Quantification and characterization of high‐affinity membrane receptors for tumor necrosis factor on human leukemic cell lines

Abstract

The expression of specific membrane receptors for TNF-alpha was determined on various human leukemic cell lines differing in their sensitivity to the growth-inhibitory activity of TNF-alpha. Binding studies with 125I-labelled TNF-alpha indicated specific binding in 8/10 cell lines with approximately 10-fold differences in the quantity of TNF-alpha bound by these distinct cell lines. Scatchard analyses of TNF-binding revealed the existence of high-affinity membrane receptors (Kd 1.5 X 10(-10) M) and approximately 3,000 binding sites/cell on both U937 and K562, representing 2 cell lines with high and low TNF sensitivity, respectively. Disuccinimidyl-suberate cross-linking of receptor-bound 125I-TNF-alpha and SDS-PAGE of membrane preparations of either U937 or K562 cells suggest a single receptor protein with an apparent molecular weight of 76 kDa. Comparison of the TNF-alpha binding capacity versus in vitro growth inhibition provides evidence that sensitivity to TNF-alpha is determined both at the level of receptor expression and at a post-receptor level. IFN-gamma strongly enhanced the TNF-alpha-mediated growth inhibition of 3 sensitive cell lines, but had no effect on 7 other leukemic cell lines with little or no TNF sensitivity. No correlation was found between this enhancement of TNF sensitivity and the IFN-gamma-mediated increase in TNF-cell membrane receptors, suggesting that IFN-gamma predominantly exerts its synergistic effect distal to TNF-binding.

Characterization of a tumor necrosis factor alpha (TNF-alpha) inhibitor: evidence of immunological cross-reactivity with the TNF receptor.

“…Cross-linking of 125I-lymphotoxin to its receptor and immunoprecipitation with the use of anti-TNF-a Inh would answer the question of whether the inhibitor also recognizes the 120-kDa structure. Alternatively, T cells represent a heterogeneous population, and it cannot be excluded that this represents TNF receptor expression of cellular subpopulations as its heterogeneity in regard to the molecular mass has been reported by several groups (31)(32)(33). This is less likely, however, because FITC-coupled TNF-a should bind its own receptor independently of the cellular subpopulation.…”

The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor

“…Both TNF-Rs have been objects of intense physiological and biochemical investigations. Likewise, the ligand binding properties of the TNF-Rs have been extensively studied (8)(9)(10)(11) although a detailed analysis became feasible only after the molecular cloning of the two individual receptor molecules. Equilibrium binding studies with 125 Ilabeled TNF ( 125 I-TNF) at 0°C defined high-affinity binding of TNF to both TNF-Rs with K d values of approximately 300-600 pM for TNF-R1 and 70-200 pM for TNF-R2 (12)(13)(14)(15)(16)(17)(18).…”

Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies.

“…Several investigators have identified TNF receptors at the surface of a variety of cell types. A single class of high-affinity sites with Kd in the range of 0.1-1.0 nM and a few hundred to a few thousand copies per cell has been reported (13)(14)(15). The approximate molecular size of the TNF-a-binding proteins, as determined in crosslinking studies, varies from '50 kDa to 140 kDa (15,16 …”

“…4). Raji cells have no detectable TNF-binding sites (15) and were used to assess the nonspecific binding of the reagents. With HEp-2 cells a positive immunofluorescence was found only with mAb htr-9 and was not found with (Fig.…”