Evaluation of the Immune System in Toxicity Studies

The various organs of the immune system such as spleen, lymph nodes, thymus and bone marrow containing the cells involved in the various immune responses offer the possibility to harvest these cells and perform in vitro assays for evaluation of effects on the immune system. When part of an in vivo animal study this may indicate a direct toxic effect of pharmaceuticals, that is, immunosuppression (Table 18.2). So, it is feasible to obtain cell suspensions for further evaluation such as determination of cellular subsets of T and B leukocytes by fluorescent activated cell sorter analysis (FACS analysis), and determination of natural killer (NK) cell activity of the spleen cell population. An advantage of this approach is that it may lead to identification of a biomarker to be used in clinical studies. In addition, in vitro stimulation of spleen cells with mitogens activating specific subsets may indicate potential effects on the functionality of splenic cell populations. Concanavalin A (Con A) and phytohemagglutinin (PHA) activate Tcells, while lipopolysaccharide (LPS) activates primarily B cell populations. Blood is collected for total white blood cell (WBC) determination and blood cell differential count. In addition, serum can be obtained for determination of serum immunoglobulins. 

In the additional functional (TIER-2) studies the immune system is more thoroughly investigated, while the animals are exposed to one or more doses of xenobiotics. Immune responses to several different types of antigens may be determined, including Tcell-dependent antigens like tetanus toxoid and ovalbumin, sheep red blood cells (SRBC) [38—41] and T cell-independent antigens like EPS [38, 

For keyhole limpet hemocyanine (KLH) both antibody responses and delayed type hypersensitivity (DTH) reactions can be determined [43—45]. In addition several infectious models, including bacterial, viral and parasitic infections may be used to challenge the immune system [18,46]. As survival and eradication of the infections is the primary function of the immune system, these models provide direct information on the functional status of the immune system. Direct immunotoxic compounds will induce immunosuppression and thus an increase in infection rate and/or severity of the infection. The number of infectious agents (bacteria, parasites, or viral colonyforming units), increased morbidity and mortality are indications for an immunotoxic effect. Also a reduction in specific antibody levels in animals treated with the test compound compared to nontreated controls indicates immunosuppression. 

Besides infectious diseases, to some extent tumor formation is also linked to the functionality of the immune system. Macrophages, NK cells and cytotoxic T cells are able to kill tumor cells. Impairment of these cell types may result in enhanced tumor take and/or growth. Thus, tumor models may be used for evaluation of the immune system [43, 44, 47]. Although these infection and tumor models may be of value in demonstrating the functionality of the immune system, contradictory effects including enhancement and reduction in infectivity or tumor growth may occur [43, 

Such differences may be explained by the immunotoxic effect on certain subpopulations of cells, and the differences in resistance mechanisms for various types of infections and/or tumor models. 

---