Inhaled environmental toxins can exert effects on the lungs by altering the function of key genes. In particular, hypersensitivity immune responses to metals, such as beryllium (Be), occur when moderate cytokine and growth factor expression. The consequences are granulomatous inflammation and fibrosis. The ability to find effective treatments of environmental lung diseases, such as chronic beryllium disease (CBD), will depend upon our understanding of molecular mechanisms underlying environmentally-induced regulation of immune cell gene function. T lymphocytes and macrophages accumulate in the lungs of CBD patients. Upon Be-stimulation in vitro, CBD lymphocytes proliferate and bronchoalveolar lavage (BAL) cells produce high levels (ng/ml) of TNF-alpha and IFN-gamma. We recently found that the -308 A TNF promote polymorphism is a functional polymorphism associated with high levels (>1.5 ng/ml) of Be-stimulated CBD/BAL cell TNF- alpha protein and that these high TNF-alpha levels correlate with disease severity in CBD. TNF-alpha is a pro-inflammatory cytokine important to granuloma formation in the lungs but the precise molecular mechanisms cytokine important to granuloma formation in the lungs but the precise molecular mechanisms by which Be regulates the production of high TNF-alpha levels our unknown. Our data shown that IFN-gamma priming of macrophages leads to enhancement of Be-induced TNF-alpha. Using a mouse macrophage cell line (H36.12j) that mimics the CBD macrophage response we found that Be up-regulates TNF-alpha protein and mRNA. Be-stimulation did not up-regulate nuclear transcription factors. However, Be+ IFN-gamma co-stimulation significantly enhances TNF-alpha production and increased nuclear levels of NF-kappaB, AP-1 and CREB. Preliminary data suggest that isolated CBD/BAL macrophages can produce lower but significant amounts of Be- stimulated TNF-alpha in the absence of BAL CD4+ T cells and without IFN-gamma. The central hypothesis of this study is that: 1) Be-antigen recognition (sensitization) alone does not result in CBD. 2) CBD results when Be triggers TNF-alpha over-expression. 3) TNF-alpha over- expression occurs because IFN-gamma and Be work in concert to up- regulate TNF-alpha mRNA splicing and to increase nuclear transcription factors. 4) TNF-alpha over-expression will be highest in people who have the -308 A, TNF promoter polymorphism because IFN-gamma up- regulated transcription factors enhance TNF gene transcription in the presence of the polymorphism. Together with Be-induced mRNA splicing, high TNF-alpha levels will be produced. The objective of our study is to test these specific hypothesis using isolated CBD macrophages and macrophage cell lines. We will determine whether the key control subjects cells that do not make Be-stimulated TNF-alpha. We will determine if the -308 A promoter polymorphism in concern with the TNF gene 3' untranslated region (3'UTR), stabilizes Be-stimulated TNF-alpha mRNA transcripts, thus boosting TNF-alpha protein expression. With improved understanding of the cellular and molecular mechanisms that result in progression of macrophages from the BeS to CBD phenotype, we hope, in the future, to design preventative strategies to modulate disease in high-risk, exposed individuals.
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