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Routine histopathology of lymphoid organs is the cornerstone in the identification of immunotoxic and immunomodulatory compounds. Enhanced histopathology is a systematic approach that can be used to further characterize, both qualitatively and semi-quantitatively, the immunomodulatory effects that may occur within both primary and secondary lymphoid organs. The lymph nodes are the major route of entry for antigens and pathogens, via the afferent lymph flow, and they can be sensitive indicators of compounds with regional or systemic immunomodulatory/toxic effects and should therefore be included in the battery of lymphoid organs to evaluate for enhanced histopathology. As with all lymphoid organs, the separate compartments should be evaluated independently and descriptive rather than interpretive terminology should be used to characterize changes within those compartments. This data, in conjunction with gross findings, clinical pathology and changes in organ weight (i.e., thymus), will enable the pathologist to determine if a significant effect on the immune system is present. Moreover, this data may enable the pathologist to determine the critical site or compartment in the targeted tissue, provide some indication of target cell population (B or T cell) and characterize a dose-response relationship.
The lymph node contains 3 major functional areas or zones that support specific immune functions. These are the cortical area (composed of predominately B-cell lymphoid follicles), the T-cell-rich paracortical area and the medulla with sinusoids and medullary cords composed of predominately plasma cells and macrophages. Each of these compartments should be evaluated individually for changes in area and cell density as well as for changes in composition and/or morphology of specific cell populations. Although not strictly a component of enhanced histopathology, the presence, location, and severity grade of apoptotic cells, tingible body macrophages, necrosis, pigmented macrophages, granulocytes, granuloma/macrophage aggregates, prominent high endothelial venules (HEV), erythrocyte rosette formation, etc. should also be indicated. An example of a checklist that can be used by the pathologist for enhanced histopathology of the lymph nodes is given in Table 1. This checklist was developed to aid the pathologist in the evaluation of the various lymph node compartments, and is not recommended for reporting results. The article by Willard-Mack may be referred to for a more comprehensive review of the normal structure, function, and histology of lymph nodes (Willard-Mack, 2006).
The plane of section is an important variable to consider when evaluating rodent lymph nodes. The relative size of the cortex, paracortex, medullary area, and the number of follicles will depend, in part, on the plane of section examined. Sections taken from smaller nodes may only contain portions of cortex and paracortex. When examining the mesenteric lymph nodes, it is preferable to examine the entire chain, sectioned longitudinally, in order to avoid cross-section variability (Figure 1A). For large mesenteric lymph node chains, multiple slides may be needed in order to evaluate the entire chain. Cross-sections through a lymph node chain at 2 different regions (Figure 1B) may give very different regional variability that is all within normal limits. A random cross-section through a normal mesenteric lymph node may have histological features that may be interpreted as either an increased or decreased area of a particular compartment when it should be interpreted as normal variability (Figure 1C). For example, one section may have an expanded paracortex and decreased medulla while the adjacent region may have decreased paracortex and follicles with an expanded medullary region. As with all histopathology evaluations, comparison of tissues from treated animals with control tissues is crucial in order to establish the range of normal tissue changes for a particular group of animals (Figure 2).
It has been demonstrated that lymph nodes, in general, show a similar systemic response after exposure to an immunomodulatory substance, although there can be individual variation (Harleman, 2000). Exposure to an immunotoxicant may result in a decrease in the size and density of the T cell rich paracortex with or without a reduction in the number of follicles with germinal centers (Figures (Figures33--9).9). Similarly, exposure to an immunostimulating substance most frequently results in hypertrophy of the high endothelial venules and an increase in follicular activity and plasmacytosis if the substance is highly antigenic (Figures (Figures1010 and and11).11). However, treatment with an immune modulating substance may result in an increase in paracortical cellularity and area with a relative decrease in the other compartments (Figures (Figures1212 and and13).13). Important for mechanistic studies, an indication can be obtained regarding the relative effect of the chemical for T-or B-cell compartments and thus the potential effects on cell-mediated versus humoral immunity, respectively. In addition to inhibitory and stimulatory effects, the other two main effects to consider when evaluating immune modulating agents are if the responses are specific (i.e., skin allergen:contact dermatitis) or nonspecific (i.e., adjuvant:increased antibody responses) (Harleman, 2000).
A recent publication by the STP Immunotoxicology Working Group (Haley et al., 2005) recommends that, as an indicator of systemic toxicity, the most proximal regional lymphoid tissues that drain the xenobiotic application site should be examined histologically. They also state that the examination of peripheral lymph nodes (i.e., popliteal, auricular, axillary, etc.) that do not drain the site of xenobiotic application should not be used for enhanced histopathology evaluation. Their position is that these peripheral nondraining lymph nodes can be highly variable with histological features that overlap with that of altered node morphology. Moreover, the minor differences in collection, embedding and sectioning also decrease the value of these small lymph nodes for detection of immunotoxicity. However, given the above caveats, one might wish to evaluate at least one node that is distant to the application route/area to ascertain systemic immunomodulatory effects. Sainte-Marie et al. (1982) and Tilney (1971) provide detailed descriptions of lymphatic drainage patterns in the rat and the nodes involved.
This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.