CELL DENSITY-DEPENDENT REGULATION OF CELL-SURFACE EXPRESSION OF 2 TYPES OF HUMAN TUMOR-NECROSIS-FACTOR RECEPTORS AND ITS EFFECT ON CELLULAR-RESPONSE

Tumor necrosis factor (TNF) is a multipotential cytokine known to regu late the growth of a wide variety of normal and tumor cells. It has be en shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on ep ithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with incr ease in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell type s at high density. The decrease in TNF receptor numbers at high cell d ensity was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF recep tors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutraliza tion of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equ ally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cyclohexim ide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overa ll decrease in protein synthesis. The density-dependent decrease in TN F receptors was accompanied by a decrease in intracellular reduced glu tathione levels. A reduction in the number of receptors on TNF sensiti ve tumor cells induced by cell-density correlated with increase in res istance to the cytokine. (C) 1994 Wiley-Liss, Inc.