Effects of 2-mercaptoethanol and buthionine sulfoximine on cystine metabolism by and proliferation of mitogen-stimulated human and mouse lymphocytes

Abstract

Cysteine is an essential amino acid for lymphocytes and its anabolic products are intimately involved in lymphocyte activation. The purpose of this study was to assess the uptake and subsequent utilization of cyst(e)ine by mitogen-stimulated human peripheral blood mononuclear cells (PBMC), to evaluate the effect of an exogenous thiol, 2-mercaptoethanol (2ME), on these processes, and to compare human and mouse lymphocyte reactivities. Unlike mouse lymphocytes, the proliferation of human T-cells was inhibited by addition of 2ME although 2ME enhanced cystine uptake. Optimal responses to T-cell mitogens (Con A and PHA) were obtained with a cystine concentration of greater than or equal to 25 and 200 microM for human and mouse cells, respectively, and 2ME enhanced DNA synthesis of Con A-stimulated mouse cells regardless of the cystine dose; however, 2ME enhanced the response of human cells only in the presence of suboptimal doses of cystine. To assess whether 2ME's inability to enhance human PBMC responses was related to their glutathione (GSH) content, the human PBMC were pretreated with buthionine sulfoximine (BSO, an inhibitor of GSH synthesis). Even when the initial intracellular GSH concentration was lowered to below that of mouse lymphocytes, 2ME still inhibited proliferation. In contrast, addition of 2ME to human PBMC maintained in the presence of BSO enhanced the proliferative response suggesting that a critical level of thiols is needed for proliferation. The ability of 2ME to enhance proliferative responses in cystine deficient medium supports this contention. Consistent with thiol involvement in activation, Con A increased [35S]cystine uptake 2-fold within 4 h of incubation and enhanced subsequent conversion of cystine into cysteine and GSH. Interestingly, BSO treatment only slightly inhibited Con A-induced protein synthesis (5%), but it significantly suppressed conversion of cystine into cysteine or GSH (80-95%) and blocked DNA synthesis (90%). Overall, the results indicate that various differential thiol characteristics must exist between human and mouse lymphocytes and that a reducing equivalent is necessary for DNA synthesis but not lymphocyte activation.