Binding and Crosslinking of 125I-Labeled Recombinant Human Tumor Necrosis Factor to Cell Surface Receptors1

Abstract

Highly purified recombinant human tumor necrosis factor (TNF) (molecular mass determined as 17 kilodaltons (kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as 36 kDa by Sephadex G-100 gel chromatography) was labeled with ¹²⁵ I to a specific activity of 5 μCi/μg without appreciable loss of activity. The binding of ¹²⁵ I-TNF to eighteen human and twelve animal cell lines was examined. The binding varied considerably among different cell lines. In most cell lines, the binding was inhibited up to >90% by the addition of a 100-fold excess of unlabeled TNF. Some human and mouse cell lines showed no significant binding above background levels, suggesting that these cell lines had no receptors for TNF. Among the TNF receptor-positive cell lines, there was no direct correlation between the level of specific TNF binding and the level of sensitivity to the cytotoxic or cytostatic effect of TNF. Some cell lines were sensitive to TNF, whereas others were not affected at all by TNF. The TNF receptor-negative cell lines were also resistant to TNF. Therefore, although the existence of TNF receptor seems to be necessary, it does not alone determine cellular sensitivity to TNF. Scatchard analysis of the binding data revealed that human HeLa S ₃ and THP-1 had about 50,000 and 10,000 receptors/cell with a dissociation constant (K _D ) of 0.3–0.5 nM, respectively. Similarly, mouse L-929 and L-M cells had about 5,000 receptors/cell with K _D of 3–5 nM. ¹²⁵ I-TNF bound to HeLa S ₃ cells was rapidly internalized at 37°C, presumably by receptor-mediated endocytosis, and degraded to acid-soluble products. The turnover of TNF receptors on HeLa S ₃ cells seemed to be rapid, since the level of specific binding quickly decreased after treatment with 100μg/ml of cycloheximide at 37°C with a half-life of about 1.5 h. The crosslinking of the cell-bound ¹²⁵ I-TNF with the use of disuccinimidyl suberate yielded a complex of 105 kDa for HeLa S ₃ and THP-1 cells, and a complex of 100 kDa for U937 cells. The crosslinking was completely inhibited by the addition of a 100-fold excess of unlabeled TNF. Assuming that the complex was due to a one-to-one association of the dimeric form of TNF (34 kDa) with the receptor, we estimated the molecular size of the human TNF receptor to be 71 kDa for HeLa S ₃ and THP-1, and 66 kDa for U937.