Cell Surface Marker Analysis of Splenic Lymphocyte Populations of the CD Rat for Use in Immunotoxicological Studies

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Abstract
The objective of this study was to enumerate splenic lymphocyte populations of CD rats using immunofluorescent cell staining along with flow cytometry. Splenic lymphocytes were examined following both direct and indirect cell staining with or without lysis of red blood cells (RBC). Indirect staining with whole IgG-fluoresceinisothiocyanate (FITC) following lysis of RBC provided results most consistent with splenic differentials. Wright-Giemsa stained differentials yielded 88 ± 0.6% lymphocytes, 8 ± 0.5% macrophages, and 4 ± 0.3% polymorphonuclear (PMN) cells. Analysis of indirectly stained splenocytes revealed 29 ± 1% W3/25 [Thelper(h)], 24 ± 1% OX8 [Tsuppressor/cytotoxic(sup/cyt)], 51 ± 1% W3/13 (pan T), 51 ± 1% 0×19 (pan T), 37 ± 1% 0×12 (pan B), and 32 ± 3% OX33 (pan B) positive cells. Experiments were also conducted with goat antimouse F(ab')2-FITC as the secondary antibody or rat serum to decrease nonspecific binding. To validate the indirect staining procedure following lysis of RBC, markers were examined along with the splenic antibody plaque-forming cell (PFC) response to sheep RBC following 4-day ip exposure of rats to cyclosporin A (CsA) or cyclo-phosphamide (CY). Treatment with 3 or 25 mg/kg CsA suppressed the PFC response by 69% and 97%, respectively, but did not alter spleen weight, spleen cell number, or any of the examined lymphocyte populations, 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 cell number by 43% and 87%, respectively. Decreases in spleen (54%) and thymus (71%) weights were observed 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, respectively. The percentages of Th, Tsup/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 assessment of splenic lymphocyte populations of CD rats, and in conjunction with other assays that assess immune function, may prove useful in identifying immunotoxic compounds.