3 Complement and cytokines

What is a complement system and its main roles?

→ major immune defense system of the body that’s central to the development of inflammatory reactions

• links innate & adaptive systems

• label pathogens for elimination

• form lytic transmembrane pores to destroy target cells

How do complement components interact with each other?

→ via a series of highly regulated proteolytic cascades

• many of these components are proenzymes that must be cleaved to become active enzymes

What are the components of complement system?

• serum

• membrane-bound proteins

Give the reaction sequence of the complement system.

 C1-C4-C2-C3-C5-C6-C7-C8-C9

List the 4 major biological effects of complement.

1. Priming of adaptive immune response

2. Opsonization

3. Chemotaxis and cell activation

4. Lysis of target cells

What is cytolysis? How does it occur?

→ lysis of cells (e.g., bacteria, tumor cells, or virus-infected cells)

• occurs via the formation of the Membrane Attack Complex (MAC)

  • made of complement proteins = C5b, 6, 7, 8, 9

• inserts into the cell membrane → makes a pore → loss of osmotic integrity → cell lysis → allows free water to freely pass across the membrane

What is chemotaxis? Give an example of a chemotactic protein and its role.

→ process where an immune cell (phagocyte) is attracted to and moves toward a soluble factor

• e.g., C5a is a potent chemotactic agent that stimulates neutrophils and monocytes to move toward sites where antigens are deposited

What is opsonization? How is phagocytosis efficiency increased?

→ coating of microorganisms or antigen-antibody complexes with molecules that bind to receptors on phagocytes

• Phagocytosis becomes more efficient in the presence of C3b b/c phagocytes have specific C3b receptors

What are anaphylatoxins? Provide examples and their roles.

→ complement fragments that promote:

• vasodilation

• ↑ vascular permeability

• promote adhesion & chemotaxis of neutrophils & monocytes

• e.g., C3a & C5a - stimulate mast cells & basophils → release vasoactive amines (histamine) → ↑ blood flow to the infection site → allows more immune components to enter

Give the 6 major roles of complement in inflammation.

1. Lysis (of bacteria)

2. Opsonization

3. Chemotaxis

4. Activation (of mast cells)

5. Degranulation (of mast cells)

6. Immune complex clearance

Name the 3 major complement pathways.

1. Classical pathway

2. Alternative pathway

3. Mannan-binding lectin (MBL) pathway

What do all 3 of these complement pathways release (Classical, Alternative & Mannan-binding lectin (MBL))?

C5 convertase - breaks down C5 into C5a + C5b

What is the role of C5a?

• anaphylatoxin

• chemotactic factor

What are the events following formation of C5b leading to the formation of MAC?

1. C5b binds with C6 + C7 → form a complex complex that inserts into the membrane bilayer

2. C8 then binds to this complex

3. Polymerization of 16 C9 to produce the complete MAC

Describe briefly the sequence of reaction and products formed during classical pathway.

1. C1 (q, r, s proteins) binds to the F_c region of an antibody (IgM or IgG) in an immune complex

2. This activates C1s

3. C1s cleaves C4 & C2 → C4b2b (active C3 convertase)

4. C4b2b cleaves C3 → C3a + C3b

5. C3b + C4b2b form C5 convertase

6. C5 convertase cleaves C5 → C5a + C5b

7. MAC formation → cell lysis

Which IgG subclass fix complement?

1, 2, 3

❌ 4

What is the Alternative pathway?

→ triggered directly by infectious agents

• uses different components like Factors B, D, and Properdin to generate its C3 convertase (C3bBb)

• C3bBb makes more C3b, where additional C3b binds to the C3 convertase to form C3bBbC3b

What does C3bBbC3b do?

Alternative pathway of C5 convertase that makes C5b → MAC production + cell lysis

What is the main constituent of the Mannan-Binding Lectin Pathway?

→ plasma protein MBL

• binds to sugar residues (mannose) on microbial LPS

• once bound it can activate C2 & C4

• remaining steps are the same as the classic pathway

Name the main microorganisms activating classical, alternative and lectin pathways.

Classical:

• HIV, retroviruses, vesicular stomatitis virus

• Mycoplasma spp.

Alternative:

• virus-infected cells (e.g., EBV),

• Gram (+) & (-) bacteria

• trypanosomes

• Leishmania spp

• fungi

Lectin:

• HIV, retroviruses

• Gram (+) & (-) bacteria

What complement pathway is activated in Herpes Virus infection? How is the infected cell destroyed?

Classical complement pathway

1. When HSV replicates, it inserts viral proteins onto the cell surface

2. This allows specific anti-HSV antibodies to bind via their Fab site

3. C1 attaches to the exposed Fc portion of this antigen–antibody complex → activates Classical pathway → destroys infected cell via the MAC

What is Herpes keratitis?

→ HSV induced viral eye infection (esp cornea)

• if it attacks epithelium → ulcers → heals w/out scarring

• if it attacks deeper layers → scarring, vision loss, blindness (rare)

What are the 2 ocular disorders that have autoimmune origin and involve complement? How does complement play a role in destruction in those disorders?

1) Cicatricial pemphigoid

• complement-mediated immune mechanisms are directed against the basal epithelial cells of the conjunctiva

2) Mooren’s Ulcer

• involves corneal tissue wasting away

• mast cells are activated by C5a

What are cytokines?

→ Potent, LMW protein cell regulators

• made transiently & locally by many cells

List the major roles of Cytokines.

HITHITC

• Hematopoiesis

• Immunity

• Tumorigenesis

• Homeostasis

• Infectious disease

• Tissue repair

• Cellular development and growth

How do cytokines act as signaling molecules?

1. Bind to specific glycoprotein receptors on cell membranes

2. This relays a signal to the nucleus

3. Signal transduction occurs via kinase-mediated phosphorylation of cytoplasmic proteins

What kind of activity do cytokine receptors have?

tyrosine kinase activity

List 4 major functional categories of cytokines.

1. Immunoregulatory

2. Proinflammatory

3. Anti-inflammatory

4. Growth & differentiation

Which cytokine has a major role in immunoregulation?

IFN-γ

What are anti-inflammatory cytokines? Name them.

→ dampen or downregulate overactive inflammatory responses

• TGF-β

• IL-4

• IL-10

• IL-11

What is the role of proinflammatory cytokines? List the 4 types.

→ seen in infectious diseases + role in developing septic shock

• TNF-α

• IL-1

• IL-6

• IFNs

Name the cytokines implicated in the development of septic shock.

• TNF-α

• IL-1

• IL-6

What are the effects of Septic shock seen based on levels of these cytokines?

Low quantities

• local inflammation

• monocyte/neutrophil activation

• complement activation (C3a, C5a)

Moderate quantities

• Cause systemic effects (e..g, fever + acute-phase reactant production in the liver)

High quantities

• Result in septic shock

• characterized by ↓ CO, ARDS & blood vessel injury/thrombosis

What are the 4 subsets of T cells?

1. Th1

2. Th2

3. Th17

4. Treg

How are the 4 subsets of T-cells formed?

→ differentiate from naïve CD4 T cells based on the cytokine environment

1. Th1: made in the presence of IFN-γ

2. Th2: Differentiated in the presence of IL-4

3. Th17: made in the presence of TGF-β & IL-6

4. Treg: made in the presence of TGF-β

How can TB reactivity be monitored based on T cell function and IFN-γ levels?

T-cell function is monitored by measuring T-cells’ ability to produce IFN-γ in vitro, which identifies TB reactivity

What are the 2 cytokines currently employed as therapeutic agents and for what disorders?

IFN-α → Hep C

IFN-β → MS

What are the ways by which IFN-α help in neutralizing Hep C virus?

1. Inhibits viral binding, entry, and uncoating

2. Inhibits viral replication via Jak-Stat signaling

3. ↑ antigen complex display

4. ↑ specific CTL (Cytotoxic T Lymphocyte) activity to trigger apoptosis

What are the visual sx of MS?

• Nystagmus

• Optic Neuritis

• Diplopia

List ways by which IFN-β is thought to work to reduce MS sx?

• Inhibits T-cell activation, proliferation & migration across BBB

• CNS inflammation

What are the 2 approaches involving cytokines to combat rheumatoid arthritis and cancer?

RA → inhibitors of TNF-α

Transplantation + cancer → inhibitors of IL-2 & IL-15

What are the ways by which infliximab helps to neutralize TNF-α?

1. Chimeric IgG1 monoclonal antibody has high affinity for TNF-α → forms a highly stable complex (neutralizing TNF-α)

2. Complex selectively lyses activated cells expressing TNF-α & targets transmembrane, soluble, and receptor-bound TNF-α

List 2 cell types that can induce damage in Dry Eye?

1. CD₄⁺ T cells (in conjunctiva)

2. CD_11b⁺ monocytic cells (in cornea)

What is the stimulus during Stage I that induces cytokine secretion? What are the cytokines secreted?

Stimulus = desiccating stress

• induces TNF-α, IL-1, IL-6 secretion by ocular surface tissues

What do the secreted cytokines do to the APCs?

→ facilitate the activation and migration of resident APCs toward regional draining lymph nodes

What are the 2 cell types that are involved in production of cytokines in the lymph node? Name the cytokines and their role if any.

APCs —| cognate naïve T cells (Th0) leading to the expansion of:

1. IL-17 secreting Th17 cells

   • IL-17 further antagonizes Treg to facilitate more Th17 expansion

2. IFN-γ secreting Th1 cells

How does IL-17 induce epithelial damage?

IL-17 interacts with receptors on the ocular surface to ↑ MMPS + more inflammatory cytokines → epithelial damage

What is the role of IFN-γ in dry eye?

• apoptosis & metaplasia of epithelia

• upregulates adhesion molecules (CAM) + chemokine ligands

• ↑ Immune cell entry into the ocular surface

Briefly summarize the key events involved in Dry Eye during stages I to III.

Stage 1:

• Desiccating stress triggers proinflammatory cytokines (IL-1, TNF-α, IL-6) → causes APCs to migrate to lymph nodes

Stage 2:

• In the lymph nodes, APCs induce Th1 & Th17 cells expansion

• IL-17 suppresses regulatory T-cells to amplify the response

Stage 3:

• Effector cells return to the ocular surface, where IL-17 and IFN-γ cause:

  • tissue damage

  • cell death (apoptosis)

  • further immune cell recruitment

List the Clinical Applications of Cytokines.

1. Biomarkers of disease

2. Monitor immune status

3. Therapeutic agents

4. Targets of therapeutics