Explain different types of innate immunity observed in vertebrate system. Innate immunity is seen to comprise 4 types of defensive barriers.

They are discussed below: Anatomic barrier: Physical and anatomic barriers that tend to prevent the entry of pathogens are an organisms 1st line of defense against infection. The skin and surface of mucous membranes are included in this category as they are effective barriers to the entry of most microorganisms. The skin consists of 2 distinct layers, a thinner outer layer called epidermis and thicker layer called the dermis. The epidermis consists of several layers of tightly packed epithelial cells. The outer epidermal layer consists of dead cells and is filled with a waterproofing protein called keratin. The dermis, composed of connective tissue, contains blood vessels, hair follicles, sebaceous glands and sweat glands. Sebaceous glands are associated with hair follicles and produce an oily secretion called sebum. Sebum contains lactic acid and fatty acids, which maintain pH of skin between 3 and 5, this pH inhibits the growth of most microbes. A few bacteria that metabolize sebum live as commensals on the skin and sometimes cause a severe form of acne. One acne drug, isotretinoin, is a vitamin A derivative that prevents sebum formation. Breaks in the skin resulting from scratches, wounds or abrasions are obvious routes of infection. The skin may also be penetrated by biting insects like mosquitoes, fleas, etc. If these harbor pathogens, they may introduce the pathogen into the body as they feed. The conjunctivae and the alimentary, respiratory and urinogenital tracts are lined by mucus membranes. These membranes consist of an outer epithelial layer and an underlying layer of connective tissue. Although many pathogens enter the body by binding to and penetrating mucus membranes, a number of nonspecific defense mechanism tend to prevent this entry. Saliva, tears and mucus secretions act to wash away potential invaders and also contain antibacterial and antiviral substances. The viscous fluid called mucus, secreted by epithelial cells of mucus membranes, entraps foreign microbes. In lower respiratory tract, mucus membrane is covered by cilia, hair like protrusions of epithelial cell membranes. The synchronous movement of cilia propels mucus entrapped microbes from these tracts. In addition, non pathogenic microbes tend to colonize epithelial cells of mucosal surfaces. These normal flora generally outcompete pathogens for attachment sites on epithelial surfaces and necessary nutrients. Some organisms have evolved ways of escaping these defense mechanisms and thus are able to invade the body through mucus membranes. Influenza virus, has a surface molecule that enables it to attach strongly to cells in mucus membranes of the respiratory tract, preventing the virus from being swept out by ciliated epithelial cells. Physiologic barriers: they include temperature, pH and various soluble and cell associated molecules. Many species are not susceptible to certain diseases simply because their normal body temperature inhibits the growth of certain pathogens. Chickens have innate immunity to anthrax because their high body temperature inhibits the growth of the bacteria. Gastric acidity is an innate physiologic barrier to infection because very few microbes can survive the low pH of the stomach contents. One reason newborns are susceptible to some diseases which do not afflict adults is that their stomach contents are less acidic than those of adults. A variety of soluble factors contribute to innate immunity, among them the soluble proteins lysozyme, interferon and complement. Lysozyme, a hydrolytic enzyme found in mucus secretions and tears is able to cleave the peptidoglycan layer of bacterial cell wall. Interferon comprises a group of proteins produced by virus infected cells. Among the many functions of the interferon is the ability to bind to nearby cells and induce a generalized antiviral state. Complement is group of serum proteins that circulate in an inactive state. A variety of specific and nonspecific immunologic mechanisms can convert the inactive forms to active forms with the ability to damage the membranes of pathogens, either destroying the pathogens or facilitating their clearance. Complement may function as an effector system that is triggered by antibodies binding to certain cell surfaces, or it may be activated by reactions between complement molecules and certain components of microbial cell walls. Reactions between complement molecules or its fragments and cellular receptors trigger activation of cells of the innate and adaptive immune system. Phagocytic barrier: Phagocytosis is one type of endocytosis, the general term for uptake by a cell of material from its environment. In phagocytosis, a cells plasma membrane expands around the particulate matter, which may include whole pathogenic microbe, to form large vescicles called phagosomes. Most

phagocytosis is conducted by specialized cells like blood monocytes, neutrophils and tissue macrophages. Most cell types are capable of other forms of endocytosis, like receptor mediated endocytosis, in which extracellular molecules are internalized after binding by specific cellular receptors, and pinocytoisi, the process by which cells take up fluid from the surrounding medium along with any molecules contained in it. Inflammatory barrier: Tissue damage caused by a wound or an invading pathogenic microbe induces a complex sequence of events collectively called inflammatory response. A molecular component of a microbe like LPS may trigger an inflammatory response via interaction with cell surface receptors. The end result of inflammation may be the marshalling of a specific immune response to the invasion or clearance of the invader by components of immune system.

PHAGOCYTOSIS

Give a detailed account on cells of immune system.

Hematopoietic stem cell can differentiate in various ways into various types of blood cells. We shall focus on each type of cell. Lymphocytes: they are small, non phagocytic mononuclear leukocytes that are immunologically competent cells. They are produced in red bone marrow by process of hematopoiesis. They leave the bone marrow, circulate in the blood and lymphatic system and some of them reside in the lymphoid organs. Lymphocytes are capable of recognizing a variety of foreign materials in a specific manner and so generate both cellular and humoral immune responses. These cells are also solely responsible for immunologic memory. The 2 major populations of lymphocytes include the T and B cells.

HEMATOPOIESIS

T lymphocytes: T cells express specific types of cell mediated immune response, carry a vast repertoire of immunologic memory, and they can regulate and directly affect a variety of immune processes. In humans the bone marrow and thymus are primary lymphoid organs. These are the sites of functional maturation of lymphocytes. The undifferentiated lymphocytes that migrate into the thymus undergo processing to become thymus dependent lymphocytes or T lymphocytes. The developing lymphocytes in the thymus are called thymocytes. Some T cells migrate away from the thymus and enter the blood stream where they comprise 7080% of circulating lymphocytes. T cells that remain in the thymus are called thymocytes. This thymus dependent differentiation of T cells occurs since early childhood and by adolescence the secondary lymphoid organs of the body generally contain a full complement of T cells. During the maturation of T cells in the thymus, these cells express unique receptor molecules on their surface called T cell receptors, capable of recognizing specific antigenic structure. Based on differences of receptors, 2 well defined subpopulations of T cells are evident, namely T helper cells and T cytotoxic cells. T cells displaying membrane bound CD4 glycoproteins function as T helper cells, whereas those that have CD8 glycoproteins are T cytotoxic cells. T cells can attack host cells that have been paracitized by viruses or microorganisms, tissue cells that have been transplanted from one host to another and cancer cells. Since T cells must physically contact foreign or infected host cells in order to destroy them, they are said to provide cell mediated immunity. T cells can recognize appropriate antigens only if the antigens are bound to cell membrane proteins called Major

Histocompatibility Complex molecules. There are 2 major types of MHC molecules- Class I MHC and Class II MHC molecules. Class I MHC molecules are expressed by nearly all nucleated cells, and they are composed of a heavy chain linked to a small invariant protein called 2 microglobulin. Cells that display antigens complexed with Class I MHC molecules are altered self cells. Class II MHC molecules consist of alpha and beta glycoprotein chains and they are expressed only on antigen presenting cells. When a T cell encounters an antigen complxed with MHC molecules, it proliferates and differentiates into memory T cells and various types of effector T cells. T helper cells can interact with antigens bound to class II MHC and get activated to become effector cells which in turn secrete various kinds of soluble immune molecules including growth factors collectively called cytokines. The cytokines play an important role in activating T cells, B cells and macrophages, etc. under the influence of TH derived cytokines, a TC cell that recognizes an antigen MHC Class I molecule complex proliferates and differentiates into an effector cell called Cytotoxic T lymphocyte. They generally do not release cytokines, but instead release cytotoxic molecules. B lymphocytes: During embryonic development of mammals, B cells differentiate in the fetal liver, later in bone marrow and then released into blood circulation. The bone marrow continues to be the major site for B cell differentiation. the letter B was originally derived from Bursa of Fabricius, an evaginated structure near the appendage of the cloaca of birds where prelymphocytes were first discovered to undergo functional differentiation. B cells make up to 20-30% of circulating lymphocytes. When B cells leave the bone marrow they express on their memebrane specific antigen recognition receptors. Such B cell receptors are called antibody molecules. Once the B cell contacts with an antigen that matches its receptor molecule, it begins to divide rapidly and its progeny differentiates into memory B cells and effector B cells and the latter called plasma cells. The memory B cells have a longer life span, more readily stimulated than the nave B cells and they are retained within the body with the same receptor profile as the parent nave B cell. Immunologic memory is the attribute of the immune system mediated by memory cells derived from B or T cells whereby a second encounter with the same antigen induces a heightened state of immune reactivity. The plasma cells are short lived and secrete antibodies into the blood and lymph. Antibodies are specifically directed against the antigens which were previously responsible for B cell stimulation leading to antibody formation. Because the antibodies are soluble immune molecules they are responsible for humoral immunity. This is active mostly against invading bacteria, bacterial toxins and viruses. Mononuclear phagocytes: The mononuclear phagocytic system consists of monocytes circulating in the blood and macrophages in the tissues. During hematopoiesis in bone marroe, granulocyte-monocyte progenitior cells differentiate into monocytes, which leave the bone marrow and enter the blood. They circulate in the bloodstream for 8 hours during which they enlarge, migrate into tissues and differentiate into tissue specific macrophages. Some of the monocytes become macrophages that remain in the blood circulation. Otherwise macrophages reside in particular tissues and are called fixed or tissue macrophages. Macrophages that remain motile in blood circulation are called free or wandering macrophages. Macrophages are named according to their tissue location. Alveolar macrophages in the lungs, Histiocytes in the connective tissue, Osteoclasts in the bone, Kuppfer cells in liver and Mesangial cells in the kidney. Macrophages initiate immune responses, serve as antigen presenting cells, and as effector cells with microbicidal and tumoricidal activity. They also produce a number of complement components or cytokines or precisely monokines like IL-1, prostaglandins, interferons and TNF that function as immune regulatory molecules. Macrophages are capable of ingesting and digesting exogenous antigens, like whole microorganisms and insoluble particles, and endogenous matter like injured or dead host cells, cellular debris and activated clotting factors. In the 1st step of phagocytosis, macrophages are attracted by and move toward a variety of substances generated in an immune response, this process is called chemotaxis. The next step in phagocytosis is the adherence of antigen to the macrophage cell membrane. Complex antigens like whole bacterial cells or

viral particles tend to adhere well and are readily phagocytosed, isolated proteins and encapsulated bacteria tend to adhere poorly and are less readily phagocytosed. Adherence induces membrane protrusions called pseudopodia, to extend around the attached material. Fusion of pseudopodia encloses the material in a membrane bound structure called phagosome, which then enters the endocytic processing pathway. A phagosome moves toward the cell interior, where it fuses with lysosome to form a phagolysosome. Lysosomes contain hydrolytic enzymes that degrade the ingested material. The digested contents are then eliminated via exocytosis. Macrophages are normally in a resting state and are activated by a variety of stimuli during the course of an immune reaction. Activated macrophages exhibit phagocytic activity and secrete various cytotoxic proteins. They show an increased capacity to secrete inflammatory mediators and activate T cells. One of the potent activators of macrophages are the gamma interferons secreted by activated helper T cells. This can lead to enhanced uptake and efficient elimination of antigens aided by a rise in activities of intracellular hydrolytic enzymes. Besides, other lymphokines can activate any responsive macrophage in the close vicinity whereby the responding cells effectively present antigens to the helper T cells or destroy tumor cells. Granulocytic cells: The granulocytes are short lived cells comprising v60-70% of leucocytes in blood circulation. The mature forms have a multilobed or bilobed nucleus and many cytoplasmic granules. Based on their histochemical staining, they are classified as follows. a. Neutrophils have a multilobed nucleus and granulated cytoplasm that stains with both acidic and basic dyes. They comprise 50-70% of the total leukocytes in circulation. Their granules are rich in microbicidal molecules. They are actively phagocytic cells and are first to arrive at a site of inflammation. During infection or injury, they are chemotactically attracted to the damaged tissues to mediate inflammatory reactions. b. Eosinophils have a bilobed nucleus and granular cytoplasm that stain with acidic dye eosin. They are motile and occasionally phagocytic cells, capable of migrating from blood to tissue spaces. They are present in low numbers in peripheral blood circulation but their count can rise on parasitic infection or allergic conditions. They release basic protein present in their granules that can damage the parasite membrane. c. Basophils also have a lobed nucleus and heavily granulated cytoplasm that stains with basic dyes like methylene blue. They are non phagocytic and release pharmacologically active substances. They play a major role in certain allergic responses. They are found in extremely small numbers in blood circulation. Mast cells: they are resident cells of several types of tissues and contain many granules rich in histamine and heparin. Although best known for their role in allergy and anaphylaxis, they are intimately involved in wound healing and defense against pathogens. These cells are found in skin, connective tissues of various organs, mucosal epithelial tissues of respiratory, urinogenital and digestive tracts. Activation of mast cells generally occurs when an antigen binds to the cell bound IgE molecule. This in turn results in mast cell degranulation and release of chemical mediators, resulting in dilation of small blood vessels and extrusion of immune molecules and cells into the desired site of action. Null cells: They are a small population of peripheral blood lymphocytes and lack the membrane markers characteristic of B and T cells. Stimulated by the presence of antibody, null cells can attack certain cellular targets directly. They kill tumor or viral infected cells and are of the following types: a. Killer cells are the effector cells of antibody dependent, cell mediated cytotoxicity. It recognizes antibodies on target cells and lyses those cells through a cell-cell interaction that does not require complement. b. Natural Killer Cells are a member of the null cell population that constitute about 5-10% of the lymphocyte in human peripheral blood. They recognize the virus infected cells apparently by a non specific mechanism and destroy them by extracellular cytotoxicity. They also play a role in killing

cancer cells. The cytoplasm of NK cells contain numerous granules containing perforin and granzyme. NK cells are constitutively cytotoxic. After an NK cell adheres to a target cell, degranulation occurs spontaneously leading to the release of perforin and granzymes directly on interacting cells. These molecules cause rapid lysis of target cells which may also involve apoptotic mechanisms. A number of cells are able to destroy foreign materials by ADCC. A cytotoxic response mediated by immune cells is dependent on the presence of an antibody. The cells that carry out this activity have a receptor for the Fc portion of the antibody and are able to bind antibody coated targets. Killer cell is one such cell. Antigen Presenting cells (APC) are specialized WBCs that help fight off foreign substances. These cells send out signals to T cells when an antigen enters the body. Each type of t cell is specially equipped to deal with different pathogens, which may be a virus, bacteria or toxin. First, the APC engulfs the antigen. Enzymes inside APC break down antigen into smaller particles. The processed antigens are transported to the surface of the APC, bound with either an MHC class I or II molecule. This complex forms epitopes which a T cell receptor recognizes and binds to. Dendritic cells: They are present in small amounts in the body tissues that frequently come into contact with the external environment. They are found in the skin, where they are often called Langerhans cells; and the inner lining of the nose, stomach, lungs and intestines. These cells have branched projections called dendrites. Immature dendritic cells called veiled cells are found in the bloodstream. They are characterized by high endocytic activity and low T cell activation potential. Their pattern recognition receptors are constantly sampling their surroundings for pathogens, such as bacteria and viruses. Once a dendritic cell comes in contact with a pathogen they are activated to engulf the invading substance. Once antigen is engulfed, the cell matures and combines the broken down antigen with MHC. This complex is presented to T cells on its surface.

Describe the structure and properties of different types of antibodies.

The immunoglobulins are classified on the basis of antigenic differences between the C regions of the H chains. These differences are due to heterogeneities in amino acid composition. Five different antisera against H chains establish 5 classes of immunoglobulins in humans. Antigenically different H chains are designated ,,,, corresponding to immunoglobulin classes IgG, IgM, IgA, IgD, IgE, respectively. Some of these classes are further divided into subclasses. IgG: This is a major class of serum immunoglobulin and constitutes about 80% of total serum immunoglobulin. It is involved in many antibacterial, antiviral and antitoxic activities. This class passes across placenta and provides passive immunity to newborn for about 6 months. The secondary response is mediated by the IgG antibody. Human IgG class is divided into 4 subclasses, IgG1, IgG2, IgG3, IgG4 based on 1,2,3,4 heavy chains respectively. IgG antibodies are large molecules of about 150kDa composed of about 4 peptide chains. It contains 2 identical heavy chains of about 50 kDa and 2 identical light chains of 25 kDa, thus tetrameric quaternary structure. The 2 heavy chains are linked to each other and to a light chain by disulfide bonds. The resulting tetramer has 2 identical halves, which together form the Y like shape. Each end of the fork contains an identical antigen binding site. IgM: this immunoglobulin accounts for 5-10% of total serum immunoglobulin with an average serum concentration of 1.5 mg/ml. Monomeric IgM, with a molecular weight of 180000, is expressed as membrane

bound antibody on B cells. IgM is secreted by plasma cells as pentamer in which five monomer units are held together by disulfide bonds that link their carboxyl terminal heavy chain domains (C4/C4 and C3/C3). The five monomer subunits are arranged with their Fc regions in the center of the pentamer and the ten antigen binding sites on the periphery of the molecule. Each pentamer contains an additional F c linked polypeptide called the J chain, which is disulfide bonded to the C terminal residues of 2 of the ten chains. The J chains appear to be needed for polymerization of monomers to form pentameric IgM. It is added just before the secretion of the pentamer. IgA: Although it consists of only 10-15% of the total Ig in serum, it is the predominant immunoglobulin class in external secretions like, breast milk, saliva, tears and mucus of the bronchial, genitourinary and digestive tracts. In serum, IgA exists primarily as a monomer, but polymeric forms are sometimes seen (di-, tri- and some tetramers) all containing a J chain polypeptide. The IgA of external secretions consists of a dimer or a tetramer, J chain polypeptide and a polypeptide chain called secretory component. The secretory component is derived from the receptorthat is responsible for transporting polymeric IgA across the cell membranes. The J chain polypeptide in IgA is identical to that found in pentameric IgM and serves a similar function in facilitating the polymerization of both serum IgA and secretory IgA. The secretory component is a 70000 MW polypeptide produced by epithelial cells of mucus membranes. IgD: It is present in trace amounts in normal humans, about 1% of the total serum Ig. IgD is also produced in a secreted form that is found in very small amounts in blood serum. Secreted IgD is produced as a monomeric antibody with 2 heavy chains of the delta class and 2 Ig light chains. IgE: The potent biological activity of IgE allowed it to be identified in serum despite its extremely low average serum concentrations (0.3 g/ml). They mediate the immediate hypersensitivity reactions that are responsible for the symptoms of hay fever, asthma, hives and anaphylactic shock. The presence of a serum component responsible for allergic reactions was 1st demonstrated in 1921 by Prausnitz and Kustner. IgE is a monomeric antibody with 4 Ig like domains. Property/activity Molecular weight Heavy chain component Normal serum level (mg/ml) In vivo serum half life in days Activates classical complement pathway Crosses placenta Present on membrane of mature B cells Binds to Fc receptors of phagocytes Mucosal transport IgG1 150000 1 9 23 + IgG2 IgG3 150000 150000 2 3 23 +/3 1 8 ++ IgG4 IgA1 150000 150000600000 4 1 0.5 23 3 6 IgA2 IgM 150000- 900000 600000 2 0.5 6 1.5 5 +++ IgE 190000 0.0003 2.5 IgD 150000 0.03 3 -

+ -

+/-

+ -

+ -

++

+/-

++

++

++

Activity levels indicated as follows: ++=high, +=moderate, +/-=minimal, -=none,? =questionable.

STRUCTURE OF DIFFERENT CLASSES OF ANTIBODIES