PRINCIPLES OF TOXICOLOGY

198 IMMUNOTOXICITY: TOXIC EFFECTS ON THE IMMUNE SYSTEM
opportunity to evaluate directly toxic endpoints difficult or impossible to assess clinically, such as the
development of immunopathology or loss of resistance to infectious disease.
Currently, a tiered approach is recommended for standardized testing for immunotoxicity in
animals. Tier I consists of a battery of tests intended to evaluate both humoral and cell-mediated
immune system integrity. An assessment of immune system pathology is also included in tier I (see
Table 10.2). If the results of tier I tests are negative, the chemical is considered not to possess significant
immunotoxic potential at the dosages tested. If effects are observed in tier I tests, additional tests are
conducted in tier II to better characterize the immunotoxic properties of the chemical. Tier II does not
consist of a rigid battery of tests, but rather the opportunity to select more specific tests to follow up
on observations made in tier I. Examples of tests that might be used in tier II are included in Table
10.2.
Many of the endpoints examined in tier I are basic. Total and differential white cell counts are
obtained from blood, body and specific organ weights are recorded, and tissues of particular relevance
for immune function (viz., spleen, thymus, and lymph nodes) are examined histologically for evidence
of injury. Humoral immunity is assessed with a plaque-forming cell (PFC) assay. In this assay, the test
animal is injected with sheep red blood cells (SRBCs) as the source of antigen. Four days later the
spleen is removed, and cells isolated from the spleen are cultured with intact SRBCs. B cells producing
IgM directed to SRBC antigens result in lysis of the red cells, producing clear areas in the culture called
plaques. The number of plaques (per spleen or per million spleen cells) provides an indication of the
ability of splenic cells to synthesize and secrete antigen-specific antibodies. This, in turn, offers
information regarding the ability of the immune system to mount a primary (IgM-mediated) response.
Cell-mediated immunity is evaluated by measuring the responsiveness of peripheral blood T and B
lymphocytes to mitogens (such as concanavalin A), and through the MLR assay. Nonspecific immunity
is evaluated in tier 1 by measuring NK cell function. These tests are essentially identical to the in vitro
methods described above for clinical assessment of potential immunotoxicity in humans.
More detailed follow-up tests are available for tier II. For example, if disturbance in the numbers
of immunocytes is suggested by tier I tests, the abundance of individual T- and B-cell types in the
spleen or blood can be measured using reagents that detect specific cell surface antigens. In the
assessment of humoral immunity, an abnormal primary response (IgM-mediated) to SRBCs detected
in the PFC assay in tier I might lead to an evaluation of the secondary response (IgG-mediated) to
SRBCs. Evidence of altered cell-mediated immunity could lead to expanded tests of T-lymphocyte
cytotoxicity in tier II, commonly using tumor cells as targets. Tier II could also include an assessment
of delayed-type hypersensitivity response. Evaluation of non-specific immunity may be extended in
tier II to include enumeration of macrophages and tests of their function. For functional tests,
macrophages are typically taken from the peritoneal or alveolar space of test animals, cultured, and
examined for phagocytic activity, secretion of cytokines, and/or production of reactive oxygen or
TABLE 10.2 Tier I and Tier II Tests for Immunotoxicity
Tier I Hematology, including CDC and differential counts
Body and organ weights, including spleen, thymus, kidney, and liver
Histology of lymphoid organs, including spleen, thymus, and lymph nodes
Humoral immunity, assessed through IgM plaque-forming cell (PFC) response
Cell-mediated immunity, assessed through T- and B-lymphocyte responses to mitogens, and the
mixed-lymphocyte response (MLR)
Nonspecific immunity, assessed through measurement of natural-killer (NK) cell activity
Tier II Quantitation of individual T- and B-cell populations in blood and spleen
Humoral immunity, assessed through IgG plaque-forming cell (PFC) response
Cell-mediated immunity, assessed through cytotoxic T-cell (CTC) activity, as well as the delayed
hypersensitivity (type IV) response
Host resistance, assessed through challenge with pathogens or tumors