Flow cytometric analysis of lymphocyte subpopulations in the spleen and thymus of mice exposed to an acute immunosuppressive dose of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD)

Abstract

The objective of the present studies was to determine if acute exposure to an immunotoxic dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces alterations in the expression of lymphocyte surface markers as measured by multiparameter flow cytometry. The immunotoxicity of a single oral dose of TCDD was assessed by the anti-SRBC PFC response; an ED50 of 0.74 micrograms/kg was determined. Subpopulations in the spleen and thymus of C57B1/6 mice were analyzed 2 days following exposure to 2 micrograms/kg TCDD. In addition, splenic lymphocyte subsets were examined on Days 1-4 following SRBC challenge of mice treated with 0, 2, or 5 micrograms/kg TCDD. T and B cells were identified by single parameter analysis of Thy 1.2 and Ig expression. T cell subsets were defined by dual parameter analysis of CD4 and CD8 expression. In TCDD-treated mice, the percentage and the total number of double-positive CD4+ CD8+ thymocytes were significantly decreased while the percentage but not the total number of double-negative CD4- CD8- thymocytes was significantly increased. No changes in the percentage or total number of single positive (CD4+ CD8- or CD4- CD8+) thymocyte subsets were observed. In contrast to the thymus, lymphocyte subsets in the spleen were not significantly altered in percentage or total number 2 days following acute TCDD exposure. When splenic lymphocytes were analyzed daily following SRBC challenge, Ig+, Thy 1.2+, and CD4+ CD8- subpopulations remained relatively unchanged in both control and TCDD-treated animals. A small but significant decrease in the percentage of CD4- CD8+ T cells was observed on Day 3 in mice treated with 2 or 5 micrograms/kg TCDD when compared to that of vehicle-treated mice. The total number of CD4- CD8+ splenocytes was also significantly lower in the 5-micrograms/kg group on Day 3. However, this effect appeared to result from an elevation of the CD4- CD8+ subset in the controls rather than from a reduction in the TCDD-treated groups. Double-positive (CD4+ CD8+) lymphocytes were not detected in either control or TCDD-treated spleens. These results indicate that an acute dose of TCDD which reduced the splenic anti-SRBC response by 65-80% did not cause detectable changes in major splenic lymphocyte subpopulations. This is an important finding from the standpoint of utilizing lymphocyte subset analysis to screen for potential immunotoxic effects of TCDD. Specifically, the absence of subset changes does not preclude the presence of functional immunosuppression.