CELL-SURFACE MARKER ANALYSIS OF SPLENIC LYMPHOCYTE POPULATIONS OF THECD RAT FOR USE IN IMMUNOTOXICOLOGICAL STUDIES

The objective of this study was to enumerate splenic lymphocyte popula tions of CD rats using immunofluorescent cell staining along with flow cytometry. Splenic lymphocytes were examined following both direct an d indirect cell staining with or without lysis of red blood cells (RBC ). Indirect staining with whole IgG-fluoresceinisothiocyanate (FITC) f ollowing lysis of RBC provided results most consistent with splenic di fferentials. Wright-Giemsa stained differentials yielded 88 +/- 0.6% l ymphocytes, 8 +/- 0.5% macrophages, and 4 +/- 0.3% polymorphonuclear ( PMN) cells. Analysis of indirectly stained splenocytes revealed 29 +/- 1% W3/25 [T-helper(h)], 24 +/- 1% OX8 [T-suppressor/cytotoxic(sup/cyt )], 51 +/- 1% W3/13 (pan T), 51 +/- 1% OX19(pan T), 37 +/- 1% OX12 (pa n B), and 32 +/- 3% OX33 (pan B) positive cells. Experiments were also conducted with goat anti-mouse F(ab')(2)-FITC as the secondary antibo dy or rat serum to decrease nonspecific binding. To validate the indir ect staining procedure following lysis of RBC, markers were examined a long with the splenic antibody plaque-forming cell (PFC) response to s heep RBC following 4-day ip exposure of rats to cyclosporin A (CsA) or cyclophosphamide (CY). Treatment with 3 or 25 mg/kg CsA suppressed th e PFC response by 69% and 97%, respectively, but did not alter spleen weight, spleen cell number, or any of the examined lymphocyte populati ons, emphasizing the importance of also including functional assays in immunotoxicology studies. Exposure to 5 or 30 mg/kg CY suppressed the PFC response by 59% and 98% and spleen eel number by 43% and 87%, res pectively. Decreases in spleen (54%) and thymus (71%) weights were obs erved only with 30 mg/kg CY. Exposure to 5 and 30 mg/kg CY resulted in significant decreases in the absolute number of W3/25 (35; 79%), Ox8 (26; 75%), W3/13 (27; 78%), and OX12 (52; 98%) positive cells, respect ively. The percentages of T-h, T-sup/cyt, and total T-cells increased, whereas the percentage of B-cells decreased with both 5 and 30 mg/kg CY. Indirect staining following lysis of RBC provides an accurate asse ssment of splenic lymphocyte populations of CD rats, and in conjunctio n with other assays that assess immune function, may prove useful in i dentifying immunotoxic compounds.