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C M S A

CMSA Medical PG Entrance Coaching Institute

Immunology Notes

Edition December 2011

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STEPS OF HOST DEFENCE MECHANICS - IMMUNITY The body has both 'innate' and 'adaptive' immune defenses When an organism infects the body, the defense systems already in place may well be sufficient to prevent replication and spread of the infectious agent, thereby preventing development of disease. These established mechanisms are referred to as constituting the 'innate' immune system. However, should innate immunity be insufficient to parry the invasion by the infectious agent, the socalled 'adaptive' immune system then comes into action, although it takes time to reach its maximum efficiency. When it does take effect, it generally eliminates the infective organism, allowing recovery from disease. There is close synergy between the two systems, with the adaptive mechanism greatly improving the efficiency of the innate response. These may be sufficient to prevent disease (1), but if not, disease may result (2). The adaptive immune system is then activated (3) to produce recovery (4) and a specific immunologic memory (5). Following reinfection with the same agent, no disease results (6) and the individual has acquired immunity to the infectious agent.

Innate immunity is referred to 'natural', and adaptive as 'acquired' -interaction between two exists 'Humoral' immunity due to soluble factors contrasts with immunity mediated by cells Primary contact with antigen produces both adaptive and innate responses, but if same antigen persists or encountered again specific adaptive response to that antigen is much enhanced Soluble factors such as lysozyme, complement with phagocytic cells contribute to innate system While lymphocyte-based mechanisms that produce antibody and T lymphocytes are main elements of adaptive immuny Memory with which lymphocytes become endowed shows very considerable specificity to that infection. E.g.infection with measles induces a memory to that microorganism alone and non else -such as rubella. Despite effective barriers, microorganisms successfully penetrate body on many occasions. Two main defensive strategies come into play based on

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 mechanism of phagocytosis, engulfing and killing microorganisms by specialized cells- 'professional phagocytes' the large macrophages; Macrophages originate as bone marrow promonocytes - develop into circulating blood Monocytes, mature macrophages develop into 'mononuclear phagocyte system'. They are particularly concentrated in lung (alveolar macrophages), liver (Kupffer cells) and lining of lymph node medullary sinuses and splenic sinusoids Other examples are the brain microglia, kidney mesangial cells, synovial A cells and osteoclasts in bone the smaller polymorphonuclear granulocytes, which are generally referred to as polymorphs or neutrophils because their cytoplasmic granules do not stain with hematoxylin and eosin. Polymorph has no mitochondria, but uses its abundant cytoplasmic glycogen stores for its energy requirements; therefore, glycolysis enables these cells to function under anaerobic conditions, such as those in an inflammatory focus destructive effect of soluble chemical factors, such as bactericidal enzymes.

Phagocytic cells. (a) Blood monocyte and (b) polymorphonuclear neutrophil, both derived from bone marrow stem cells

Phagocytosis cannot occur unless bacterium first attaches surface of phagocyte, and clearly this cannot happen unless both have become physically close to each other. There is therefore a need for a mechanism that mobilizes phagocytes from a-far and targets them onto the bacterium. Many bacteria produce chemical substances, such as formyl methionyl peptides, which directionally attract leukocytes, a process known as 'chemotaxis'. However, this is a relatively weak signaling system, and evolution has provided the body with a far more effective 'magnet' that uses a complex series of proteins collectively termed 'complement'.
Activation of the complement system Complement resembles blood clotting, fibrinolysis and kinin formation in being a major triggered enzyme cascade system. Such systems are characterized by their ability to produce a rapid, highly amplified response to a trigger stimulus mediated by a cascade phenomenon in which product of one reaction is enzymic catalyst of next. Most abundant component is C3, and cleavage of this molecule is heart of all complement-mediated phenomena. (Complement synergizes with phagocytic cells to produce an acute inflammatory response) In normal plasma, C3 undergoes spontaneous activation at a very slow rate to generate split product C3b. This is able to complex with another complement component, factor B, which is then acted upon by a normal plasma enzyme, factor D, to produce C3-splitting enzyme C3bBb. This C3 convertase can then split new molecules of C3 to give C3a (a small fragment) and further C3b. This represents a positive feedback circuit with potential for
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runaway amplification; however, overall process is restricted to a tick-over level by powerful regulatory mechanisms, which break unstable soluble-phase C3 convertase into inactive cleavage products Activation of the alternative complement pathway with the consequent splitting of very large numbers of C3 molecules has important consequences for the orchestration of an integrated antimicrobial defense strategy Large numbers of C3b produced in the immediate vicinity of the microbial membrane bind covalently to that surface and act as opsonins-molecules that make particle they coat more susceptible to phagocyte

Defensive strategy of the acute inflammatory reaction initiated by bacterial activation of the alternative complement pathway. Activation of the C3bBb C3 convertase by the bacterium (1) leads to the generation of C3b (2) (which binds to Activation of complement by the bacterium (3)), C3a and C5a (4), which recruit mast cell microorganisms.Convertase is heavily regulated by (MC) mediators. These in turn cause capillary dilation (5), factors H and I but can be stabilized on the surface exudation of plasma proteins (6), and chemotactic attraction (7) of microbes and properdin. Horizontal on indicates engulfment. C3b with C3 convertase acts barnext component C5 to produce a small fragment C5a; which together an enzymically active complex. iC3b, to cause their and adherence of polymorphs to the C3b-coated bacterium (8). with C3a has a direct effect on mast cellsinactive C3b. degranulation. The polymorphs are then activated for the final kill.

Vascular permeability mediators increase permeability of capillaries by modifying intercellular forces between endothelial cells of the vessel wall. This allows exudation of fluid and plasma components, including more complement, to the site of the infection. These mediators also up-regulate molecules such as intercellular adhesion molecule-1 (ICAM-1) and endothelial cell leukocyte adhesion molecule-1 (ELAM-1), which bind to specific complementary molecules on the polymorphs and encourage them to stick to capillaries walls- a process termed 'margination'.
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Chemotactic factors, on the other hand, provide a chemical gradient which attracts marginated polymorpho nuclear leukocytes from their intravascular location, through the walls of the blood vessels, and eventually leads them to the site of the C3b-coated bacteria that initiated the whole activation process. Processes of capillary dilation (erythema), exudation of plasma proteins and of fluid (edema) due to hydrostatic and osmotic pressure changes, and the accumulation of neutrophils, are collectively termed the 'acute inflammatory response', and result in a highly effective way of focusing phagocytic cells onto complement-coated microbial targets. (C9 molecules form the 'membrane attack complex', which is involved in cell lysis) Acute phase proteins Certain proteins in the plasma, collectively termed 'acute phase proteins', increase in concentration in response to early 'alarm' mediators such as the cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor (TNF), released as a result of infection or tissue injury. Many acute phase reactants such as mannose binding protein and Creactive protein (CRP) increase dramatically during inflammation. Like the professional phagocytes, both use pattern recognition receptors to bind to molecular patterns on the pathogen (PAMPs), to generate defensive effector functions. Other acute phase reactants show more moderate rises, usually less than five-fold. In general, these proteins are thought to have defensive roles.

Acute phase proteins, here exemplified by C-reactive protein (CRP), are serum proteins that increase rapidly in concentration (sometimes up to 100-fold) following infection (graph) They are important in innate immunity to infection. CRP recognizes and binds in a calcium (Ca2+) dependent fashion to molecular groups found on a wide variety of bacteria and fungi In particular, it uses its pattern recognition to bind the phosphocholine moiety of pneumococci CRP acts as an opsonin and activates complement with all the associated sequelae
C-reactive protein is an antibacterial agent produced by liver cells in response to cytokines. Among the acute phase proteins produced in the course of most inflammatory reactions, C-reactive protein (CRP) is particularly interesting in being an antibacterial agent, albeit of very restricted range. CRP is a pentameric globulin, somewhat resembling a miniature version of IgM (molecular weight 130 000 compared with 900 000 for IgM).

ACUTE PHASE PROTIENS PRODUCED IN Acute phase reactant Dramatic increase in concentration

RESPONSE TO INFECTION IN HUMAN Role Fixes complement, opsonizes

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C reactive protein

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 Mannose binding protein, Alpha 1 acid Fixes compliment, opsonizes transport protein glycol protein Serum amyloid A protein Complex chondroitin sulphate
Moderate increases in concentration

Alpha 1 proteinase inhibitors Alpha 1anti chymotrypsin C3, C9, Factor B Ceruloplasmin Fibrogen Angiotensin Hepatoglobin Fibronectin

Inhibit bacterial proteases Inhibit bacterial proteases Increase complement function O2 scavenger Coagulation Blood pressure Binds hemoglobin Cell attachment

When cells are infected by a virus, they synthesize and secrete IFNs, which bind to specific receptors on nearby uninfected cells. The bound IFN exerts its antiviral effect by facilitating the synthesis of two new enzymes, which interfere with the machinery used by the virus for its own replication The cellular basis of adaptive immune responses Adaptive immune responses are generated by lymphocytes which are derived from stem cells differentiating within the primary lymphoid organs (bone marrow and thymus). From there they colonize the secondary lymphoid tissues where they mediate the immune responses to antigens. The lymph nodes are concerned with responses to antigens which are carried into them from the tissues, while the spleen is concerned primarily with antigens which reach it from the bloodstream. Organized lymphoid tissue. Stem cells (S) arising in the bone marrow differentiates into immune-competent B and T cells in the primary lymphoid organs. These cells then colonize the secondary lymphoid tissues where immune responses are organized. (MALT,- mucosaassociated lymphoid tissue.)

NATURAL KILLER CELLS Natural killer cells are a rapid but non-specific means of controlling viral and other intracellular infection Natural killer (NK) cells provide an early source of cytokines and chemokines during infection, until there is time for the activation and expansion of antigen-specific T cells Immune defenses in action

Skin, mucosa and their adjuncts such as cilia are first barrier to infections Antimicrobial peptides protect skin against invading bacteria, e.g. Lysozyme is one of the most abundant antimicrobial proteins in lung
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 Back-up mechanisms rapidly act when an organism has penetrated these barriersnamely complement, phagocytic and cytotoxic cells, and a variety of cytotoxic molecules (Cytokines contribute to both infection control and infection pathology. Early studies with supernatants from cultures of lymphocytes and macrophages revealed a family of nonantigen-specific molecules with diverse activities that were involved in cell-to-cell communication. These are now collectively known as 'cytokines'. They play many crucial roles in protection against infectious diseases) While they lack the dramatic specifity and memory of adaptive (i.e. lymphocyte-based) immune mechanisms, these natural defenses are vital to survival In addition to these non-specific mechanisms, the immune system enables specific recognition of antigens by T and B cells as part of adaptive immunity. Broadly speaking, antibodies are particularly important in combatting infection by extracellular microbes, particularly pyogenic bacteria, while T cell immunity is required to control intracellular infections with bacteria, viruses, fungi or protozoa.
Antibody mediated immunity

Speed, amount and duration - Because of the cell interactions involved and the need for proliferation of a small number of specific precursor lymphocytes, a primary antibody response can be dangerously slow in reaching protective levels. The classic example, before penicillin, was lobar pneumonia, where the race between bacterial multiplication and antibody production was 'neck-and-neck' for about a week, at which point one side or the other dramatically won. Nowadays, of course, vaccines and antibiotics have intervened to improve the patient's chances Replication rates indicated by doubling times vary from less than an hour (most viruses, many bacteria) to days or even weeks (mycobacteria, T. pallidum). Microorganisms tend to grow more slowly in vivo than in vitro, which shows that the host environment is generally hostile. When incubation period is only a few days (e.g. rhinovirus, rotavirus, cholera) antibody response is too slow to affect the initial outcome, and rapidly produced cytokines such as interferons are more important. Antibody response continues as long as antigen is present to limit immunopathology. Lifelong immunity follows many virus infections may often be due to regular boosting, e.g. by measles and mumps virus still circulating in the community, but sometimes (e.g. yellow fever) there is no obvious boost yet antibodies persist for decades. Such persistence of immunological memory may be due to the non-specific stimulation of memory B and T cells by cytokines during responses to other antigens, a process called bystander activation. Polysaccharides T-independent antigens induce only IgM antibodies. T cells being required for the switch to IgG, IgA or IgE. IgG anti-polysaccharide responses tend to be mainly IgG2, whereas anti-protein IgG is mainly IgG1. The poor development of IgG2 in children below the age of about 2 years explains their lack of response to bacteria with polysaccharide capsules (e.g. Strep. pneumoniae, Haemophilus influenzae) results vaccines that would induce other subclasses of IgG. Antibodies to viruses are predominantly IgG1 and IgG3, and to helminths IgG4 and IgE, while antigens that reach digestive tract induce mainly IgA, the only type of antibody that can function in proteaseConfidential & Proprietary 2011 Chandigarh Medical Science Academy | www.cmsaonline.com Page 8 of 13

rich intestinal environment; T cells and cytokines also play important roles in these isotype preferences. Blocking and neutralizing effects of antibody- Simple binding of antibody molecules to a microbial surface is often enough to protect the host. It may physically interfere with receptor interaction necessary for microbial entry (e.g. of a virus into a cell) or with the binding of a toxin to its host receptor. This is the basis of many life-saving vaccines against viruses or bacterial toxins Immobilization and agglutination - Immunoglobulin antibodies, particularly the large, pentameric IgM, are of size as some of smaller viruses, and larger than thickness of a bacterial flagellum, thereby simple physical attachment of antibody can considerably restrict activities of motile organisms. In addition multivalent design of the antibody molecules enables it to link together two or more organisms, as can readily be demonstrated in the bacterial agglutination tests. . Agglutination reactions in vitro are very useful in diagnosis Opsonization- Whether by direct binding of immunoglobulin CH2 and CH3 regions to Fc receptors, or via activation of complement to allow C3b to bind to its receptor, opsonization represents the most important overall function of the antibody molecule. It is estimated that the rate of phagocytosis is enhanced by up to a thousand-fold by antibody and complement acting together
CELL-MEDIATED IMMUNITY

T cells form second main component of adaptive immune response. Some act by producing cytokines that induce macrophage activation or help antibody production, others by their direct cytotoxic action on infected target cells. In both cases, T cell needs to 'see' combination of specific peptide and MHC molecule that is recognized by its T cell receptor T cell immunity correlates with control of bacterial growth in leprosy Is a positive delayed type hypersensitivity skin test an indicator of immunity? Cytotoxic T lymphocytes kill by inducing 'leaks' in the target cell
Antibody-Mediated (Humoral) Immunity (AMI)

On exposure to antigenic determinants in lymphatic organs, B lymphocytes are activated and differentiated to form plasma cells Plasma cells are specialized, differentiated cells that synthesize and secrete antibodies specific for an antigen Other activated B lymphocytes form memory cells. These cells can be activated later to differentiate to plasma cells for rapid antibody production This antibody production will occur on future reentry of the antigen to body and is the basis of long-term immunity The products of plasma cells are antibodies An antibody is a specialized protein substance produced by the host cells in response to an antigen in the host's tissues. Antibodies are capable of reacting specifically with the antigen that provoked their production. Antibodies are often referred to as
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immunoglobulins. They circulate in the blood and are associated with the gamma globulin fraction of the plasma. Five classes of antibodies (immunoglobulins) are produced by the plasma cells First class, called IgM, is the major component of the primary antibody response in adult humans and is the first antibody to appear in the immune reaction. IgM cannot diffuse through cell membranes and is found almost exclusively in the blood Because of its many binding sites, it is more reactive with antigens than are other kinds of antibodies IgM is also formed by the fetus during uterine development and is active against the A and B factors of red blood cells Many antitoxins formed against bacterial toxins are composed of IgM.
KEY FACTS

Protection against infectious organisms that penetrate the outer barriers of the skin and mucous membranes is mediated by a variety of early defense mechanisms, which constitute innate immunity. These early defense mechanisms occur more rapidly but are less specific than the adaptive mechanisms based on lymphocyte responses. Important early defense mechanisms include the acute phase response, the complement system, IFNs, phagocytic cells and NK cells. Together these act as a first line of defense during the initial hours or days of infection. Adaptive immunity, mediated by antibody and T cells, is responsible for recovery from infection in many cases, although these mechanisms take days to weeks to reach peak efficiency. Sometimes, as in the common viral infections, cell-mediated immunity is responsible for recovery from infection, and antibody for the maintenance of immunity. Failure to recover from infection may be due to some deficiency of host immunity or to successful evasion strategies used by the microorganism.
The many activities of bacterial endotoxin. Lipopolysaccharide (LPS) activates almost every immune mechanism as well as the clotting pathway and as a result LPS is one of the most powerful immune stimuli known. (DIC, disseminated intravascular coagulation; IFN, interferon; IL, interleukin; M, macrophage; PMN, polymorphonuclear leukocyte; TNF, tumor necrosis factor.)

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INTERLEUKIN

An important cytokine is interleukin-1, which activates T-lymphocytes, causing them to proliferate further and form clones Helper T cells (TH) secrete cytokines that promote the proliferation and differentiation of cytotoxic T cells, B cells and macrophages and activation of inflammatory leukocytes

TH cells are identified by the presence of the CD4 marker. They recognize antigen when presented along with Class II MHC molecules. TH cells are further subdivided into the TH1 and TH2 subsets on the basis of the kinds of cytokines they produce TH1 cells produce interleukin-2 (IL-2), interferon-gamma (IFN gamma), and tumour necrosis factor-beta (TNF-beta) while TH2 cells produce IL-4, IL-5, IL-6, IL-10 and TGF-beta

PATHOLOGIC CONSEQUENCES OF THE IMMUNE RESPONSE

Overreaction of the immune system is known as 'hypersensitivity'. Adaptive immune responses are vital to defense against infection, as witnessed by increased susceptibility to infectious disease of immune-deficient patients
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 Each of the four main types of hypersensitivity can be of microbial or non-microbial origin
Type I hypersensitivity Mediated through IgE allergic reactions are a feature of worm infections. Most dramatic allergic (type I) reaction is that following the rupture of a hydatid cyst. Anaphylaxis. Anaphylaxis, or type I hypersensitivity, is a whole body, immediate hypersensitivity also known as anaphylactic shock.

Type II hypersensitivity - Type II reactions are mediated by antibodies to the infectious organism or autoantibodies. Strictly speaking, type II reactions are mediated by antibody -usually IgG leading to cytotoxicity Type III hypersensitivity - Immune complexes cause disease when they become lodged in tissues or blood vessels. The formation of immune complexes can lead to phagocytosis and removal of antigen, but also to complement activation. Complications occur when the complexes escape removal by the phagocytes of the reticuloendothelial system and become lodged in the tissues or blood vessels, attracting complement and neutrophils. Release of lysosomal enzymes then results in local damage, which is particularly serious in small blood vessels, especially in the renal glomeruli. Immune complex disease is a major cause of both acute and chronic glomerulonephritis, and the majority of cases are probably the result of infection. In tissue examples are allergic alveolitis, actinomycosis while in blood glomerulo nephritis, malaria, streptococci, hepatitis B, syphilis Type IV hypersensitivity - Cell-mediated immune responses invariably cause some tissue destruction, which may be permanent. Despite examples of antibody-mediated tissue damage discussed above, the antibody response generally achieves its purpose in eliminating invading organisms without any trace of damage to host. Cell-mediated (type IV) responses activation of both T cells and macrophages invariably causes some tissue destruction, which may be reparable if not too prolonged, but can also lead to fibrosis and even calcification with serious permanent loss of tissue. From medical viewpoint granuloma formation and organ transplant histo compatibility are most important type IV response
Immunodeficiency Diseases

Immune system disorder may also be due to deficiencies of the system These deficiencies may be congenital or iatrogenic (induced by immunosuppressive drugs), or they may result from malignancies occurring in the lymphatic system. Abnormalities of the B-lymphocytes result in an immunodeficiency accompanied by abnormal production of antibodies. These abnormalities may result from infections such as diseases of the liver, from immune complex disorders such as systemic lupus erythematosus, or from malignancies Multiple myeloma is a malignant disease of plasma cells in which certain clones of Blymphocytes produce an over abundance of antibodies. The antibodies are excreted into urine, where they are known as Bence Jones proteins. Decreased ability of the B-lymphocytes to produce antibodies is called hypo-gammaglobulinemia. Such a condition may result from a genetic defect or the failure of the body to produce B-lymphocytes.

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 Bone marrow disorders may also be a cause abnormalities of the T-lymphocytes result in reduced capacity for cell-mediated immunity. This condition, called DiGeorge's syndrome, often develops from malformation or absence of the thymus gland An individual with this syndrome is highly susceptible to infection by fungi, protozoa, and viruses. When both B-lymphocytes and T-lymphocytes are deficient, the condition is called severe combined immunodeficiency Recent procedures employing transplantation of bone marrow tissue have attempted to relieve this condition

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