Chapter 12. Toxic Responses of the Immune System

• Immunity is a series of delicately balanced, complex, multicellular, and physiologic mechanisms that allow an individual to distinguish foreign material from “self” and to neutralize and/or eliminate that foreign matter.
• Innate immunity, which eliminates most potential pathogens before significant infection occurs, includes physical and biochemical barriers both inside and outside of the body as well as immune cells designed for specific responses.
• Acquired immunity involves producing a specific immune response to each infectious agent (specificity) and remembering that agent so as to mount a faster response to a future infection by the same agent (memory).
• Autoimmunity occurs when the reactions of the immune system are directed against the body's own tissues, resulting in tissue damage and disease.
• Hypersensitivity reactions require prior exposure leading to sensitization in order to elicit a reaction on subsequent challenge.
• Xenobiotics that alter the immune system can upset the balance between immune recognition and destruction of foreign invaders and the proliferation of these microbes and/or cancer cells.

Immunity is a homeostatic process, a series of delicately balanced, complex, multicellular, and physiologic mechanisms that allow an individual to distinguish foreign material from “self” and to neutralize and/or eliminate the foreign matter. Decreased immunocompetence (immunosuppression) may result in repeated, more severe, or prolonged infections as well as the development of cancer. Immunoenhancement may lead to immune-mediated diseases such as hypersensitivity responses, and if some integral bodily tissue is not identified as self, an autoimmune disease may be the end result.

The immune system comprises numerous lymphoid organs and numerous different cellular populations with a variety of functions. The bone marrow and the thymus support the production of mature T and B lymphocytes and myeloid cells, such as macrophages and polymorphonuclear cells (PMN), and are referred to as primary lymphoid organs.

Within the bone marrow, the cells of the immune system developmentally “commit” to either the lymphoid or myeloid lineages. Cells of the lymphoid lineage make a further commitment to become either T cells or B cells. T-cell precursors are programmed to leave the bone marrow and migrate to the thymus, where they differentiate further.

Mature naive or virgin lymphocytes (those T and B cells that have never undergone antigenic stimulation) are first brought into contact with exogenously derived antigens within the spleen and lymph nodes, otherwise known as the secondary lymphoid organs.

Lymphoid tissues associated with the skin and the mucosal lamina propria of the gut, respiratory tract, and genitourinary tract can be classified as tertiary lymphoid tissues. Tertiary lymphoid tissues are primarily effector sites where memory and effector cells exert immunologic and immunoregulatory functions.

Antigen Recognition

Immunity

Innate immunity is a nonspecific first-line defense response with no associated immunologic memory. The innate immune response to a foreign organism is the same for a secondary or tertiary exposure as it is for the primary exposure. Acquired immunity is characterized by both specificity and memory, resulting in a much greater immune response on secondary challenge.

Antigen

A nonself-molecule, including foreign DNA, RNA, protein, carbohydrates, and even mutated self-proteins, that can be recognized by the immune system is called an antigen. Antigens are usually rearranged for presentation to other immune cells to induce the immunologic response.

Each antigen is recognized by specific antibodies produced by B cells. There are several light-chain genes and several heavy-chain genes coding for antibody protein, and when rearranged in various combinations, contribute to the immense genetic diversity of the produced antibodies.

Innate Immunity

Innate immunity acts as a first line of defense against infectious agents, eliminating most potential pathogens before significant infection occurs. It includes physical and biochemical barriers both inside and outside of the body as well as immune cells designed for specific responses. There is no immunologic memory associated with innate immunity.

Most infectious agents enter the body through the respiratory system, gut, or genitourinary tract, whereas the skin provides an effective barrier. Innate defenses include mucus secreted along the nasopharynx, the presence of lysozyme in most secretions, and cilia lining the trachea and main bronchi. In addition, reflexes such as coughing, sneezing, and elevation in body temperature are also a part of innate immunity. Pathogens that enter the body via the digestive tract are met with severe changes in pH (acid) within the stomach and a host of microorganisms living in the intestines.

Cellular Components: NK, NKT, PMN, and Macrophage

Two general types of cells are involved in nonspecific (innate) host resistance: natural killer (NK) cells and professional phagocytes. NK cells can recognize virally infected and malignant changes on the surface of cells as well as an antibody-coated target cell. The latter recognition is utilized in cell-mediated immunity (CMI). Using surface receptors, the NK cell binds, expels cytolytic granules, and induces apoptosis of the target cell.

NKT cells express surface markers characteristic of both NK and T cells and play a role in innate immunity, as their activation is early and they do not possess immunologic memory. They also produce cytokines and mediate cytolysis of target cells.

Phagocytic cells include PMN (neutrophil) and the monocyte/macrophage, which develop from pluripotent stem cells that have become committed to the myeloid lineage (Figure 12–1). PMNs are excellent phagocytic cells and can eliminate most microorganisms and induce an inflammatory response.

Macrophages are terminally differentiated monocytes. On exiting the bone marrow, monocytes distribute to the various tissues where they can then differentiate into macrophages. Within different tissues, macrophages have distinct properties and vary in extent of surface receptors, oxidative metabolism, and expression of major histocompatibility complex (MHC) class II, which present the antigen.

Should PMNs be unable to contain an infection, macrophages are then recruited to the site of infection. Macrophages have both phagocytic and bactericidal functions, and can also function as antigen-presenting cells (APCs). They are recruited to sites of inflammation by chemotactic factors, can be activated by cytokines to become more effective killers, and can produce cytokines. Macrophages also play critical roles as scavengers in the daily turnover of senescent tissues such as red cell nuclei from maturing red cells, PMNs, and plasma cells.

Soluble Factors: Acute-Phase Proteins and Complement

Soluble components of innate immunity (Table 12–1) are the acute-phase proteins and complement. On infection, macrophages become activated and secrete various cytokines, which are carried by the bloodstream to distant sites. This global response to foreign agents is termed the acute-phase response and consists of fever and large shifts in the types of serum proteins synthesized by hepatocytes. These proteins can bind to bacteria via a process called opsonization and facilitate the binding of complement and the subsequent uptake of the bacteria by phagocytic cells.

The complement system consists of about 30 proteins whose primary functions are the destruction of the membranes of infectious agents and the promotion of an inflammatory response. Complement activation occurs with each component sequentially acting on others to coat the foreign cell and disrupt membrane integrity without harming the host cells. The final components that can enter the membrane and disrupt its integrity are termed the membrane attack complex (MAC). The complement-coated material is targeted for elimination by interaction with complement receptors on the surface of circulating immune cells.

Acquired (Adaptive) Immunity

If the primary defenses against infection (innate immunity) are breached, the acquired arm of the immune system is activated and produces a specific immune response to each infectious agent. This branch of immunity can protect the host from future infection by the same agent. Therefore, two key features that distinguish acquired immunity are specificity and memory. This means that in a normal healthy adult, the speed and magnitude of the immune response to a foreign organism are greater for a secondary challenge than they are for the primary challenge (Table 12–1).

Essential to the development of specific immunity is recognition of antigen and generation of an antibody that can bind to it. An antigen is defined functionally as a substance that can elicit the production of a specific antibody and can be specifically bound by that antibody. Small antigens are termed haptens and must be conjugated with carrier molecules (larger antigens) in order to elicit a specific response.

Antibodies, proteins classified as immunoglobulins (Igs), are produced by B cells and are also defined functionally by the antigen with which they react (i.e., anti-sheep red blood cell [anti-sRBC] IgM). There are five types of Ig that are related structurally (Table 12–2): IgM, IgG (and subsets), IgE, IgD, and IgA. All Igs are made up of heavy and light chains and of constant (Fc) and variable regions. It is the variable regions that determine antibody specificity. The variable region interacts with antigen, whereas the Fc region mediates effector functions such as complement fixation and phagocyte binding (via Fc receptors). Antibodies also coat foreign cells to help with opsonization, initiate the complement cascade to lead to cell lysis, bind to viral particles, and bind to antigens on target cells to help NK cells and cytotoxic T lymphocytes (CTL) destroy them.

During an immune response, the cells of the immune system communicate via a vast network of soluble mediators, the cytokines. Nearly all immune cells secrete cytokines, which may have local or systemic effects. Table 12–3 provides a brief summary of the sources and functions of cytokines.

Cellular Components: APCs, T Cells, and B Cells

In order to elicit a specific immune response to a particular antigen, that antigen must be taken up and processed by accessory cells, called APCs, for presentation to lymphocytes. The macrophage plays a critical role as an APC in acquired immunity. Although thought of more for its ability to produce Ig, the B cell can also serve as an APC.

APCs and lymphocytes interact during the immune response. APCs absorb the antigen, cut it into pieces, and then display a piece of the antigen on its cell surface attached to a complex of proteins called MHC II.

Besides serving as APCs, B lymphocytes are also the effector cells of humoral immunity, producing a number of isotypes of Ig with varying specificities and affinities. On antigen binding to surface Ig, the mature B cell becomes activated and, after proliferation, undergoes differentiation into either a memory B cell or an antibody-forming cell (AFC; plasma cell), actively secreting antigen-specific antibody.

T cells undergo a complex process of maturation wherein only cells that do not recognize self but do bind to MHC II proteins and recognize foreign antigens survive. These become either T-helper cells (which carry a certain protein on their surface called CD4 and work by facilitating the B-cell response) or T-cytotoxic cells (which carry CD8 and mediate cell killing).

Humoral and Cell-Mediated Immunity

The activation of antigen-specific T cells begins with the interaction of the T-cell receptor (TCR) with MHC class II peptide. On activation and in the presence of IL-1 secreted by the APC, T cells begin to produce the T-cell growth factor IL-2 and express receptors for them. As T cells begin to proliferate, they secrete numerous lymphokines (Table 12–3), which can influence many aspects of the immune response. The next step in the generation of the humoral response is the interaction of activated T cells with B cells. This may be a direct interaction of the T cell with B cell (antigen-specific) or may simply involve the production of lymphokines, which lead to B-cell growth and differentiation into AFCs or memory B cells. A general diagram of the cellular interactions involved in the humoral immune response is given in Figure 12–2. The production of antigen-specific IgM requires 3 to 5 days after the primary (initial) exposure to antigen (Figure 12–3). On secondary antigen challenge, the B cells undergo isotype switching, producing primarily IgG antibody, which is of higher affinity. In addition, there is a higher serum antibody titer associated with a secondary antibody response.

There are two general forms of CMI, referred to as delayed-type hypersensitivity (DTH) and cell-mediated cytotoxicity. DTH is presented later in this chapter in the section titled “Immune-mediated Disease.” In cell-mediated cytotoxicity, the effector cell (CTL or NK) binds in a specific manner to the target cell (Figure 12–4), and the effector cell then releases the contents of its cytolytic granules onto the target cell, causing it to undergo programmed cell death.

Developmental Immunology

Developmental immunotoxicology involves investigation into the effects that xenobiotics have on the ontogeny of the immune system and includes prenatal (in utero), perinatal (<36 h of age), and neonatal periods of exposure. Immune development in humans and other species may be altered after perinatal exposure to immunotoxic chemicals. It has also been suggested that these effects may be more dramatic or persistent than those following exposure during adult life.

The immune system develops initially from a population of pluripotent hematopoietic stem cells that are generated early in gestation from uncommitted mesenchymal stem cells. This early population gives rise to all circulating blood cell lineages. Immunocompetent cells are produced in the bone marrow and thymus and then leave via the blood to the secondary immune organs: spleen, lymph nodes, and mucosal lymphoid tissue.

As thymic function wanes throughout life and thymocytes are no longer produced here, it is the pool of memory B and T cells that maintains immunocompetence for the life of the individual.

Neuroendocrine Immunology

Cytokines, neuropeptides, neurotransmitters, and hormones are an integral and interregulated part of the central nervous system, the endocrine system, and the immune system. The triad of influence these three systems exert over one another is bidirectional.

Xenobiotics can have significant effects on the immune system. Among the unique features of immune cells is their ability to be removed from the body and to function in vitro. This unique quality offers the toxicologist an opportunity to comprehensively evaluate the actions of xenobiotics on the immune system.

Many medical devices may have intimate and prolonged contact with the body. Possible immunologic consequences of this contact could be envisioned to include immunosuppression, immune stimulation, inflammation, and sensitization.

Methods to Assess Immunocompetence

General Assessment

All studies of immunocompetence should include toxicologic studies (such as organ weights, serum characteristics, hematologicparameters, and bone marrow function) to investigate the effects of immune modulation on other body organs. Histopathology of lymphoid organs also may provide insight into potential immunotoxicants. Moreover, use of fluorescently labeled monoclonal antibodies to cell surface markers in conjunction with a flow cytometer enables accurate enumeration of lymphocyte subsets and whether the xenobiotic may affect maturation.

Functional Assessment

Innate Immunity

Innate immunity encompasses all those immunologic responses that do not require prior exposure to an antigen and that are nonspecific in nature. These responses include recognition of tumor cells by NK cells, phagocytosis of pathogens by macrophages, and the lytic activity of the complement system.

To evaluate phagocytic activity, macrophages are placed in culture plates and incubated with radiolabeled red blood cells. Those cells that are not bound by the macrophages are removed, as are the cells that are bound but not phagocytized. The macrophages are then lysed to determine the amount of cells that were phagocytized. This test provides information about both the binding and phagocytizing activity of the macrophages and can also be performed in vivo by measuring the uptake of the radiolabeled red blood cells by certain tissue macrophages.

Another method to evaluate phagocytosis in vitro is to evaluate the uptake of latex spheres by macrophages. Evaluation of the ability of NK cells to lyse tumor cells is achieved by incubating radiolabeled target cells with NK cells and measuring the amount of radioactivity released into solution from the target cells.

Acquired Immunity: Humoral

The plaque (antibody)-forming cell (PFC or AFC) assay tests the ability of the host to mount an antibody response to a specific antigen, which requires the coordinated interaction of several different immune cells: macrophages, T cells, and B cells. Therefore, an effect on any of these cells (e.g., antigen processing and presentation, cytokine production, proliferation, or differentiation) can have a profound impact on the ability of B cells to produce antigen-specific antibody.

A standard PFC assay involves immunizing mice with sRBC. The antigen is taken up in the spleen and an antibody response occurs. Four days after immunization, spleens are removed and splenocytes are mixed with RBCs, complement, and agar, the mixture plated, and incubated until the B cells secrete anti-sRBC IgM antibody. This antibody then coats the surrounding sRBCs, and areas of hemolysis (plaques) can be seen.

The PFC assay can be evaluated in vivo using serum from peripheral blood of immunized mice and an enzyme-linked immunosorbent assay (ELISA; Figure 12–5). Serum from mice immunized with sRBCs is incubated in microtiter plates that have been coated with sRBC membranes to serve as the antigen for sRBC-specific IgM or IgG to bind. After incubation, an enzyme-conjugated monoclonal antibody (the secondary antibody) against IgM (or IgG) is added. This antibody recognizes the IgM (or IgG) and binds specifically to that antibody. Then, the enzyme substrate (chromogen) is added. When the substrate comes into contact with the enzyme on the secondary antibody, a color change occurs that can be detected by measuring absorbance with a plate reader.

Acquired Immunity: Cell-Mediated

Of numerous assays of CMI, three routinely performed tests are: the CTL assay, the delayed hypersensitivity response (DHR), and the T-cell proliferative responses to antigens.

The CTL assay measures the in vitro ability of splenic T cells to recognize allogeneic or antigenically distinct target cells by evaluating the ability of the CTLs to proliferate and then lyse the target cells. CTLs are incubated with target cells that have been treated so that they cannot themselves proliferate. CTLs recognize the target cells and proliferate until they are harvested. Then, they are incubated with radiolabeled target cells. CTLs that have acquired memory recognize the foreign MHC class I on target cells and lyse them.

The DHR evaluates the ability of memory T cells to recognize foreign antigen, proliferate and migrate to the site of the antigen, and secrete cytokines in vivo. Mice are sensitized by a subcutaneous injection of the chemical. Radiolabeled iodine is allowed to be incorporated into the mouse's mononuclear cells by injecting it into the mouse's bloodstream. Then, some of the sensitizing chemical is injected into the ear, and, after euthanizing the mouse, the ear is evaluated for the presence of radiolabeled mononuclear cells.

Several mechanisms exist to evaluate proliferative capacity of T cells in CMI. The mixed lymphocyte response (MLR) measures the ability of T cells to recognize foreign MHC class I and undergo proliferation.

Flow Cytometric Analysis

Flow cytometry employs light scatter, fluorescence, and absorbance measurements to analyze large numbers of cells on an individual basis. Usually, fluorochrome-conjugated monoclonal antibodies raised against a specific protein are employed for detection. This approach can be used to provide insight into which specific T-cell subsets are targeted after exposure to a xenobiotic, and to identify putative effects on T-cell maturation.

Molecular Biology Approaches to Immunotoxicology

Proteomics (the study of all expressed proteins in a particular cell, and thus the functional expression of the genome) and genomics (the study of all genes encoded by an organism's DNA), combined with bioinformatics, facilitate the evaluation of xenobiotic-induced alterations in the pathways and signaling networks of the immune system.

Mechanistic Approaches to Immunotoxicology

Once an agent has been identified as being an immunotoxicant, it may be necessary to further characterize its mechanism. A general strategy involves the following steps: (1) identifying the cell type(s) targeted by the agent; (2) determining whether the effects are mediated by the parent compound or by a metabolite of the parent; (3) determining whether the effects are mediated directly or indirectly by the xenobiotic; and (4) elucidating the molecular events responsible for altered leukocyte function.

Regulatory Approaches to the Assessment of Immunotoxicity

The NTP Tier Approach

The National Toxicology Program screens for potential immunotoxic agents using a tier approach. Tier I provides assessment of general toxicity (immunopathology, hematology, and body and organ weights) as well as endline functional assays (proliferative responses, PFC assay, and NK assay). Tier II was designed to further define an immunotoxic effect and includes tests for CMI (CTL and DHR), secondary antibody responses, enumeration of lymphocyte populations, and host resistance models.

Health Effects Test Guidelines

Guidelines for functional immunotoxicity assessments in regulatory studies recommend conduct of three tests. Assessment of immunotoxicity begins by exposure for a minimum of 28 days to the chemical followed by assessment of humoral immunity (PFC assay or anti-sRBC ELISA). If the chemical produces significant suppression of the humoral response, surface marker assessment by flow cytometry may be performed. If the chemical produces no suppression of the humoral response, an assessment of innate immunity (NK assay) may be performed.

Animal Models in Immunotoxicology

Rats and mice have been the animals of choice for studying the actions of xenobiotics on the immune system because: (1) there is a vast database available on the immune system, (2) rodents are less expensive to maintain than larger animals, and (3) a wide variety of reagents (cytokines, antibodies, etc.) are available. Many reagents that are available for studying the human immune system can also be used in rhesus and cynomolgus monkeys. Chicken and fish are being used to evaluate the immunotoxicity of xenobiotics as alternative animal models with heightened environmental consciousness.

The manipulation of the embryonic genome, creating transgenic and knockout mice, may allow complex immune responses to be dissected into their components. In this way, the mechanisms by which immunotoxicants act can be better understood. Severe combined immunodeficient mice (SCID) have been used to study immune regulation, hematopoiesis, hypersensitivity, and autoimmunity.

Evaluation of Mechanisms of Action

Direct effects on the immune system may include chemical effects on immune function, structural alterations in lymphoid organs or on immune cell surfaces, or compositional changes in lymphoid organs or in serum. Xenobiotics may exert an indirect action on the immune system as well. They may be metabolically activated to their toxic metabolites, and may also have effects on other organ systems (e.g., liver damage) that then impact the immune system.

Immunosuppression

Immunosuppression can be produced by numerous natural and synthetic chemicals as listed in Table 12–4.

Tobacco Smoke

Pulmonary defenses against inhaled gases and particulates are dependent on both physical and immunologic mechanisms. Immune mechanisms primarily involve the complex interactions between PMNs and alveolar macrophages and their abilities to phagocytize foreign material and produce cytokines.

In humans, the number of alveolar macrophages is increased three- to fivefold in smokers compared with nonsmokers and the macrophages present appear to be in an activated state but have decreased phagocytic and bactericidal activity. Decreased serum Ig levels and decreased NK-cell activity have been reported. Concentration-dependent leukocytosis (increased numbers of T and B cells) is well defined in smokers when compared with nonsmokers. Numerous immunologic studies conducted in animals exposed to cigarette smoke have demonstrated suppression of antibody responses.

Recombinant DNA-Derived Proteins

Biologics (e.g., blood or vaccine products) and recombinant DNA-derived proteins may elicit the production of neutralizing antibodies. The effects of neutralizing antibodies may also lead to hypersensitivity reactions.

Ultraviolet Radiation

Ultraviolet radiation (UVR) suppresses DHR in both animals and humans and results in decreased host resistance to infection. Induction of suppressor T cells and alterations in homing patterns have been suggested as possibilities. One plausible explanation is that UVR induces a switch from a predominantly Th1 response (favoring DHR) to a Th2 response (favoring antibody responses).

The purpose of the immune system is to protect the individual from disease states, whether infectious, parasitic, or cancerous, through both cellular and humoral mechanisms. In so doing, the ability to distinguish “self” from “nonself” plays a predominant role. However, situations arise in which the individual's immune system responds in a manner producing tissue damage, resulting in a self-induced disease, either (1) hypersensitivity, or allergy, or (2) autoimmunity. Hypersensitivity reactions result from the immune system responding in an exaggerated or inappropriate manner. In the case of autoimmunity, mechanisms of self-recognition break down and Igs and TCRs react with self-antigens, resulting in tissue damage and disease.

Hypersensitivity

Classification of Hypersensitivity Reactions

All four types of hypersensitivity reactions require prior exposure leading to sensitization in order to elicit a reaction on subsequent challenge. Figure 12–6 illustrates the mechanisms of hypersensitivity reactions as classified by Coombs and Gell.

Type I (Immediate Hypersensitivity)

Sensitization occurs as the result of exposure to appropriate antigens through the respiratory tract, dermally, or by exposure through the gastrointestinal tract and is mediated by IgE production. IgE binds to appropriate cells and sensitizes an individual; reexposure to the antigen results in degranulation of the mast cells with the release of preformed mediators and cytokines that promote vasodilatation, bronchial constriction, and inflammation.

Type II (Antibody-Dependent Cytotoxic Hypersensitivity)

Type II hypersensitivity is IgG-mediated. Tissue damage may result from the direct action of cytotoxic cells or by antibody activation of the classic complement pathway. Complement activation may result in cell lysis.

Type III (Immune Complex-Mediated Hypersensitivity)

Type III hypersensitivity reactions also involve IgG Igs. Ig may form complexes with soluble antigen and the complex may deposit (lodge) in various tissues, causing tissue damage. The most common location is the vascular endothelium in the lung, joints, and kidneys. Macrophages, neutrophils, and platelets attracted to the deposition site contribute to the tissue damage.

Type IV (Cell-Mediated Hypersensitivity)

Type IV is a DTH response. Contact hypersensitivity is initiated by topical exposure, and consists of two phases: sensitization and elicitation. Sensitization results in development of activated and memory T cells when the chemical is presented on an APC to T-helper cells in local lymph nodes, leading to generation of memory T cells.

On second contact, antigen-presenting Langerhans–dendritic cells present the processed hapten–carrier complex to memory T cells. These activated T cells then secrete cytokines that bring about further proliferation of T cells and facilitate the movement of inflammatory cells into the skin, resulting in erythema and the formation of papules and vesicles. Cells of the cell-mediated immune response may cause local tissue damage.

Whereas separation of hypersensitivity responses into types I to IV is helpful in understanding the involved mechanisms, it is important to realize that often pathology is the result of a combination of these mechanisms.

Assessment of Hypersensitivity Responses

Assessment of Respiratory Hypersensitivity in Experimental Animals

Methods for detecting pulmonary hypersensitivity can be divided into two types: (1) those for detecting immunologic sensitization and (2) those for detecting pulmonary sensitization. In the case of types I to III, immunologic sensitization occurs when antigen-specific Ig is produced in response to exposure to an antigen or, in the case of type IV, when a population of sensitized T lymphocytes is produced. Pulmonary sensitization is determined by a change in respiratory function subsequent to the challenge of a sensitized animal.

Guinea pig models have been most frequently used because the lung is the major shock organ for anaphylactic response. Immunologic sensitization may be determined by obtaining sequential blood samples throughout the induction period and measuring antibody titer. Pulmonary sensitization is evaluated by detecting the presence of pulmonary reactivity (either visible respiratory distress or changes in respiratory function) following challenge.

Assessment of IgE-Mediated Hypersensitivity Responses in Humans

Two skin tests available for immediate hypersensitivity testing measure a “wheal and flare” reaction. The prick–puncture test introduces very small amounts of antigen under the skin. For test compounds not eliciting a reaction in the less sensitive skin test, the intradermal test using dilute concentrations of antigen may be used, but there is a higher risk of systemic reactions.

In vitro serologic tests, ELISAs, and radioallergosorbent tests (RASTs) may also be used to detect the presence of antigen-specific antibody in the patient's serum.

Bronchial provocation tests may be performed by having the patient inhale an antigen into the bronchial tree and evaluating his or her pulmonary response.

Assessment of Contact Hypersensitivity in Experimental Animals—The two most commonly utilized guinea pig models are the Büehler test and the guinea pig maximization test. In the Büehler test, the test article is applied to the shaven flank and covered with an occlusive bandage for 6 h once a week for 3 weeks. On day 28, a challenge dose of the test article is applied to a shaven area on the opposite flank; the area is evaluated for signs of edema and erythema for 2 days afterwards. In the guinea pig maximization test, the test article is administered by intradermal injection, an adjuvant is employed, and irritating concentrations are used. These assays evaluate the elicitation phase of the response in previously sensitized animals.

The mouse local lymph node assay is a stand-alone alternative to the guinea pig assays for use in hazard identification of chemical sensitizers. Animals are dosed by topical application of the test article to the ears for 3 consecutive days. A few days later, the animals are injected with radiolabeled thymidine, which is incorporated into proliferating lymphocytes. Later, the animals are sacrificed and the local lymph nodes assayed for radiolabeled lymphocytes to see if the test article induced an immune response.

Assessment of Contact Hypersensitivity in Humans

Human testing for contact hypersensitivity reactions is by skin patch testing. Patches containing specified concentrations of the allergen in the appropriate vehicle are applied under an occlusive patch for 48 h. Once the patch is removed, the area is read for signs of erythema, papules, vesicles, and edema. Generally, the test is read again at 72 h and in some cases signs may not appear for up to 1 week or more.

Hypersensitivity Reactions to Xenobiotics

Numerous xenobiotics illicit hypersensitivity reactions. Polyisocyanates, and toluene diisocyanate in particular, used in the production of adhesives and coatings are known to induce the full spectrum of hypersensitivity responses, types I to IV, as well as nonimmune inflammatory and neuroreflex reactions in the lung. Inhaled acid anhydrides, which are used in the manufacturing of paints, varnishes, coating materials, adhesives, and casting and sealing materials, may conjugate with serum albumin or erythrocytes leading to type I, II, or III hypersensitivity reactions on subsequent exposure.

Metals

Metals and metallic substances, including metallic salts, are responsible for producing contact and pulmonary hypersensitivity reactions. Platinum, nickel, chromium, beryllium, and cobalt are commonly implicated.

Drugs

Hypersensitivity responses to drugs are among the major types of unpredictable drug reactions. Drugs are designed to be reactive in the body and multiple treatments are common. This type of exposure is conducive to producing an immunologic reaction. Immunologic mechanisms of hypersensitivity reactions to drugs include types I to IV. Penicillin is the most common agent involved in drug allergy.

Pesticides

Pesticides have been implicated as causal agents in both contact and immediate hypersensitivity reactions.

Latex

Natural rubber latex is used in the manufacture of over 40,000 products from balloons to surgical gloves. Dermatologic reactions to latex include irritant dermatitis and contact dermatitis.

Cosmetics and Personal Hygiene Products

Contact dermatitis and dermatoconjunctivitis may result from exposure to many cosmetic and personal hygiene products. These agents contain paraben esters, sorbic acid, phenolics, organomercurials, quaternary ammonium compounds, and formaldehyde.

Enzymes

Subtilin and papain are enzymes capable of eliciting type I hypersensitivity responses. Subtilin is used in laundry detergents. Both individuals working where the product is made and those using the product may become sensitized. Subsequent exposure may produce signs of rhinitis, conjunctivitis, and asthma. Papain is another enzyme known to induce IgE-mediated disease. It is most commonly used as a meat tenderizer and a clearing agent in beer production.

Formaldehyde

Formaldehyde exposure occurs in the cosmetics and textile industries, and in the furniture, auto upholstery, and resins industries. Occupational exposure to formaldehyde has been associated with the occurrence of asthma.

Autoimmunity

In cases of autoimmunity, self-antigens are the target, and in the case of chemical-induced autoimmunity, the disease state is induced by a modification of host tissues or immune cells by the chemical and not the chemical acting as an antigen/hapten.

Mechanisms of Autoimmunity

Three types of molecules are involved in the process of self-recognition: Igs, TCRs, and the products of MHC. Igs and TCRs are expressed clonally on B and T cells, respectively, whereas MHC molecules are present on all nucleated cells.

The process of negative selection against autoreactive T cells in the thymus is important in the prevention of autoimmune disease. T cells expressing receptors that bind to self-antigens undergo apoptosis (negative selection), whereas those that do not recognize self-proteins proliferate (positive selection) and migrate to the peripheral lymph organs. Some cells that recognize self-molecules do not die, but undergo anergy, where they stay in the body but are inactive.

Several mechanisms may break down self-tolerance, leading to autoimmunity. First, if exposure to antigens is not available in the thymus during embryonic development, such as to myelin, which is not produced until later in development, then antigen-specific T-cell-reactive lymphocytes not subjected to negative selection could induce an autoimmune reaction. Breakdown of self-tolerance to these antigens may be induced by exposure to adjuvants, chemicals used to enhance immunogenicity, or to another antigenically related protein. Second, T-cell anergy can be overcome with chronic lymphocyte stimulation. Third, interference with normal immunoregulation by CD8+ T-cell suppressor cells may create an environment conducive to the development of autoimmune disease.

As is the case with hypersensitivity reactions, autoimmune disease is often the result of more than one mechanism working simultaneously. Therefore, pathology may be the result of antibody-dependent cytotoxicity, complement-dependent antibody-mediated lysis, or direct or indirect effects of cytotoxic T cells.

Autoimmune Reactions to Xenobiotics

Table 12–5 lists chemicals known to be associated with autoimmunity, showing the proposed self-antigenic determinant or stating adjuvancy as the mechanism of action. Table 12–6 shows chemicals that have been implicated in autoimmune reactions, but in these cases the mechanism of autoimmunity has not been as clearly defined or confirmed.

Multiple Chemical Sensitivity Syndrome

Multiple chemical sensitivity syndrome (MCS) has been associated with hypersensitivity responses to chemicals. The syndrome is characterized by multiple subjective symptoms related to more than one system. The more common symptoms are nasal congestion, headaches, lack of concentration, fatigue, and memory loss. Many mechanisms have been suggested to explain how chemicals cause these symptoms. A major hypothesis is that MCS occurs when chemical exposure sensitizes certain individuals, and, on subsequent exposure to exceedingly small amounts of these or unrelated chemicals, the individual exhibits an adverse response.

New technology has brought more questions to be answered and new tools to assess these questions. The significant immunotoxicologic challenges needing to be addressed include: (1) how to interpret the significance of minor or moderate immunotoxic effects in animal models in relation to human risk assessment; (2) how to better integrate a consideration of exposure, especially to multiple agents simultaneously, into immunotoxicologic risk assessment; (3) how to design better human studies to assess the impact on the immune system in the species of greatest interest in the context of risk assessment; (4) how to identify and establish sensitive human biomarkers of immunotoxicity; and (5) how to gain a better understanding of the role of genetics in identifying sensitive subpopulations to immune-altering agents.

Systemic hypersensitivity is a frequent cause for withdrawal of drugs that have made it to the market. These findings are generally unexpected in that they were not predicted in preclinical toxicology and immunotoxicology studies. Assays are needed that are more predictive of drug antigenicity, food allergy, or hypersensitivity in humans.

Use of computational toxicology methods to predict potential biological/toxicologic activity of chemicals is growing. The premise is that the structure of a chemical determines the physiochemical properties and reactivities that underlie its biological and toxicologic properties. Being able to predict potential adverse effects will aid the designed development of new chemicals and will potentially reduce the need for animal testing.

Efforts to exploit biomarkers to indicate exposure to a specific chemical, susceptibility to adverse effect, and/or predict disease associated with chemical exposure are ongoing. The most desirable biomarkers are those that indicate exposure in the absence of immediate adverse effect. Biomarkers of effect would indicate subclinical effects of chemical exposure. Potential biomarkers include cytokinegene expression patterns, cell population quantification using flow cytometry, and serum antibody titers.

Assessments of the use of immunotoxicology data in animals as predictors of risk for human clinical effects have limitations, including the fact that no single immune test has been observed to be highly predictive of altered host resistance. Variability in the virulence of infectious agents in the human population, the complexity of the immune system, and the redundancy (multiple components capable of responding to a foreign challenge) in the immune system may all contribute to the difficulty in quantifying relationships between chemical-induced alterations in immune status and alterations in host resistance in humans.

The balance between immune recognition and destruction of foreign invaders and the proliferation of these microbes and/or cancer cells can be a precarious one. Validated methods to detect xenobiotics that produce adverse effects related to the immune system must be continually improved using the latest knowledge and technologies in order to provide a safe environment.