The Immune System: Complement, Cytokines

Overview

• This lecture covers the complement system, cytokines, immunomodulation, and the sequence of events in an immune response.
• At the end of this lecture you should be able to identify complement components, describe complement activation, describe complement functions, classify cytokines, identify cytokine functions, describe immunopotentiation, identify adjuvant types, describe immunosuppression, describe immunosuppression methods, and describe immune response event sequences.

Case Scenario

• A 14-month-old male infant presents with high fever and no response to antipyretic therapy.
• The illness started with an abrupt onset of fever, a severe cough, and increased work of breathing.
• The mother reports the child is frequently ill.
• Two months prior, the infant was hospitalized for pneumococcal pneumonia with right upper lobe consolidation and pneumococcal bacteremia.
• The infant is hospitalized for possible sepsis and treated with intravenous antibiotics, resulting in slow improvement.
• A blood culture is positive for pneumococcus.
• An immunologic workup reveals markedly decreased C3 and C4.

Complement System

• Circulating and membrane-associated proteins function in both innate and adaptive immunity.
• Components include C1-C9, factor B, D, and P.
• Fragments of these components can be cleaved into two fragments, indicated by lowercase letters (e.g., C5a, C5b).

Complement Activation

• In innate immunity, complement can be activated by the alternative pathway and the mannan-binding lectin pathway.
• In adaptive immunity, complement can be activated via the classical pathway

Classical Pathway

• Activated by antigen-antibody (IgG, IgM) complex.
• C1 (C1q, r, s) binds the Ag-Ab complex, leads to the activation of C1.
• C1 acts as an enzyme and cleaves both C2 and C4.
• C4b + 2b forms C3 convertase C4b2b, which cleaves C3.
• C3b binding to C4b2b leads to the formation of C4b2b3b, which is the C5 convertase, which cleaves C5 to C5a and C5b, initiating the membrane attack complex.

Mannan-Binding Lectin (MBL) Pathway

• MBLs are serum proteins that bind to specific carbohydrates.
• The pathway activates via MBL binding to mannose residues of glycoproteins on microbes.
• MBL interacts with 2 MBL-activated serine proteases (MASP1 & MASP2).
• MASP activation leads to activation of C2, C4, and C3, similar to the classical pathway.

Alternative Pathway

• Initiated by cell-surface components of microbes recognized as foreign.
• Spontaneous breakdown of C3, the most abundant serum complement component, occurs.
• C3b binds to factor B.
• Factor B is cleaved by factor D, producing C3bBb, an unstable C3 convertase.
• C3bBb binds properdin factor (factor P) to produce stabilized C3 convertase.
• Additional C3b fragments are added to form C5 convertase C3bBb3b.
• C5 convertase cleaves C5 into C5a and C5b.
• C5b inserts into the cell membrane of microbes to begin the formation of the membrane attack complex and cell lysis.

Terminal or Lytic Pathway

• Can be entered through the classical, MBL, or alternative pathway.
• Attachment of C5b to the bacterial membrane initiates the formation of the membrane attack complex (MAC) and lysis of the cell.
• C5b attaches to the cell membrane.
• C6, C7, and C8 are added to form C5b678.
• Multiple C9 molecules (poly C9) are added to form pores in the cell membrane of the microbe.
• C9 corresponds to "perforin".
• The MAC is C5b6789(n).

Complement System Functions

• Opsonization and phagocytosis: C3b (or C4b) acts as opsonins.
• Complement-mediated lysis: MAC creates pores in cell membranes and induces osmotic lysis.
• Stimulation of inflammatory reactions: C5a, C3a, and C4a bind to receptors on neutrophils and stimulate inflammatory reactions; C5a, C3a, and C4a are chemoattractants for neutrophils.
• Providing stimuli for B cell activation and the humoral immune response.

Cytokines

• Proteins secreted by immune cells in response to microbes.
• Cytokines stimulate lymphocyte growth and differentiation, activate immune cells to eliminate microbes and antigens, stimulate hematopoiesis, and are used in medicine as therapeutic agents.

Cytokine Nomenclature

• Monokines: from macrophage/monocyte.
• Lymphokines: from lymphocyte.
• Interleukins: from leukocytes that act on other leukocytes (e.g., IL-1, IL-2, IL-3).
• Cytokines: a preferred name, as they may be produced by lymphocytes, monocytes, or other cells.

Cytokine Classification

• Mediators and regulators of innate immunity: produced mainly by macrophages and NK cells.
  • Includes TNF-α for neutrophil activation and inflammation, IL-1 for neutrophil activation and inflammation, IL-12 for T & NK cell activation, IFN-α, IFN-β for antiviral action and increased expression of class I MHC, and chemokines for chemotaxis and migration of leukocytes into tissues.
• Mediators and regulators of adaptive immunity: produced by T lymphocytes.
  • Includes IL-2 for proliferation of T, NK, and B cells, IL-4 for B cell isotype switch to IgE and mast cell proliferation, IL-5 for B cell proliferation and eosinophil activation, and IFN-γ for macrophage activation and increased microbicidal functions.
• Stimulators of hematopoiesis: GM-CSF, IL-3 and IL-7

Cytokine Therapy

• Interferon α: used in viral hepatitis HCV and melanoma treatment.
• Interferon β: used in treating multiple sclerosis.
• IL-2: used in clinical trials for tumors.
• GM-CSF: used to promote BM recovery and correct neutropenia in cancer patients.
• TNF and IL-1 antagonists: for rheumatoid arthritis treatment.

Immunomodulation

• Adjustment of the immune response to a desired level, including immunopotentiation or immunosuppression.

Immunopotentiation

• Enhancement of the immune response by increasing its rate or prolonging its duration.
• Immunopotentiation is achieved through the administration of an adjuvant

Adjuvants

• Agents that stimulate the immune system and increase the response to a vaccine without having any specific antigenic effect itself.
• Inorganic adjuvants: aluminium salts.
• Organic adjuvants: squalene.
• Oil-based adjuvants: Complete Freund's adjuvant (CFA) is a water-in-oil emulsion containing killed Mycobacteria.
• Incomplete Freund's adjuvant: Water-in-oil emulsion without Mycobacterium.
• Virosomes: phospholipid bilayer vesicles containing hepatitis A and influenza antigens
• Cytokines: IL-12

Adjuvant Mechanisms of Action

• Prolong retention of the immunogen.
• Increase the size of immunogen, promoting phagocytosis and presentation by macrophages.
• Stimulate the influx of macrophages and other immune cells to the injection site.
• Increase local cytokine production.

Immunosuppression

• Suppression of the immune system.
• Indications include: hypersensitivity responses, autoimmune disease, and prevention of rejection after transplantation.
• Induction of immunosuppression: drugs, radiation, and anticancer drugs.

Immunosuppression - Methods in Clinical Use

• Cyclosporine and tacrolimus (FK-506): fungal macrolides that inhibit T cell activation by blocking T cell cytokine production (IL-2); tacrolimus is less nephrotoxic than cyclosporin.
• Azathioprine and mycophenolate mofetil: antiproliferative drugs that inhibit purine synthesis required for cell division, blocking lymphocyte proliferation.
• Corticosteroids: anti-inflammatory drugs that inhibit proinflammatory cytokine secretion (IL-1, IL-3, IL-4, IL-5, IL-8, TNF-α) and decrease inflammatory cell migration.
• Anti-CD3 monoclonal antibody: depletes T cells by binding to CD3 molecules.
• Anti-IL-2 receptor antibody: inhibits T cell proliferation by blocking IL-2 binding to its receptor.

Prototypical Immune Response Events

• APCs capture a minute amount of antigen by phagocytosis.
• APCs process the antigen into small fragments (peptides).
• APCs present peptides plus class II MHC molecules to T helper cells.
• Binding of the peptide-MHC complex to TCRs activates helper T lymphocytes.
• Activated TH cells secrete cytokines such as IL-2, which encourages TH cell proliferation and activation, and IFN-γ, which activates macrophages and phagocytosis.
• TH cells are being activated while B cells recognize and bind antigen through their BCRs. B cell activation requires two signals: the first signal is binding of the Ag to the BCR, and the second is provided by helper factors (cytokines) secreted by TH cells, such as IL-2, IL-4, and IL-5.
• Activated B lymphocytes can differentiate into either memory B lymphocytes or plasma cells, which secrete antibodies.
• Tc lymphocytes recognize Ag on the surface of target cells expressing class I MHC molecules. Tc cell activation also requires IL-2 and IFN-γ from a nearby activated Th cell. The activated CTLs kill the target cell.