Immune Response to Biomaterials

1. Immune Response to Biomaterials • Topics: • Acquired Immunity • Antigens and Lymphocytes • B-cells, T-cells and Antibodies • The Complement System • Assays for Immune Response

2. Acquired Immunity • Acquired Immunity • Body responds to biomaterial or adsorbed proteins • This is Acquired Immune Response (AIR) mediated by lymphocytes in blood and tissue • Immunotoxicity: • Adverse effects on the function of the immune system or other body systems as a result of alterations in the immune system function • e.g.: allergies, autoimmune diseases • Design criteria: • How the immune system will react to and be affected by the biomaterial

3. 4 Criteria of AIR: • Specificity • Diversity • Self/non-self recognition • Immunologic memory • 2 types of acquired immunity: • Humoral: based on action of antibodies and foreign substances (e.g. bacteria) • Cellular: utilizes specialized lymphocytes (T-cells); detects altered self-cells (cancer, viruses)

4. Specificity:comes from the fact that lymphocytes (B, T cells) are activated by an antigen. Antigen:a substance (usually foreign, with a molecular weight > 8000 g/mol) that binds specifically to an antibody (e.g., immunoglobulin glycoprotein) or T-cell receptor (TCR) to initiate AIR. A specific site on the antigen recognized by the antibody is called the epitope. Hapten:low molecular weight substance that combines with a larger molecule (e.g. protein) to produce much greater immune response than with either the hapten or carrier alone Adjuvant:substance that non-specifically enhances immune response to antigens, possibly by increasing their uptake by phagocytic cells or prolonging the time an antigen remains in the body.

5. 2 main types of lymphocytes response to antigens: • B cells are mediators of humoral immunity and produce highly specific antibodies. They are formed in bone marrow and mature in lymph system (lymph nodes and spleen) • T cells (both Th and Tc) are involved in cellular immunity and produce proteins with high specificity (the TCRs). TCRs are bound to the cell membrane. T cells are produced in bone marrow and mature in the thymus. T lymphocyte Each lymphocyte has about 10,000 antibodies/TCRs on its surface, all of which bind to the same antigen. Each cell is reactive toward a different antigen, determined by the arrangement of the genes that encode for antibody/TCR B lymphocyte

6. T cells • Tc cells: • Cytotoxic lymphocytes (CTLs) • Have CD8, interact with MHC class I • Secrete “perforins” that lyse cells • Can remove antigen from lysed cell and move to another – very efficient in removing “self altered” cells • Important in removing viral and cancerous cells • Play little role in acquired immune response

7. Th cells: • Have surface glycoprotein CD4 to recognize antigens present on MHC class II molecules • Activated by MHC class II molecules along with B7-1 or B7-2 glycoproteins on surface of antigen presenting cells (APCs), creating effector and memory cells when activated. • Stimulate B cell growth (IL-4, 5, 6) • Stimulate Tc proliferation (IL-2) • Stimulate further Th cell activation (IL-2) • Promote chemotaxis and activation of macrophages (MIP, IL-8) • Bind to B cells to stimulate humoral response

8. Major Histocompatibility Complex Molecules 2 types: Class I and Class II A Class I MHC is a transmembrane glycoprotein found on almost all nucleated cells in conjunction with a smaller b2 macroglobulin protein. It is composed of two noncovalently associated chains (a and b). The far end of the a chain forms a cleft to interact with the antigen, and are recognized by cytotoxic T cells.

9. Class II MHCs are similar to Class I, having a binding cleft at the far end of both a and b chains. These are recognized by the T-helper cells (Th cells) which include macrophages and B lymphocytes.

10. Genes that make up MHC in cells are inherited from both parents and are recognized by body. Foreign cells don’t contain proper MHCs, are recognized by body and cause cell-based immune response Cells contain both Class I and II MHCs, occupying different regions of affected cells. Note that class I MHCs attach to Tc cells, while Th cells attach to Class II MHCs.

11. Intracellular processing of antigens. (a) Endogenous antigens, e.g. proteins produced as a result of virus, degrade into peptide fragments in cytoplasm, then bind with MHC Class I molecules in rough ER and are transported to the cell membrane. (b) Exogenous antigens enter the cell through phagocytosis. They are degraded during endocytosis and bind to Class II molecules in the endocytotic vesicles and transported to cell membrane.

12. Antigen presentation with class II MHC activates Th lymphocytes by promoting binding of the antigen to the T cell receptor. Tc cells and B cells can also bind the antigen depending on how it is presented. Cytokines released by Th cells aid in activation of Tc cells after antigen binding. Co-stimulation by binding with and/or secreted products from Th cells is required for full B cell activation. B and Tc cells rapidly multiply forming large populations of cells specific to that antigen. Both types of cells produce 2 variations: effector cells and memory cells.

13. Proper antigen presentation activates T lymphocytes by promoting binding of that antigen to the TCR. Cytokines released by the cells that help activate the Tc cells after antigen binding include: IL-1, IL-6, TF-a secreted by macrophages. These activate both B and T cells, aiding in communication between innate and acquired immune response.

14. The structure of antibodies produced by B cells are composed of four polypeptide chains. The two larger (heavy) chains have MW ~ 55-70 kDa, and are joined by disulfide bonds. The smaller (light) chains have MW ~ 24 kDa and are linked via disulfide bonds to each heavy chain. The cleft formed at the end of the heavy/light chain combination serves as the antigen binding site. The chains are made up of two portions: the constant portion, Fc, and the variable portion, Fab. Fc is responsible for recognition of the antibody receptors in phagocytic cells or by the complement complex. The structure of Fab is determined by the arrangement of amino acids (genes) in the heavy and light chains.

15. Mechanisms of antibody action: • Agglutination: clumping together of multiple large particles with antigens on surface • Precipitation: occurs when antibody/antigen complex is no longer soluble, interfering with function of the foreign substance • Neutralization: antibodies bind and cover the active or toxic sites on a foreign substance. Antibodies capable of direct attack on the cell membrane of an invading organism, resulting in cell lysis. • Lysis: cell death

16. There are 5 main classes of antibodies, each name beginning with “Ig” (immunoglobulin) followed by a capital letter. IgG, IgD and IgE can bind with two antigens. IgA has a “J” joining chain allowing for dimerization and can bind with 4 antigen molecules. IgM can bind with 10 antigens and is the most effective class for cell or bacterial agglutination.

17. Th cells are activated in response to antigen-MHC Class II complexes in combination with a co-stimulatory signal supplied by a antigen-presenting cell (APC). This co-stimulatory signal is usually the presence of B7-1 or B7-2 glycoproteins on the surface of the APC that can interact with specific receptors on the Th cells.

18. The Complement System • The Complement System: • Plays a role in both innate and acquired immune response • Is composed of over 20 plasma proteins that ultimately activate Membrane Attack Complex (MAC) and eliminate foreign elements (cause cell death) • Two pathways: Classical and Alternate

19. Structure of the complement protein C1, a complex of C1q and two molecules each of C1r and C1s. C1q is composed of 18 polypeptide chains forming six arms, each of which can bind to the Fc portion of an antibody through its globular “heads”. C1r and C1s are both enzymes (serine protease).

20. Classical Pathway: • IgG or IgM antibody binds to an antigen on suitable target site (e.g. bacterial cell or biomaterial) • C1 protein →C1q + 2 enzymes: C1r & C1s • C1s → C4a (smaller) & C4b (larger) • C4b acts as receptor for C2 • C2 cleaved by C1s →C4b2a (aka C3 convertase. This is a big amplification step. • C4b2a acts on C3 → C3a and C3b • C3b binds to C4b2a → C4b2a3b (aka C5 convertase) • C4b2a3b → C5a (mediates inflammation, chemotactic agent for neutrophils) & C5b (activates MAC)

21. Alternate Pathway: • C3 → C3a and C3b • C3b binds to foreign host cells along with “B” factor, which exposes a binding site for “D” factor enzyme. • D acts on B → Ba (diffuses away) & C3bBb (another C3 convertase) • C3bBb cleaves more C3 to C3b (amplification step) • C3b & C3bBb → C3bBb3b (aka C5 convertase) • C3bBb3b cleaves C5 → C5b & formation of MAC

22. Membrane Attack Complex Both classical and alternate paths lead to creation of C5b on foreign surface. C5 binds to C6 C5b6 binds to C7 This complex changes conformation to expose hydrophobic regions for interaction with phospholipid bilayer on target cell. After binding with C8, C5b678 creates 10 Å pores in cell membrane C5b678 joins with C9 → becomes “Membrane Attack Complex” MAC forms pores 70 – 100 Å that allow ion and molecule leakage from cell and cell dies.

23. Regulation of the Complement System • How to confine complement cascade to foreign pathogens? • Short half life of certain enzymes (e.g. C3b) limits distance they can migrate to 40 nm from where it is cleaved by C3 convertase, limiting its binding to neighboring cells. • Regulatory proteins limit actions that occur in cascade process, functioning at C3 (amplification) or MAC stages • C3b is especially regulated as it has a huge amplification effect and will kill host cells if unregulated • In MAC process, if C5b67 cannot insert properly to target it can attack bystander cells

24. Regulation of the Complement System Regulatory proteins (a) compete for binding sites or (b) encourage dissociation of MACs. e.g. Regulator of complement activation proteins (RACs) bind with C4b to block its attachment to C2a and prevent formation of C3 convertase RCAs bind with C8 on MAC, preventing formation of poly C9 complexes RCAs “decay accelerating factor” (DAF) binds to C3 convertase forcing dissociation of enzymatic component from cell-bound component.

25. Type I hypersensitivity response, caused by plasma cells (effector B cells) that secrete IgE molecules specific for the allergen. The IgE molecules bind to receptors on basophils and mast cells, resulting in sensitization of these cells. A second exposure to the antigen from the allergen causes crosslinking of these membrane-bound antibodies. Crosslinking provides a signal to induce degranulation of mast cells and the release of soluble mediators (e.g. histamine). Mediators cause localized and systemic events, including smooth muscle contraction and vasodilation.

26. Type IV hypersensitivity reaction, caused by TDTH cells. Upon first exposure to the antigen presented on an APC, Th cells become sensitized and mature into TDTH cells. Further exposure causes the activation of the TDTH cells and secretion of cytokines to attract macrophages to the region. After their accumulation (1 to 3 days later) release of lytic agents by the inflammatory cells results in local tissue damage and clinical symptoms.

27. The End