Abstract
Enhanced histopathology of the immune system is a tool that the pathologist can use to assist in the identification of immunomodulatory drugs and chemicals. This assessment is based on the assumption that compound induced-alterations (drug or chemical) may produce changes in cell production and cell death as well as cellular trafficking and recirculation, which will be histologically expressed as alterations in cellular density or compartment sizes in lymphoid organs (Vos, 1980; Vos and Dean, 1990; Schuurman et al., 1994; Basketter et al., 1995; Harleman 2000; Ruehl-Fehlert et al., 2005).
Enhanced histopathology involves the subjective and semi-quantitative histological evaluation of various lymphoid organs and their respective tissue compartments to identify specific cellular and architectural changes. Although this methodology cannot directly measure immune function, in conjunction with gross changes, organ weights, hematologic measurements, and clinical chemistry, it does have the potential to determine whether or not exposure to a specific compound may cause suppression or enhancement of the immune system (ICICIS, 1998; Schulte et al., 2002; Germolec et al., 2004). An inherent limitation of any histological evaluation is its static representation of a dynamic process, and this is especially true for lymphoid tissues. As with all screening tests, evaluation of, and comparison with, control tissues is crucial in order to establish the range of normal tissue changes for a particular group of animals (Kuper et al., 2000).
Laboratory animals include species other than rat and mouse; therefore, recognition of species differences in the structure and function of the immune system should be noted, as well as identification of which differences are biologically relevant for the endpoint being considered (Haley, 2003). Consideration should also be given to the overall health, nutritional status, antigen load, age, spontaneous lesions, steroid hormone status, and apparent stress for each strain and group of animals (Odio et al., 1987; Levin et al., 1993; Gopinath 1996).
It is important to note that the distinction between a direct immunotoxic effect of a chemical as compared to secondary stress-related tissue changes can be challenging. Stress-induced lymphoid lesions and lesions caused by direct effects of chemical treatment can be identical. Interpretation of stress-related changes should be based on the combination of clinical signs and other study data, such as decreased body weight gain and decreased activity, as well as complete blood cell count (CBC) results, increased adrenal gland weights, decreased thymus weight, decreased thymic cortical cellularity with associated lymphocyte apoptosis, and changes in spleen and lymph node cellularity.
The basic requirements to perform enhanced histopathology include organ (spleen and thymus) and body weights, hematology and clinical chemistry parameters, and the gross examination of lymphoid tissues. In addition, before microscopic evaluation of the immune system is performed, information on dosing regimen should be evaluated. The lymphoid tissues that should be evaluated are thymus, spleen, bone marrow, lymph nodes (especially nodes draining the site of xenobiotic application) and mucosa-associated lymphoid tissues (emphasis on GALT for oral xenobiotic administration and NALT and BALT for inhalation). General guidelines to the examination of each of these tissues are provided in this monograph. The reader is encouraged to consult the published approaches for “enhanced histopathology” provided by Kuper and coworkers (Kuper et al., 1995, 2000, 2002).
Some practical consideration in conducting enhanced histopathology of lymphoid tissues are important. For any given study, the pathologist must first define “normal.” The practical consequences of this dictate that the initial evaluation should not be blinded. Because of the dynamic nature of lymphoid tissue responses, the range of normal appearance is often quite large. Blinded reevaluation of a study is always an option once the range of control animal changes is fully appreciated. A particular compartment of a lymphoid tissue may have changes in cellular density and/or changes in compartment size. Both are reflective of the movement of lymphocytes and macrophages through the particular compartment. Thus, an increase in cellular density in the paracortex of a lymph node, for example, may reflect an increased number of lymphoid cells arriving in that compartment or, alternatively, an increased retention time for the lymphoid cells already present in that compartment. Other changes in the lymph node, such as increased prominence of high endothelial venules, changes in other lymphoid tissues, such as the thymus and spleen, and lymphocyte counts in the peripheral blood will help in the pathologist’s interpretation of morphological aspects of the immune system response. If enhanced histopathology indicates that a certain chemical may be immunomodulatory, then additional sectioning and immunohistochemistry can be done and more specific immune function studies may be performed; these are reviewed in the following citations and not covered in this Monograph (Luster et al., 1988, 1993; Van Loveren et al., 1989; IPCS, 1996; ICICIS, 1998; Hinton, 2000).
My colleagues and I had struggled with how to apply enhanced histopathology on standard hematoxylin and eosin ((H&E)-stained sections of bone marrow. I am clearly convinced that bone marrow cytological evaluations based on smears, cytospins, and differential counts are entirely useful, although these are not typically done on all toxicity studies. While the bone marrow is a critical primary lymphoid tissue responsible for development of uncommitted B and T cells throughout adult life, enhanced histopathology on standard H&E-stained sections of bone marrow, in contrast to other lymphoid organs, is likely to be of minimal utility for detection of immunomodulatory effects. While between 20 and 30% of the nucleated cells in the bone marrow are of the lymphoid lineage (Jain, 1986; Picker and Siegelman, 1999), the anatomic pathologist typically cannot unequivocally identify a lymphoid cell in standard H&E-stained sections of bone/bone marrow.
This causes pragmatic concerns regarding the proposed best practices guidelines relative to enhanced histopathology on the bone marrow. Unlike other lymphoid tissues, the bone marrow does not have distinct subcompartments for cataloging changes in cellularity. In addition, in most adult species a decrease in marrow lymphopoiesis has little or no effect on lymphocyte numbers in a CBC (Jain, 1986). Since the myeloid:erythroid (M:E) ratio is traditionally a comparison of relative numbers of granulocytic versus erythroid cells, an M:E estimate derived from an H&E-stained section is of uncertain relevance to immune system perturbations. Even in a bone marrow with severely depleted cellularity, it is not possible to unequivocally identify the lineage of the few remaining cells in an H&E-stained section. This certainly argues for use of cytological preparations for a more informative evaluation of the bone marrow.
Enhanced histopathology on bone marrow in a conventional toxicity study can, however, identify changes that reflect systemic toxicity. A similar set of cytokines associated with fibroblastic stromal cells orchestrate the differentiation, maturation and migration of developing lymphoid cells as well as hematopoietic cells in the bone marrow. Because of their relatively large numbers, significant changes in hematopoietic cell numbers would be relatively easy to detect in H&E-stained bone marrow sections. Since it is reasonable to expect that exposure to toxicants could affect the controlling stromal cells, there would be consequences for production and integrity of hematopoietic as well as lymphoid cells. Therefore, decrements (adverse effects) on hematopoiesis imply a high probability that lymphoid cells are affected as well. Thus, even the subjective histoanatomic evaluation of bone marrow that is possible in standard H&E-stained sections might give alerts to potential pertubation in the lymphoid system.
The STP Immunotoxicology Working Group (Haley et al., 2005) published a “best practice” concept for the examination and reporting of specific lymphoid organs using an enhanced histopathology approach. Their position is that the separate compartments of each lymphoid organ should be evaluated individually and reported in a semiquantitative descriptive, rather than interpretive fashion. The final interpretations and conclusions should be presented within the pathology narrative. The following illustrated papers on enhanced histopathology evaluation of various lymphoid organs are consistent with this approach. Standard operating procedures should be established within each laboratory for grading schemes, initial screening procedures and follow-up studies and functional tests. The appropriate regulatory guidelines should be followed or an alternative should be justified.