Secretion of macrophage migration inhibitory factor differs from interleukin-6 in hydrogen peroxide- and...

To investigate the mechanism for secretion of macrophage migration inhibitory factor (MIF) in cultured human fibroblasts, we compared it with the secretion of interleukin-6 (IL-6) after stimulation with lipopolysaccharides (LPS) and H2O2. MIF content of the medium of 2.0 x 10(6) cells/20 ml after 20 h culture of nonstimulated fibroblasts was 0.30 +/- 0.06 ng/ml, whereas LPS-stimulation (10 microg/ml) only led to a 1.5-fold increase 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, higher concentrations of H2O2 (0.6-1.2 mM) caused an increase of MIF secretion into the culture medium irrespective of LPS-stimulation; with 1.2 mM H2O2-stimulation for 20 h, it was increased to 40-fold as compared with the nonstimulated cells. However, lower concentrations (0.1-0.4 mM) did not cause this. Interestingly, H2O2-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 H2O2-stimulated fibroblasts. From analysis of trypan blue exclusion, formazan formation, morphological changes, and intracellular MIF content by Western blotting, we found that MIF secretion by H2O2 seemed to be mainly due to cell death and subsequent leakage of intracellular MIF. Taken together, these results suggest that MIF secretion differs from IL-6 via LPS-mediated signaling pathways.