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The spleen is the largest secondary lymphoid organ, is considered the draining site for compounds that are administered intravenously, and is therefore considered an important organ to evaluate for treatment-related lesions. Due to the presence of B and T lymphocytes, the immunotoxic effects of xenobiotics or their metabolites on these cell populations may be reflected in the spleen. Therefore it is one of the recommended organs to evaluate for enhanced histopathology of the immune system. The two major functional zones of the spleen are the hematogenous red pulp and the lymphoid white pulp (periarteriolar sheaths, follicles and marginal zones). For enhanced histopathology, these splenic compartments should be evaluated separately for changes in size and cellularity, and descriptive rather than interpretive terminology should be used to characterize any changes (Haley et al., 2005). Moreover, germinal center development within the lymphoid follicles should be noted as increased or decreased.
The spleen is considered the draining site for compounds that are administered intravenously (US FDA, 2001). The two major functional zones of the spleen are the hematogenous red pulp and the lymphoid white pulp. The white pulp, located around a central arteriole, is composed of the periarteriolar lymphoid sheath (PALS, T-cell area), the adjacent follicles (B-cell area), and marginal zone (B-cell area) (Figure 1). The marginal zone lies between the white and red pulp and the junction between the marginal zone and red pulp is not always distinct. In addition to lymphocytes, the marginal zone contains specific populations of macrophages. The splenic architecture is similar across species; however the marginal zone of the rat is comparatively prominent.
According to the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005), the separate compartments in each lymphoid organ should be evaluated separately and descriptive rather than interpretive terminology should be used to characterize changes within those compartments. Therefore, the PALS, lymphoid follicles, marginal zone, and red pulp should be evaluated separately for changes in size and cellularity. Germinal center development within the lymphoid follicles should be noted as increased or decreased. Typical cellular changes that can be observed after exposure to an immunomodulatory agent are an alteration in the size and density of the PALS and/or marginal zone, and a change in the number of follicles with germinal centers (Gopinath, 1996; Harleman, 2000; Kuper et al., 2000). Measures of follicle cellularity and germinal center development have been reported to be the most sensitive predictors for potential immunotoxicity whereas subtle changes in the red pulp are often difficult to detect (Germolec et al., 2004). See Cesta for more detailed information on the normal structure and function of the spleen (Cesta, 2006).
Decreased cellularity of the PALS region can occur after exposure to irradiation, viruses or drugs that can cause necrosis or apoptosis of the T cells (Figure 2). Athymic animals also have decreased cellularity in this T cell-rich region. There can also be specific decreased cellularity of the B cell-rich marginal zone, although this is a less frequent treatment-related effect but may be increasingly encountered with the development of novel immunomodulatory drugs (Haley, personal communication) (Figure 3). Such a change may be suggestive of deficits in T-independent humoral immune responses. Some chemicals, such as N,N-dimethyl-p-toluidine and AZT/Methadone HCL will cause an overall decrease in the cellularity and area of the white pulp (Figures 4 and and5).5). Increased cellularity of the PALS region may be seen with certain chemical treatments such as Citral and androstenedione (Figures 6 and and7)7) but there can also be increased cellularity of this region in early metastasis of thymic T-cell lymphomas, and other lymphomas and leukemias.
An acute immune response to antigens may result in increased cellularity in the B-cell areas and an increase in secondary follicles with prominent germinal centers. Immature B cells, or immunoblasts, will proliferate in response to antigenic stimuli. These cells are larger and paler-staining than the mature lymphocytes and have rounded, regular nuclear membranes. They will mature to immunoglobulin-producing plasma cells and migrate into the red pulp. F344 rats with mononuclear cell leukemia (MCL) may have an apparent increased cellularity in the marginal zone due to infiltration or sequestration of the large granular lymphocyte leukemic cells within this region. Marked increased in cellularity of the white pulp is sometimes difficult to distinguish from neoplasia. See Suttie for a comprehensive review of splenic lesions and discussion of borderline diagnoses of splenic neoplasia (Suttie, 2006).
The red pulp should also be evaluated for changes in lymphocyte numbers. Reactive extramedullary hematopoiesis may be seen in conjunction with conditions that target the destruction of lymphocytes. This condition must be differentiated from the infiltration of the red pulp with large granular lymphocyte leukemic cells in F344 rats with MCL. It is also possible to identify decreased lymphocyte cellularity of the red pulp in those cases where systemic lymphocyte numbers are significantly decreased. Although the red pulp contains lymphocytes and macrophages, it is predominantly composed of red blood cells. For this reason there can be variation in splenic size and weight as well as histological variation in red pulp size and erythrocyte cellularity depending on the method of euthanasia and the efficacy of exsanguination at necropsy. However, variations in spleen weight due to methods of exsanguination are more likely to be encountered in dogs and monkeys, rather than rats. The available evidence suggests that splenic weight is a relatively insensitive indicator of immunotoxicity (Luster et al., 1992). In order to be toxicologically significant, the changes in spleen weight should be dose related and be confirmed with histopathological evaluation.
Increased cellularity in the B cell-rich follicles or marginal zones can be seen in aging rats and mice without any apparent increase in plasma cells. Enlarged spleens with decreased lymphocyte cellularity and increased myelopoiesis may be seen in aging rats, especially Sprague-Dawley rats, without an apparent etiology.
The presence, severity grade and location of plasma cells, apoptotic cells, tingible body macrophages, pigmented macrophages, granulocytes and hematopoietic cells should also be noted. Although granulomas, macrophage aggregates, fibrosis and necrosis are not strictly a component of enhanced histopathology, they should be noted during the evaluation. An example of a checklist for the changes to be noted in the spleen for enhanced histopathology is given in Table 1. This table is intended to be an example of a guideline that the pathologist can use during histological evaluation rather than a format for reporting lesions. The diagnoses listed in this table are descriptive rather than interpretive, consistent with the STP position paper: Best Practice Guideline for the Routine Pathology Evaluation of the Immune System (Haley et al., 2005).
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.