Characterization of Protein Phosphorylation by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Murine Lymphocytes: Indirect Evidence for a Role in the Suppression of Humoral Immunity

ABSTRACT

Studies were undertaken to more thoroughly characterize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced stimulation of kinase activity in murine lymphocytes. In female B6C3F1 mice, TCDD-induced phosphorylation of 29, 45, 52 and 63 KDa proteins was selective for B cells, with little or no enhancement observed in T cells. When B cells were purified and separated by density on a percoll gradient, phosphorylation was only observed in the band composed of activated B cells, and was not enhanced in the band composed of resting B cells. TCDD-stimulated phosphorylation was associated with both the cytosol (45 and 52 KDa species) and membrane (52 KDa species) fractions. Purified B cells from both DBA/2 (Ah^dd) and C57B16 (Ah^bb) mice demonstrated equivalent enhancement of phosphorylation in response to TCDD. Administration of human gamma interferon (Hu-IFNg) at concentrations from 0.5 to 500 Units/ml produced a dose-related reversal of TCDD-induced suppression of in vitro antibody responses to both the polyclonal B cell activator, LPS, and the T-dependent antigen, sRBC in whole splenocytes isolated from female B6C3F1 mice. These concentrations of Hu-IFNg did not affect the magnitude of either response in the absence of TCDD, and did not reverse dexamethasone-induced suppression of either in vitro antibody response. TCDD-induced suppression of the T-dependent response was reversed only when Hu-IFNg was added to culture within the first 18 hours after treatment with TCDD and sRBC. These studies demonstrate that Hu-IFNg can reverse TCDD-induced in vitro Ab response suppression if it is administered during the period of susceptibility to TCDD. TCDD-induced phosphorylation in isolated B cells was also antagonized following co-incubation with Hu-IFNg. The profile of TCDD-induced increases in protein phosphorylation, including the selective effect on activated B cells, the general involvement of both cytosolic and membrane proteins, the lack of segregation with the Ah -dependent processes, and the ability of Hu-IFNg to reverse both the suppression of the Ab response and the increase in phosphorylation, supports the interpretation that such phosphorylation is involved in TCDD-induced suppression of the Ab response.