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Transcript
Chapter 2. Immunology System I. Immunology A. Nonspecific defense (innate immunity, no memory) 1. Body surface (Physical defense) a) first line of defense b) antimicrobial chemicals found in sweat, sebaceous, and lacrymal glands. c) mucous: Particles adhere to it and are prevented from entering the blood d) hairs of nose, cough and sneeze reflex, acid secretion by stomach, and natural flora of microbes. e) Chemical elements : pH, Digestive enzymes.. 2. Phagocytosis : natural Killer cell(bacteria, cytolysis of virus-infected host cell) Note: Natural Killer (NK) Cells At least two types of lymphocytes are killer cells—cytotoxic T cells and natural killer cells.To attack, cytotoxic T cells need to recognize a specific antigen, whereas natural killer or NK cells do not. Both types contain granules filled with potent chemicals, and both types kill on contact. The killer binds to its target, aims its weapons, and delivers a burst of lethal chemicals. 3. Inflammation: A process in which blood vessels dilate and become more permeable, thus enabling body defense cells and defense chemicals to leave the blood and enter the tissues. Acute inflammation is usually a localized, protective response to tissue injury. Excessive or chronic inflammation, however, may lead to tissue destruction. Note : Inflammation 1. The Mechanism of Inflammation As a result of proinflammatory cytokine and chemokine release in response to injury or infection, mast cells in the connective tissue as well as basophils, neutrophils and platelets leaving the blood from injured capillaries, release or stimulate the synthesis of vasodilators such as histamine, leukotrienes, bradykinins, and prostaglandins. Certain products of the complement pathways (C5a and C3a) can also trigger mast cells to release their vasodilators. Vasodilation is a reversible opening of the junctional zones between endothelial cells of the blood vessels and results in increased blood vessel permeability Tissue and Blood Vessel During Inflammation 2. Benefits of Inflammation a. Plasma flows out of the blood into the tissue. This has the following benefits: 1. clotting factors cause fibrin clots to form to localize the infection, stop the bleeding, and chemotactically attract phagocytes; 2. antibodies enter the tissue to help remove or block the action of microbes 3. proteins of the complement pathways enter the tissue to: 1) stimulate more inflammation (C5a, C3a, and C4a), 2) stick microorganisms to phagocytes (C3b and C4b), 3) chemotactically attract phagocytes ( C5a), and 4) lyse membrane-bound cells displaying foreign antigens (membrane attack complex or MAC) 4. nutrients enter the tissue to feed inflamed tissue 5. lysozyme and beta-defensins enter the tissue to degrade peptidoglycan and cytoplasmic membranes 6. transferrin enters the tissue to deprive microbes of needed iron. b. Leukocytes enter the tissue through a process called diapedesis. 1. First, molecules called selectins cause the circulating leukocytes to slow their flow and roll along the inner blood vessel wall. 2. Proinflammatory cytokines such as Il-1 and TNF-alpha produced by cells at the injured or infected site then stimulate the endothelial cells that form blood vessels to produce chemokines such as interleukin-8 (IL-8). The chemokines are held on the inner surface of the endothelial cells where they interact with chemokine receptors on the surface of the rolling leukocytes. This interaction, in turn, trigger the activation of molecules called integrins on the surface of the leukocytes. 3. Activation of these integrins by chemokines enables the slowly rolling leukocytes to strongly bind to adhesion molecules such as ICAMs (intercellular adhesion molecules) and VCAMs (vascular cell adhesion molecules) on the inner surface of the vascular endothelial cells. (Macrophage-produced cytokines such as IL-1 and TNF-alpha induce the expression of these adhesion molecules on the vascular endothelium.) 4. Once bound to the endothelial cells, the leukocytes then flatten and squeeze between the endothelial cells to leave the blood vessels and enter the tissue. 5. The leukocytes are then chemotactically attracted to the injured site by the chemokines and other chemotactic factors mentioned earlier. The process of diapedesis, also known as emigration. Benefits of diapedesis include: 1. phagocytosis (neutrophils, monocytes/macrophage, and eosinophils phagocytes); are 2. more vasodilation (basophils, neutrophils, and platelets promote inflammation); 3. exposure of immunocompetent cells (B-lymphocytes and T-lymphocytes) to microorganisms and other antigens (discussed in Unit 3); 4. killing of infected cells and cancer cells by cytotoxic T-lymphocytes (CTLs) and NK cells (discussed in Unit 3). Cytokines called chemokines are especially important in this part of the inflammatory response. They play key roles in enabling white blood cells to adhere to the inner surface of blood vessels, migrate out of the blood vessels into the tissue, and be chemotactically attracted to the injured or infected site. They also trigger extracellular killing by neutrophils. 5. Fever :Activated macrophages and other leukocytes release leukocytic pyrogens (cytokines such as TNF-alpha and Il-1) which stimulate the anterior hypothalamus of the brain to produce prostaglandins which lead to an increase in body temperature. Up to a certain point, fever is beneficial. It can inhibit the growth of some microorganisms but more importantly the elevated body temperature increases the rate of enzyme reactions in the body thus speeding up the body's metabolism. An increase in the rate of metabolism can increase the rate of phagocytosis, the immune responses, and tissue repair. Too high of a body temperature, however, may cause damage by denaturing the body's enzymes. Finally, within 1 to 3 days, macrophages release the cytokines Il-1 and TNF-alpha which stimulate a proliferation of endothelial cells and fibroblasts. The endothelial cells form a fine network of new capillaries into the injured area to supply blood, oxygen, and nutrients to the inflamed tissue. The fibroblasts deposit the protein collagen in the injured area and form a bridge of connective scar tissue to close the open, exposed area. This is called fibrosis or scarring, and represents the final healing stage. Inflammation is regulated by cytokines. Proinflammatory cytokines such as interferon-gamma and interleukin-12 enhance the inflammatory response whereas the cytokine interleukin-10 inhibits inflammation by decreasing the expression of proinflammatory cytokines. So as can be seen, acute inflammation is essential to body defense. Chronic inflammation, however, can result in considerable tissue damage and scarring. With prolonged increased capillary permeability, neutrophils continually leave the blood and accumulate in the tissue at the infected or injured site. As they discharge their lysosomal contents and oxidizing agents, surrounding tissue is destroyed and eventually replaced with scar tissue. Antiinflammatory agents such as antihistamines or corticosteroids may have to be given to relieve symptoms or reduce tissue damage. 4. Enzyme degradation : the lytic enzymes released from granulocytes (e.g. lysozyme, muramidase), variety of serum proteins including the acute phase proteins, complement and kinins. 5. Interferon : inhibit viral replication inside host cells B. Specific defense (Acquired or adaptive immunity) In contrast to innate immunity, acquired immunity is specific and has memory: upon second contact with an infectious agent, the immune response will occur faster and will be stronger than upon first exposure to the same agent. The elements of the acquired immune response include the B lymphocytes and plasma cells, both of which secrete antigen-specific antibodies, and the T lymphocytes, which secrete cytokines and are cytotoxic for virally infected cells, tumors and foreign tissue grafts. II. Cells mediating immune responses Cells that mediate immune responses (leukocytes) fall loosely into four categories, the phagocytes, granulocytes, lymphocytes, and cytotoxic cells. Some cells fall into more than one category. For example, neutrophils contain granules and are phagocytic, so they are considered both granulocytes and phagocytes. A. Phagocytes: engulf and destroy foreign particles 1. Monocytes (mononuclear phagocytes) have horseshoe-shaped nuclei and circulate freely in the blood. When a monocyte passes out of the blood vessels and becomes fixed in a tissue, it is referred to as a macrophage. Tissue-fixed phagocytic cells form a multi-organ network called the mononuclear phagocyte system, formerly known as the reticuloendothelial system. a. b. c. d. e. f. g. h. i. Kupffer cells in the liver Alveolar macrophages in the lung Splenic macrophages Peritoneal macrophages Microglial cells in the brain Osteoclasts in the bones Dendritic cells in the lymphoid organs Langerhans cells in the skin Mesangial cells in the kidneys 2. Neutrophils (polymorphonuclear neutrophils, PMNs) are characterized by their multilobed nucleus and azurophilic granules. These cells account for 70% of all leukocytes and are vital for combatting bacterial infections. Because they disintegrate rapidly upon emigrating to tissue, they are most often found circulating in the blood. 3. Eosinophils destroy parasitic worms and participate in inflammatory responses. B. Granulocytes 1. Mediate inflammation by releasing their granule contents 2. Neutrophils, eosinophils, basophils and mast cells (histamine) C. Cytotoxic Cells; Cytotoxic cells kill other cells by producing lytic enzymes or reactive oxygen metabolites. Several of the cell types previously mentioned under other categories also have cytotoxic capabilities, including monocytes, macrophages, neutrophils, eosinophils and Tc cells. In addition, the natural killer (NK) cell can kill tumor cells and virus-infected cells. NK cells are also known as large granular lymphocytes (LGLs) but do not express antigen-specific receptors as B and T lymphocytes do. Note Phagocytes and Granulocytes Phagocytes are large white cells that can engulf and digest foreign invaders. They include monocytes, which circulate in the blood, and macrophages, which are found in tissues throughout the body, as well as neutrophils, cells that circulate in the blood but move into tissues where they are needed. Macrophages are versatile cells; they act as scavengers, they secrete a wide variety of powerful chemicals, and they play an essential role in activating T cells. Neutrophils are not only phagocytes but also granulocytes: they contain granules filled with potent chemicals. These chemicals, in addition to destroying microorganisms, play a key role in acute inflammatory reactions. Other types of granulocytes are eosinophils and basophils. Mast cells are granule-containing cells in tissue. III. Humoral immune response The definition of humoral immunity has evolved over the years. In the past, humoral immunity was synonymous with antibody-mediated immunity. However, the term now refers to any extracellular, soluble factor found in the blood or lymph that contributes to host protection. A. Acute phase proteins: These are a group of plasma proteins, many of which are produced by the liver, whose concentrations increase several-fold during an inflammatory response. Plasma proteins can be separated according to charge by electrophoresis through a gel matrix, yielding an electrophoretogram with 5 major peaks—albumin and the globulins alpha-1 (1), alpha-2 (2),beta () and gamma (). Acute phase proteins are found in all serum fractions except albumin. Albumin levels fall as acute phase protein levels rise during an infection; this is the body's attempt to keep serum protein concentration within the normal range (60-83 mg/ml). Electrophoretic patterns of the acute phase proteins vary with disease state. For example, an increase in the a2 fraction can indicate acute inflammation, while elevated levels of gamma globulins signal chronic infections, lymphocyte neoplasms and autoimmune diseases. Two important acute phase proteins are C-reactive protein (CRP), which binds to phosphorylcholine (the C-protein) of pneumococci, and complement, a heterogeneous group of serum proteins that are chemotactic for phagocytes and lytic for bacteria. Both CRP and complement are opsonins that facilitate phagocytosis of coated bacteria. B. Cytokines: "Cytokine" is a general term for a large group of protein molecules that signal between cells during immune responses. Cytokines produced by T lymphocytes are lymphokines, while those made by mononuclear phagocytes are monokines. 1. Interferons (IFNs) are produced very early during viral infections and are the first line of resistance against many viruses. One group (IFNand IFN) is produced by virusinfected cells. Another type (IFN) is released by T lymphocytes and NK cells. 2. Interleukins (IL-1 to IL-18) and colony-stimulating factors (CSFs) direct the division and differentiation of many cell types. Most ILs are made by T cells, while monocytes and macrophages are the usual sources of CSFs. 3. Tumor necrosis factor enhances inflammation and cytotoxic reactions, while transforming growth factor- suppresses them. C. Antibodies (immunoglobulins) are glycoproteins made by B cells and plasma cells that bind specifically to a single antigen. They are most important in combatting extracellular pathogens, since host cells are impervious to entry by antibodies and thus shield intracellular pathogens from their effects. - Memory - Specificity - Amplification 1. Antigens :as any foreign molecule that will stimulate antibody (Ab) formation when injected into vertebrate host. The new formed antibody will react (bind) specifically to the immunizing antigen. Antigen can be defined more broadly as any substance that can bind to specific antibody molecule or specific antigen receptors on lymphocytes. A distinct site (epitope) on the antigen can react with an antigen binding site (paratope) on the antibody. Types of Antigens Soluble and cell-bound proteins constitute the largest group of naturally occurring antigens. In addition polysaccharides, lipids and nucleic acids (often associated with proteins) are also antigenic. Complete antigens are multideterminant which posses several epitopes. Generally, there is one epitope for every molecular weight of 10,000 daltons. An antigen with a molecular weigh of 60,000 can have six different epitopes and react with six different paratopes and different antibodies. There can be as many different species of antibodies (or effector T cell clones) produced as there are functional epitopes on an antigen. Hapten An incomplete antigen (hapten) is a chemically defined substance of low molecular weight that cannot induce an adaptive immune response by itself. The hapten can react with the paratope on an antibody. Haptens can produce antibodies if they are conjugated to larger carrier molecule (normally proteins). Haptens contain a single epitope. The hapten is too small to engage T and B cells simultaneously. If the hapten is conjugated to a carrier protein, a T cell recognizes epitopes of this protein and a B cell recognizes the hapten. Antihapten antibodies are produce as well as other antibodies to the carrier molecule. The antihapten antibody will react with the hapten with out the carrier protein molecule. Landsteiner demonstrated that virtually any chemical substance can serve as an antigenic determinant or epitope if conjugated to a suitable carrier. He also found that antibodies can distinguish between structural similar haptens. Antibodies recognize overall threedimensional shapes of an antigenic determinant, rather than any specific chemical property. Epitope Characteristics The paratope of an antibody that is specific for an antigen binds noncovalently to a region of the molecule know as the epitope. Naturally occurring epitopes are relatively small about four to six amino acids or sugar residues. The convex epitope area might be only 2 X 3 nm across. At these dimension epitopes can fit into the antigen binding site (paratope) of the antibody. Large antigens (proteins or other macromoleculse) can possess many different epitopes on the same molecule. If an antigens has four different epitopes it could stimulate the production of four different antibody molecules with different paratopes. The antigen binding site (paratope) is concave pocket which match the convexity of the epitope. The paratope and epitope must come in close contact with each other since the binding forces are short (4 A) and weak. Forces of the Antigen Antibody Reaction The forces are hydrophobic, and Van der Waals forces which are spherical symmetrical. Hydrogen bonds are also involved which are directional and require matching reactants. Antigen and antibody reactions only take place when the epitope and paratope fit tightly. The hydrophilic regions of protein surface are the most likely to be antigenic; however the entire exposed surface could be antigenic. Normally the functional epitopes are exposed epitopes and are hydrophilic. These protruding parts of the molecular surface are generally most accessible to antibody molecules. Some components of an epitope are more immunodominant than others. When the epitope is a terminal sequence, the terminal residue of that sequence is almost invariably the immunodominant subunit. Some amino acids contribute to the epitope more than others. Some Amino Acids Are More Important Than Others Since antibodies bind to antigen by noncovalent binding through charged groups and hydrophobic groups then amino acids like lysine and glutamic acid (charged amino acids) and alanine leucine, tyrosine, phenylalanine, and valine (hydrophobic amino acids) are important in the formation of the epitope. Sequential and Conformational Epitopes Epitopes whose specificity is determined by the sequence of subunits are designated sequential determinants. Sequential determinants can be composed of terminal or internal sequences of the antigen. Sequential epitopes are located in the hydrophilic part of the molecule. Conformational determinants depend on the folding of the molecule. Conformational epitopes are normally associated with globular proteins and helical structures. Epitopes can be continuous or noncontinuous. Discontinuous epitopes are epitopes which are separated by amino acid sequence and are brought together by folding. Conformational epitopes are determinants which can be sequential discontinuous but sequential epitopes are always continuous. 2. Immunogenicity Immunogenicity refers to the relative ability of an antigen to stimulate the immune responses. In order for a molecule to be considered an antigen, it need only be capable of binding to a specific antibody molecule (or T cell receptor). Antigens that provoke the immune responses are immunogenic and can be called immunogens. Characteristic of immunogens 1. Size 10,000 daltons or greater. 2. Foreignness 3. Molecular complexity 4. Concentration. Every antigen has an optimal immunogenic dose range. The amount of antigen can be quite critical. Too much or too little antigen might not elicit an immune response. 5. Adjuvants : The response to antigens can be increased by use of adjuvants. Ajuvants contribute to greater and prolonged antibody production and increased effector cell counts. Note Adjuvants can react in several ways 1. Alter the distribution and persistence of antigen within the host. 2. Stimulate lymphocytes production non specifically. 3. Activate macrophages. 4. Alter traffic of circulating lymphocytes. * Freund adjuvant consist of oil and Mycobaterium tuberculosis. This adjuvant can not be use in humans. 6. Routes of injection: 1. Into the muscle (intramuscular IM) 2. Under the skin (subcutaneous) 3. Into a vein (intravenous IV) 4. Into the abdominal cavity (intrapeitoneal IP) 5. Into the skin (intradermal, Usually multiple injections) Antibody I. Isolation and characterization Antibody structure is difficult to study because antibodies are very heterogenous. Structural studies were facilitated by the discovery of myeloma proteins, homogeneous immunoglobulins produced by the progeny of a single plasma cell that has become neoplastic and reproduced itself endlessly in the disease called multiple myeloma. Another aid to structural studies of antibodies was the discovery of Bence-Jones proteins in the urine. These homogeneous proteins, produced in large quantities by some patients with multiple myeloma, are dimers of antibody light chains. II. Structure of heavy and light chains Even before purified preparation of antibodies were readily available, early workers deduced considerable information about the structure of immunoglobulins. In the 1950, Rodney Porter treated IgG from rabbits with papain which cleaved the molecule into three fragments. Two of the fragment are called fragment antigen binding (Fab) and one fragment is called fragment crystilizable (Fc). The Fab fragment retains its antigen binding ability and Fc can be crystillized which means that it must be pure. Fc has carbohydrates and has many effector functions (binding complement and binding to cell receptors) expressed by antibodies. The antigenic determinant that distinguish one class of antibody from another is located on the Fc fragment. The digested material was separated on a carboxymethylcellulose (CM) column, which separates molecules on the basis of charge. A CM column has negative charges and binds positively charged molecules. The fragments that bind are eluted with a salt gradient. Each fragment (Fab and Fc) has a sedimentation coefficient of 3.5 S and a molecular weight of 50 kD. Pepsin was also found to digest IgG into fragments (F(ab')2 and pFc fragments). By 1960 Gerald Edelman treated IgG with reducing agents, such as mercaptoethanol, in the presence of urea and split the molecule into four peptides (disulfide bonds were reduced). Reassociation was prevented with an alkylating agent such as iodoacetate. The chains were separated by size on Sephadex column. They found two L chains with a molecular weight of 25 kD and two heavy chains with 50 kD. With the availability of homogeneous Igs, it soon became evident that the two H chains were always identical in a given molecule, as were the L chains. H chains contain about 440 amino acids, whereas L chains possessed about 220 amino acids. The variable (V) region different between two L chains from different molecules. The constant region of the L chain is the same amino acid sequence in different amino acids. A similar pattern was found in the H chain. The variable region in the H chain is only about 25% as long as the C region. Each domain consist of about 110 amino acids. Each domain Contains a intrachain disulfide bridge of about 60 residues. Heavy Chains are distinguishable on the basis of amino acid sequences in their C region and by the number of domains. L chains are distinguished by the amino acid sequences in their C regions. Antibodies contain kappa or lambda chains but not one of each. Only about 5% of mouse antibodies have lambda chains where as 40% of humans contain kappa chains. Human Kappa chain has about four subgroups, while lambda has about six sub groups. Each subgroup contains certain amino acids in defined positions that are never changed, while other amino acids that localize nearby may display pronounced variability Light Chain Variable Regions Hypervariable subregions were observed in three sites within Kappa and lambda variable subregions. Each hyper variable region contains about 10 amino acids, which serve as contact residues for antibody binding. They function as complementary determining regions (CDRs) with respect to the binding of an antigen. The three hypervariable sub regions (CDRs) of the L chain and the CDRs on the H chain determine antibody specificity for antigen. In the variable region there are areas that are fairly constant in between the CDRs. Index of variability Measurement of variability at any amino acid position may be calculated by the following equation. Variability = Number of different amino acids found at that point/Frequency of the most common amino acid at that position. Thus, the index can range from 1, for a completely invariant position, to a maximum of 400 for a position in which all 20 amino acids are different. V = 1/1 =1 V= 20/(1/20) = 400 III. Ig and Ig Antibody molecules anchored to the surface of B cells are closely associated with secondary components Iga and Igb, which are thought to couple the antibody to intracellular signalling pathways. If an antigen binds to surface-bound antibody, tyrosine kinases become activated and phosphorylate target sequences in the Iga and Igb chains. These then bind another kinase that activates phospholipase C, which in turn acts on membrane phosphatidyl inositol to generate the second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release of internal Ca+2 stores and DAG activates protein kinase C. Together with other kinases, protein kinase C phosphorylates a number of surface molecules, leading to induction of transcription factors and subsequent expression of B cell genes. IV. Classes of Antibodies Since antibodies have a high molecular weight they make very good antigens and we can produce antibodies against another antibody. Differences in amino acid sequences between antibody classes, subclasses, and types can be determined by antigenic specificity of antibodies. Antibodies carry three major categories of antigenic determinants: isotypic, allotypic, and idiotypic. A. Allotypic Determinants : The second group of antigenic determinants are those found on the Igs of some, but not all, members of a particular species. Allotypic determinants reflect genetic polymorphism of Igs within one species. Antibodies can be formed against an allotypic determinant by injecting Igs into another member of the species that does not possess the alloantigen. In human the Gm antigens are a good example. B. Idiotypic Determinants : The third group of antigenic determinants of Igs exist as a result of unique structures generated by the hypervariable subregions (or complementarity-determinging regions CDR) on L and H chains. The antigenic determinants are called idiotopes, analogous to epitopes of classical antigens. Alpha-idiotopes lie outside the Ag-binding site. Beta-idiotopes are close to the antigen binding site. Gamma-idiotopes are formed by the antigen binding site. Antiidiotypic antibodies may be formed in self naturally or induced artificially in self. The significance of these molecules relates to their importance for immune regulation. C. Isotypic Determinants : These determinants distinguish the C regions of the H chain classes (and subclasses) and the L chain types. Each isotype is encoded by a distinct gene that is characteristic for a particular mammalian species and is present in all members of that species. Antibodies to these determinants may be raised by injecting IgG from one species into another species. Each Ig class (IgG. IgM, IgA, IgD, and IgE) possesses a specific isotypic determinant on its H chain. IgG IgG is the major immunoglobulin in normal human serum, accounting for about 70% of total immunoglobulin. There are four subclasses (subisotypes) in humans, ranging in size from 146,000 to 170,000 daltons. About 10% of the amino acids in the constant regions of the IgG subclasses differ from one subclass to another. All IgG heavy chains possess 3 constant regions. The table below lists the most important features of the IgG subclasses. In addition to those functions listed, IgG is the major antibody class that can neutralize toxins; IgA may play a minor role in neutralizing some toxins within the gastrointestinal tract. Important Differences Between Human IgG Subclasses IgG1 IgG2 70 20 Occurrence (% of total IgG) Half-life (days) Complement binding Placental passage Binding of monocytes IgG3 7 3 23 23 + + +++ - ++ + ++ ++ + +++ + +++ 7 IgG4 23 IgM IgM accounts for ca. 10% of the immunoglobulin pool and is the predominant immunoglobulin of the primary immune response. It is the largest antibody, with a MW of 970,000 daltons. IgM is composed of five IgG-like structures joined together by additional disulfide bonds between the Fc portions and by the J chain polypeptide (15,000 daltons). Mild reduction with mercaptoethanol reduces the binding efficiency of IgM to antigen. The maximum valence of IgM is 10. Because of its pentameric structure, IgM can form extensive latticeworks with complex antigens. Therefore, IgM is especially well suited for the agglutination of microbes. It is also quite efficient at fixing complement. IgM antibodies include the natural isohemagglutinins--the naturally occurring IgM antibodies against the red blood cell ABO antigens. IgM is the only class produced by the fetus. IgA IgA exists either as serum IgA or as secretory IgA (sIgA). Secretory IgA is the predominant antibody in seromucous secretions such as saliva, colostrum, milk, sweat, mucus, tracheobronchial and genitourinary secretions. There are two subclasses, IgA1 and IgA2. IgA1 is the predominant subclass in serum. IgA2 predominates in secretions because many microorganisms in the respiratory and gastrointestinal tracts produce proteases capable of cleaving IgA1. In humans, secretory IgA always occurs as a dimer of the basic four-chain structure and has a MW of 385,000. It is associated with a second protein, the secretory component, MW 70,000 daltons. Polymeric serum IgA and all sIgAs contain a J chain (15 kDa). The secretory component of sIgA is synthesized by epithelial cells. It facilitates the transport of sIgA into secretions as well as protecting the molecule from proteolytic enzymes. Secretory IgA traverses mucosal membranes and prevents entry of infectious microorganisms through the barriers of the respiratory and gastrointestinal tracts. IgD IgD is similar to IgG in structure. IgD accounts for less than 1% of total plasma Ig. It is present in large quantities on circulating B lymphocytes and is believed to play a role in antigen-triggered lymphocyte differentiation. IgD has a tendency to undergo spontaneous proteolysis. IgE (reagin) IgE is similar to IgG in structure, except that the e heavy chain contains four constant regions instead of three. IgE is a trace serum protein found on the surface membranes of basophils and mast cells in all individuals. This class originally evolved to combat helminthic infections, but in developed countries it is more commonly associated with immediate hypersensitivity diseases such as asthma and hay fever. * Summary Molecular weight(monomer) Additional protein subunit mg/ml in serum % total Ig Half-life (days) Placental passage Presence in milk Fixes complement Binds to FcR on MO&PMNs Agglutinating capacity Antiviral activity Antibacterial (gram -) IgG 150K None 12 80 23 ++ + + ++ + +++ +++ IgA 160k J & SC 1.8 13 5.5 + ++ +++ ++ IgM 970k J 1 6 5 0-trace +++ +++ + +++ IgD 180k None 0-0.4 0.2 2.8 - (+lysozyme)(+ complement) Antitoxin activity +++ Allergic activity - + (GI tract) - - - - ++ IgE 200k None 0.00002 0.002 2.0 - V. Type of Antigen-Antibody Reaction Serological reactions. 1) Agglutination reactions : Ab and particulate antigens (RBC or bacteria) Agglutination of bacteria has been a key method in identifying and classifying microorganisms. 2) Precipitation reactions: Ab and a fluid antigen. Precipitation reactions are most useful since most antigens are soluble or can be solubilized by simple procedures. In addition, there are many types of precipitation test which can be adapted to many types of antigens. In many cases nonprecipitating antigen-antibody reactions can be detected indirectly with other antibodies, isotopes, or enzymes or fluorescent dyes. Reaction Phase The antigen-antibody reaction takes place in two phases . In the first phase, combination of the reactants occurs; this is followed by second phase, an aggregation. The first phase stages take place almost instantaneously. In the second stage of the reaction, aggregation of the antigenantibody complex occurs. This phase does not occur when monovalent antibodies or haptens are involved in the reaction. The lattice hypothesis, which is based on multivalent antigens and bivalent antibodies, is useful in explaining the zone phenomena. At the equivalence point all of the antigen and all the antibody molecules are consumed in the lattice formation. With multivalent antigen where the antigenic site is not repeated, it requires two different antibodies directed at different antigenic sites to form a lattice. When there is an excess of antibody (prozone) , preciptitation is not observed. Each antigen is surround by antibodies. Essentially the reverse situation occurs in ( postzone) , where too little antibody is present to produce an aggregation each antibody molecule only binds to one antigen. The size of the aggregation complex increases as the optimal ratio of antigen to antibody is achieved. This is referred to as the (equivalence zone) . Antigen-Antibody binding The antigen-antibody binding take place by noncovalent bonding between the epitope (active site on antign) and paratope (antigen binding site on antibody). The antigen-antibody binding is strong because of the multiple hydrogen bonds, ionic bonds, van der Waal force, and hydrophobic interactions. The reactants must be close and their structures (antigen and antibody) must be complementary. The reaction is reversible and neither the antigen or antibody has changed. Antibody affinity refers to the strength of binding between the paratope of the antibody and the epitope of monovalent antigen or hapten. The term Avidity is used to designate the strength of binding of a multivalent antigen with an antibody possessing at least two combinding sites or antiserum containging antibodies of differing specificities. IgM with ten binding sites or an IgG molecule, with two binding sites, might become bound to an antigen possesing seral identical determinants. The avidity of these Ab-Ag binding reaction is greater than the sum of the intrinsic affinities, since all bonds must be broken simultaneously. Measurement of Antibody Affinity The intrinsic affinity of an antibody can be expressed in terms of an affinity constant (K). This constant can be used to express antigens affinity for an antibody. r/c = - Kr + Kn Equation 1 Equation 1 is a equation of a straight line r/c = - Kr + Kn y =mx + b In equation 1 r and c are variables, while K and n are constants. Thus , if r/c is plotted against r, the slope will be -K, the y intercept on the vertical axis will be Kn and the x intercept on the horizontal axis will be n, the valence. Plotting of compartments equilibrium is ligand between I II until reached. The and difference between the two curves reflects the percntage of total ligand bound to antibody compartment I. Precipitation reactions Most commonly used methods to characterize monoclonals and soluble antigens : 2D immunoelectrophoresis rocket electrophoresis countercurrent electrophoresis radial immunodiffusion immunoelectrophoresis Western blot,. radioimmuno assay fluorescent antibody assay Special Topic : Techniques in Immunology 1. Fluorescence-Activated Cell Sorting Cells can now be analyzed and isolated on the basis of their distinct surface antigens, their size, or both by a process known as flow cytometry. Flow cytometery are instruments that can analyze properties of single cells as they pass throug an orifice at high velocity. These intruments measure light scatter, volume, and fluorescence. FACS can analyze and sort lymphocyte sub populations, as identified by fluorescein-labeled monoclonal antibody. A suspension of leukocytes is incubated with labeled monoclonal antibody against the CD4 helper cells. The washed sample is then introduced into the sample chamber of the cytometer and the cells are forced through a nozzle in a liquid jet. With vibration at the nozzle tip, the stream breaks up into droplets. The size of the droplet can be regulated so that each contains a single cell. The cells pass in front of a laser beam, which excites the fluorescent dye (which is monitored by fluorescent detector). The droplets that emit appropriate fluorescent signals electrically are charged in a high-voltage field between the detector and deflection plate and separated into collection tubes. CD4 helper cells can be separated from other leukocytes of the suspension on the basis of the fluorescence of the bound antibody. 2. Monoclonal Antibody Production. The desired cells are found and separated in a three-step process: selection, screening, and cloning The only application presented in this paper will be the use of monoclonal antibodies in immunoassays. The major advantages of monoclonal Abs are: (1)highly specific antibodies can be produced in large quantities; (2)pure antigens are not necessary for production of monoclonals; (3) clones can be frozen and reused at later date as a standardized reagent.The disadvantages of monoclonal antibodies are: (1) the level of technology required; (2) the cost; (3) the time required for production; (4) and in some cases the antibody may be too specific. 3. Enzyme-Linked Immunosorbent Assay (ELISA) : - diagnostic tests have been available commercially since 1981 (microorganisms, hormones, aflatoxins, drugs, tumor markers and pesticides) - sensitive, inexpensive, safe, and easy-to-use assays. 4. Western Blot Analysis VI. Cells and Tissue of the Immune Response 1. Organ - Primary Lymphatic Organ : a) Bone Marrow : In adult animals, red bone marrow serves as the major source of all blood cell, including lymphocytes. The bone marrow is found in most bones including the skull, ribs, sternum femur, an spine. The distribution of red marrow diminishes with age. The bone marrow is the major site of hemopoiesis (production of red blood cells). Within the sinusoids are found blast cell which are the precursors of the different blood cells. Bone marrow contains various intermediate and mature forms of erythrocytes, monocytes, granulocytes, lymphocytes and megakaroyocytes. Bone marrow is not only the source of all blood cell classes, but it provides the environment for the antigen-independent differentiation. of B cells. The bone marrow may provide an antigen-processing environment, as does the spleen. b) Thymus - Secondary Lymphatic Organ; Organs of the secondary lymphoid system are spleen, Adenoids, tonsils, peyer's patches of the gut, and appendix. The secondary lymphoid organs are where mature T and B cells have the opportunity to bind antigen and undergo further antigen dependent differentiation. The active immune response both cell mediated and humoral immunity begins. All of the secondary lymphoid organs are capable of trapping antigen which normally involves macrophages. These organs contain recirculating lymphocytes, which pass from the blood through the lymphoid tissues back to the blood, it virtually ensures that circulating antigen react with lymphocytes with the proper receptors. The size of the lymphocyte pool suggest that there are lymphocytes available to fit any type of antigen. During the course of normal lymphocyte circulation, lymphocytes leave the blood and accumulate in lymphatic tissues, as if by a homing instinct, to set up B and T cell areas. Here they reside temporarily before returning to the circulation. The steadily input and output of cells among the lymphoid organs is disrupted during a primary immune response. Within 24 hours the antigen becomes localized in the lymph nodes and spleen and this depletes antigen-reactive cells from the circulating pool of lymphocytes. This temporary cessation of lymphocyte recirculation is called lymph node shutdown. Three day later, activated lymphocytes are released into the circulation. This delivers cells of the immune response (B and T cells) to tissue and blood stream. The B cell release antibodies and the T cell release lymphokines and antigen-specific effector T cells. a) Spleen: The spleen is a large encapsulate lymphoid organ which filters out antigen from the blood stream. The cortex of the spleen contains packed lymphocytes with germinal centers and a medulla containing a variety of cell types. Basically there are two types of tissues in the spleen referred to as the lymphoid, or white pulp, of the cortex and the erythoid or red pulp, of the medulla. The T cells are diffusely packed in the white pulp, with the follicles primarily composed of B cells. Most lymphocytes enter and leave the spleen by way of the bloodstream. The red pulp is involved in removing old red blood cells and is reserve site for hemopoiesis during pathological states. b) Lymph nodes : Lymph nodes are small, bean-shaped organs that act as filters. They are located at major junctions of the network of lymphatic channels. They consist of an outer cortex and an inner medulla. If antigens enter the body via the respiratory or gastrointestinal tracts, they must pass through the lymph nodes. B cells and plasma cells are accumulated in subcapsular follicles, while T cells occupy space between them in the diffuse cortex. c) Adenoids d) Mucosa-associated lymphoid tissue (MALT) :Tonsils, Peyer's patches and appendix.: The bulk of the body's lymphoid tissue (>50%) is found associated with the mucosal system. MALT is composed of gut-associated lymphoid tissues (GALT) lining the intestinal tract, bronchus-associated lymphoid tissue (BALT) lining the respiratory tract, and lymphoid tissue lining the genitourinary tract. The major effector mechanism at these sites is secretory immunoglobulin A (sIgA) secreted directly onto the mucosal epithelial surfaces. Examples of MALT include the Peyer's patches lining the small intestine, the tonsils and the appendix. Diffuse accumulations of lymphoid tissue are seen in the lamina propria of the intestinal wall. The intestinal epithelium overlying the Peyer's patches is specialized to allow the transport of antigens into the lymphoid tissue. This particular function is carried out by epithelial cells termed "M" cells, so called because they have numerous microfolds on their luminal surface. M cells absorb, transport, process and present antigens to subepithelial lymphoid cells. 2. Cell lineages : All leukocytes originate in the bone marrow from self-renewing haemopoietic stem cells. These stem cells give rise to four major cell lineages: erythroid (erythrocytes), megakaryocytic (platelets), myeloid (granulocytes and monocytes/macrophages), and lymphoid (T and B lymphocytes and natural killer cells). VI. Major Histocompatibility Complex Histocompatibility Systems Transplantation from one person to another are generally not successful because of the immune system. In an attempt to elucidate the genetic basis of graft rejection, investigators became interested in histocompatibility gene products that determine resistance or rejection. Later it became clear that these gene products participate not only in transplantation rejection, but are significantly involved in regulating immune responsiveness involving cell-to-cell interactions and in turn, susceptibility to a number of diseases. - Structure of MHC molecules There are two main kinds of MHC molecules, class I and class II. Both kinds of molecules belong to the immunoglobulin superfamily, but they are NOT immunoglobulins. Class I MHC molecules are found on all nucleated cells, while class II molecules show a more restricted expression pattern. Class II molecules are always expressed by B lymphocytes, interdigitating dendritic cells, and thymic epithelial cells. Other cells, such as macrophages, activated T lymphocytes, and endothelial cells can be induced to express class II molecules by cytokines such as IFN. Class I MHC molecules are composed of an chain (45 kDa) and a small peptide called 2-microglobulin (12 kDa). Class II MHC molecules are composed of an chain (30-34 kDa) and a chain (26-29 kDa). The structure of MHC class I and class II molecules The extracellular domains of both class I and class II molecules show variability in their amino acid sequences, yielding grooves with different shapes. The grooves cradle processed antigens, holding the antigens steady for interaction with the TCR on T cells (figure, next page). CD8 TC cells recognize antigen in association with class I MHC molecules, while CD4 TH cells recognize antigen in association with class II MHC molecules. A scheme for the structure of the ternary complex between MHC class I, foreign antigenic peptide, the TCR and CD8. The peptide is located in the groove formed by the The region contacts the binding site of the TCR formed by the six hypervariable regions of the V and V domains. - Antigen processing and presentation The antigen-presenting cell (APC) degrades antigen into small peptides (processing), links the peptides to MHC molecules, and then expresses the peptide/MHC complex on its cell surface (presentation). Whether an antigen peptide binds preferentially to class I or class II MHC molecules depends upon how that antigen entered the cell. Antigens that are produced within the cytoplasm of the APC tend to associate with class I MHC molecules. A good example of a cytoplasmic (endogenous) antigen is a viral protein synthesized by an infected cell. Exogenous antigens (those produced outside the APC, e.g. most extracellular bacterial proteins) that are taken into the APC by phagocytosis, endocytosis or pinocytosis tend to associate with class II MHC molecules. A. MHC class I-peptide association (endogenous processing) Within the APC, cytoplasmic antigens are that are denatured or tagged by ubiquitin are degraded by organelles called proteasomes, which have a variety of endopeptidase activities. Two genes, LMP2 and LMP7 (low molecular-mass polypeptide), encode proteasome components. The degraded peptides are then transported by proteins known as TAP-1 and TAP-2 (transporter of antigenic peptides), to the rough endoplasmic reticulum (RER). Within the RER, the antigen peptides slip into the grooves of class I MHC molecules which have been synthesized in the RER. The class I/antigen peptide complex is then transported to the APC surface where it is available for interaction with CD8 TC cells. B. MHC class II-peptide association (exogenous, endosomal or endocytic processing) Like class I molecules, class II MHC molecules are synthesized in the RER. The class II and chains reside there as a complex with an additional polypeptide called the invariant chain (Ii). The invariant chain blocks the groove of the class II molecule and prevents endogenous antigens from binding there. The /invariant chain complex is transported to an acidic endosomal or lysosomal compartment that contains a degraded antigen peptide. The invariant chain comes off the complex, exposes the groove of the class II molecule, and allows the antigen peptide to slip into the groove. The class II/antigen peptide complex is then transported to the surface of the APC where it is available for interaction with CD4 TH cells. Model of separate antigen-presenting pathways for endogenous and exogenous antigens. Routing of class I and class II MHC molecules to separate intracellular compartments may determine the type of antigenic peptide that each class of MHC molecule binds. Endogenous (cytoplasmic) pathway. Class I MHC molecules are thought to interact with peptides that have been degraded by proteasomes, part of a large cytoplasmic proteolytic complex called the low-molecular-mass polypeptide (LMP). The degraded peptides are carried into the rough endoplasmic reticulum (RER) by the transporter of antigenic peptides (TAP). Upon peptide binding, the interaction between a class I MHC chain and 2-microglobulin is stabilized and the complex is routed through the Golgi to the plasma membrane. Exogenous (endosomal) pathway. The presence of the invariant chain prevents peptide binding to class II MHC molecules within the endoplasmic reticulum and facilitates their routing to an endosomal compartment. Late in the exogenous processing pathway, endosomes containing class II MHC molecules and the invariant chain fuse with lysosomes. Enzymes within the lysosomes degrade the invariant chain, enabling the class II MHC molecules to bind peptides. C. Clinical applications employing knowledge of processing pathways The accumulating knowledge about different antigen-processing pathways has important implications for the design of new vaccines. If a class I-restricted TC response is desired, then a vaccine must be processed via the endogenous pathway. This requirement is met by attenuated live viral vaccines capable of limited growth within the cytoplasm of infected cells, and by vaccine molecules delivered to the cell's cytoplasm packaged in liposomes or immunestimulating complexes (ISCOMs). Vaccines intended to elicit a humoral antibody response must possess epitopes that are recognizable by B cells. The B cell binds to the antigen via surface antibody, endocytoses the antibody-antigen complex, and then processes the antigen via the exogenous pathway. The B cell presents the processed antigen complexed to class II molecules to TH cells, which are in turn stimulated to secrete cytokines necessary for B cell proliferation and differentiation into plasma cells. D. The role of accessory molecules in antigen presentation The binding between the TCR and the MHC/antigen peptide complex is highly specific and acts as the first signal to induce T cell activation. Activated T cells differ from resting T cells in that they proliferate and secrete lymphokines and/or lytic substances. However, the affinity of the TCR for the MHC/antigen complex is often too low to fully activate the T cell. There are a number of accessory molecules that increase the avidity between the T cell and APC by performing an adhesive function. As mentioned previously, the CD4 and CD8 accessory molecules on the T cell have a weak affinity for MHC class II and class I molecules, respectively. CD4 and CD8 interact with the MHC/antigen peptide complex, thus stimulating intracellular events that cause T cell activation. Additional co-stimulatory molecules found on APCs, illustrated on the next page, include intercellular adhesion molecule-1 (ICAM-1), which is known to interact with lymphocyte functional antigen-1 (LFA-1) present on all immune cells, and LFA-3, which interacts with CD2 on the T cell. The most potent co-stimulatory molecules known are termed B7-1 (CD80) and B7-2 (CD86). These molecules are constitutively expressed on interdigitating dendritic cells and can be up-regulated on monocytes, B cells and other APCs. They are the ligands for CD28 and its homolog CTLA-4, a molecule expressed on activated T cells. CD28 prolongs and augments production of several cytokines including interleukin-2 (IL-2), which causes T cell proliferation. CTLA-4 has the opposite effect; it downregulates IL-2 production by activated T cells to turn off the immune response. Co-stimulatory molecules that reinforce the signal from the TCR and induce positive activation of T cells have been termed the second signal, without which T cells cannot respond to antigen. - The genetics of the MHC (HLA) In humans, the MHC is known as the HLA (human leukocyte antigens) and is located on chromosome 6. The organization of the genes that encode histocompatibility molecules is illustrated below. You will note that genes important in antigen processing (LMP and TAP) are located in the class II region. 2-microglobulin is encoded on chromosome 15. A. Class I HLA genes In humans there are three major loci (HLA-A, HLA-B and HLA-C) that code for class I molecules. Within a locus, there are different allelic forms of the molecules. For example, HLA-A2 and HLA-Aw68 class I molecules differ from one another at 13 amino acid residues. These differences give rise to dramatic differences in the shape of the groove of the molecule and the foreign peptides it can accommodate. The alleles are expressed codominantly, so a cell can express both HLA-A2 and HLA-Aw68 simultaneously if both genes are inherited. B. Class II HLA genes The genetic locus encoding class II molecules is known as the D region in humans. The D region is further subdivided into DP, DQ, and DR. Like the HLA class I genes, HLA-D class II genes exhibit polymorphism and codominant expression. C. Class III HLA genes Class III HLA genes encode complement components that show no structural similarity to either class I or class II molecules. These genes, along with genes encoding tumor necrosis factor (TNF), separate HLA class II and class I genes on the chromosome. - MHC restriction T cells can only recognize antigen when it is complexed to a self-MHC molecule. This is known as MHC-restriction. CD4 TH cells are MHC class II-restricted, while CD8 TC cells are MHC class I-restricted. MHC restriction ensures a division of labor within the immune system: T cells are directed toward abnormal host cells expressing MHC molecules and foreign peptides, while antibody molecules synthesized by B cells direct their efforts toward the elimination of extracellular antigens. So, in the case of a viral infection, it is the job of the B cells to neutralize free viral particles, while T cells are dedicated to destroying the host cells where those virions are being produced. - The role of the MHC in "thymic education" The education process by which T cells in the thymus learn to recognize antigenic peptides in the context of self-MHC molecules is a two-step process involving both positive and negative selection. Initially, immature thymocytes within the thymic cortex express low levels of TCR, but high levels of both CD4 and CD8 (double-positive cells). They interact with thymic epithelial cells that express high levels of both class I and class II MHC molecules. Thymocytes with moderate affinities for these self-MHC molecules are allowed to develop further, while thymocytes with affinities too high or too low for self-MHC are induced to die by apoptosis. The thymocytes that survive are said to have been "positively selected" through their interaction with self-MHC. The positively selected thymocytes then begin to express high levels of TCR, some of which r recognize self components other than self-MHC. These cells must be deleted to prevent autoimmune destruction of healthy host tissues. Negative selection is the elimination of T cells reactive with self components OTHER THAN the MHC. Negative selection occurs in the deeper cortex, at the corticomedullary junction, and in the medulla of the thymus. The thymocytes interact with antigen processed and presented by interdigitating cells and macrophages. Only thymocytes that fail to recognize self antigens are allowed to survive and proceed along the maturation process, with the remainder undergoing apoptosis. Eventually, T cells that survive the negative selection process lose either CD4 or CD8, becoming "single positive" cells. Fewer than 5% of thymocytes survive selection and leave the thymus to take up residence in the secondary lymphoid organs. - Association of disease with MHC haplotype Expression of certain allelic HLA genes predisposes individuals to specific diseases. For example, 90% of people with ankylosing spondylitis carry the HLA-B27 allele. In addition, expression of HLA-DR4 is associated with rheumatoid arthritis, while expression of HLA-DR2 is associated with both Goodpasture's syndrome and multiple sclerosis. It is hypothesized that in some cases MHC molecules serve as receptors for the attachment and entry of pathogens into the cell; this makes individuals with a certain HLA type more susceptible to infection by a particular intracellular pathogen using that HLA molecule as a receptor. Alternatively, an infectious agent might possess antigenic determinants that resemble an MHC molecule (molecular mimicry). Such resemblance might allow the pathogen to escape immune detection because it is seen as "self," or it may induce an autoimmune reaction. Because MHC molecules differ in their ability to accomodate different peptides, some individuals who express certain MHC genes may lack the ability to present microbial epitopes capable of inducing protective T cell responses. Finally, there may simply be no T cells capable of recognizing a particular MHC/antigen combination, leading to a "hole in the T cell repertoire." - The T Cell Receptor for Antigen (TCR) I. TCR : Polypeptides that comprise the TCR belong to the immunoglobulin superfamily (even though the TCR is not an immunoglobulin). There are two kinds of TCR, the first composed of a disulfide-linked ab heterodimer and the second composed of a gd heterodimer that may or may not be disulfide-linked. The chains vary in size from 35 to 47 kDa. An individual T cell can express either ab or gd as its receptor but never both. Each chain has a distinct constant (C) and variable (V) region. 95% of T cells express the ab heterodimer. The remaining T cells expressing gd are concentrated in the skin, intestinal epithelium, pulmonary epithelium, uterus and tongue. II. CD3 Always found closely associated with TCR is the CD3 marker, composed of at least 5 distinct, invariant polypeptides (g, d, e, z and h ). CD3 is necessary for signal transduction following antigen recognition by the TCR. Target sequences in the z chain are analogous to those in Iga and Igb, becoming phosphorylated through the activities of tyrosine kinases. Once the sequences are phosphorylated, another tyrosine kinase, ZAP-70, binds to them and becomes activated, in turn activating phospholipase C to initiate the phophatidyl inositol signalling pathway. A schematic of the TCR-CD3 structure is shown below. Schematic drawing of the TCR-CD3 complex. The figure shows the ab T cell receptor and the CD3 complex consisting of a zz homodimer plus the ed and ge heterodimers. The a and b chains of the TCR are joined by a disulfide bond. Some T cells express the zh heterodimer rather than the zz homodimer as part of the CD3 molecule. VII. Antibody Production, Regulation & Diversity The exposure to foreign antigen yields a biphasic response. The first phase is associated with production of IgM, followed by production of IgG. The second phase is characterized by a reduction of IgM followed by a reduction of IgG. The antigen will select and expand a clone of effector B cells which will develop into plasma cells and produce antibodies. In subsequent encounters with the same antigen, the response might differ qualitatively and quantitatively since memory cells are involved. The immune memory B cells are long-lived with the ability to react upon the second contact with the original antigen by more rapid and stronger response. The anamnestic response is the result of many more specific clones of memory B cells. When an individual or animal is exposed the antigen a second time the response has shorter lag period, an extended plateau, and a slower decline. The antibodies are in a much higher concentration. The differences in maximal serum concentration of IgG is a 10 to 100 fold. The average affinity of the induced antibody is increased in the secondary response and this is know as affinity maturation. B cell precursors are derived from a stem cell. Through Ag-independent differentiation in a B cell environment, these cells acquire the competence to function a B cells. They migrate to secondary lymphoid organs, where they can differentiate into active B cells through agdependent differentiation. This leads to clones of plasma cells for each class of antibody. Memory cells can arise that will respond rapidly and more strongly upon subsequent encounter with same antigen. Antigen dependent differentiation proceed in two stages: (1) activation and (2) proliferation and differentiation. Antigen acts as specific signals that activate particular B cells. In addition, nonspecific signals are also provided by other cells to promote and modulate the differentiation of activated B cells. B cell activation by T-Independent Antigen T-independent antigen usually are polymeric, with repeating identical antigenic determinants (lipopolysaccharide) which degrade slowly. They can stimulate B cells directly by crosslinking surface receptors (IgG or TCR) for the antigen. The antigen interaction with membrane-bound surface antibodies triggers a cascade of reactions that produce change in the cell membrane, cytoplasm and nucleus. The membrane depolarizes leading to an influx of extracellular Ca ions. This, in turn, contributes to the activation of three membrane-associated response pathways: (1) stimulation guanylate cyclase with the production of cyclic quanosine monophosphate (cGMP), (2) activation of phospholipase C with the subsequent generation of Diacylglycerol (DAB) and inositol triposphate (ITP), and (3) activation phospholipase A2, which promote the metabolism of arachidonic acid (formation of protaglandins and leukotrienes). ITP, DAG, Ca and cGMP are substances that act as second messengers in the activation of several cellular systems. These factors are responsible for increasing the metabolic activity of the activated B cells. Specifically, these intracellular messengers activate protein kinases that promote the first steps of activation: (1) entry into B1 (2) production of interleukin 2 (Il-2) and other growth factors, (3) increased expression of IL-2 receptors (IL-2Rs) and increased expression for MHC Class II molecules. Passage into the S phase of the cell cycle, with subsequent DNA replication and cellular proliferation is apparently regulated by events mediated through IL-2Rs. Binding of antigens to specific receptors (antibodies) activates B cells. This triggers entry into the cell division cycle. Cytokines Il-1, Il-2, Il-4, Il-6 and IFNs) help promote this. Other cytokines, like IL-2 and BCFGs further promote cell division. This leads to clonal expansion of activated B cells. Cells that are induced to undergo terminal differentiation early by BCGFs undergo class switching early. These become antibody secreting plasma cells. Those that do not receive this signal early continue to repeat this divisional cycle. During each cycle , the opportunity exist to modify variable (V) regions through somatic mutation. Nonspecific antigen processing cells (APCs) take up antigen in the lymphoid centers like spleen or lymph nodes through a process called phagocytosis . The antigen becomes internalized and is processed. Different determinants are returned to the cell surface. Antigenic determinants are presented by the APC to THcells which in turn help stimulate the B cells divide and differentiate into clones of B cells. The displayed determinant are associated with major histocompatibility complex (MHC) class II antigens, and then presented to T lymphocytes by way of TCR. The APCs display antigens that possess two domains. The first is an epitope capable of binding to the paratope of TH cell receptor (TCR). The second is agrotope that is intimately associated with a class II MHC molecule. The TCR consist of transmembrane proteins that bind antigen. The TCR is noncovalently associated with five additional integral membrane proteins. The relative ability of each individual to respond to the antigenic universe is inherited to a large extent. Lymphocytes neither reproduce in uncontrolled fashion nor continuously produce increasing quantities of antibodies or lymphokines. A decline in functional expression follows primary and secondary immune responses. Hence specific immune responses must be downregulated in some fashion. Several mechanisms operate to modulate immunological expression. Positive regulators include antigens and cytokines (IL-1, IL-2, BCGF, and BCDF) from macrophages and helper/inducer T Cells. Negative regulators include formed antibodies, immune complexes, suppresser factors (T suppresser cells) and Ig isiotype networks. Genetic Control Two broad categories of genes contribute to the control of immune responses. The first category of genes, receptor genes, controlling B and T cell receptors to a particular foreign antigen. The second category, immune response (Ir) genes, influence the quality and extent of the possible immune response. This category is composed of two types of genes. Ir genes either can be part of the MHC or be unrelated to it. Ir genes of the MHC, which encode class II molecules, primarily influence responses involving T helper cells and sensitized T delayed hypersensitivity cells, While those that encode class I molecules, principally control T suppresser and T cyotoxic cells. Non-MHC Ir genes appear to encode several products that might influence both arms of the immune response. There are numerous Ir gene loci (MHC and non-MHC) that influence the level of the immune response to any particular antigen. Ir MHC class II products are believed to act as the stage when Ag is processed. They influence cell interaction and the activation process. Malfunctioning class II MHC might not provide the proper signals for B cell activation for a normal humoral response. The humoral response is controlled, in part, by antibody production itself. This appears to be negative feedback regulation that is highly specific. The removal of blood from an immunized animal can stimulate antibody production. Passive immunization can suppress antibody formation to a certain antigen without effecting the response to other antigens. Antibodies can bind to antigen and prevent them from binding to receptors to T and B cells. Feedback regulation of antibody production Activation of B cells by antigen leads to production of antibody specific for those antigens. Antigen B stimulates the production of anti-B antibody. Subsequently, the antibody reduces the concentration of that antigen. This diminishes stimulation of further production of the specific antibody. Eventually, antigen B is completely removed and no further stimulation of the production of anti-B antibody occurs. These interactions, however, have no effect on the level of antigen A or its influence in stimulating anti-A antibody. Influence of immune complexes on immune responses Antigen characteristically triggers an immune response. An antigen that is complexed with antibody has differential effects on immune responses. At low Ab/Ag ratios, suppression of antibody production can occur. Under these conditions, immune complexes can bind to both mIg(ag) and FcRs (Ab) of the B cells. This suppresses antibody production. In contrast, at high Ab/Ag ratios, immune responses can be stimulated. This occurs through binding to FcRs on APCs that leads to activation of T helper Cells and subsequent enhancement of immune responses. Idiotype Network Antigen triggers antibody production. The antibody produced (Ab-1) not only react against the antigen, but they also serve as antigen themselves. This triggers the production of a second level of antibodies (Ab-2). In turn, these antibodies react against Ab-1 but serve to induce the anti-Ab-3 antibodies, and the process continues. At each step, the concentration of antibody available to provoke formation of an antibody is less than in the preceding step. As a result, a level is eventually reached at which there is insufficient antibody produced to provide and antigenic signal. Idiotype An idiotype consists of a set of epitopes associated with the V regions on the Ab antibody molecule. Each individual epitope is designated an idiotope. An idiotype is a set of epitopes displayed by the variable regions of a set of antibody molecules. Idiotopes can be associated with the paratope or lie outside it. Actions of anti-Id antibodies Antibodies against Ids that lie within a paratope can crosslink mIgs. In so doing, they can activate the affected B cell. On the other hand, antibodies against Ids lying outside a paratope can interact with mIg through their Fab region and with Fc receptors of the same B cell. In this configuration . Antibody production is suppressed. The result is tolerance. T and B cell activity can be regulated by T helper and T suppresser cells. Both cell types are stimulated during a normal immune response. They play a continuous homeostatic role in regulation of the response. T helper cells collaborate with B cells, providing the positive signals for T-dependent humoral responses. The T suppresser cells on the other hand provide negative signals. These signals limit or inhibit the development of antibody producing cells. T suppresser cells release suppresser factors (TsF) that promote most of the functions. For most suppresser cells there are no clear-cut MHC restrictions, as exist for T helper, cytotoxic and delayed hypersensitivity cells. The immune response is regulated by two major classes of factors; positive factors that promote the expression of cellular or humoral immunity and negative factors that diminish or suppress immunological expression. Antigen binding to mIg on B cells can induce activation. In addition, processed antigen by APC cells with the help of MHC and T cell receptor molecule can give a boost to the humoral response by stimulation of B cell proliferation and differentiation. The interaction between APC and T cells can ultimately suppress response by stimulation of T suppresser cells. Theory of Germline Gene Rearrangement When myeloma proteins were first sequenced, it became clear that both light (L) and heavy (H) chains varied markedly in their amino-terminal regions, but not in their carboxyl-terminal regions. It is hard to understand how a single gene might encode both V regions and C regions. It was proposed by Dreyer and Bennett that the V and C regions of antibody might be encoded by two different genes. This was the first speculation that genes might be split. They envisioned the possibility that many different B Germline genes existed and that any one of these might become associated with a single C region gene at the DNA level. They predicted that these antibodies genes were brought together during B cell differentiation. Their proposal became known as a recombinational germline theory. VI. Antibody Production, Regulation & Diversity Immunoglobulin Genes Somatic cell hybridization techniques have been used to determine the chromosomal location of antibody genes. Murine B lymphocytes may be fused to another mammalian cell line to form unstable hybrids, whose chromosomes are randomly lost. The DNA of each hybrid cell line is then probed with radiolabeled complementary, or copy, cDNA corresponding to kappa, lambda, or H chain regions for the presence of complementary H or L chain antibody genes. By correlating loss of a particular mouse chromosome with loss of a particular antibody gene, we can determine the chromosomal location of gene families. Using this approach, it became clear that three unlinked families of genes encode H and L chains and are located on separate chromosomes. Each family consists of a cluster of gene segments capable of encoding V regions separated from one or more genes capable of encoding C regions. ______________________________________ Chain Class Human Mouse ______________________________________ Kappa 2 16 Lambda 22 6 H chain 14 12 ______________________________________ Hybridization experiments revealed that V region and C region gene segments were separately encoded and located on distinct DNA fragments in embryonic cells, but on the same fragment in mature plasma cells. These studies suggested that DNA rearrangements must have occurred during maturation and differentiation of B lymphocytes. These rearranged DNA segments were destined to be expressed as specific monospecific receptors by antibody forming plasma cells. From the collection of gene segments available in each family, several may be specifically joined to encode an entire antibody chain L or H. It should be note that genes of one family were never intermingled with genes of either of the other families. Extracted germline DNA from mouse embryos and differentiated DNA from myeloma cells were cleaved with restriction enzymes. Fragments were separated by electrophoresis, denatured to yield single-stranded fragments, and extracted. Fragments were hybridized with kappa chain radiolabled probe (mRNA), derived from myeloma cells. The entire mRNA molecule (encoding the V and C regions) or only the 3' half of mRNA (encoding the C region) was employed. Results showed that mRNA for Ig L chain hybridized to germline DNA by its V region to one DNA fragment and by its C region to a totally separate DNA fragment. In contrast, the same mRNA hybridized to both V and C regions in a single DNA fragment from fully differentiated cells. With the advent of recombinant DNA technology, it became possible to clone specific antibody genes. Sufficient quantities of specific DNA segments could be obtained, permitting analysis of their organization. Studies of DNA sequence showed that discontinuous segments existed that did not code for peptides. Some noncoding segments were deleted from the DNA before transcription and others from the mRNA after transcription. The pioneering work of Kohler and Milstein in producing hybridomas has given rise to entirely new areas of immunodiagnosis and immunotherapeutics. It is now possible to produce monoclonal antibodies for use as probes and potential therapeutic agents. There are numerous technical problems that make production of therapeutic human monoclonals generally unsatisfactory. Mouse monoclonals are foreign proteins. Antibody genes form mice and humans can be isolated, cloned and recombined. The tailor-made genes can then be inserted into suitable vector and transfected into meylomas. The resulting products have been called transfectomas. These chimeric monoclonals can be administered to humans Light Chain Gene Organization There are three peptide segments in each L chain and are coded by three distinct genes, V, J, and C. In the kappa family there are a large number of V gene segments and five joining (J) segments and a single C segment. By contrast, in the lambda family, there are fewer V gene segments and a similar number of J genes but several C genes. The V and J gene segments encode V regions and C genes encode C regions. The L chain is encoded by three distinct gene segments that must be joined to form a functional antibody gene. Joining of Gene Segments During the process of differentiation of a pluripotent stem cell into a mature antigen reactive B cell, DNA rearrangements take place. In the kappa family one of the many B gene segments will become joined directly to one of the J gene segments. The V-J segment will be joined to the C segment to form a functional antibody gene. The joining of these segments is brought about through the activity of recombinase enzymes. Each germline V gene contains a region encoding the V segment and a leader sequence. The leader sequence encodes a leader peptide that controls passage of new protein products across the rough endoplamic reticulum The leader peptide is cleaved and removed before the antibody molecule is secreted. The V segment encodes the first 95 N-terminal amino acids of the V region of the antibody L chain. The J segment consist of five alternative forms encode the remaining 12 or more amino acids of B region of the L chains. All segment are separated by short introns. In contrast, the single C region is preceded by a long intervening sequence. Recognition Signals for V-J joining Analysis of DNA sequence has revealed that a set of conserve nucleotides border germline B and J segments. These sequences of DNA occur at the site where B-J fusion takes place and may provide recognition signals to facilitate the joining process. After each V segment is a heptamer (7) with identical sequence (5'CACAGTG 3"), followed by a spacer of 11 or 12 nucleotides of unconserved sequence. The spacer is followed by a nonanucleotide (9) (5'ACAAAAACC3'), or its analogue. Preceding the J segments there are two signal sequence complementary to those following the B segments. The first is nonanucleotide (5'GGTTTTTTGT3') occurs, then a spacer of 23 or 24 nucleotides, which is followed by the heptanucleotide (5'CACTGTG3'). When two segments can join, one always has a 11 or 12 nucleotide-long spacer between its conserved heptanucleotide and nonanucleotide, while the other has a 23 or 24 nucleotide-long spacer. Heavy Chain Variable Region Diversity H chain diversity is generated by mechanisms quite similar to those previously described for L Chains. There are many V genes in the H chain gene family (perhaps over 300), which may encode the leader and the main region to the 95 residue, as seen for L chains. There are also a number of functional J segments (dour in mice, and five in humans), which may encode part of the CDR3 and all of the last frame work region (FR4) described for the L chains. The process is somewhat more complex, since 10 to 20 short DNA segments, known as D, or diversity, segments are located between the H and J segments. D segments encode two to 14 amino acids in the hypervarible region, or complementary region 3 (CDR3), and, therefore, are critically involved in determining the specificity of an antibody. Recombination of VH-DH-JH segments During differentiation of pluripotent stem cells, a DH segment, JH segment, and B segment, in this example DH4, JH2 and BH2, become joined. The rearranged genes are then transcribed. A second transcription follows, whereby introns re removed and mRNA is formed and translated into specific Ig H chains. These gene segments obviously contribute significantly to the generation of hypervariablility in the V regions. When three segments of DNA randomly join to encode the entire H chain V region, they enhance the recombination possibilities. Thus, if there were 300 VH, 12 D, and four alternative J Gene segments and one of each becomes randomly associated during the differentiation, 14,400 different chains could be formed (300 x12 x 4 =14,400) H chains have five chains as previously described. Each isotype and homology region is encoded by genes separated by intervening sequence or introns. Thus, CH genes are composed of multiple exons in contrast to L chain constant domain (CL) genes. Interestingly, all of the different classes and subclasses of Ifs us the same set of V region genes. Thus if the class or subclass is change, only the C region is switched. VII. Complement Complement. Serum proteins, found in the blood, along with cells of the immune system work together to eliminate foreign cells. Once activated, the complement system can bring about a variety of responses. This complement system consists of three separate activation triggers: (1) Ab binding to a cell surface, (2) formation of immune complexes, and (3) a carbohydrate component of a microbe's cell membrane. Along with this triggers, there are also two sets of mechanisms. Both of these mechanisms, classical pathway and alternative or properdin pathway, make MAC (Membrane Attack Complex), which can lyse and destroy the cell. Structure of the C1 Molecule. The C1 protein is composed of three proteins: C1q, which binds to the Fc portion of the Ab molecule; C1s, which can enzymatically cleave the next complement component, C4; and C1r, which acts as a bridge connecting C1q to C1s. Classical Complement Pathway. The classical complement pathway requires the presence of antibodies, either as immunoglobulin (IgG or IgM) bound to cell surface Ag or as an Ag-Ab immune complex. The serum protein C1 binds to the antibody, which in turn results in activation of C4, C2, and C3, and leads to the formation of C5, C6, C7, C8 and C9. This eventually leads to the formation of the C5-C9 membrane attack complex (MAC), which lyses and destroys the cell. Found in the classical complement pathway are three phases or stages. These are (1) a recognition phase, during which the complement system becomes activated; (2) an enzymatic phase, which is characterized by amplification of complement activation; and (3) an attack phase, during which cell destruction actually occurs. Recognition Phase. The recognition phase initiates the classical complement pathway when the inactive C1 serum protein interacts with the Fc portion of either cell-bound Ig (IgG or IgM) or an immune complex. Even though only one IgM-bound molecule is necessary to fix the C1 complex, two molecules of IgG bound in close proximity are required for activation to occur. This C1 recognition complex requires calcium to form the inactive unit, C1, which consist of one molecule of C1q and two molecules of each of C1r and C1s. When the Ab is bound, C1q connects with the Ab receptor sites located on the Fc portions of two adjacent Ab molecules. These sites are exposed when the Ab binds to the Ag. After binding to the sites, C1q undergoes a conformational change, which causes C1r to activate itself by limited self-cleavage. C1r now sees C1s as its substrate and activation of C1s gives rise to its ability to cleave the next two complement proteins, C4 and C2. Enzymatic Phase Activation of C4 and C2 involves the cleavage of a specific peptide bond in each molecule, resulting in the dissociation of peptide fragments, C4a and C2a, and the exposure of a binding site in the larger fragment, C4b2b. The C4b peptide binds to the cell membrane, while C2b attaches itself to the C4b fragment. This C4b2b complex is enzymatically active, requires magnesium ions, and is called C3 convertase since it can bind and cleave the next inactive complement component in the sequence, C3. Activation of C3 initiates the generation of a second convertase enzyme. This occurs when C3 is cleaved into C3a and C3b. The larger C3b fragment attaches to both the cell membrane and C4b2b complex, while the smaller fragment, C3a, is released to the body fluids. C4b2b3b is termed C5 convertase (or C3/C5 convertase), and its newly created catalytic site now accommodates C5, the next component in the sequence. Attack Phase The attack phase begins when C5 convertase (C3/C5) cleaves C5 into two products, C5a and C5b. C5a is released when formed and C5b attaches to the cell membrane. The binding of C5b leads to the uncovering of a binding site for C6 and C7 on the molecule, producing a stable complex, C5b67, which attaches to the membrane surface and enables C8 to bind. C8, in turn, binds several C9 molecules. This stable complex is called C5b6789, or MAC. The membrane attack complex (MAC) can produce a lesion in the plasma membrane causing cell lysis. The classical complement cascade must be carefully regulated. The series of events must be of limited duration in order to prevent the complete consumption of inactive complement components in the serum. Each stage in this pathway contains regulatory proteins. The first stage of complement activation is the formation of recognition complex, C1. C1 inhibitor is responsible for the inactivation of C1. Its main function is to combine with activate C1 by binding to sites of C1r and C1s, causing these subunits to dissociate from Ab-bound C1q. The inactivation of C3/C5 convertase is accomplished in two stages. The first stage consists of activity lost by dissociation of C2 from cell-bound C4. This is done by C4-binding protein (C4BP) and decay-accelerating factor (DAF). In the second stage, the generation of C3 convertase is blocked. This occurs through the further degradation of C4b into the inactive fragments C4c and C4d, and also by the cleavage enzyme, Factor I. Factor I requires a cofactor, these may be factor H and CRI. Alternative or Properdin Pathway The alternative or properdin pathway can be initiated with the complete absence of antibodies. This pathway can be activated by nonimmune activators, for example, repeating polysaccharide units or the LPS found on the cell walls of some microbes. This alternative pathway differs from the classical pathway in that it has substitutes for the early acting C1, C4, and C2 components. Therefore, it can directly initiate the reactions of the late-acting C3-C9 components. It also differs in that it provides an immediate line of defense that does not require immunological memory. This pathway is characterized by a two-phase system in which six proteins participate. Initiation is the first phase, particle-bound C3b acts like the C1 recognition complex. The second phase is called amplification, driven by a positive feedback loop involving bound C3b, Factor B, and Factor D. Initiation Phase. C3 is a two-chain molecule, which upon cleavage, a highly reactive thiol-ester group is exposed on the C3b fragment. This reactive group could be attacked by water, by the surface of an organism, or by foreign substances to which it might bind. If the acceptor surfaces are chemically suitable, interaction with C3b can occur and the alternative pathway can be activated. C3b bound to foreign substances can interact with plasma protein Factor B, which is then cleaved into membrane-bound fragment Bb and soluble fragment Ba, through the action of an enzyme called Factor D. The C3bBb is a C3/C5 convertase. Amplification Phase As C3bBb converts more C3 to C3b, the amplification process is initiated. This newly formed C3b binds more factor B. This positive feedback cycle continues until the membrane surface is saturated with C3bBb. The result is opsonization (enhanced engulfment) of the cell or particle by neutrophils. In addition, the soluble C3a that is released upon cleavage of C3 has anaphylatoxic activity, which can initiate an inflammatory response. The C3 convertase can bind additional C3b to produce C5 convertase (C3bBb3b), which, in turn, activates the terminal lytic complement sequence, C5-C9. The activation of either the classical complement pathway or the alternative pathway leads to the formation of C3 convertase (C4b2b or C3bBb). Cleavage of C3 and binding of C3b to C3 convertases results in the formation of C5-converting enzymes (C4b2bC3b or C3bBb3b). At the end, both pathways form MAC, which mediates lysis of target cells. Biological Consequences of Complement Activation Complement activation not only causes cell lysis, but other effects as well. Some of these include: contraction of smooth muscle, release of histamine from mast cells and platelets, enhanced phagocytosis, chemotaxis of phagocytes, and activation of lymphocytes and macrophages. The main activity of C3a and C5a is anaphylaxis. These cause histamine release from mast cells and basophils, which can affect the activity of smooth muscle. Spasmogenicity, accounts for the ability of these molecules to induce an anaphylactic response in animals. In addition to spasmogenicity, histamine release induced by the anaphylatoxins as effects on inflammation. The cellular responses of neutrophils and monocytes to C5a include (1) degranulation and lysosomal enzyme release, (2) cell adherence, and (3) chemotactic migration. The acute inflammatory response is characterized by symptoms of redness, pain, swelling, and heat due to the action of C4a, C3a, C5a, and histamine. Inflammation's primary goal is to set into motion a series of events that result in the elimination of foreign and damaged cells. This response can be mediated by the anaphylatoxins and their byproducts. Opsonization. Facilitated phagocytosis is referred to as opsonization. In this process, C3b, which coats the particle, is known as an opsonin. This process occurs when cells, viruses, or immune complexes are made ready for enhanced phagocytosis by becoming coated with C3b or C4b. This leads to binding of bacteria to phagocyte C3 receptors and the subsequent clearance of the bacteria by phagocytosis. It can either take place in the presence or absence of antibodies. Complement -Dependent Viral Neutralization Part of the body's natural defense against viral attack is the production of antibodies, which can bind to the surface of the infecting virus. This, in turn, can activate complement, which could neutralize the virus. In fact, the complement system appears to play an important role in neutralizing viruses since it can be activated against certain viruses and virus-infected cells in the absence of antibodies. Viral neutralization ban be accomplished by the complement system through four mechanisms: (1) viral aggregation, (2) coating of viral surfaces, (3) lysis of virally infected cell, and (4) activation of inflammatory cells.
