module 4 IS

MODULE OUTCOMES

• Describe the mechanism and laboratory tests involving the complement system.
• Trace the pathways of activation of the complement system, including:
    - Activators/initiators
    - Inhibitors
    - Products
    - Biological effects
• Discuss the concepts of hypersensitivity and the immunological mechanisms of each type.
• Discuss the associated disorders and laboratory methods for each type of hypersensitivity.

INTRODUCTION

• The complement system is a series of over 30 proteins that enhance host defense against foreign cells.
• Activated complement proteins promote:
    - Opsonization
    - Lysis of foreign cells
    - Immune complexes
    - Inflammation
• Chronic activation can lead to tissue damage; some proteins serve as regulators.
• Mechanisms of complement activation:
    1. Classical Pathway: Triggered by antigen-antibody complexes.
    2. Alternative Pathway: Initiated by microbial cell walls.
    3. Mannose-Binding Lectin (MBL) Pathway: Initiated by binding of MBL with mannose or related sugars in a calcium-dependent manner.
• Abnormal complement levels can indicate deficiencies or extreme activation, leading to infection risk or autoimmune manifestations.
• Laboratory assays for complement levels include:
    - CH50, AH50
    - Use of RID (Radial Immunodiffusion) and ELISA (Enzyme-Linked Immunosorbent Assay) techniques
    - Complement fixation tests, detecting antigen-antibody complexes.

KEY CONCEPTS

I. COMPLEMENT

A. General Information

• The complement system
    - Involves lysis of cell membranes
    - Mediation of inflammation
    - Enhancement of phagocytosis
    - Metabolism of immune complexes.
• Activation mechanisms:
    - Classical pathway
    - Alternative pathway
    - Lectin pathway
• Composed of over 30 proteins:
    - Components identified as C1 to C9, based on discovery.
    - Includes additional proteins in the alternative pathway:
      * Factor B
      * Factor D
      * C3bBb
      * Cobra venom factor (CVF)
      * Properdin (P)

B. General Properties

• Activation is a cascade process.
• Heat destroys complement proteins at 56°C for 30 minutes.
• IgM and IgG are the only antibodies that interact with complement.
• Found in all mammalian sera.
• Migration occurs in the beta region.
• Produced by the liver, except:
    - C1 produced by intestinal epithelial cells
    - Factor D produced by adipose cells.

C. Functions of Complement

1. Cell lysis
2. Anaphylatoxins: C4a, C3a, C5a
      - Bind to mast cells/basophils causing degranulation (release of histamine)
      - Result in vasodilation and increased vascular permeability
3. Chemotaxin: C5a
4. Opsonin: C3b (promotes immune adherence)

D. Destruction of Complement in vitro

1. Anticoagulants: do not use during complement testing.
      - EDTA removes Ca++
      - Heparin inhibits cleavage of C4
2. Heating: Heat serum to 56°C for 30 min.
3. Normal Serum Inhibitor
4. Storage:
      - Principally affects C4
      - Serum must be separated from RBCs ASAP, kept at 4°C for up to 48 hours.

II. COMPLEMENT CLASSICAL PATHWAY

A. General Information

1. Initiated by immune (Ag-Ab) complexes
2. The first complement pathway discovered
3. Activated by most antibody-sensitized cells
4. Complement activation cascade order: C1, C4, C2, C3, C5, C6, C7, C8, C9

B. Complement Activation

1. Recognition unit: C1
2. Activation unit: C4, C2, C3
3. Membrane Attack unit: C5, C6, C7, C8, C9

C. Recognition Unit

1. C1 is a trimolecular complex (C1q, C1r, C1s) held together by Ca++
2. C1q appears as 6 fused globes at a common base
3. Initial cascade requires attachment of C1q globes to 2 Fc fragments:
      - IgM: requires 1 molecule
      - IgG: requires 2 molecules (IgG3 > IgG1 > IgG2)

D. Activation Unit

1. C4: Second most abundant, activated by C1s
      - Cleaved into C4a (anaphylotoxin) and C4b (binds to cell surface)
2. C2: Activated by C1s after C1-C4 interaction
      - Forms C3 convertase (C4b2a) in the presence of Mg++
3. C3: Most abundant, cleaved/activated by C3 convertase
      - C3a (anaphylotoxin), C3b (opsonin and activates alternative pathway)
      - C3b attaches to C3 convertase forming C5 convertase (C4b2a3b)

E. Membrane Attack Unit

1. Also known as the Membrane Attack Complex (MAC)
2. C5b-C9 complex inserts into the lipid membrane, forming a transmembrane protein channel, leading to:
      - Influx of Na+ and H2O, causing cell lysis.
3. C5: Activated by C5 convertase, results in C5a (anaphylotoxin and chemotaxin) and C5b (first element in MAC)
4. C6 and C7 bind to C5b
5. C8 disrupts the cell membrane
6. C9 enhances C8 activity; in absence of C9, cells lyse, but at a slower rate.

III. COMPLEMENT ALTERNATIVE PATHWAY

A. General Information

1. Known as the Properdin pathway
2. Identified after classical pathway
3. Discovered by Pillemer et al.
4. Antibody-independent pathway
5. Bypasses C1, C4, and C2, starting at C3
6. Initiated by C3b deposited on bacterial cell surfaces in association with Factor B.

B. Steps

1. C3 hydrolysis produces C3b binding to Factor B, forming C3bB complex.
2. Factor D cleaves Factor B forming C3bBb complex (C3 convertase).
3. C3 convertase is stabilized by properdin (C3bBbP), cleaving more C3 into C3a and C3b.
4. Some C3b binds C3 convertase (C3bBbP) forming C5 convertase.

C. Cobra Venom Factor (CVF)

1. Complement-activating protein from cobra venom, structurally and functionally similar to C3.

IV. COMPLEMENT LECTIN PATHWAY

A. General Information

1. Lectin pathway, aka MBL pathway.
2. Antibody-independent.

B. Steps

1. MBL binds to mannose or related sugars in a calcium-dependent manner, initiating this pathway:
      - Found in glycoproteins/carbohydrates of various microorganisms (bacteria, yeasts, viruses, parasites).
      - MBL structure resembles C1q, associated with three MBL-serine proteases (MASPs): MASP-1, MASP-2, MASP-3.
      - MASP-2 autoactivates upon binding to a cellular surface, cleaving C4 and C2, and then follows the classical pathway.

V. REGULATION OF COMPLEMENT

A. Reasons for Regulation

1. Limit wide-ranging inflammation.
2. Avoid excessive activation.
3. Protect host cells from unintended injury.

B. Plasma Complement Regulators

1. C1 inhibitor (C1INH): dissociates C1r and C1s from C1q.
2. Factor I: Cleaves C3b and C4b.
3. Factor H: Co-factor with I to inactivate C3b; prevents Factor B binding to C3b.
4. C4-binding protein: Acts as a co-factor with I to inactivate C4b (C4bp).
5. S protein (vitronectin): Prevents attachment of C5b67 complex to cell membranes.
6. Anaphylotoxin inactivator: Removes an amino acid from C4a, C3a, C5a, inactivating them.
7. MAC inhibitors: Prevent formation by binding S protein to C5b-7 complex.
8. Complement receptor type I (CRI or CD35): Binds C3b and C4b to inhibit amplification loop.

VI. COMPLEMENT DEFICIENCIES AND LABORATORY DETECTION

A. Causes

1. Genetic alterations may lead to complement protein deficiencies.
2. Consumption can occur in infections and collagen vascular conditions.
      - C3 and C4 levels indicate consumption and disease status; C2 deficiency is most common, while C3 deficiency is severe.

B. Deficiencies of Complement Components

C. Decay-Accelerating Factor (DAF or CD55)

1. Affects both classical and alternative pathways, dissociating C3b.
2. Inhibitory effects:
      - Classical pathway: DAF dissociates C2a from C4b.
      - Alternative pathway: DAF helps dissociate Bb from C3b.
3. Missing/defective DAF contributes to paroxysmal nocturnal hemoglobinuria (PNH).

D. Laboratory Detection of Complement Abnormalities

1. Immunologic Assays of Individual Components: Methods such as RID and nephelometry.
2. Classical Pathway Hemolytic (CH50) Assay: Measures serum needed to lyse 50% of antibody-sensitized sheep erythrocytes, expressed in CH50 units.
3. Alternative Pathway Hemolytic Assay (AH50): Same as CH50, but uses a chelating buffer preventing classical pathway activation, with rabbit red cells as an indicator.
4. ELISA: Enzyme-linked immunosorbent assay for complement detection.

HYPERSENSITIVITY

A. Definition

• Hypersensitivity refers to an exaggerated immune response leading to tissue injury and disease.

B. Gell and Coomb’s Classification of Hypersensitivity

• Type 1: Anaphylactic hypersensitivity
• Type 2: Cytotoxic hypersensitivity
• Type 3: Immune complex hypersensitivity
• Type 4: Delayed hypersensitivity
• Both humoral and cell-mediated responses involved.
    - Humoral mediated: Type 1, 2, 3
    - Cellular mediated: Type 4

VIII. TYPE I HYPERSENSITIVITY (ANAPHYLACTIC HYPERSENSITIVITY)

A. General Information

1. Also known as immediate hypersensitivity.
2. Occurs seconds to minutes post-exposure.
3. Involves basophils/mast cells sensitized with IgE.
4. Common in allergic reactions and helminth infections.

B. Mechanism

1. Sensitization: Formation of antigen-specific IgE on first exposure, attaching to mast cells.
2. Activation: Upon reexposure, IgE binds allergen, causing mast cell degranulation and mediator release, leading to allergic symptoms.

C. Preformed Mediators

1. Histamine: Causes contraction of smooth muscles, increases vascular permeability, and mucus secretion.
2. Eosin chemotactic factor of anaphylaxis (ECF-A)
3. Neutrophil chemotactic factor

D. Newly Synthesized Mediators

1. Prostaglandin D2: Causes vasodilation and increased vascular permeability.
2. Leukotrienes:
      - Cause erythema and wheal formation.
      - 30-1000x more potent than histamine for bronchospasms and mucus secretion.
      - LTB4: chemotactic for neutrophils and eosinophils.
      - LTC4, LTD4, LTE4: Increase vascular permeability, cause bronchoconstriction and mucus secretion.
3. Cytokines: Produce increased inflammatory cells, and elevate IgE production.
      - Include: IL-1, TNF-α, IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, IL-14, IL-16, GM-CSF.
   

E. Allergens and Disease

1. Allergens (atopic antigens) trigger IgE production in susceptible individuals.
2. Atopy: An inherited tendency to produce IgE in response to specific allergens.
      - 25% of the population experiences allergies.
3. Symptoms vary by exposure route:
      - Inhalation: respiratory symptoms (asthma, rhinitis)
      - Ingestion: gastrointestinal symptoms (food allergies)
      - Injection: systemic response.
4. Anaphylaxis: Systemic form of Type I hypersensitivity; severe allergic reaction leads to symptoms like:
      - Bronchospasm
      - Laryngeal edema
      - Vascular congestion
      - Skin reactions (urticaria, angioedema)
      - Diarrhea
      - Vomiting
      - Intractable shock.
   

F. Management/Treatment of Immediate Hypersensitivity

1. First line: Avoidance of known allergens.
2. Medications: Antihistamines, decongestants, bronchodilators, inhaled corticosteroids, epinephrine.
3. Hyposensitization: Small doses of allergens to build up IgG defenses.
4. Anti-IgE monoclonal antibodies: e.g., Omalizumab.

G. Testing for Immediate Hypersensitivity

1. In vivo tests: Cutaneous and intradermal tests. Positive reaction: wheal 3 mm larger than negative control.
2. In vitro tests:
      - Total IgE (RIST): screening test
      - Antigen-Specific IgE Testing (RAST)
      - Microarray Testing: Patient serum reacts with allergen microarray, followed by anti-IgE with fluorescent tag.

IX. TYPE II HYPERSENSITIVITY (CYTOTOXIC HYPERSENSITIVITY)

A. Mechanism

1. IgM or IgG binds to cell-bound antigens, causing cytolysis, complement activation, and destruction by phagocytosis or ADCC.

B. Examples

1. Hemolytic Transfusion Reaction: Significance of ABO blood groups.
2. Hemolytic Disease of the Newborn (HDN):
      - More common with ABO incompatibility; Rh HDN commonly due to D antigen.
3. Organ-specific Autoimmune Diseases: Examples include:
      - Goodpasture’s syndrome (anti-GBM).
      - Hashimoto’s disease (anti-Tg).
      - Myasthenia gravis (anti-AChR).
      - Insulin-dependent diabetes mellitus.
4. Autoimmune Hemolytic Anemia:
      - Warm-reactive antibodies (IgG) at 37°C associated with idiopathic/secondary due to infections, CLL, drugs.
      - Cold-reactive antibodies (IgM) at 4°C associated with Mycoplasma pneumonia, infectious mononucleosis, CLL, non-Hodgkin’s lymphoma.
      - Complement activation varies.

C. Testing for Type II Hypersensitivity

1. Direct Antiglobulin Testing (DAT): Detects transfusion reactions, HDN, autoimmune hemolytic anemia.
      - Polyspecific AHG: Anti-IgG, anti-C3b, anti-C3d.
2. Indirect Antiglobulin Testing (IAT):
      - Crossmatching blood to prevent transfusion reactions.
      - Determines presence of specific antibodies.

X. TYPE III HYPERSENSITIVITY (IMMUNE COMPLEX HYPERSENSITIVITY)

A. Mechanism

1. Antigen-antibody complexes deposit in tissues, causing inflammation and complement activation, leading to PMN accumulation and tissue damage.

B. Examples

1. Arthus Reaction: Localized inflammatory reaction after intradermal antigen injection in immunized rabbits.
2. Serum Sickness: Occurs after passive immunization; immune complexes deposit in tissues.
3. Autoimmune Diseases:
      - Systemic lupus erythematosus (SLE): complex deposition in multiple organs, mainly affects kidney GBM.
      - Rheumatoid arthritis: complex deposition in inflamed joint membranes.
4. Glomerulonephritis
5. Bacterial Endocarditis

C. Testing for Type III Hypersensitivity

1. Detect antibodies in specific diseases.
2. Fluorescent staining: To determine immune complex deposition in tissues.
3. Measurement of complement levels.

XI. TYPE IV HYPERSENSITIVITY (CELL MEDIATED HYPERSENSITIVITY)

A. Mechanism

1. T cells (Th1) respond to antigens at the injection site.
2. T cells release lymphokines (IL-3, IFN-gamma, TNF), recruiting macrophages and neutrophils, creating inflammation.
3. This is also known as delayed-type hypersensitivity (1-3 days post-exposure).

B. Examples

1. Contact Dermatitis: from cosmetics, adhesives, plant toxins, topical medications, latex.
      - Can last 3-4 weeks post-exposure.
2. Hypersensitivity Pneumonitis: T cells respond to inhaled allergens, predominantly microorganisms (bacterial/fungal spores).
3. Tuberculin-Type Hypersensitivity: An intradermal challenge leads to erythema and induration in sensitized individuals; PPD injection is read at 48-72 hours.

C. Testing for Delayed Hypersensitivity

1. Patch Test: Gold standard for contact dermatitis; adhesive patch with allergen applied for 48 hours, assessed for redness/papules.
2. Mantoux Method Skin Test: Intradermal injection measured at 48 and 72 hours; induration of 5 mm or more is positive.

FURTHER READING

• Stevens, C. D., & Miller, L. E. (2016). Clinical Immunology and Serology: A Laboratory Perspective. FA Davis.
• PER handbook: A Review Manual for Clinical Laboratory Examinations.
• McPherson, R. A., MSc, M. D., & Pincus, M. R. (2021). Henry's Clinical Diagnosis and Management by Laboratory Methods E-book. Elsevier Health Sciences.