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The Immune Sustems

THE IMMUNE RESPONSE

by T.C. McGuire

Cells of the Immune System

All lymphocytes arise from a common bone marrow stem cell but follow different matura-tional pathways after leaving the bone mar­row.¹'² One pathway is for lymphocytes destined to provide cell-mediated immunity, the T-Iym-phocytes. The T-lymphocytes migrate from the bone marrow to the thymus, where their ma­turation is influenced by the thymic environ­ment, including soluble hormones secreted by thymic epithelial cells.³ They then leave the thymus to populate the thymic-dependent areas of the lymph nodes (paracortical) and spleen (periarteriolar lymphocytic sheaths).

The mature T-lymphocyte population is het­erogeneous, especially with regard to func­tion.⁴ One population, referred to as T-helper cells, interacts with cells of the second major pathway, the B-lymphocytes, to produce anti­body to most antigens (T-dependent antigens). A second T-lymphocyte population, called T-cy-totoxic cells, is responsible for such functions as graft rejection, protection against infection, and the destruction of tumor cells and cells in­fected with viruses, bacteria or other patho­gens. A third type of T-lymphocyte is the T-suppressor cell, which depresses function of other lymphocytes involved in cell-mediated and antibody-producing functions. There may be other T-lymphocyte populations that regu­late immune reactions.

The other major immune pathway involves B-lymphocytes, the cells that become plasma cells and secrete antibodies. In chickens, cells programmed to become B-lymphocytes leave the bone marrow and migrate to the bursa of Fabricius, where they mature,³ The mammal­ian equivalent of this bursa is unknown; how ever, mature B-lymphocytes eventually populate the B-lymphocyte-dependent areas of the mammalian spleen (follicles with adjacent ar-terioles) and lymph nodes (follicles). The B-lymphocytes respond to a few antigens without T-lymphocyte help to produce antibody. Such antigens (T-independent antigens) are not nu­merous and include such things as bacterial lipopolysaccharides. Most antigens are T-de­pendent and require B-lymphocytes and T-helper lymphocytes to produce antibody.

A third cell, the macrophage, functions in host defense independent of lymphocytes or by interacting with lymphocytes. Macrophages are thought to present antigen to B- and T-lym-phocytes in the initial step of the immune re­sponse. The macrophage/monocyte system arises in the bone marrow from promonocytes, which enter the blood as monocytes.⁵ Monocytes re­main in circulation a few days before entering various organs to become macrophages.

In summary, antibody formation is initiated by macrophage presentation of antigen to B-lymphocytes and T-helper lymphocytes, result­ing in proliferaton and differentiation of B-lymphocytes to plasma cells that secrete anti­body. Cell-mediated immunity is initiated by macrophage presentation of antigen to T-cyto-toxic lymphocytes, which proliferate to expand this population of killer cells. Antigen inter­action with T-suppressor lymphocytes creates cells that can regulate antibody production and some T-lymphocyte function.

B-Lymphocytes and Immunoglobulins

Immunoglobulin on the surface of B-lympho­cytes is the receptor for antigen. Clones of B-lymphocytes, each having surface immuno-globulin with antibody activity against a dif­ferent antigen, are present in animals at birth. Antigen entering the body reacts with the B-

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lymphocytes- This reaction causes prolifera­tion of the B-lymphocytes, which then differ­entiate to plasma cells and secrete antibody capable of reacting with the antigen. Several features of B-lymphocvtes allow antigen rec­ognition, including surface immunoglobulin, surface receptors for complement, and cyto-plasmic immunoglobulin.⁶"¹⁰

Plasma cells secrete immunogiobulins, which have antibody activity against specific anti­gens. The 5 major classes of immunogiobulins include IgG (in serum and colostrum), IgM (in serum), IgA (in secretions), IgE (in serum and mast cells), and IgD (on the surface of B-lym-phocytes).⁶ The horse has 5 subclasses of IgG, the most important of which are IgG and IgG(T).^11-21

Immunoglobulin Function

To be effective against most infectious or­ganisms, antibody must not only bind to the organism but must also interact with other parts of the host defense system. The most im­portant of these are the inflammatory cells (monocytes/macrophages, neutrophils and eo-sinophils) and the complement system. The complement system consists of at least 20 serum proteins that, when activated, interact in a cascading sequence similar to the clotting system. Binding of antibody to antigen allows a portion of the antibody molecule to interact with the first component of complement sys­tem. Complement activation results in lysis of microorganisms, increased vascular permea­bility, chemotaxis in WBC and a variety of other inflammatory reactions. The WBC, es­pecially macrophages and neutrophils, can also attach to bound antibody, resulting in phagocytosis or degranulation. In horses, IgG mediates these reactions.¹³

Of the immunogiobulins described, IgG and IgM are most commonly measured. Several technics are available for quantitation.

Radial Immunodiffusion: This technic re­quires the use of monospecific antibody against the class or subclass being measured. Antibody is added to melted agar and poured onto glass slides. Wells are punched in the cooled agar and the serum or fluid to be tested is placed in the wells. The size of the circle of precipitate that develops around the well is proportional to the concentration of the immunoglobulin being measured. Results are calculated from a standard curve of known immunoglobulin con­centrations prepared with each test and results are expressed as mg/dl.

Radial immunodiffusion is the only test de­scribed here that can measure various immu­noglobulin classes and subclasses specifically. For instance, if an IgM determination is re­quired, radial immunodiffusion is the only suitable test. The same is true when values of IgG(T), IgA and AI are required. The level of IgG can be quantitatively measured by radical immunodiffusion and semiquantitatively measured by other technics discussed below. The disadvantage of radial immunodiffusion is that monospecific antisera for each immuno­globulin to be tested are required, in addition to a calibrated standard serum and at least 24 hours to obtain results. Some types of monospe­cific antisera can be purchased and eventually all may be commercially available. Anti-equine IgG and IgM sera are available and fortunately some available anti-human IgM sera react with equine IgM.²² Other antisera are avail­able only from laboratories studying equine immunogiobulins.

Zinc Sulfate Turbidity Test: This test for im­munoglobulin levels is commonly performed on bovine sera but also works well on equine sera.^{23 -25} The test involves the addition of equine serum to a solution of zinc sulfate. The result­ant turbidity is measured in a spectrophoto-meter and compared with a standard curve to determine the level of immunoglobulin. The test measures primarily IgG levels but is not specific for IgG. Because hemoglobin interferes with the test, a correction factor must be used when hemolyzed serum is tested.

Under defined conditions, the zinc sulfate turbidity test can be used without a spectro-photometer to determine if passive transfer of immunogiobulins to a foal has occurred.²⁵ Five ml of zinc sulfate solution (205 mg/L H₂0), boiled to remove CO₂, are mixed with 0.1 ml test serum and incubated at room temperature for an hour. Appearance of cloudiness or opac­ity in the tube indicates the foal has an im­munoglobulin level above 400-500 mg/dl.

Serum Electrophoresis and Total Serum Pro­tein: Measurement of the percentage of gamma-globulin by serum electrophoresis on cellulose acetate strips and determination of the total serum protein level of the same sample allows calculation of the amount of gamma-globulin in serum. This procedure, like the zinc sulfate turbidity test, measures primarily IgG but is not specific for IgG since portions of other classes are included. Also, as with the zinc sul­fate turbidity test, no information about the IgM level is obtained by serum electrophoresis.

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Measurement by serum electrophoresis and total protein determination is reasonably ac­curate. The technic requires specialized equip­ment usually available only in commercial and research laboratories.

T-Lymphocytes

The best in vivo test of T-lymphocyte func­tion is intradermal injection of antigen into a. sensitized host. To check the delayed hypersen-sitivity response of horses, dinitrochloroben-zene (DNCB) is used for sensitization.²⁶ Five daily skin applications of 2 mg DNCB in di­methyl sulfoxide are used to sensitize normal horses. Horses are challenged a week after the last sensitization dose by application of 0.2 ml of 0.2% DNCB in olive oil. A raised area of thickened skin occurs at the challenge site in sensitive animals within 24 hours. Biopsy of this site reveals primarily lymphocytes with a smaller number of other inflammatory cells. Lack of response indicates a deficiency in cell-mediated immune function.

If time is not available to sensitize a horse to evaluate the delayed hypersensitivity re­sponse, a simple qualitative test is available. Intradermal injection of 50 ixgphytohemagglu-tinin-P results in a lymphocyte reaction in 12-24 hours that resembles the delayed hypersen­sitivity reaction to DNCB. This reaction re­quires no prior sensitization and apparently requires T-lymphocytes for expression.²⁶ Lack of response indicates a defect in cell-mediated immunity. In vitro measurement of equine T-lymphocytes and their products is described elsewhere.²⁷³²

Passive Immunization

Passive immunization is any transfer of im­munity from a resistant to a susceptible ani­mal. An example of passive immunization is the injection of tetanus antitoxin, which pro­vides immediate protection for the recipient. However, the immunity lasts only until the transferred antibodies are catabolized; the half-life of IgG in horses is 23 days.³³ Active immun­ization is the production of immunity in re­sponse to antigen administration. At least several days are required before protection is achieved with active immunization. Neverthe­less, a distinct advantage of active immuniza­tion is persistence of memory lymphocytes that react to subsequent antigen exposure with an accelerated secondary immune (anamnestic) response.

Transfer of Maternal Antibodies

Normal foals are born with a fully immuno-competent lymphoid system. However, patho­genic microorganisms might kill the foal before antibody production and/or cell-mediated im­munity can be stimulated were it not for trans­fer of antibodies to foal via colostrum.

Nearly all transfer of human maternal an­tibodies occurs transplacentally. The major protective role of human colostral antibodies is in the GI tract of the newborn. Dogs and cats have some transplacental antibody transfer but the majority is through colostrum. Horses have no transplacental antibody transfer and all transfer is by colostrum.³⁴

The amount of transplacental passage of an­tibody is related to the placentation of the spe­cies, especially to the amount of tissue between fetal and maternal circulation. Horses have an epitheliochorial placenta, which presents the greatest barrier between the 2 circulations.

Regardless of the fact that the equine fetus receives no maternal antibodies transplacen­tally, the serum of foals that have not suckled contains IgM; some have IgG and possibly other immunoglobulins.^{35 ar} However, these im-munoglobulins are present in small amounts and have no significant protective function for the neonate. The serum IgM and IgG levels in calves that have not suckled are elevated in cases of in utero infection.³⁸ Since equine fe­tuses can respond to some antigens several months before birth and to many antigens just prior to birth, evaluation of immunoglobulin levels in serum from aborted fetuses or dis­eased foals that have not suckled might pro­vide clues to the cause of the abortion or disease.³⁹