THE HUMAN MYELOMONOCYTIC CELL-LINE U-937 AS A MODEL FOR STUDYING ALTERATIONS IN STEROID-INDUCED MONOKINE GENE-EXPRESSION - MARKED ENHANCEMENT OF LIPOPOLYSACCHARIDE-STIMULATED INTERLEUKIN-1-BETA MESSENGER-RNA LEVELS BY 1,25-DIHYDROXYVITAMIN-D3

The active metabolite of vitamin D, 1,25-dihydroxy-vitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1-beta (IL-1-beta) mRNA and IL-1-beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1-beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1-beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1-beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1-beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone. No IL-1-beta protein was found in U-937 cell supernatants under any conditions; however, cell associated IL-1-beta protein was detected in LPS-stimulated cells. The LPS-induced IL-1-beta protein level was increased 8-fold by preincubation of the cells with 1,25-(OH)2D3. These studies establish U-937 as an important cell line for use in biochemical studies of 1,25-(OH)2D3-regulated monokine synthesis and demonstrate heightened sensitivity of 1,25-(OH)2D3-treated cells to LPS.