------------------------------------------------------------------------------ TITLE: CELLULAR AND HUMORAL IMMUNOLOGY SOURCE: Dept. of Otolaryngology, UTMB, Grand Rounds DATE: April 15, 1992 RESIDENT PHYSICIAN: Robert Hoffman, MD FACULTY: Karen Calhoun, MD DATABASE ADMINISTRATOR: Melinda McCracken, M.S. ------------------------------------------------------------------------------ "This material was prepared by resident physicians in partial fulfillment of educational requirements established for the Postgraduate Training Program of the UTMB Department of Otolaryngology/Head and Neck Surgery and was not intended for clinical use in its present form. It was prepared for the purpose of stimulating group discussion in a conference setting. No warranties, either express or implied, are made with respect to its accuracy, completeness, or timeliness. The material does not necessarily reflect the current or past opinions of members of the UTMB faculty and should not be used for purposes of diagnosis or treatment without consulting appropriate literature sources and informed professional opinion." I. Characteristics of the Immune system A. Nonspecific Immunity 1. Barrier provided by the skin and lining of the aerodigestive tract 2. Acid in the stomach 3. Lysozyme (mucolytic enzyme that cleaves bacterial cell walls) 4. Complement cascade a. Directly lyses bacteria b. Is chemotactic for phagocytes, especially C5a c. Opsonizes bacteria (coats bacteria, makes them more readily recognized and destroyed by phagocytes), especially C3b d. Increases blood flow and capillary permeability 5. Acute phase proteins, ie, C-reactive protein which binds to a wide variety of bacteria and fungi and increases binding of complement 6. Commensal organisms in nose, mouth, throat, intestines, and vagina which colonize the mucosal surfaces and prevent overgrowth by pathogens 7. Phagocytes (neutrophils, monocytes, and macrophages) 8. Natural killer cells B. Specific Immunity Immune reactions distinguish between non-cross-reacting antigens. Specificity is mediated through the antigen- specific receptors on the surfaces of T and B lymphocytes and through antibodies. This immunity has the properties of memory, mobility, replicability and cooperativity. The immunologic response to an antigen stimulus usually leaves the immune system changed. Memory T and B cells are generated after initial contact with a foreign antigen. They produce either a faster and more vigorous response during the next encounter with the foreign substance (positive immunologic memory) or a lesser or no response (acquired tolerance or negative immunologic memory). II. Major Histocompatibility Complex While searching for antigens that are involved in the acceptance or rejection of transplanted tissue, a group of antigens were found which, when matched greatly improved the ability of a graft to survive. The name histocompatibility antigens was coined. This region, the Major Histocompatibility Complex (MHC), is the HLA gene cluster on chromosome 6. The MHC codes for three different types of proteins as determined by their structures and functions. Class 1 proteins consist of two polypeptides. The larger is encoded by the MHC, and this is non-covalently associated with the polypeptide Beta2-microglobulin which is encoded outside the MHC. Class 2 proteins consist of two non-covalently associated peptides referred to as Alpha chains and Beta chains both of which are encoded by the MHC. Class 3 proteins are those complement components which are coded by the MHC. In the MHC the main regions are D, B, C and A. It appears that the A, B, and C regions encode Class 1 proteins which act as cell surface recognition molecules which can be identified by cytotoxic T cells. The D region encodes for Class 2 proteins which are involved in cooperation and interaction between cells of the immune system. In man essentially all nucleated cells carry the antigens of the A, B and C regions in varying amounts, but the antigens coded by the D region have a restricted distribution. Region D tissue antigens occur only on B lymphocytes, macrophages, monocytes, activated T cells and possibly epithelial cells. III. Cellular Immune System All the cells of the immune system arise from pluripotent stem cells through two main lines of differentiation, the lymphoid lineage and the myeloid lineage. Lymphocytes are the unique bearers of immunologic specificity which depends on their antigen receptors. The full development and expression of immune responses, however, requires non- lymphoid cells and molecules primarily to act as amplifiers and modifiers. The antigen-presenting cell either internalizes antigen or presents antigen on its surface to T or B cells in various lymphoid compartments of the body. A. Myeloid cells 1. Polymorphonuclear Granulocytes Granulocytes represent about 60 to 70% of the total normal blood leukocytes but are also found in extravascular sites. Polymorphs are able to adhere to and penetrate the endothelial cells lining the blood vessels. The mature cell forms usually contain a multi-lobed nucleus and many granules. Although these cells do not show any specificity for antigens they play an important role in acute inflammation and together with antibodies and complement, in protection against microorganism, predominantly by phagocytosis. -Neutrophils - These represent over 90% of the circulation granulocytes. They possess two main types of granules. The primary granules (lysosomes) contain acid hydrolases, myeloperoxidase and muraminidase (lysozyme). The secondary or specific granules contain lactoferrin in addition lysozyme. Ingested organisms are contained within vacuoles termed phagosomes which fuse with the enzyme containing granules to form the phagolysosomes -Eosinophils - These comprise 2 to 5% of blood leukocytes. The granules in mature eosinophils are membrane bound organelles with a crystalloid core. They can be triggered to degranulate by appropriate stimuli. Degranulation releases the contents of the granules including histaminase and aryl sulphatase which inactivate the mast cell products histamine and Slow Reactive Substance of Anaphylaxis (SRS-A). The net effect is to dampen down the inflammatory response and reduce granulocyte migration into the site invasion. -Basophils and Mast Cells - These constitute less than 0.2% of circulating leukocytes. The granules in both basophils and mast cells contain heparin, Eosinophil Chemotactic Factor of Anaphylaxis, and SRS-A and these are released on degranulation initiated by the appropriate stimulus. This is usually an allergen which cross-links specific IgE molecules bound to the surface of the mast cell or basophil via Fc receptors for IgE. -Platelets - In addition to their role in blood clotting, platelets are also involved in the immune response, especially in inflammation. The possess receptors for both IgG and IgE. Following endothelial injury, platelets adhere to and aggregate at the endothelial surface releasing permeability increasing substances and factors responsible for activating complement components to attract leukocytes. 2. Antigen-Presenting cells Macrophages and dendritic cells constitute two broad groups of nonlymphoid mononuclear cells involved in immune responses. The monocyte/macrophages predominant role is to remove particulate antigens. They contain several lysosomes which contain acid hydrolases and peroxidase which are important in the intracellular killing of microorganisms. Adherence and ingestion is promoted when the cells bind the microorganisms through specialized receptors for IgG and complement with which the microorganism is coated. The function of these cells can be enhanced by factors released from T cells. In addition, they produce complement components, prostaglandins, interferons and Interleukins. Dendritic cells include Langerhans' cells, interdigitating dendritic cells (IDCs) and follicular dendritic cells (FDCs). IDCs are found in the T cell areas of lymphoid tissue, particularly the tonsils and adenoids, and FDCs are found in follicles of the tonsils and adenoids as well as the lymph nodes of the head and neck. Presentation of antigen in suitable form for either T or B cell recognition is required for the initiation of the primary immune response. B. Lymphoid Cells 1. T Cells Lymphoid precursor stem cells arise from the bone marrow and commit to development into one of two lymphoid pathways of development. Those that migrate to the thymus to mature are known as T cells. Intensive lymphocyte mitosis occurs within the thymus. Most of the cells generated die in situ. A few leave the cortex, enter the medulla and complete their maturation. They re- enter the circulation and proceed to the peripheral lymphoid tissue, lymph nodes, and spleen and take up characteristic locations in the pericortical sections of these tissues. The emerging cell has a dedicated specificity as expressed by the presence of a surface receptor structure for the recognition of a single antigenic determinant. The cells are therefore clonally restricted. The T cell receptor is a two chain, disulfide linked heterodimer which has many similarities to Ig. T lymphocytes are activated when their receptors detect fragments of foreign proteins that have complexed with MHC proteins of the surface of antigen presenting cells. T cells carry out a variety of functions, and, to some extent, these functions are associated with certain subsets of T cells, often defined by surface markers that react with certain monoclonal antibodies. All T lymphocytes react with OKT3 and Leu-4 antibodies and are given the nomenclature CD3. The T cells can then be subdivided into two major groups, the CD4 and CD8 cells. The CD4 group includes helper and inducer T cells. The helper cells participate in T-T cell and T-B cell interactions. These cells recognize antigen in the context of MHC class II components and help B cells produce antibody. These cells may also produce interleukin-2 (IL-2) when activated. Delayed-type hypersensitivity or inducer T cells carry out type IV delayed hypersensitivity reactions. They recognize antigen in the context of the class II components of the MHC. They carry out their effector, proinflammatory reactions mainly through production of T-cell cytokines such as IL-2. The CD8 group includes suppressor and cytotoxic T cells. The suppressor T cells also participate in T-T cell and T-B cell interactive mechanisms. The suppressor cells negatively regulate immune responses. Although they function in antigen specific ways, by down regulating T helper cells, it is not clear what sort of receptor they use for antigen recognition. These cells may be important in acquired immunologic tolerance and perhaps in autotolerance. The cytotoxic T lymphocytes recognize antigens in the context of the Class I MHC. When activated, they can lyse a target cell of that HLA specificity bearing the antigen. If a host is immune to a virus, a cytotoxic T cell can lyse cells that are infected with the virus. This are how these cells are classified: Classification Reactivity MHC recognition CD3 All T lymphocytes CD4 T Helper Cells Class II T Inducer Cells Class II CD8 T Suppressor Cells Unknown Cytotoxic Cells Class I T-Cell Activation Peripheral T cells are long lived and generally exist in resting states. When activated by the proper signals, they may carry out one or more of the following functions: proliferation, differentiation, production of lymphokines, and development of effector function. T cells can be activated specifically or nonspecifically. Specific activation occurs when a relevant clone of T cells meet the appropriate antigen in the context of the proper MHC component. An APC presents the antigen in the context of the MHC to the T cell that has a receptor for that antigen- MHC complex. An auxiliary signal, Interleukin-1 (IL-1), is also delivered by the APC. The T cell responds to activation by clonal expansion and often by production of lymphokines such as IL-2. Nonspecific activation occurs when many clones of T cells are induced by polyclonal activators such as the plant lectins or concanavalin A. These mitogens can stimulate large numbers of T cells by interacting with receptors on the T cell surface that are different from the antigen specific T cell receptor. Thus the response is polyclonal. A patients response to such a mitogen is a rough measure of that person's T cell capabilities. 2. B Cells B cells have their origin in stem cells, which give rise to cells destined for B cell development in the bone marrow. The earliest cells of the B cell series are recognizable by the fact that they bear certain B cell specific surface determinants detected by monoclonal antibodies. These early B cells have only a heavy chain for IgM in their cytoplasm and are called pre-B cells. They do not contain whole IgM molecules that bear surface IgM. Immature B cells, in turn, become mature B cells, which synthesize both IgM and IgD and bear these immunoglobulins on their surface. Further development of the B cell involves interaction of the cell with antigen as well as several antigen nonspecific factors derived from T cells. Such interactions initiate isotype differentiation, a process during which the B cell becomes a definitive IgM, IgG, IgE, or IgD B cell. Finally, B cells differentiate into immunoglobulin secreting plasma cells under the influence of other T cell factors. The resting, mature B cell uses its surface IgM as a receptor for the antigen with which it is clonally selected to react, but the function of surface IgD is unknown. B cells have other surface polypeptides including Class I and II products of the MHC and receptors for the Fc part of some Ig classes. B cell, like T cells, can be stimulated in their resting state to enlarge, to develop synthetic machinery, to divide, to mature, and to secrete antibody. The proper signals for this sequence depend on the type of triggers, which can be specific or nonspecific and polyclonal. Specific activation involves the antigen that is complementary to the particular Ig on the surface. Nonspecific activation occurs with B cell mitogens. Efficient antibody production to complex protein antigens requires T cell assistance. T cells, which were previously activated by APC presentation of a antigen, secrete a variety of lymphokines that when combined with the specific antigen trigger the B cell to develop into an antibody-secreting cell. This process also involves immunoglobulin class switching because the first B cells to make antibody make IgM, while later in the primary response and also in the anamnestic response, the preponderant Ig isotype classes are IgG, IgA, and sometimes IgE. Some antigens, particularly polysaccharide polymers can trigger B cells without help from T cells. These are called T independent antigens. In general, they are not strong, they provoke mainly IgM responses, and they induce little immunologic memory. 3. Null Cells These cells are large granular lymphocytes that are neither T cells nor B cells. They are characterized by the possession of Fc receptors for IgG and although probable of bone marrow origin their exact lineage is uncertain. It is believed that this population of cells contains the majority of natural killer and antibody dependent cellular cytotoxic effectors. Natural killer (NK) cells non-specifically kill tumor cells and virally infected cells and play a role regulating the immune response. Antibody dependent cellular cytotoxic cells (ADCC) also kill non-specifically but this occurs via antibodies bound to their cells. IV. Structure and Function of Immunoglobulins Immunoglobulins are B cell products. The Ig molecules are composed of poly peptide chains that are bound to each other, usually by disulfide linkages. Heavy and light chains are basic building blocks of Igs. IgG can serve as a prototype Ig molecule and consists of two identical heavy and two identical light chains. This basic four chain immunoglobulin subunit is referred to as a monomer. The N terminal regions of the heavy and light chains are the variable parts of the antibody as they differ in structure from one antibody to another. They form a uniquely shaped cavity into which the appropriate antigen fits. This end of the molecule is the fragment for the antigen binding portion and it confers immunologic specificity. The fragment for the antigen binding portion from each antibody is different and is called an idiotype. The C-terminal end of the molecule has a constant structure for each Ig class and subclass. This constant region confers various biologic functions on the Ig classes. For instance, the unique ability of IgG to be transported across the placenta depends on the structure of the that crystallizes (Fc) part of the IgG molecule. The C-terminal of the heavy chains exist as five classes or isotypes, namely, gamma, alpha, mu delta, and epsilon for IgG, IgA, IgM, IgD, and IgE, respectively. Differences between the constant regions of the heavy chains account for the different biologic functions of these Ig classes. Light chains themselves have both variable and constant regions, the former being at the N terminal end of the molecule. Light chains exist in two classed, kappa and lambda. Both heavy and light chains are made up of domains. Each domain contains 110 to 120 amino acid residues and also has an intrachain disulfide bond spanning 55 to 65 amino acid residues. Each light chain has two domain, variable and constant, and each heavy chain has one variable and 3 to 4 constant domains, depending on its class. IgA - This immunoglobulin is secreted by many tissues of the head and neck. It is certainly the principal class of immunoglobulin in most external secretions. It is made by plasma cells in glands and mucous membranes that oppose the outside world. IgA occurs in primarily two forms: a serum form and a secretory form. Secretory IgA has a distinctive extra polypeptide chain known as the secretory component. This helps it pass through cell membrane barriers. In addition, the two monomers of the IgA molecule that make up the secretory dimer are held together by a J chain. This J chain also exists on IgM molecules. J chains appear to be important in the assembly of polymeric forms of the immunoglobulins. The J chain is covalently bound to the monomers and allows them to acquire new biologic properties. It is important to note that IgA is a critical first line defense system that protects the body against invasion by microorganisms and the entrance foreign molecules. IgA antibodies are not absorbed into the bloodstream from the digestive tract and therefore exert all the beneficial effect locally. IgA molecules do not mediate anaphylaxis, chemotaxis, or fix complement. It functions as an anti- phagocytic that prevents phagocytosis and adherence to the mucous membrane. Thus it prevents binding of bacteria and especially viruses to the mucous membrane and their penetration into the mucosa. IgG - This is the most abundant Ig in the serum. It is an important antiviral and antibacterial isotype and a potent opsonin and toxin neutralizer. It is the only Ig that crosses the placenta and can provide passive immunity for the newborn for three to six months. It has an approximate half-life of three weeks and is one of the two Ig isotypes that fixes complement by the classic pathway. Four subclasses of IgG exist: IgG1, IgG2, IgG3 and IgG4. These differ because of small changes in the Fc portion of the heavy chain. Each of these subclasses of IgG has a definite function. For example, IgG1 and IgG3 appear to fix complement better than IgG4. IgG4 appears to be the most important immunoglobulin directed against polysaccharide capsules of bacteria. IgM - This large molecule is composed of five monomers. The subunits are arranged in a pinwheel-like array, with the Fc portions in the center, held together by joining chains. It is the isotype present in the cytoplasm and on the surface of B cells in the early stages of the maturation and it is the first antibody produced by activated B cells in the primary antibody response. IgM is an efficient activator of the classic complement pathway. IgE - This group contains the classic skin sensitizing, anaphylactic antibodies important in type I hypersensitivity. Most of its unique biologic properties depend on the fact that the Fc portion of the chain binds this molecule with high avidity to FcE receptors on mast cells and basophils. When these cell associated IgE molecules of a particular antigenic specificity are cross-linked by their appropriate antigen, the cells degranulate and the pharmacologic mediators of anaphylaxis and the type I reaction are released. IgE is present in small quantities in plasma and tissue but, because of its affinity for basophils and mast cells, it is an antibody class of potent biologic abilities. IgD - This immunoglobulin consists of two delta chains and two light chains. It is found on immature B cell surfaces and in low concentrations. Little is known about the true biologic significance of IgD. Function of Ig Classes: IgA Predominant Ig in seromucous secretions (saliva, tracheobronchial secretions) Dimer associated with "secretory component" (prevents proteolysis by digestive enzymes) and a "J" chain IgG Major antibody of secondary (anamnestic) responses Important activity against viruses, bacteria, parasites, and some fungi Only Ig class that crosses the placenta (provides 3-6 months immunity after birth) Fixes complement by classic pathway IgM Predominant antibody in early immune response Pentamer in association with a "J" chain Fixes complement by classic pathway IgE Found on basophils and mast cells Involved in response to helminthic infections and immediate hypersensitivity IgD Found in large quantities on circulating B cells May be involved in antigen induced lymphocyte proliferation V. Complement Antibody was discovered between 1880 and 1890, but it was revealed soon after that the ability of antibody to inactivate foreign material depended upon the collaboration of another factor, complement. Complement consists of a complex series of proteins many of which are proteinases. This system of enzymes non-specifically complements the immunologically specific effects of antibody by the opsonization and lysis of foreign matter. The complement system performs three vital functions: cell activation, cytolysis, opsonization (rendering cells vulnerable to phagocytosis by the adherence of opsonins). The proteins of the complement system form two interrelated enzyme cascades, termed the classical and alternative pathways. The classical complement system involves the activation of nine major protein components designated as C1 through C9. The first component activated is the C1 complex. It can be activated by IgG and IgM. Activation of C1 leads to sequential activation of C4 and C2. C2 becomes the C3 convertase which constitutes an important amplification step in the cascade by cleaving C3 into C3a and C3b and propagating the reaction. Subsequent activation of C5, C6, and C7 by C3b focuses the activities of the next components, C8 and C9, onto the target cell membrane. The C8 and C9 molecules insert themselves into the membrane and produce transmembrane channels. The passage of ions through these channels disturbs the cell membrane and the cell is lysed. The alternative pathway of complement activation does not require antigen-antibody complexes for initiation. The pathway may be triggered by lipopolysaccharides or endotoxin from the cell walls of gram negative bacteria, by the cell walls of some bacteria, by cell walls of some yeasts and by aggregated IgA. C3b exists in trace amounts in normal serum. It combines with a serum factor called factor B, forming a complex, C3bB. This complex is further activated by serum factor D, which cleaves factor B while it is attached to C3b to generate the enzyme complex C3bBb. This acts as a C3 convertase and continuation down the classic pathway is propagated. VI. The Interleukins Interleukin I (IL-1) or lymphocyte activation factor is secreted by activated macrophages. The antigen presenting cells produce IL-1 when they present antigen to the appropriate T cell. Thus, the T cell receives two signals; the antigen-MHC complex and IL-1. Without IL-1, T cell activation is minimal. It is therefore, a co-mitogen together with antigen. It promotes the production of another hormone, interleukin-2 (IL-2) or T cell growth factor by antigen-activated T cells and stimulates the expression of IL-2 receptor on T helper cells. IL-1 also affects other cells of the immune system such as the neutrophil. It stimulates their release from bone marrow and enhances their local accumulation, and degranulation and reactive oxygen intermediate secretion. It is also effective on fibroblasts, synoviocytes, hepatocytes, and osteoblasts. Interleukin 2 (IL-2) or T cell growth factor is a true T cell lymphokine produced by activated T cells in the presence of IL-1. The hormone acts on the T cells to stimulate clonal proliferation in the production and secretion of other lymphokines that are active on B cells. It also stimulates the clonal proliferation of natural killer cells and enhances their activity. Interleukin 3 (IL-3) or hemopoietic growth factor is a multiple colony stimulation factor and is also produced by activated helper T cells. It stimulates the proliferation of granulocytes, mast cells, monocytes, basophils, eosinophils, megakaryocytes, and erythroid cells in vitro but inhibits the development of NK cells. Interleukin 4 (IL-4) is a T cell derived, B cell active lymphokine. It is involved in stimulation of anti-IgM- activated B cells and can deliver a proliferative signal to the B cells that are activated in this way. It is therefore felt to be an important potential B cell activating factor. VII. The Interferons The interferons are a family of related cell regulatory glycoproteins produced by many cell types in response to viral infection, double stranded RNA, endotoxin, and variety of mitogenic and antigenic stimuli. Human interferons have been classified at alpha, beta and gamma. Alpha interferon is produced by leukocytes and decreases viral replication, increases cell membrane proteins, and decreases lymphocyte mitogenesis. Beta interferon is produced by fibroblasts and epithelial cells. Its functions are similar to alpha interferon. Gamma interferon or immune interferon is produced by activated lymphocytes and it increases expression of cell membrane antigens, including class I and class II MHC. This interferon plays an important role in the modulation and control of the immune response. It may enhance or suppress the immune response and its immunomodulatory roles include the regulation of antibody production, expression of cell surface antigens, regulation of NK activity, and enhancement of macrophage functions. ------------------------------------------------------------------------------- BIBLIOGRAPHY 1. Ballenger JJ. Diseases of the Nose, Throat, Ear, Head and Neck. Lea and Febiger, 1991. Chapter 4. 2. Benjamini E. Immunology. Wiley-Liss. 1991. 3. Claman HN. "The Biology of the Immune Response". JAMA 1987;258:2834-2840. 4. Cummings CW, et. al. Otolaryngology - Head and Neck Surgery. C.V. Mosby. 1986. Chapter 8. 5. Lee KJ. Essential Otolaryngology. Medical Examination Publishing Company. 1991. Chapter 14. 6. Roitt IM, et. al. Immunology. C.V. Mosby. 1985. ---------------------------END-----------------------------------------