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 rode nts. 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 popu lations using flow cytometry in nonimmunized animals; (2) to optimize and e stablish baseline values for the primary humoral immune response to sheep r ed blood cells (SRBC) using either an enzyme-linked immunosorbant assay (EL ISA) or the plaque-forming cell (PFC) assay; and (3) to determine the impac t of SRBC immunization on the histology of the spleen. Responses for each a ge group were compared both within and between litters. Spleen cell counts and absolute and relative (to body weight) spleen and thymus weights were a lso obtained. Analysis by flow cytometry indicated the following overall me an absolute and relative (%) numbers of splenic lymphocytes in nonimmunized 10-day-old rats, respectively: 0.18 +/- 0.08 x 10(8) (13 +/- 5) CD45(+); 0 .13 +/- 0.04 x 10(8) (9 +/- 3) OX12(+); 0.07 +/- 0.01 x 10(8) (5 +/- 1) W3/ 25(+) CD3(+); 0.03 +/- 0.01 x 10(8) (2 +/- 0.5) OX8(+)CD3(+) cells. Because of the difficulty in administering SRBC by tail vein injection, the pups r eceived 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 spleen s of 10-day-old rats consist predominantly of functionally immature lymphoc ytes. Analysis by flow cytometry indicated the following mean absolute and relative (%) numbers, respectively, for nonimmunized 21-day-old rats: 0.96 +/- 0.25 x 10(8) (48 +/- 5) CD45(+); 0.77 +/- 0.16 x 10(8) (39 +/- 3) OX12( +); 0.23 +/- 0.02 x 10(8) (12 +/- 2) W3/25(+) CD3(+); 0.14 +/- 0.02 x 10(8) (7 +/- 1) OX8(+)CD3(+) cells. Results when using the ELISA indicated that the SRBC-specific IgM antibody log(2) titer for rat weanlings was considera bly less than the titer obtained for young adult rats, whereas results with the PFC assay indicated a response in weanlings that was within the histor ical range of responses for adult rats. The latter results were consistent with the histological analysis in that prominent germinal centers were obse rved in weanlings immunized with SRBC. Based on our flow cytometric, ELISA, and PFC assay data, a litter can be used as the unit of comparison for dev elopmental 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 weanlings suggest that an antibody r esponse to SRBC of sufficient magnitude can be demonstrated with the PFC as say, but that a high background may preclude the use of the SRBC-specific I gM ELISA.