Downregulation of tumor necrosis factor receptors on macrophages and endothelial cells by microtubule depolymerizing agents.

Abstract

Exposure of murine and human macrophages and human umbilical vein endothelial cells to micromolar concentrations of five microtubule (MT)-depolymerizing agents (colchicine, nocodazole, podophyllotoxin, vincristine, and vinblastine) resulted in a loss of binding sites for iodinated TNF-alpha. The reduction amounted to 40-60% by 1 h and approximately 75% by 2-4 h. In 1 h, specific binding was reduced 50% by 0.1-5 microM of these drugs at 37 degrees C, but not at 4 degrees C. Inactive isomers of colchicine were ineffective, as were microfilament-destabilizing cytochalasins. The active agents did not compete with TNF-alpha R for binding. Antiserum against TNF-alpha did not neutralize the effect of colchicine and nocodazole. PGE1 and dibutyryl-cAMP could not mimic, and cyclooxygenase inhibitors could not prevent the drug effects. All the binding sites were regenerated within 3 h after removal of nocodazole, which binds tubulin reversibly, whereas little recovery was found even 18 h after the removal of colchicine, which binds tubulin irreversibly. These findings suggested that MT disassembly was responsible for the observed downregulation of TNF-alpha R. The protein synthesis inhibitor cycloheximide inhibited binding of TNF-alpha to a similar extent and with a similar time course as colchicine in the absence of added ligand. Neither drug affected binding of IFN-gamma to macrophages, nor binding of TNF-alpha to human polymorphonuclear leukocytes. Thus, an intact MT network appears to be important in maintenance of the steady state of TNF-alpha R on those cells in which TNF-alpha R turns over rapidly in the absence of ligand. The antiinflammatory actions of MT-depolymerizing agents may result in part from their interference with the ability of such cells to respond to TNF-alpha.