We have demonstrated multiple effects of ethanol on thymus differentiation and signal transduction using in vitro exposure of fetal thymus organ culture (FTOC) or dispersed thymocytes. Ethanol treatment leads to acceleration of differentiation, alteration of subpopulations identified by CD4 and CD8 antigen expression, increased apoptosis, and induction of a rise in intracellular calcium levels. The in vitro data suggest that it perturbs processes of positive and negative selection, a physiological process that molds the T cell repertoire by eliminating self-reactive thymocytes by apoptotic cell death and rescuing thymocytes that are restricted to recognizing antigen in the context of self major histocompatibility complex products. The mechanisms by which ethanol alters thymocyte differentiation in FTOC and induces apoptosis are not known. However, we have data suggesting that ethanol-induced thymocyte apoptosis involves protein kinase C (PKC); furthermore, chronic ethanol up-regulates membrane density of GM, a ganglioside that increases the disordering effect of ethanol on membranes. Studies are therefore proposed using both in vivo and in vitro ethanol exposure of thymocytes to address the following questions: (1) Does chronic ethanol exposure affect positive or negative selection of thymocytes in young mice exposed to ethanol in vivo in a model of neonatal alcohol exposure, or in several mouse strains characterized for in vivo positive selection or clonal deletion of T cells expressing specific V, T cell receptors (TCRs). Sublethally irradiated adult mice will be fed a liquid diet containing ethanol during thymic regeneration, and their thymocytes will be analyzed by flow cytometry for expression of specific V TCRs as well as CD4 and CD8 differentiation antigens. Thymuses from fetuses of the same strains will be treated with ethanol in organ cultures to determine in vitro effects of ethanol on positive and negative selection; (2) Does ethanol exposure of thymocytes alter density of other gangliosides besides GM. and does chronic in vivo ethanol exposure alter thymocyte cell surfaces gangliosides?; and (3) Does ethanol exposure affect signal transduction pathways or alter expression of early activation genes in thymocytes? To address this questions, thymocytes chronically exposed to ethanol (both in vitro and in vitro) will be evaluated for total PKC activity and for expression of specific PKC isoenzymes by Western and Northern blot procedures. Fetal and adult thymocytes will also be tested in the presence and absence of ethanol for expression of early activation genes implicated in processes of clonal deletion. Data obtained from the proposed experiments will provide insights into the nature and mechanisms of alterations to the immune system produced by fetal alcohol exposure.
