Estrogen up-regulates inducible nitric oxide synthase, nitric oxide, and cyclooxygenase-2 in splenocytes activated with T cell stimulants: role of interferon-gamma.

Estrogen is implicated in many autoimmune diseases and is a robust immunomodulator. For example, it regulates interferon (IFN)-gamma, a cytokine believed to up-regulate inducible nitric oxide synthase (iNOS). A notable gap in the literature is a lack of information on the regulation of nitric oxide in immune tissues by estrogen. We now show that activation of splenocytes with T cell stimulants [concanavalin-A (Con-A) or anti-CD3 antibodies] results in copious release of nitric oxide in splenocyte cultures from estrogen-treated but not placebo-treated mice. Moreover, even a low dose of T cell stimulants induced nitric oxide in splenocytes from estrogen-treated, but not placebo-treated, mice. Con-A-activated splenocytes from estrogen-treated mice also have up-regulated iNOS mRNA, iNOS protein, and cyclooxygenase-2 (a nitric oxide-regulated downstream proinflammatory protein) when compared with controls. Our studies suggest that the induction of nitric oxide by activated splenocytes from estrogen-treated mice is mediated in part by IFNgamma. First, blocking costimulatory signals mediated through interactions of CD28 and B7 molecules by CTLA-4Ig markedly decreased not only IFNgamma but also nitric oxide. Second, estrogen treatment of IFNgamma-knockout (IFNgamma(-)/(-)) mice did not induce iNOS protein or nitric oxide. Finally, in vitro addition of recombinant IFNgamma to Con-A-activated splenocytes from IFNgamma((-)/(-)) mice induced iNOS protein primarily in estrogen-treated mice. Overall, this is the first report to show that estrogen treatment up-regulates IFNgamma-inducible-iNOS gene expression, iNOS protein, nitric oxide, and cyclooxygenase-2 as an indirect consequence of activation of T cells. These findings may have wide implications to immunity and inflammatory disorders including female-predominant autoimmune diseases.

Altered splenocyte function in aged C57BL/6 mice prenatally exposed to diethylstilbestrol.

The linkage between in utero exposure to diethylstilbestrol (DES) and the manifestation of a variety of reproductive disorders and possibly immune alterations in adults (i.e., human and mice) is suggestive of a fetal basis of adult disease. While the long-term adverse consequences of prenatal DES-exposure on reproductive disorders are well known, there is a paucity of data with regard to immune outcome. We hypothesize that prenatal DES-exposure "imprints" the immune system, altering the response to subsequent exposure to DES in adult mice. In this pilot study, C57BL/6 mice were prenatally exposed to DES or vehicle only (oil) and then exposed to DES at 1 year of age. Potential alterations in the spleen were then examined. Female DES-exposed mice (DES(prenatal)/DES(adult)) or female(DES) had higher serum levels of interferon-gamma (IFNgamma) in response to administration of an IFNgamma -inducer (soluble proteins-derived from Toxoplasma gondii), compared to female controls, which received oil during prenatal life (Oil(prenatal)/DES(adult)). Splenic lymphocytes from female DES(prenatal)/DES(adult) mice, when activated with Concanavalin A (ConA), also secreted higher levels of IFNgamma compared to female controls (Oil(prenatal)/DES(adult)) when examined at 14-months of age. This increase in IFNgamma in prenatal DES-exposed mice is not due to enhanced numbers of splenocytes or increased relative percentages of CD4(+) or CD8(+) cells. ConA-activated T-cells from female DES(prenatal)/DES(adult) had increased expression of the co-stimulatory molecule, CD28. These above immune changes were not evident in the males prenatally exposed to DES. Prenatal DES exposure also did not induce autoimmunity in non-autoimmune C57BL/6 mice. Overall, results from these prefatory studies suggest that prenatal DES exposure may have long-term immune alterations, which become evident following a secondary exposure to DES in adult life.