Clinical Veterinary Immunology

Laurel J. Gershwin

Department of Pathology, Microbiology, and Immunology
School of Veterinary Medicine
University of California, Davis
Davis, California

The field of clinical immunology has evolved from serological testing for the presence of antibodies to infectious agents to a multifaceted discipline that utilizes some of the traditional techniques in addition to many newer more sensitive assay systems. Yet it is still involved with evaluation of the immune system of patients and the ability of the immune system to respond to antigenic stimuli. Assays developed to target specific parts of the immune system enable the clinician not only to determine if a patient has normal immune responsiveness but also to target those parts of the immune system that are suspect of inadequate function. Serology has historically been used to determine retrospectively if a patient were infected with a particular disease agent; antibody titers continue to have importance in diagnostics.

This chapter reviews some basic principles of immunology and presents current methodologies used in the clinical immunology laboratory.

Entry into the body of a pathogen is the first stimulus for immunity. Pathogens contain pattern recognition receptors called pathogen associated molecular patterns (PAMP), which are recognized by Toll-like receptors (TLR) on the surface of host cells. There are at least 10 such receptors, each recognizing a different motif. For example, TLR 2 recognizes the peptidoglycan of the Gram-positive bacterial cell wall, TLR 4 recognizes lipopolysaccharide from the Gram-negative bacterial cell wall, TLR 6 recognizes flagella protein present on motile bacteria, and TLR 9 recognizes DNA containing cytosine-guanine repeating motifs (CpG). The binding of these TLRs with their ligands stimulates production of proinflammatory cytokines that jumpstart the immune response (Takeda, 2005).

The immune response is generally divided into innate and acquired responses. This division is based on the need for the host to have previously been exposed to the antigen/pathogen in order to rapidly mount a protective response. Innate immunity does not require previous exposure to a pathogen for it to be effective. It is thus not an antigen-specific response. Innate immunity is a broad category that includes protective barriers such as skin and mucosa. Reflexes are included, such as the cough coupled with the anatomical/physiological function of the mucociliary apparatus, which moves inhaled material out of the respiratory system. Dogs with inherited ciliary dyskinesis have nonfunctional cilia and suffer from repeated respiratory infections because of their inability to remove inhaled particles (such as bacteria) from the lung.

Phagocytes are important components of innate immunity. The initial responder to infection is usually the polymorphonuclear leukocyte or neutrophil. These cells participate in phagocytosis and killing of bacteria. Dogs with inherited cyclic neutropenia develop cyclic bouts of bacterial disease that coincide with the episodes during which the bone marrow shuts down its production of these essential phagocytes. Other defects, such as that seen in calves with bovine leukocyte adhesion deficiency (BLAD), occur when production is good, but the neutrophils lack the CD18 part of the adhesion molecule that allows them to adhere to blood vessel endothelium and then exit into the tissue by diapedesis. These animals develop even more critical disease because their defect is not cyclic, but constant. These calves generally succumb to overwhelming bacterial disease within the first 6 months of life. These experiments in nature demonstrate the importance of the innate defense provided by the neutrophil.

The other population of phagocyte is the macrophage. This cell plays a role not only as a phagocyte, generally entering an area of inflammation after the neutrophil, but also as a vital link to the acquired immune response. Macrophages function as antigen-presenting cells. As such they engulf a pathogen, digest it within a vacuole, and then display peptides generated from the engulfed organism on their cell surface. This antigen presentation function relies on the presence of a cell surface molecule called major histocompatibility complex antigen class II (MHC II). Lymphoid cells of the CD4 + T cell lineage then bind to the peptide and to the MHC II for initiation of the immune response. This is a critical step in immune responses. However, there is another cell type, the dendritic cell, that performs the antigen presentation function more efficiently than the macrophage. These cells are pivotal to induction of the acquired immune response and serve as an effecter for innate immunity.

The need for acquired immunity is demonstrated by certain bacterial species that are able to live and divide after being ingested by a macrophage. These organisms, called facultative intracellular bacteria, are able to overcome the macrophage and prevent their own digestion in the phagosome. To overcome the infection, the macrophages infected with these bacteria require signals from cytokines that are secreted by T cells stimulated in an acquired immune response. Infection of cattle with Mycobacterium bovis subspecies paratuberculosis causes a chronic wasting disease because of the ability of the bacteria to overcome the killing function of the macrophages. The acquired immune response required for killing these organisms is discussed with cellular immunity.

There is a population of lymphocytes that are neither T nor B cells; they lack the receptors for antigen recognition. These cells are natural killer cells (NK cells). The NK cells have the ability to recognize cells that lack or have depressed levels of the MHC class I molecule on the cell surface. Many tumor cells and some viral infected cells fall into this category. It is an evasion technique employed by some viruses to down-regulate the expression of the MHC molecules, which are required for recognition of the effector cells of the immune system. These NK cells are part of the innate immune system, because they are available to act on target cells without prior exposure.

There are two major types of T cells: CD4 + helper T cells and CD8 + cells, usually called cytotoxic T cells. This latter group has the capacity to kill target cells that are infected with antigens, such as viruses. There are two main subsets of CD4 + T helper cells: T helper 1 and T helper 2. The Th1 cells assist in cellular immune responses, such as activation of the macrophages infected with facultative intracellular bacteria. The Th2 cells provide “help” to B cells by provision of cytokines and co-stimulatory molecules (as described earlier). This T cell help initiates clonal expansion into mature B cells and ultimately into memory B cells and plasma cells. The plasma cells are the end cell that makes the immunoglobulin (antibody) that is so important in humoral immunity.

These Th1 and Th2 cells are primarily identified by the cytokines that they produce. Th1 cells make IL-2, IL-12, and interferon γ. The former activates T cells to divide and proliferate, and the latter activates macrophages to become more efficient killers. Cytokines produced by the Th2 subset include IL-4, IL-5, and IL-13. IL-4 is a B cell growth factor; and in conjunction with IL-13, they can facilitate development of an allergic type response (in which plasma cells produce lots of IgE). The T helper cell subsets were originally described in the inbred mouse, where the division between the two is distinct. However, in many of out bred species, such as humans and cattle, the distinction is less clear, with a T helper 1 or 2 skew more commonly identified than a complete polarization of the immune response.

One additional T cell subset that is described is the regulatory T cell (T reg). These cells are CD4 + and CD25 + and contain the nuclear activation factor FoxP3. T regs produce IL-10 and TGF-β, which depress the T helper 2 response. These cells may have a role in control of autoimmunity and allergy.

Electrophoretic separation of serum proteins separates the proteins into four broad categories: albumin, alpha globulins, beta globulins, and gamma globulins. The antibody activity is present in the gamma globulin fraction, with a slight amount in the beta fraction. These immunoglobulins are heterogeneous, having different molecular weights and functional properties.

There are five classes (isotypes) of immunoglobulins: IgG, IgM, IgA, IgD, and IgE. They share a basic structure, which consists of four polypeptide chains bound together by disulfide bonds. Two of these chains are called light chains, because with a molecular weight of about 22K each they are lighter than the other two heavy chains (approximately 55K each). At the nitrogen terminal of the polypeptide chains on all four chains is a portion of variable amino acid sequences. This is the antigen-binding end of the immunoglobulin. The hinge region of the immunoglobulin provides for flexibility of the molecule for binding to antigenic epitopes.

In the serum, IgG is the antibody class with the greatest concentration, approximately 1 to 2 g/100 ml, with some species differences (Tizard, 2008). Subclasses of IgG are recognized in most species. IgG has a four polypeptide-chain structure with a total molecular weight of 180,000 daltons. The heavy chains in IgG are called gamma chains and are unique to IgG. Immunoglobulin G is important in host defense because it can exit the vascular system and distribute throughout the extravascular tissue fluid where it has many protective functions. For example, IgG can agglutinate bacteria, causing them to clump; it can opsonize bacteria, by binding to the bacteria by the Fab fragment and to the phagocyte by receptors for the Fc fragment, thereby facilitating engulfment of the bacteria by the phagocyte. The complement system (a series of serum proteins to be discussed later in this chapter) can be activated by two IgG molecules bound near each other on a cell membrane and target cells can be lysed by this mechanism. In addition, IgG can participate with several different effector cells in antibody-dependent cellular cytotoxicity (ADCC). This mechanism allows destruction of virus-infected cells by lymphocytes that lack specific antigen receptors. The ability of IgG to neutralize toxins, such as those produced by Clostridium tetani, is an important protective mechanism for bacterial diseases.

Immunoglobulin M (IgM) is the first antibody to be synthesized in response to an immunogenic stimulus and is the first antibody seen in ontogeny. In serum, IgM is present in the second greatest concentration, generally between 100 and 400mg/100ml (species dependent). The structure of IgM consists of five of the basic four polypeptide units held together by a J chain. The large size of IgM (900,000 daltons) keeps it confined to the intravascular space. There are a total of 10 potential antigen-binding sites on IgM. Even though in reality, because of steric hindrance, only five to seven of the antigen-binding sites are functionally active, this large capacity to bind antigens makes IgM an efficient antibody at agglutination, precipitation, opsonization, complement fixation, and virus neutralization.

IgD is the is usually of not generally quantitated in the serum, although serum levels are reported for humans are greater than those measured for IgE. IgD is a four-polypeptide chain configuration (heavy chains are called δ) with a molecular weight of 180,000 daltons. IgD serves as a B cell receptor for antigen. Early in an immune response, immature B cells express IgD. As the cell matures in response to antigen, the IgD is replaced with monomeric IgM. Although IgD has been demonstrated in humans, mice, pigs, horses, cattle, dogs, and chickens, information is lacking in the cat. The existence of IgD in the animal species (other than the mouse) is based primarily on genome sequencing.

Immunoglobulin E is recognized and characterized in dogs, cattle, sheep, pigs, horses, and functionally recognized in cats. IgE has never been documented in avian species. IgE occurs normally in very small amounts in the serum (nanogram quantities). In allergic or parasitized individuals, the serum concentration of IgE is greatly increased. The basic four-polypeptide chain structure of IgE, with epsilon heavy chains that contain one additional domain, has a molecular weight of 196,000 daltons. Functions of IgE are mediated through its ability to bind via the high affinity Fcε receptors on tissue mast cells and blood basophils. When an antigen cross-links these cell-bound antibodies, the cell degranulates, releasing vasoactive amines, stimulating leukotriene synthesis, and resulting in potent pharmacological effects. IgE can also bind to the low-affinity IgE receptor, CD23. Binding of IgE to the CD23 stimulates a regulatory function. IgE can participate in parasite killing by binding to low-affinity IgE receptors on eosinophils and then to the parasite by specific Fab regions. This allows the eosinophils to deposit their toxic granule contents on the cuticle of the worm.

The cellular immune response is important for viral pathogens, tumor immunity, and for defense against bacterial pathogens that are able to evade killing by macrophages (these are facultative intracellular bacteria). The T cell response is important for cellular immunity. The CD8 + T cells are cytotoxic cells. They are able to recognize peptides derived from antigens that grow in an intracellular location and are processed and “presented” on the cell surface with the major histocompatibility molecule (MHC class I). Once the T cell has recognized the peptide from the antigen, it is able to respond by killing the infected cell and others infected with the same pathogen. The killing is mediated by interaction of surface molecules called “death receptors,” Fas and Fas-ligand to initiate apoptosis. The polymerization of perforins from the T cell onto the surface of the infected target cell allows for the entry of granzymes, which are molecules that are able to initiate cell death. This mechanism of immunity is particularly effective for viruses such as herpesviruses that are primarily cell associated and therefore not very accessible to antibodies.

The immune response to the facultative intracellular pathogens is primarily mediated by the T helper 1 subset of CD4 + T cells. These cells make interferon γ, which activates the macrophage and helps it to become a better killer. It does this by increasing a variety of metabolic activities, such as synthesis of cytokines (tumor necrosis factor α, IL-1α, IL-12), by increasing the ruffled membrane activity and increasing nitric oxide production.

When the clinician is concerned that there may be some defect in the innate immune system of a patient, the concern is usually initiated by repeated infection in the patient. If the infection is primarily bacterial, the focus of the immune system investigation will be on phagocytes and humoral immunity. Other types of innate defenses that may be perturbed, such as the ciliary dyskinesis described earlier, require other diagnostic assays such as bronchial biopsy and radio isotopic clearance studies.