<I>Secretion mechanism of macrophage migration inhibitory factor in hydrogen peroxide-and LPS-stimulated human fibroblasts differs from interleukin-6</I>
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In order to investigate the mechanism for secretion of macrophage migration inhibitory factor (MIF) in cultured human fibroblasts, we compared with the secretion of interleukin-6 (IL-6) as stimulated with lipopolysaccharides (LPS) and H<SUB>2</SUB>O<SUB>2</SUB>. MIF content of the medium of 2.0×10<SUP>6</SUP> cells/20 ml after 20 h culture of non-stimulated fibroblasts was 0.30±0.06 ng/ml, whereas LPS-stimulation (10μg/ml) led to increase only 1.5 fold as compared with the nonstimulated cells. In contrast, a significant increase of IL-6 was induced by LPS-stimulation (6048±488 pg/ml in LPS-stimulated cells vs 58±36 pg/ml in control cells) . On the other hand, the higher concentrations of H<SUB>2</SUB>O<SUB>2</SUB> (0.6 mM-1.2 mM) caused an increase of MIF secretion into the culture medium irrespective of LPS-stimulation, showing that 1.2 mM H<SUB>2</SUB>O<SUB>2</SUB>-stimulation for 20 h was increased to 40 fold as compared with the nonstimulated cells. However, the lower concentrations (0.1 mM-0.4 mM) did not cause this. Interestingly, H<SUB>2</SUB>O<SUB>2</SUB>-stimulation not only failed to increase IL-6 production from fibroblasts, but also repressed induction of IL-6 by LPS-stimulation in a dose dependent manner. Genistein, a tyrosine kinase inhibitor and H-7, a protein kinase C inhibitor also inhibited IL-6 secretion but not MIF secretion in both LPS-and H<SUB>2</SUB>O<SUB>2</SUB>-stimulated fibroblasts. From analyses of trypan blue exclusion, formazan formation, morphological changes, and intracellular MIF content by Western blot, we found that MIF secretion by H<SUB>2</SUB>O<SUB>2</SUB> seems to be mainly due to cell-death and subsequently leakage of intracellular MIF. Taken together, these results suggest that MIF secretion differs from IL-6 via LPS-mediated signaling pathways.
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