DESFERRIOXAMINE REGULATES TUMOR-NECROSIS-FACTOR RELEASE IN MESANGIAL CELLS

Cultured rat mesangial cells have been demonstrated to express tumor necrosis factor alpha (TNF-alpha) mRNA and to release TNF activity into the medium upon stimulation by bacterial lipopolysaccharide (LPS). The present study was undertaken to determine whether TNF was only secreted by mesangial cells or was also present as a cell-associated molecule. LPS-activated mesangial cells which had been fixed in paraformaldehyde lysed the TNF-sensitive L-929 fibroblasts, as assessed by Cr-51 release. This cytotoxic activity was inhibited by anti-TNF-alpha antiserum. Cell-associated TNF expression was demonstrable after less than one hour of exposure to LPS, peaked at two hours and decreased progressively thereafter, while TNF activity increased in the medium. Mesangial cell-associated TNF was localized at the cell surface, as shown by immunohistochemical demonstration and by the ability of plasma membranes purified from LPS-activated mesangial cells to lyse L-929 fibroblasts. Flow cytometry experiments revealed that two-thirds of LPS-activated mesangial cells were stained by anti-TNF-alpha antiserum. The major part of these cell-associated TNF molecules persisted after low pH treatment, indicating that they were integral membrane proteins. As assessed by immunoprecipitation analysis, these proteins were 26 kDa molecules, whereas the released forms of TNF were 17 kDa molecules. Pretreatment of mesangial cells with desferrioxamine (DFX), an iron chelator preventing the synthesis of hydroxyl radicals (OH bullet), delayed the release of TNF from the membranes into the medium, and enhanced its cell surface expression. It also subsequently accelerated its decay in the medium. This indicates that OH bullet could be involved in the mechanism of TNF detaching from the membranes and in that of TNF preservation. Taken together, these data suggest that during endotoxic shock DFX could represent a new way to prevent TNF release from mesangial cells into the blood stream while maintaining the local response to TNF.