BASIC IMMUNOLOGY AND IMMUNOTOXICOLOGY

To understand immunotoxicology, a certain amount of basic immune system immunology is desirable for those who are not exposed to this field. The immune system can be seen as a more or less concerted system of armies carrying different varieties of weapons to act against different enemies. It defends the body but can also turn against it. It is among the fastest dividing organs together with gut mucosa (under permanent digestion) and hair follicles. The immune system also has enormous overcapacities, as it must be the last to fail. This characteristic will predictably buffer chemical impacts on this organ system and indeed a functional deficit often only manifests under the additional stress of mass infection or chemical insult of an animal/human.

I. IMMUNOLOGY:A. What is the Immune System? The immune system is a network of cells, tissues, and organs that work together to defend the body from harmful agents. When chemicals, bacteria, viruses, and other germs invade our body, they multiply and attack. This Invasion causes the diseases that make us sick. Our immune system protects us from the disease by fighting off the invading agents. B. The Immune System Recognizes and Eliminates Pathogens Immunology may be described as the summation of all those physiologic processes that endow the host with the capacity to recognize materials as foreign to itself and to neutralize, eliminate, or metabolize them with or without injury to its own tissue(s). This ability to differentiate ‘‘self’’ from ‘‘non-self’’ constitutes the basic hallmark of the immune response and the basis for an understanding of clinical immunology in health and disease. If the foreign substance cannot be blocked by natural barriers such as skin and mucous secretions, the substance comes into contact with the immune system. Foreign substances that enter the body through the natural portals of entry, i.e., skin, respiratory, gastrointestinal, or genitourinary tracts, encounter components of the external immune system found in collections of lymphoid elements found at these sites. Since most of these organ systems are lined by mucosa, this system is referred to as the mucosa-associated lymphoid tissues (MALT). Foreign substances that penetrate these mucosal and skin-site barriers enter the body through the blood or lymphatics and encounter components of the internal immune system found in the lymph nodes, thymus, and spleen. C. How does the Immune System Work? Our immune system is always on patrol in our body. When it comes across an invading agent, it attacks. This is called an immune response.Here’s how an immune response works: • Our immune system sounds the alarm so our body knows there’s an infection/exposure to a xenobiotic agent. It begins releasing antibodies to fight the xenobiotic — think of antibodies as soldiers designed to fight off the specific germs we have. This process can take a few days. The antibodies work to attack, weaken, and destroy the germ/chemical. Afterward, our immune system remembers the chemical/germ. If the xenobiotic invades again, our body can recognize it and quickly send out the right antibodies so we don’t get sick! This protection against a certain disease/agent is called immunity. In many cases, immunity lasts your whole life. The immune system is typically divided into two categories--innate and adaptive--although these distinctions are not mutually exclusive. D. Innate Immunity Innate immunity refers to nonspecific defense mechanisms that come into play immediately or within hours of an antigen's appearance in the body. The first set of responses to foreign substances are called innate immune responses because they are present without the requirement for specific induction and are present upon initial and subsequent encounters with a foreign substance. The innate immune responses are primitive, stereotyped, and lack the form of memory associated with adaptive immunity or the ability to respond in an enhanced manner upon subsequent encounters with the same foreign substance. The innate immune system recognizes certain structures on a foreign substance—referred to as pathogen-associated molecular patterns (PAMPs) that are mediated utilizing receptors called pattern recognition receptors (PRRs) located on the surfaces of a variety of cells of the innate immune system. Macrophages and dendritic cells are examples of cells bearing these receptors. E. Adaptive Immunity Adaptive immunity refers to antigen-specific immune response. The adaptive immune system includes a complex set of genetically controlled, interdependent, and interactive responses, and is also referred to as acquired (specific) immunity. In contrast to the innate immune system, the adaptive immune system is more expansive and diverse and is characterized by: a. Specificity: The recognition of the foreign substance (i.e., antigen or immunogen) by antigen-recognition molecules on the surfaces of lymphocytes in a highly precise and selective manner; b. Heterogeneity: The cells and cell products that comprise the adaptive immune system consist of a variety of different types; c. Memory: The ability to recognize an antigen upon subsequent encounters with the foreign substance in a more rapid and highly augmented fashion. Because the adaptive immune system is composed of relatively small numbers of cells with specificity to recognize an individual immunogen, the responding cells must proliferate, forming a cell clone, and differentiate into effector cells. After encountering a foreign substance, the effector cells attain sufficient numbers to mount an effective response commensurate with the quantity of the foreign agent being presented. Thus, the adaptive immune response generally expresses itself temporally, usually several days after the innate response, in the encounter with foreignness. • A key feature of the adaptive immune response is that it produces massive quantities of long-lived cells (i.e., memory cells) that persist in an apparently dormant state, but that can re-express effector functions rapidly after subsequent encounters with the same antigen. This provides the adaptive immune response with the ability to manifest immune memory, permitting it to contribute to a more effective host response against specific pathogens when they are encountered a second time, even decades after the initial sensitizing encounter. F. Immunological Response When first exposed to antigen "A", we begin to make low levels of antibody in about a week However, the second exposure to antigen "A" produces a much faster response and several orders of magnitude higher levels of antibody. The ability of the antibody to bind antigen also increases dramatically in the secondary response. Injecting a new antigen "B" with "A" shows that a memory or prior exposure is required for the accelerated response. The memory of antigen and the stimulated response is the basis for success in vaccination programs. This is explained by the clonal selection theory of the immune response (see below).  

1. The Clonal Selection Theory The immune system produces billions of kinds of B-cells each making one kind of antibody receptor. The presence of antigen leads to the proliferation and differentiation of clones that have antibody capable of binding the antigen. In the diagram below, the "green" antigen binds to the green antibody on a B cell. The color code means that only this antibody receptor on the cell binds free antigen. The "green" helper T-cell must give a stimulatory signal to allow a specific B cell to be selected. This step allows a regulation or control of the process. The antigen-driven selection produces memory cells and plasma cells secreting antibody capable of binding the original selecting antigen with high affinity. If antigen appears in the organism a second time, then the memory cells are already present at high levels and produce a more rapid and much stronger immune response. 2. The Two Major Components of the Adaptive Immune System The two major components of the adaptive immune response are humoral immunity and cell-mediated immunity (CMI). i. Humoral immunity is a process carried out by antibodies (immunoglobulins), produced by B lymphocytes in response to and capable of reacting with antigen ii. Cell-mediated immunity is the other arm of the adaptive immune response carried out by T lymphocytes. The cells of the adaptive immune system, in contrast to those of the innate immune system, interact with the environmental agent in a highly discriminative way, i.e., they display specificity, heterogeneity, and memory (discussed earlier). a. These functions are primarily carried out by two types of cells that are involved in the recognition of antigen: i. The thymus-dependent or T lymphocytes, which participate in cellular responses against intracellular pathogens, organ transplants, and malignant cells ii. The bone marrow or bursal-dependent B lymphocytes, which provide humoral immunity, i.e., antibody-mediated immunity against extracellular pathogens, their toxins, and other environmental substances. 

b. The third group of cells involved in the presentation of antigen to T cells, i.e., APCs, include dendritic cells, macrophages, and B cells. APCs take up predominantly protein antigens, cut them into peptides, bind the peptides to major histocompatibility complex (MHC) molecules, and display these presented antigens on their cell surface, where they can be recognized and bound by antigen receptors on T lymphocytes. c. T lymphocytes are identified by a surface Cluster of Differentiation (CD) molecule named CD3 and are comprised of two major groups: the CD4 and CD8 populations d. The CD4 cells display helper activities on other populations of cells, and in turn are subdivided into at least Th1, Th2, Th9, Th17 and T regulatory (Treg) groups, each with a characteristic profile of production cytokines. e. The CD8 T cytotoxic population is the second major group of T lymphocytes that function in killing target cells; they are comprised of Tc1 and Tc2 subpopulations with similar cytokine profiles as Th1 and Th2 cells. f. Collectively, the T lymphocytes play or facilitate a significant role in the orchestration of all functions of the adaptive immune system and perform four important tasks: i. promotion of inflammation by cytokine production (Th1 and Th17 cells) ii. helping B lymphocytes (Th2 cells) iii. regulating immunosuppressive responses (T regulatory cells) iv. the killing of unwanted target cells (CTL) Type Effector cells Effector mechanism(s) Outcome Humo ral B cells Antibody Neutralization of foreign antigen and coating substances for opsonization Cellmedia ted T cells Cytokines, cell-cell interaction & Cytotoxic activity Promotion or inhibition of inflammation, and/or humoral function & Lysis of infected cells 1. Antibodies are proteins produced by and secreted from B cells and specifically bind extracellular antigen.In humans, there are five major classes (i.e., isotypes) of immunoglobulins: IgM, IgG, IgA, IgD, and IgE, each differing in physical, chemical, and biologic properties.The primary function of antibody is to directly bind with the foreign substance/pathogen. 2. T cells can recognize intracellular infections (viruses and bacteria that can survive inside the cells that have ingested them). 

3. Cell-mediated immunity is a process carried out by T cells through the production of cell-regulating molecules (cytokines) or through inducing cell death (cytotoxicity) without the participation of antibody.

II. IMMUNOTOXICOLOGY:  Immunotoxicology can be defined as the study of adverse effects on the immune system resulting from occupational, inadvertent, or therapeutic exposure to drugs, environmental chemicals and, in some instances, biological materials. Immunotoxicity can be divided into two areas depending on whether the immune system is activated (such as in allergies or in chemical-induced autoimmune diseases) or suppressed by xenobiotics (foreign or nonendogenous chemicals, including drugs and environmental chemicals). Xenobiotics may interfere with normal immune system homeostasis by affecting the formation of immune cells; modifying cell-to-cell interactions; modifying cell activation, proliferation, or differentiation; altering cell selection; and enhancing or suppressing the release of immune products such as cytokines, chemokines, antibodies, and complement factors. The immune system is a target following exposure to a diverse group of xenobiotics including chemical pollutants, recreational drugs, ultraviolet radiation, and therapeutics. There are well-established cause and effect relationship between suppression of the immune response and reduced resistance to infections. The immune dysfunction may take the form of immunosuppression or alternatively, allergy or autoimmunity or any number of inflammatory-based diseases Xenobiotic exposure may result in stimulation of immune function. Xenobiotics can act as allergens and elicit hypersensitivity responses, or they can modulate hypersensitivity responses to other allergens. Immunotoxicity can be regarded as one of several distinct immunopathologies that include allergic disease, immunodeficiency, and autoimmunity. In immunopathologies, the immune system responds to the agent as an allergen of low (hapten) or high molecular weight, that result in allergic contact dermatitis, food hypersensitivity or respiratory hypersensitivity. In immunodeficiency, the immune system acts as a passive target for the agent and this may lead to increased incidence or severity of infectious disease. Autoimmunity which is a break-down in self-tolerance occurs when an agent directly or indirectly induces an immune response to autologous constituents that result in pathological consequences. As the immune system plays a critical role in host resistance to disease as well as in normal homeostasis of an organism, identification of immunotoxic risk is significant in the protection of human, animal and wildlife health.  

Mechanisms of Immunotoxicity: 1. Chemicals can kill immune cells, resulting in bone marrow toxicity and immunosuppression. Compounds that can damage or destroy the bone marrow will often have a profound immunotoxic effect since the effectors of the immune system will no longer be available. Antitumor drugs, benzene, and ionizing radiation are examples of myelotoxic compounds. 2. Chemicals can interfere with general or immune specific signaling pathways, resulting in changes in the expression of surface markers, cytokine production, cell differentiation and activation. Immunotoxic compounds can act via a receptor-mediated or non-receptor mediated effect. Examples of chemicals acting through a receptor-mediated effect include glucocorticoids, polycyclic aromatic hydrocarbons, and cannabinoids, while immunotoxic compounds acting through a nonclassical receptor-mediated event include calcineurin inhibitors, metals, and some pesticides. 3. Many substances exert immunosuppressive effects by inhibiting bone marrow stem cell proliferation (cyclophosphamide, methotrexate), or spleen/thymus (organotin, TCDD), or by directly affecting mature leukocytes (glucocorticoids, TCDD, cyclophosphamide, methotrexate), e.g., by inhibiting lymphocyte proliferation or triggering their apoptosis. Cyclophosphamide was shown to selectively deplete a regulatory T cell population. Humans appear to be much less sensitive to the immunosuppressive effects of TCDD than rodents or even monkeys. Because of the non-specific nature of some of these immunosuppressants, several modes of action are observed, e.g., for steroids suppression pro-inflammatory cytokine and chemokine production, up-regulation of TGF-β, shift to anti-inflammatory TH2responses, suppression of NK cell functions, and impaired dendritic cell activation and differentiation. Cytotoxic drugs also have various immunostimulatory effects, such as increases in effector T cell stimulation and tumor immunogenicity as well as decreases in tumorinduced immune suppression. 4. Immunosuppression of the granulocytes of the innate immune system appears to be rare, likely due to the rapid renewal of granulocytes from bone marrow, which can be dramatically accelerated by the induction of colony-stimulating factors. There is no evidence for environmental chemicals playing a major role, though case reports can be found. 

Therefore, macrophage effects might be more critical as they control granulocyte recruitment and activation. The most advanced test here is the whole blood monocyte cytokine release assay, though variants using isolated peripheral blood mononuclear cells (PBMC) are also available for similar evaluation.

B. In vitro Testing for Immunosuppression General recommendations for in vitro immunotoxicity testing are as follows: 1. Before starting with in vitro tests, bioavailability should be considered. If the compound does not have appreciable bioavailability, immunotoxicity is unlikely to occur. 2. It is recommended to use a flow chart/decision tree approach to evaluate whether or not a compound is immunotoxic (initial screening). Detection of compounds as potential immunotoxicants can then be followed up by more detailed in vitro mechanistic assays (e.g., antigen-specific or redirected CTL). 3. To maximize human relevance, and due to the lack of species limitations for these assays, it is recommended that human cells be used for all in vitro test systems. With the exception of bone marrow assays, the source of cells should be PBL (peripheral blood leukocytes) from donors prescreened for health, immune reactivity, etc. 4. As a general strategy, an initial evaluation of myelotoxicity should be performed (Tier 1). If a compound is myelotoxic, there may be no need to proceed with additional evaluation. 5. Compounds that are not overtly myelotoxic may still selectively damage or destroy lymphocytes, which are the primary effectors and regulators of acquired immunity. Compounds are therefore tested for lymphotoxicity (Tier 2). This toxicity may result from the destruction of rapidly dividing cells by necrosis or apoptosis; alternatively, chemicals may interfere with cell activation, affecting signal transduction pathways. An in vitro test to determine lymphotoxicity should be carried out (cell death by necrosis or apoptosis). 6. After myelotoxicity and overt cytotoxicity are excluded as endpoints, basic immune cell functionality should be assessed by performing specific functional assays, i.e., proliferative responses, cytokine production, NK cell activity, etc. (Tier 3), using non-cytotoxic concentrations of the tested chemicals (viability >80%). For T cells, the stimulatory agent could be a combination of anti-CD3 and antiCD28 or mitogens such as concanavalin A (ConA) and phytohemagglutinin (PHA). 7. Potential effects of chemicals on cytokine expression should be determined. The role of cytokine transcription or production should be evaluated as well as the modulation of cytokine receptors. It should also be investigated if cytokine transcription or production is skewed (TH1/TH2 shift). It will require careful consideration of which cytokines should be measured to obtain the most useful information (e.g., proinflammatory, specific immunoregulatory cytokines). Both basal and activated cytokine production should be measured, and for activated cytokine production, anti-CD3 and anti-CD28, LPS, or allergens should be used. 8. Many assay systems are available for measuring cytokine expression (e.g., ELISA, flow cytometry, molecular biology techniques, such as RT-PCR). 9. Potential effects on NK cells should be determined. The cytolytic function should be measured (this is important for innate immunity). There are a variety of systems available for measuring cytolytic function (e.g., whole blood, radiolabel release, flow cytometry). The immunoregulatory function of NK cells should be evaluated due to the key regulatory nature of these cells. 10. An assessment of the functionality of immune cells could also include the measurement of other mediators, e.g., histamine, cytokines, eicosanoids, or activation of the complement cascade leading to hypersensitivity reactions. The use of the whole blood assay can also address the release of mediators by basophils (histamine) and monocytes (cytokines). Finally, the use of mast cell models also needs to be considered. At the moment, there is no strong evidence for a role of eosinophils being directly activated by compounds. Assessment of the functionality of immune cells could also include the measurement of other mediators, e.g., histamine, cytokines, eicosanoids, or activation of the complement cascade leading to hypersensitivity reactions. The use of the whole blood assay can also address the release of mediators by basophils (histamine) and monocytes (cytokines). Finally, the use of mast cell models also needs to be considered. Now, there is no strong evidence for a role of eosinophils being directly activated by compounds. Models are available

C. Determining Immunotoxicity in vivo There are relatively few guidelines for testing compounds for immunotoxicity. The earliest guidelines were developed for pesticides in 1996 by US EPA (OPPTS 880.3550 followed by 880.3800 and 870.7800). They reflect the NTP’s tier-testing approach and typically request in vivo tests in rodents.

I. For drugs, International Conference on Harmonization (ICH) Safety Number 8 guidance recommends a “weight-of-evidence approach”, i.e., alerts of immunotoxicological potential in standard tests should trigger specific tests.  

II. Typical immunotoxic parameters that can be obtained in routine toxicology studies are identified below:

Table : 1 Immunotoxic Alerts in Standard Toxicology Studies (i.e., 28-day repeated dose toxicity testing study in rodents) 1. Changes in total and differential white blood cell counts, i.e.,leukocytopenia/leukocytosis, granulocytopenia/granulocytosis, or lymphopenia/ lymphocytosis 2. Changes in clinical chemistry, i.e., serum immunoglobulin levels and albumin/globulin ratios; 3. Alterations in organ weights, i.e., thymus and spleen, and/or histology of primary and secondary lymphoid organs, i.e., bone marrow, thymus, spleen, draining and distant lymph nodes; 4. Increased incidence of infections; 5. Increased occurrence of tumors, in the absence of genotoxicity, hormonal effects, or liver enzyme induction; 6. Chemical retention in organs/cells of the immune system.

iii. For environmental chemicals, no dedicated OECD test guidelines exist, but extensions were made to 28-day repeat dose toxicity testing (TG 407). The most recent guidance document comes from WHO 2012 as Harmonization Project Document No. 10 Guidance for Immunotoxicity Risk Assessment for Chemicals. Latest OECD guideline 443 on Extended One Generation Reproductive Toxicity recommends Immunotoxicity assessment as one ofthe cohorts. iv. The most common assay used to assess immunotoxicity in rodents is the antibody response to sheep red blood cells (SRBCs) because it was found to be the most predictive single assay for immunotoxicity and requires cooperation between B, T, and antigen-presenting cells. Animal tests constitute the current gold standard for immunotoxicology. Interpreting data from animal immunotoxicology

D. Immuno-Risk Assessment The immune system can be the target of many chemicals, with potentially adverse effects on the host's health. The extent to which immunotoxicity of chemicals represents a health problem at low levels of exposure is not clear.It appears that responses of healthy individuals to immunological challenges differ widely and most immunomodulators have little adverse effects. The enormous overcapacity of immune defense, the presence of compensatory mechanisms and their fast restoration contribute to limiting health threats for the individual, although on a population base minor immunomodulation might also result in increased morbidities. 

Immunotoxicity risk assessment of chemicals is an evaluation of the potential for unintended effects of chemical exposure on the immune system. These effects manifest as following principal types of immunotoxicity: immunosuppression involving infection and carcinogenesis etc, immuneaccentuation involving sensitization and autoimmunity, or immunostimulation. Such immune dysregulation may lead to diverse types of illnesses. Included among them are illnesses that are associated with a dysfunctional immune system, such as infections, inflammatory diseases, allergic diseases, autoimmune diseases, etc, although all of them are not induced by chemical exposure.

No immunotoxicity tests have been conducted on 86% of high production volume (HPV) chemicals. However, it isnot clear whether a certain immunomodulation observed in a study model will result in a clinical manifestation, especially in a low dose, prolonged exposure scenario. This is a very critical issue because an extrapolation from the high doses used in the laboratory model to the low doses of mixtures of compounds entering the human system through environmental and/or occupational exposure can be very difficult.

Immunotoxicity risk assessment should be performed according to the same principal approaches as applied in risk assessment for other toxicological endpoints, because the immune system or each type of immunotoxicity manifests many special aspects that need specific consideration in risk assessment. Furthermore, the guidance recommends that a weight of evidence approach is most suited for risk assessment of immunotoxicity and that the approach should include clinical and epidemiological information, equally as information from animal experiments and other information.

Any meaningful change induced by xenobiotic exposure on the functionality of immune cells must be considered a hazard, whose effective risk for human beings should be carefully evaluated during the risk assessment phase. If a chemical is immunotoxic and if it affects NK cell activity and cell-mediated immune responses, this may represent a risk for decreased immune surveillance and cancer. Immunotoxicity assays might, therefore, play a role in future integrated testing strategies for carcinogenicity, representing a nongenotoxic mechanism of carcinogenicity.

In industrialized countries, hypersensitivity reactions represent the most frequently reported immunotoxic effects of chemicals. Immuno-stimulation or “immunoenhancement”, i.e., an exaggerated immune response, is known primarily in sensitization (allergies, including contact dermatitis) and auto immunity. The clearest disturbance of the immune system, indeed, is the dramatically increasing incidence of allergies. There are examples of drugs associated with autoimmune phenomena: autoimmune hepatitis (dihydralazine, halothane, tienilic acid), drug-induced lupus (dihydralazine, procainamide, propylthiouracil), glomerulonephritis (gold thiomalate), and occulomucocutaneous syndrome (practolol). This side effect can be quite frequent, e.g., 10-20% of patients receiving procainamide and 5-20% receiving hydralazine develop systemic lupus erythematosus. There is some evidence for food and environmental chemicals causing autoimmunity, e.g., autoimmune thyroiditis (iodine), scleroderma (L-5hydroxytryptophan), and SLE (alfalfa seeds). Vinyl chloride, trichloroethylene, aniline, tryptophane, silica, paraffin, and silicones are among chemicals leading to autoimmune manifestations, especially sclerotic and lupus-like diseases. Pesticides also have been suggested to play a role.

Interpreting data from animal immunotoxicology studies for risk assessment has proven challenging, especially when the immunological effects are minimal-to-moderate in nature. Currently, a tiered approach has been proposed, since useful information can be obtained from regular 28-day general toxicity tests if increased attention is paid to the study of the histopathology of a large variety of lymphoid tissues, coupled with immunohistochemical measurements and the determination of antibody classes. Furthermore, it was established that the in vitro test should be validated against information gained from humans rather than the results from laboratory animal species. 

III. CONCLUSIONS Mammalian immune systems have innate and adaptive components that play important roles in resistance to infections and cancer. The immune systems of mammals are formed by primary lymphoid organs, including yolk sac, fetal liver, bone marrow, and thymus. Secondary lymphoid organs (e.g., lymph nodes, spleen, mucosa-associated lymphoid tissues) store differentiated cells that await activation by environmental antigens or undergo endogenous selection processes to discriminate self from non-self. T and B cells are activated in clonally restricted (antigen-specific) ways, and they demonstrate a memory response. One feature of innate immunity is that the responding cells (macrophages, natural killer cells, granulocytes) do not demonstrate clonal specificity. Xenobiotics may interfere with normal immune system homeostasis by affecting the formation of immune cells; modifying cell-to-cell interactions; modifying cell activation, proliferation, or differentiation; altering cell selection; and enhancing or suppressing the release of immune products such as cytokines, chemokines, antibodies, and complement factors. The immunotoxicity of chemicals is evaluated in animal models, in vitro studies, and occasionally in humans after occupational or environmental exposures. Because of the complexity of the innate and adaptive immune systems, no single assay can be used to study the potential toxicity of xenobiotics. Instead, a tiered approach has been developed and validated by several laboratories for studies in animals. Although there is no single immune assay or parameter that can be used to determine whether a xenobiotic exerts a toxic effect on the immune system, certain combinations of markers and functional assays can predict immunotoxicity. The nonclinical evaluation of the toxicity, as well as the immunotoxicity of drugs and other chemicals, must take notice of a number of important but sometimes conflicting considerations. The final strategy to be selected not only always depends on the nature of the drug or chemical being tested and the expected conditions of human treatment or exposure, but it should also take into account specific regulatory requirements when available or applicable. Therefore, the selection of optimal strategies for the identification of immunotoxicity, especially for regulatory purposes, is always difficult and even impossible with regard to most hypersensitivity reactions and autoimmune reactions.