Gallium arsenide, a semiconductor used in the electronics industry, exposure of mice induces profound immunosuppression. The major objective of this research proposal is to define the mechanism by which gallium arsenide interferes with antigen processing by macrophages. Antigen processing is the conversion of exogenous foreign proteins to antigenic peptides for the activation of helper CD4+ T cells. Our previous toxicological study showed that exposed macrophages are impaired in processing the particulate antigen, sheep erythrocytes, but not the soluble antigen, cytochrome c. The proposal will focus on the biochemical property of arsenic to bind sulfhydryl moieties. Mice will be exposed to gallium arsenide or vehicle by an intratracheal route and macrophages will be isolated one day later. The ability of splenic and peritoneal macrophages exposed to vehicle and gallium arsenide in vivo to process various antigens will be analyzed by in vitro stimulation of antigen- specific CD4+ T cell lines to secrete interleukin-2. Disruption of disulfide bonds is an important step in processing ovalbumin, lysozyme, and insulin, unlike cytochrome c. Synthetic peptides of ovalbumin and lysozyme, which do not require processing, will distinguish between effects on antigen processing and presentation. Carboxymethylated ovalbumin and lysozyme will detect a problem in unfolding antigens. Second, the relationship between the mode of antigen internalization and inhibition of processing by gallium arsenide will be examined by changing the nature of the antigens. Cytochrome c will be converted to a particulate antigen by coupling onto latex beads. Treatment of macrophages with cytochalasin D should prevent phagocytosis of the beads and eliminate the T cell response. Conversely, the T cell response to solubilized sheep red blood cells, which will be endocytosed, will be analyzed. Third, endosomal and lysosomal acidic thiol proteases require a reduced cysteine residue in their active site. The enzymatic activities of cathepsins B, H and L in cell lysates from vehicle- and gallium arsenide-exposed cells will be measured by the use of a combination of specific substrates and inhibitors. The aspartyl protease, cathepsin D, will serve as a control. Proteolytic activities in isolated low (endosomes) and high (lysosomes) density subcellular fractions will detect any change in the intracellular distribution of the proteases. Finally, the effect of gallium arsenide on various aspects of the intracellular reducing environment will be examined by comparing its status in vehicle- and gallium arsenide-exposed cells. The efficiency of lysosomal cysteine and cystine transporters will be determined by the specific influx and efflux of radio labeled cystine during a time course. The levels of intracellular cysteine and glutathione, the physiological reducing agents, will be measured in cell lysates. The understanding of the mechanism by which gallium arsenide impairs antigen processing may provide insights into circumventing gallium arsenide-mediated.
