Characterization of an approach to developmental immunotoxicology assessment in the rat using SRBC as the antigen

Although developmental immunotoxicology is an area of emerging importance, few data exist that compare profiles of activity in neonatal and adult rodents. One of the factors contributing to the paucity of comparative results is that it is unclear whether immunotoxicological test procedures optimized to evaluate the immune system of adults can be integrated into studies of younger animals. Therefore, the following objectives were addressed in this study for both male and female Crl:CD(SD)BR rat pups and weanlings: (1) to quantify baseline values for absolute and relative splenic lymphocyte populations using flow cytometry in nonimmunized animals; (2) to optimize and establish baseline values for the primary humoral immune response to sheep red blood cells (SRBC) using either an enzyme-linked immunosorbant assay (ELISA) or the plaque-forming cell (PFC) assay; and (3) to determine the impact of SRBC immunization on the histology of the spleen. Responses for each age group were compared both within and between litters. Spleen cell counts and absolute and relative (to body weight) spleen and thymus weights were also obtained. Analysis by flow cytometry indicated the following overall mean absolute and relative (%) numbers of splenic lymphocytes in nonimmunized 10-day-old rats, respectively: 0.18 ± 0.08 x 10⁸ (13 ± 5) CD45⁺; 0.13 ± 0.04 x 10⁸ (9 ± 3) OX12⁺; 0.07 ± 0.01 x 10⁸ (5 ± 1) W3/25⁺ CD3⁺; 0.03 ± 0.01 x 10⁸ (2 ± 0.5) OX8⁺ CD³+ cells. Because of the difficulty in administering SRBC by tail vein injection, the pups received SRBC intraperitoneally but were still unable to generate a primary IgM response. Histologically, no germinal centers were observed in rat pups immunized with SRBC. Taken together, these results suggest that the spleens of 10-day-old rats consist predominantly of functionally immature lymphocytes. Analysis by flow cytometry indicated the following mean absolute and relative (%) numbers, repectively, for nonimmunized 21-day-old rats: 0.96 ± 0.25 x 10⁸ (48 ± 5) CD45⁺; 0.77 ± 0.16 x 10⁸ (39 ± 3) OX12⁺; 0.23 ± 0.02 x 10⁸ (12 ± 2) W3/25⁺CD3⁺; 0.14 ± 0.02 x 10⁸ (7 ± 1) OX8⁺CD3⁺ cells. Results when useing the ELISA indicated that the SRBC-specific IgM antibody log₂ titer for rat weanlings was cosiderably less than the titer obtained for young adult rats, whereas results with the PFC assay indicated a response in weanlings that was within the historical analysis in that prominent germinal centers were observed in weanlings immunized with SRBC. Based on ouir flow cytometric, ELISA, and PFC assay data, a litter can be used as the unitmof comparison for developmental immunotoxicity. Our results with SRBC suggest that it may not be possible to evaluate a humoral immune functional parameter in rat pups due to the apparent immature status of their immune cells; however, additional antigens must be examined. Results in wealings suggest that an antibody response to SRBC of sufficient magnitude can be demonstrated with the PFC assay, but that a high background may preclude the use of the SRBC-specific Igm ELISA.