Quantitative Analyses of Lymphoid Tissue in the Spleen, Lymph Nodes and Peyer s Patches in Cynomolgus Monkeys

Transcription

1 FULL PAPER Pathology Quantitative Analyses of Lymphoid Tissue in the Spleen, Lymph Nodes and Peyer s Patches in Cynomolgus Monkeys Akiko MORIYAMA 1,2), Junko FUJISHIMA 1), Tomohiro FURUKAWA 1), Tsuyoshi YOSHIKAWA 1), Rinya KODAMA 1), Yuji SASAKI 1), Takaharu NAGAOKA 1), Yasuhiro KAMIMURA 1), Hiroshi MAEDA 1), Takuya HIRAI 2,3) and Ryoji YAMAGUCHI 2,3) * 1) Drug Safety Research Laboratories, Shin Nippon Biomedical Laboratories, Ltd., 2438 Miyanoura, Kagoshima , 2) The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi and 3) Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, 1 1 Gakuen Kibanadai-nishi, Miyazaki , Japan (Received 28 April 2011/Accepted 2 July 2011/Published online in J-STAGE 15 July 2011) ABSTRACT. To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer s patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer s patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer s patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer s patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies. KEY WORDS: cynomolgus monkey, immunotoxicity, lymph node, Peyer s patches, spleen. J. Vet. Med. Sci. 73(11): , 2011 Immune system toxicity encompasses a variety of adverse effects including the suppression or enhancement of immune response. Suppression of immune response can lead to decreased host resistance to pathogens or tumor cells. Enhancement of immune responses may result in hypersensitivity and autoimmune disease. Drugs or drugprotein adducts recognized as foreign substances by the immune system can induce an immune response and hypersensitivity from subsequent exposure. Histopathological examination of the lymphoid organs is one of the most sensitive means of evaluating immunotoxicity in routine safety tests [7]. It is important to improve the precision of immunotoxicologic evaluations and the quality of histotechnical procedures [9]. Furthermore, immunohistochemistry and flow cytometry are useful for evaluating immunotoxicity in primates [10, 15]. To promote international harmonization with regard to new drug applications, the ICH S8 guidelines were developed in In addition, a position paper on pathological evaluation methods for the immune system published by the Society of Toxicologic Pathology [6] describes practical points for evaluating changes in the immune system. This position paper focused on rodents, but stated that when a nonrodent species such as the monkey or dog is used, attention must be paid to variability in organ weights and gross and microscopic observations. *CORRESPONDENCE TO: YAMAGUCHI, R., Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, 1 1 Gakuen Kibanadai-nishi, Miyazaki , Japan. a0d402u@cc.miyazaki-u.ac.jp. The importance of nonhuman primates is widely recognized in biomedical research, and their use for preclinical toxicologic and safety evaluations is increasing because of their phylogenetic similarity to humans [1 3]. Among these, the cynomolgus monkey (Macaca fascicularis) is the most frequently used in toxicity studies [2]. It is clear that species-specific characteristics of immune organs must be considered during pathological evaluations of immunotoxicity [5]. However, anatomical and pathological research in cynomolgus monkeys has been limited. Until now, only histological examinations of the spleen and thymus in cynomolgus monkeys have been reported [14], and detailed quantitative compartmental analyses have yet to be published. Therefore, the aim of this study was to clarify the morphological characteristics of the cynomolgus monkey immune system by quantitative analyses of the spleen, lymph nodes and Peyer s patches and to provide a basis for histopathological evaluation of lymphoid organs when performing standard toxicology studies in this species. MATERIALS AND METHODS Animals: Naïve three-to eight-year-old cynomolgus monkeys (33 males and 30 females) purpose-bred in China and maintained at Shin Nippon Biomedical Laboratories, Ltd. were used. The animals were housed in individual stainless steel cages (680 mm 620 mm 770 mm) in a conventional facility at a temperature of 23 C to 29 C with a relative humidity of 35% to 75% and a 12-hr light/dark cycle (lights on from 07:00 to 19:00). Approximately 108 g (approxi-

2 1460 A. MORIYAMA ET AL. mately 12 g 9 pieces) of pellet food was provided daily to each animal, and water was available ad libitum. Ethics: The study was approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. and conducted in accordance with the bylaws of the committee. Tissue sampling, organ weight and dimensions and specimen preparation: The animals were anesthetized by an intravenous injection of sodium pentobarbital solution (64.8 mg/ml, 0.4 ml/kg) into the cephalic vein and euthanized by exsanguination. Spleens, lymph nodes and Peyer s patches were sampled. Lymph nodes were removed from the submandibular, mesenteric, retropharyngeal, mediastinal, superficial cervical, deep cervical, axillary, bronchial, splenic hilum, pancreaticoduodenal, large intestine, inguinal, iliac and popliteal regions. The spleen and lymph nodes were weighed, and the major axes of the lymph nodes were measured. These organs and tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H & E). Immunohistochemistry: After sections had been deparaffinized and rehydrated, antigens were activated by heating the sections in an autoclave (121 C, 3 min) with ready-touse Target Retrieval Solution (DAKO, Carpinteria, CA, U.S.A.). Nonspecific staining was blocked with 3% hydrogen peroxide and protein block (DAKO). Sections were stained using the Ventana NX System (Ventana Medical Systems Inc., Tucson, AZ, U.S.A.). T cells were immunolabeled with a 1:1 diluted anti-cd3 rabbit polyclonal antibody (Zymed Laboratories, Inc., South San Francisco, CA, U.S.A.), and B cells were immunolabeled with a 1:1 diluted anti-cd20 human monoclonal antibody (Ventana Medical Systems Inc.). The anti-rabbit secondary antibody was labeled and visualized with 3,3-diaminobenzidine. Sections were counterstained with hematoxylin, dehydrated, cleared and mounted on coverslips. Selection of lymphoid follicles: H & E-stained sections were used for measurement of the areas of lymphoid follicles and germinal centers. For the spleen, intermittent serial sections were cut, and three slides at intervals of approximately 25 µm were prepared. Lymphoid follicles of uniform size in three serial slides were regarded as the largest. Five lymphoid follicles per animal were measured by image analysis as described later. For the submandibular and mesenteric lymph nodes, one slide per animal was cut along the minor axis, and three lymphoid follicles in which the germinal centers were clearly observed were selected for image analysis in order of size. For the Peyer's patches observed macroscopically in the ileum, one slide per animal was cut along the minor axis. All lymphoid follicles in which germinal centers were clearly observed were selected for image analysis. Image analysis: All the selected histological slides were scanned for imaging. The images were analyzed with analysis software (Soft Imaging System GmbH, Muenster, Germany). The areas of lymphoid follicles and germinal centers were determined from delineated H & E-stained Fig. 1. H & E-stained image. The lymphoid follicle is indicated by the green circle, and the germinal center is indicated by the yellow circle. A: Spleen. The lymphoid follicle includes the germinal center (light-stained area), mantle zone (dark-stained area) and the marginal zone. B: Lymph node (submandibular). C: Peyer s patches. Bar=500 μm. images (Fig. 1). The lymphoid follicle included the germinal center (light-stained area), mantle zone (darkstained area) and marginal zone. Percentages of CD3- and

3 LYMPHOID TISSUE IN PRIMATES 1461 CD20-positive areas were calculated by automatic identification of the colors of positive cells using anti-cd3 and anti-cd20 stained images (Fig. 2). Figure 2 shows images before identification on the left side and those after identification, for which positive areas are shown in red, on the right side. When multiple lymphoid follicles were measured, mean values were expressed as individual values. Statistical analysis: Pairwise comparison by gender or organ was performed by t-test based on a one-way ANOVA model. Pearson s product-moment correlation coefficient was calculated between the lymphoid follicle and germinal center areas or between the lymphoid follicle area and splenic weight. P<0.05 was considered statistically significant. RESULTS Spleen: Lymphoid follicle areas of male and female cynomolgus monkeys are shown in Fig. 3. The mean lymphoid follicle area was 0.20 mm 2. The maximum and minimum Fig. 2. Examples of images measured for each parameter. Positively stained areas are shown in red on the right side of the image. A: Spleen. Anti-CD3 staining. B: Spleen. Anti-CD20 staining. C: Lymph node (submandibular). Anti-CD3 staining. D: Lymph node (submandibular). Anti-CD20 staining. E: Peyer s patches. Anti-CD3 staining. F: Peyer s patches. Anti-CD20 staining. Bar=500 μm.

4 1462 A. MORIYAMA ET AL. areas of individual lymphoid follicles varied by greater than 10-fold. Values generally ranged between 0.1 mm 2 and 0.3 mm 2, but larger and smaller areas were widely distributed. The maximum and minimum individual coefficients of variation were 73.1% and 13.6%, respectively. Germinal center areas for male and female cynomolgus monkeys are shown in Fig. 4. The germinal center was observed for each animal, and the mean germinal center area was 0.08 mm 2. The maximum and minimum individual coefficients of variation were 93.0% and 12.1%, respectively. No statistically significant sex difference was observed in the size of the lymphoid follicles or germinal centers (data not shown). The ratio of the germinal center to the lymphoid follicle is shown in Fig. 5. Values ranged from 20% to 70%. The mean percentage was 35.64%, and the maximum and minimum individual coefficients of variation were 64.2% and 4.5%, respectively. No statistically significant sex difference was observed (data not shown). The size of the germinal center correlated with that of the lymphoid follicle (correlation coefficient: 0.92, Fig. 6). However, there was no correlation between the lymphoid follicle area and splenic weight (correlation coefficient: 0.11, Fig. 7). Lymph nodes: The sizes and weights of lymph nodes are shown in Table 1. Lymph nodes were selected for pathological evaluation in accordance with the following criteria: 1. Major axis of at least 5 mm in length to facilitate sampling at necropsy 2. Minimal variation in weight and size 3. All essential components (cortex including germinal center, paracortex and medulla) clearly visible The major axes of the submandibular, mesenteric, axillary, bronchial, pancreaticoduodenal and large intestine lymph nodes all exceeded 5 mm in length. Variations in the weight and size of the submandibular, mesenteric and large intestine lymph nodes were minimal. Almost all components of the submandibular, mesenteric, axillary, splenic hilum and iliac lymph nodes were clearly visible. Representative submandibular and mesenteric lymph nodes were selected, and detailed morphometric analyses were performed (Table 2). The lymphoid follicle and germinal center areas in the mesenteric lymph nodes were significantly smaller than those in the spleen. The submandibular lymph nodes were larger than the mesenteric lymph nodes. CD3-positive and CD20-positive areas, representative of the T cell and B cell areas, respectively, were calculated. In contrast to the spleen, both lymph nodes had larger T cell areas than B cell areas. Peyer s patches: The morphometric parameters of Peyer s patches are shown in Table 2. The lymphoid follicle areas in Peyer s patches were not significantly different from those in the spleen, while the germinal center areas in Peyer s patches were significantly smaller than those in the spleen. As observed in the spleen, the B cell areas in Peyer s patches were larger than the T cell areas. Fig. 3. Variations in the splenic lymphoid follicle areas in cynomolgus monkeys. The individual coefficients of variation ranged from 13.6% to 73.1%. Fig. 4. Variations in the splenic germinal center areas in cynomolgus monkeys. The individual coefficients of variation ranged between 12.1% and 93.0%. Fig. 5. Ratio of germinal centers to lymphoid follicles in cynomolgus monkeys. The individual coefficients of variation ranged from 4.5% to 64.2%. DISCUSSION An understanding of individual characteristics of lymphoid organs is required for immunotoxicologic evaluations. In a previous report, the spleens and thymuses of cynomolgus monkeys were analyzed semiquantitatively [14]. Quantitative analysis is a valuable method, since the results enable comparison between organs. The results of spleen analysis indicated that the lymphoid

5 LYMPHOID TISSUE IN PRIMATES 1463 Fig. 6. Correlation between lymphoid follicle and germinal center areas in cynomolgus monkeys. The correlation coefficient was Fig. 7. Correlation between lymphoid follicle areas and splenic weight in cynomolgus monkeys. Lymphoid follicle areas were calculated as the mean for each individual. The correlation coefficient was follicle and germinal center areas were widely distributed with large individual coefficients of variation. The spleen is an important organ for evaluating toxicity, and intra- and interindividual variations of lymphoid follicles and germinal centers are large. Interindividual variations are also large for the lymph node and Peyer s patches. As the position paper stated [6], an awareness of concrete individual variation is essential for reliable evaluation in preclinical studies. Lymphoid follicles and germinal centers were clearly observed by H & E staining in the spleen, submandibular lymph nodes, mesenteric lymph nodes and Peyer s patches. Lymphoid follicle sizes did not markedly differ among the analyzed organs except the mesenteric lymph nodes. Germinal center sizes in the mesenteric lymph nodes and Peyer s patches were significantly smaller than those in the spleen. For compounds that induce immunosuppression, the spleen or submandibular lymph nodes may provide the most information when examining changes in the germinal centers. However, this is not the case for compounds that enhance immune function. As shown in Table 2, the T cell area was largest in the lymph nodes, while the B cell area was largest in the spleen and Peyer s patches, suggesting that the lymph nodes are more useful for detailed evaluation of T cell changes, whereas the spleen and Peyer s patches are better suited for evaluation of B cell changes. From the viewpoint of comparisons between different animal species, splenic lymphoid follicles in cynomolgus monkeys are larger than those in rats [11], in which the germinal center does not develop and the B cell area is relatively small. These interspecies differences in lymphoid tissue are partly attributable to the maintenance of cynomolgus monkeys in a conventional environment, whereas rats are maintained in a specific pathogen free environment. These respective maintenance environments are common to toxicity studies, and species-specific characteristics should be recognized as data for laboratory animals. By confirming immune system changes when administering new compounds, consideration of environmental differences may enable more significant extrapolation of preclinical data to humans. The standard deviation of the splenic lymphoid follicle in Table 1. Long axis, weight and microscopic examination of lymph nodes of cynomolgus monkeys Long axis Weight Microscopic examination Mean Minimum Standard Coefficient of Mean Standard Coefficient of Component Lymph node (mm) (mm) deviation variation (%) (mg) deviation variation (%) (%) Submandibular a) b) b) 100 c) Mesenteric a) b) b) 100 c) Retropharyngeal Mediastinal Superficial cervical Deep cervical Axillary c) Bronchial a) Splenic hilum c) Pancreaticoduodenal a) Large intestine b) b) 81.8 Inguinal a) Iliac c) Popliteal a) Indicated lymph nodes all exceeded 5 mm in length. b) Variations in the weight and size of the indicated lymph nodes were minimal. c) Almost all components of the indicated lymph nodes were clearly visible.

6 1464 A. MORIYAMA ET AL. Morphological parameters of lymphoid organs in cynomolgus monkeys (mean standard deviation/coefficient of varia- Table 2. tion) Spleen Submandibular Ly. d) Mesenteric Ly. d) Peyer s patch Number of animals (n) n=63 n=14 n=14 n=14 Lymphoid follicle area (mm 2 ) / / b) 0.05/ /37.9 Germinal center area (mm 2 ) / / c) 0.02/ c) 0.03/63.6 Number of animals (n) n=14 n=14 n=14 n=14 CD3 positive area a) (%) / / / /41.2 CD20 positive area a) (%) / / / /27.6 CD3 positive /CD20 positive ratio a) The percentages of the CD3 or CD20 positive areas in the area of the entire tissue section are shown. b) Indicates a statistically significant smaller area compared with the lymphoid follicle area of the spleen. c) Indicates a statistically significant smaller area compared with the germinal center area of the spleen. d) Ly; lymph node. cynomolgus monkeys is greater than that in humans, even though the mean size of the lymphoid follicle is similar between the two species [12]. This difference is not considered attributable to the environment, since the conventional environment for monkeys is similar to the human environment in that it is almost free of microbiological control. Genomic sequences of major histocompatibility complex class II in rhesus monkeys are more diverse than in humans [4]. This may be a cause of the observed individual specificity, although this remains to be confirmed for cynomolgus monkeys. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. When evaluating data including large individual variations, analysis of multiple parameters for lymphoid organs, such as size and weight, and routine H & E, IHC or morphometry is advantageous. As observed for substance-induced alterations in the immune system, with regard to extrapolation to humans, nonhuman primates provide better model systems for evaluation of changes in blood and tissue cells, surface receptors and immunoglobulins [13]. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in a standard toxicity study. REFERENCES 1. Bondarenko, G. I., Dambaeva, S. V., Grendell, R. L., Hughes, A. L., Durning, M., Garthwaite, M. A. and Golos, T. G Characterization of cynomolgus and vervet monkey placental MHC class I expression: diversity of the nonhuman primate AG locus. Immunogenetics 61: Buse, E., Habermann, G., Osterburg, I., Korte, R. and Weinbauer, G. F Reproductive/developmental toxicity and immunotoxicity assessment in the nonhuman primate model. Toxicology 185: Committee SCHER The need for non-human primates in biomedical research, production and testing of products and devices. [cited 2010 April 8]. Available from ec.europa.eu/health/ph_risk/committees/04_scher/docs/ scher_o_110.pdf. 4. Daza-Vamenta, R., Glusman, G., Rowen, L., Guthrie, B. and Geraghty, D. E Genetic divergence of the rhesus macaque major histocompatibility complex. Genome Res. 14: Haley, P. J Species differences in the structure and function of the immune system. Toxicology 188: Haley, P., Perry, R., Ennulat, D., Frame, S., Johnson, C., Lapointe, J. M., Nyska, A., Snyder, P., Walker, D. and Walter, G STP position paper: best practice guideline for the routine pathology evaluation of the immune system. Toxicol. Pathol. 33: Harleman, J. H Approaches to the identification and recording of findings in the lymphoreticular organs indicative for immunotoxicity in regulatory type toxicity studies. Toxicology 142: The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Immunotoxicity studies for human pharmaceuticals. S8. [cited 2009 September 7]. Available from Guidelines/Safety/S8/Step4/S8_Guideline.pdf. 9. Kuper, C. F., Harleman, J. H., Richter-Reichelm, H. B. and Vos, J. G Histopathologic approaches to detect changes indicative of immunotoxicity. Toxicol. Pathol. 28: Lappin, P. B. and Black, L. E Immune modulator studies in primates: the utility of flow cytometry and immunohistochemistry in the identification and characterization of immunotoxicity. Toxicol. Pathol. 31: Milićević, N. M. and Milićević, Z Stereological study of splenic tissue compartments in FK506-treated rats. Histol. Histopathol. 12: Milićević, Z., Cuschieri, A., Xuereb, A. and Milićević, N. M Stereological study of tissue compartments of the human spleen. Histol. Histopathol. 11: Neubert, R., Helge, H. and Neubert, D Nonhuman primates as models for evaluating substance-induced changes in the immune system with relevance for man. pp In: Experimental Immunotoxicology (Smialowicz, R. J. and Holsapple, M. P. eds.), CRC Press, Boca Raton, FL. 14. Spoor, M. S., Radi, Z. A. and Dunstan, R. W Characterization of age- and gender-related changes in the spleen and thymus from control cynomolgus macaques used in toxicity studies. Toxicol. Pathol. 36: Vugmeyster, Y., Howell, K., Bakshi, A., Flores, C., Hwang, O., and McKeever, K B-cell subsets in blood and lymphoid organs in Macaca fascicularis. Cytometry A 61: