DIMETHYLNITROSAMINE EFFECTS ON CELLULAR IMMUNITY

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Experiments outlined in this competitive renewal are aimed at determining how the tissue response to dimethylnitrosamine (DMN)-induced hepatotoxicity leads to immune dysfunction. Our previous work demonstrated that DMN exposure resulted in altered bone marrow differentiation, induction of serum borne cytokines, and tissue macrophages with increased prostaglandin synthesis, and enhanced respiratory burst activity and tumor necrosis factor alpha gene expression. Studies outlined in this proposal will continue to focus on the role of the macrophage since this cell is involved in both the inflammatory response to tissue injury as well as regulating cell- mediated immunity (CMI). Molecular methodologies developed in prior studies will be utilized to elucidate the mechanism(s) by which DMN exposure affects the immune system. The hypothesis to be addressed in this proposal is that the lack of adequate down-regulation of the chemically-induced inflammatory response results in inappropriate cytokine expression, thus, affecting the regulation of the immune response. Consequently, the overproduction of specific cytokines leads to immune dysfunction. To address this hypothesis we will perform qualitative and quantitative studies to characterize the induction and resolution of the DMN-induced inflammatory response. These studies will monitor the cellular influx, induction of acute phase proteins and adhesion molecules, and changes in cytokine expression by utilizing immunodetection, in situ hybridization, enzyme-linked immunosorbent- and bio-assays as well as solution hybridization and polymerase chain reaction (PCR) gene amplification. Secondly, experiments will determine the mechanisms responsible for elevated serum cytokine activity and the induction of "primed" macrophage populations, and altered immune responses (in vitro). These efforts will utilize the "molecular phenotyping" and quantitative PCR procedures which we have developed to focus on our continued interest in the generation of macrophage heterogeneity and why DMN-induced changes in differentiation results in macrophages with altered responses to regulatory signals. Specific questions which will be addresses include: Are specific "molecular phenotypes" associated with tissue and inflammatory macrophages following DMN exposure? What is the correlation between changes in macrophage markers with functions? Are T cell regulatory signals absent in DMN-induced inflammation? Answers to these questions will be used in our third objective to determine whether the cytokine profiles (biomarkers) associated with DMN exposure are directly responsible for immunotoxicity and can be used to predict altered host resistance (in vivo). Hence, these studies will determine the cause and effect relationships between identified biomarkers and DMN-induced immunotoxicity. The objectives of these efforts are to determine whether administration of cytokines can reverse DMN-induced immunotoxicity or that monitoring of cytokine expression can be used to predict altered immune responses in vivo. Thus, this approach will permit us to establish a model for making risk assessments following DMN exposure. Finally, experiments will be conducted to determine whether there is a genetic component to DMN-induced changes.