Infectious diseases are a major cause of morbidity and mortality in the elderly population. For example, Gram negative sepsis may exceed 50% in the aged population. This is due particularly to improved medical technology that allows individuals to live longer, yet ironically, places them at increased risk to secondary infection. There is substantial evidence implicating endotoxin as a major virulence determinant in Gram negative septicemia. Cytokines, particularly tumor necrosis factor, (TNF), appear to be major effectors of endotoxic reactions. Glucocorticoid hormones have been shown to regulate the synthesis of TNF as well as influence the response of target cells to it. The focus of this proposed study will be on age associated differences in TNF-glucocorticoid interaction in sepsis. Results of preliminary studies revealed that senescent C57BL/6NNia mice (24 months old) died much earlier than young (4-6 months old) or mature (12 months old) mice after the induction of acute peritonitis and sepsis by cecal ligation and puncture (CLP). We propose that the increased sensitivity of senescent mice to sepsis and endotoxemia is related to the interaction of glucocorticoid hormones with tumor necrosis factor. Senescent animals produced 20 to 30-fold more serum TNF than mature mice when administered E.coli endotoxin. Furthermore, the usual down-regulation of TNF production by exogenous glucocorticoids was not observed in endotoxic senescent mice. WE HYPOTHESIZE THAT DISRUPTED GLUCOCORTICOID REGULATION OF TNF GENE EXPRESSION MAY UNDERLIE THE INCREASED SENSITIVITY OF AGED ANIMALS TO SEPSIS. In the proposed study we will define the role of glucocorticoids in age associated differences in TNF production by endotoxic mice. We will utilize the glucocorticoid antagonist, RU486, for in vivo experiments in which the effect of glucocorticoid receptor blockade on TNF production will be examined. Peritoneal macrophages will be isolated from young, mature and senescent mice for in vitro studies of the influence of dexamethasone on TNF synthesis.
A second Aim i s to investigate if altered glucocorticoid regulation of TNF production in aged animals occurs at the TNF gene level. We will develop and utilize transgenic mice carrying a TNF:CAT gene construct to study the influence of glucocorticoids in vivo on TNF gene expression. For in vitro studies, a variety of TNF:CAT gene constructs will be used to transfect primary peritoneal macrophages to examine the effect of glucocorticoids, RU486 and actinomycin D on TNF production. Our long term goal is to understand TNF-glucocorticoid interactions so that the increased sensitivity of aged mammals to bacterial sepsis can be reduced through more effective therapeutic intervention.
