Modulation of two forms of tumor necrosis factor receptors and their cellular response by soluble receptors and their monoclonal antibodies.
Recently, two different receptors for human tumor necrosis factor (TNF) with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. In this report, we investigated the effect of the soluble forms of these receptors and monoclonal antibodies against them on ligand interaction, receptor down-regulation, and mediation of cellular response in U-937 cells. Our results indicate that p60 and p80 constitute 20-30 and 60-80% of the total TNF-binding sites on U-937 cells, respectively. However, by cross-linking, only the p80 form of the receptor could be detected. In contrast to unlabeled TNF, the anti-p60 and anti-p80 antibodies together only partially inhibited ligand binding, and this inhibition was not additive. Lack of additive inhibition of binding was found to be not due to stereo-chemical hindrance. TNF binding to cells can be completely displaced by soluble forms of either the p60 or p80 receptor. However, 100-fold more of the p80 than the p60 form of the soluble receptor is needed for equivalent displacement. Under optimum conditions, TNF and the anti-p80 and anti-p60 antibodies down-regulated 30, 80, and 20% of the TNF receptors, respectively. The anti-p60 and anti-p80 antibodies down-regulated not only their own receptors, but also reciprocal receptors, suggesting a cross-communication between the p60 and p80 forms of the TNF receptor. In spite of inhibiting as much as 80% of TNF binding, none of the receptor antibodies significantly inhibited the cytotoxic response to TNF in U-937 cells. Soluble forms of both receptors, however, completely abrogated the cellular response to TNF. Thus, overall, our results indicate that the antibodies against both receptors together inhibit the majority of the receptor-ligand interaction without any significant effect on the biological response to TNF.
Higuchi
M
M
Department of Clinical Immunology and Biological Therapy, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Aggarwal
B B
BB
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology; D015536:Down-Regulation; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008970:Molecular Weight; D011869:Radioligand Assay; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D013388:Succinimides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D013388:Succinimides; D014409:Tumor Necrosis Factor-alpha; C028722:ethylene glycolylbis(succinimidyl succinate)