Selective Release of Tumor Necrosis Factor Binding Protein II by Malignant Human Epidermal Cells Reveals Protection from Tumor Necrosis Factor α-mediated Cytotoxicity

Abstract

Most tumor cells produce both tumor necrosis factor (TNF) receptors, the M_r 55,000 TNFRI and the M_r 75,000 TNFRII, but they are mostly resistant to TNF-α-induced cytotoxicity. To gain further insight into the escape mechanisms of tumor cells from the harmful effect of TNF-α, we investigated the production of TNF-binding proteins (TNF-BPI, TNF-BPII, both M_r 30,000) by malignant and normal epidermal cells and studied their functional role in TNF-α-induced cytotoxicity. Malignant human keratinocytes (A431, KB, HaCaT) and malignant human melanoma cells (KRFM) produced significant levels of both TNF-BPI and TNF-BPII on stimulation with phorbol myristate acetate. In contrast, normal human keratinocytes (HNK) and normal human melanocytes (HNM) released TNF-BPI but not TNF-BPII. The specific production of TNF-BPII in concert with TNF-BPI by the malignant cell lines revealed an inhibitory effect of supernatants on recombinant human TNF-α-mediated cytotoxicity of the TNF-dependent murine cell line L929, while supernatants of normal epidermal cells had no effect. Preincubation of supernatants with anti-TNF-BPI monoclonal antibody htr-9 or anti-TNF-BPII monoclonal antibody utr-1 reversed this inhibitory effect additively, indicating that the production of both TNF-BPs is necessary to protect cells from TNF-α-mediated cytotoxicity. A TNF-α scavanging effect of TNF-BPs resulting in subsequent inhibition of TNF-α binding to L929 cells could be demonstrated by ligand blotting and fluorescence-activated cell sorting analysis. Thus the production of TNF-BPII by epidermal tumor cells in concert with TNF-BPI appears to demonstrate a specific mechanism by which malignant epithelial cells escape from TNF-α-mediated cytotoxicity.