of MAC to prevent it from going out of control.

• Factor H: Alt. pathway - blocks binding of Factor B → prevents C3 convertase format.

• C1-INH & Factor I: classical & lectin path - Breaks C3 into unusable pieces

What are the 3 cell-bound regulators? What are the 5 soluble regulators?

Cell-bound: Cr1, MCP, DAF

Soluble: C1-INH, C4BP, Factors H, I & S

Complement also has receptors. What cell types trigger upon interaction with CR3 and CR4 of complement? What are the receptors’ functions?

CR3:

• Cells: phagocytic - mono/macro, neutro, NK

• Funct: Adhesion & increased activity of phagocytic cells

Cr4:

• Cells: phagocytic: mono/macro, neutro, NK, activ T & B, dendretic

• Funct: Adhesion & increased activity of phagocytic cells

Aside from complement lysing cells, is also increases the inflammatory response (OIL). What proteins of the complement system activate anaphylatoxins, chemotaxins, and opsonins?

Anaphylatoxins: C5a & C3a

Chemotaxins: C5a

Opsonins: C3b & C4b

What do the inflammation proteins of complement help WBCs do to activate the adaptive?

Helps WBCs take in and present antigens to adaptive system → facilitates lymphocyte activation

What two things can occur that makes complement activation dangerous? (Think about what diseases could cause problems).

• Systemic activation → Gram (-) septicemia

• RBC lysis → cold agglutination disease or hemolytic anemia (autoimmune)

  • Body makes ab’s that target RBCs → activates complement

What disease(s) is associated with a C2, C3, and MBL deficiency? Briefly describe why/how they cause issues.

C2: Lupus-like syndrome, recurrent infections, atherosclerosis

• C2 loss leads to lack of classical & lectin complement activation

C3: Severe recurrent infections, glomerulonephritis

• Rare condition → caused by C3 getting trapped in kidney → inflammation

MBL: Pneumococcal diseases, sepsis, Nisseria infections

• Lack of MBL = lack of sugar/carb recognition on pathogens

What 3 immunological assays are done to check for individual complement component abnormalities?

• ELISAs

• Automates Nephelometry (or immunoturbidimetry)

• Radial immunodiffusion (RID)

What 2 lab tests check for function (or lack of) in the classical pathway?

1. Hemolytic titration (CH50) assay

2. ELISA to check C9 epitopes after binding MAC

What 2 lab tests check for function (of lack of) in the alternative pathway?

1. AH50

2. ELISA to check for C3bBbP or C3bP

The CH50 assay is used to check for function (or lack of) the classical pathway. Explain how this test works. Interpret what it means if different dilution activate the lysis.

1. Patient serum is serially diluted (which naturally has complement proteins)

2. Sheep RBCs that are coated with Ab’s (a.k.a sensitized RBCs) are added to patient serum

3. C1qrs (classical) binds RBC w/ Ab’s and lysis them

Complement functions normally if: hemolysis of 50% of RBCs occurs at a lab designated normal dilution.

• Ex. Dilute 1/20 (least dilute serum), 1/40, 1/80 (50% RBCs lysed here), 1/60, and 1/320 (most dilute serum)

  • 1/80 is the point at which RBCs should be 50% lysed and if so, complement works as intended

  • If 50% lysed at dilution 1/20 → Doesn’t work well, depleted complement proteins

  • If 50% lysed at 1/320 → Works potentially too well, very dilute while still getting 50% lysis

How is the CH50 reported?

As the reciprocal of the dilution that caused 50% RBC lysis.

• if 1/80 dilution caused the lysis = 80 U/mL

What pathway(s) are impacted if there is an issue/deficiency of C1qrs, MBL/MASP2, B/D/P factors, C1-INH, and H & I factors?

C1qrs: classical

MBL/MASP2: Lectin

Factors B/D/P: Alternative

C1-INH: Classical & Lectin

H & I factors: Classical, Lectin, & Alternative

What are the 3 different complement therapies used to fix a deficient complement?

1. Target key areas in complement path to treat disease

2. Recombinant complement proteins to treat deficiencies (ex. recombinant C1-INH to treat HAE)

3. Monoclonal Ab (Mab’s) to block formation of certain complement components (ex. C5 Mab blocks C5a → prevents MAC = no lysis)