Zinc Deficiency and Thl Functions: Molecular Mechanisms

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Zinc plays an important role in immune functions in humans. Decreased thymulin activity, decreased production of IL- 2, decreased NK cell lytic activity, decreased cytolytic T cells, anergy, decreased CD4/CD8 and decreased CD4+ CD45RA+/CD4+ CD45RO+ ratios, have been observed in zinc deficient humans and these abnormalities are corrected by zinc supplementation. In order to understand the mechanism of zinc action on IL-2 production, we propose to utilize HUT-78, a ThO human malignant lymphoblastoid cell line for our studies. The human IL-2 gene promoter contains one binding site for genuine Rel/NF-kB factors and binding of NF- kB to DNA is specifically blocked by a zinc chelator and is reconstituted by addition of zinc. NF-kB also binds to the regulatory gene of IL-2 receptor alpha. We hypothesize that in zinc deficient HUT-78 cells, the activation and translocation of NF-kB to nucleus and the gene expression of NF-kB will be decreased and this will lead to decreased gene expression of IL-2 and IL-2 receptor alpha and decreased production of IL-2, sIL-2 receptor alpha and total sIL-2 receptors. The effect of different concentrations of zinc on NF-kB activation in HUT-78 cells will be determined by i) nuclear binding of NF-kB by gel shift assay and confocal imaging ii) gene transfection of cells with luciferase reporter gene vector containing NF-kB enhancer element and iii) assays of phosphorylated, unphosphorylated and ubiquitinated forms of IkB (the inhibitory molecule of the NF-kB complex) in cellular cytosolic fraction. The functional role of NF-kB on transcriptional activation of IL-2 and IL-2 receptor alpha as affected by zinc will be determined by using antisense p105 expression vector in HUT-78 cells. We are also hypothesizing that NF-kB activation, IL-2 production and IL-2 mRNA will be decreased in PHA stimulated zinc deficient human mononuclear cells and that these abnormalities will be corrected by in vivo and in vitro zinc supplementation. For this study we will select appropriate number of healthy ambulatory elderly subjects from a nursing home. Our previous experience indicates that approximately 30 percent of these healthy elderly are zinc deficient.