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

Abstract

Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100-500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37 degrees C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37 degrees C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1; Kd = 1.9 x 10(-11) M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2; Kd = 4.2 x 10(-10) M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand-receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF-TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin-TNF-R1 complex at 37 degrees C.

Figure 1

Dissociation and association kinetics of ¹²⁵I-TNF binding at 37°C. (A) Approximately 4 × 10⁵ KYM-1 cells (□, approximately 30,000 TNF-R2 per cell) and HeLa cells (▪, approximately 3,000 TNF-R1 per cell) were preincubated with anti-TNF-R1 antibody H398 (KYM-1) or left untreated (HeLa) and then incubated with 300 pM of ¹²⁵I-TNF for 1 hr at 4°C. Subsequently, dissociation of ¹²⁵I-TNF from TNF-Rs at 37°C was followed by dilution of the cells in a 200-fold excess of unlabeled TNF and determination of cell-bound radioactivity by centrifugation of the cells through a layer of phthalate oil at the indicated time points. Shown is a typical experiment (KYM-1, n = 4; HeLa, n = 7) with 31,535 cpm and 2,017 cpm of specific binding at time 0 for KYM-1 and HeLa cells, respectively. Nonspecific binding was 3,482 cpm and 182 cpm, respectively, and has been subtracted. (B) For association kinetics 4.5 × 10⁵ HeLa cells (▪) and KYM-1 cells (□) were incubated at 37°C for different times with 300 pM ¹²⁵I-TNF. Unbound ligand was then removed by centrifugation of the cells through phthalate oil and cell bound radioactivity was measured. A typical experiment (KYM-1, n = 4; HeLa, n = 7) is shown with 41,640 cpm and 2,451 cpm of maximum specific binding for KYM-1 and HeLa cells, respectively. Nonspecific binding determined in the presence of 60 nM unlabeled TNF has been subtracted. The continuous curves passing through the data in A and B were calculated from the best-fit parameter values by using single-exponential time-dependency curves.

Figure 2

Alternative methods to determine association and dissociation rate constants. (A) Association experiments with HeLa cells were performed as in Fig. 1 by using various ¹²⁵I-TNF concentrations (100, 200, 400, 800, and 1,600 pM) (Inset). The obtained net association rate values were plotted against the ¹²⁵I-TNF concentration initially present in the assay to determine the dissociation rate constant (ordinate intercept) and the association rate constant (slope). Shown is a representative experiment (n = 3). (B) HeLa cells were incubated at 37°C with 250 pM ¹²⁵I-TNF to obtain the association kinetics shown (▪). After 2, 5, and 7 min of incubation (arrows), aliquots were supplemented with a 200-fold excess of unlabeled TNF in prewarmed binding buffer, and cell-bound radioactivity was followed for an additional 7 min (○). The dotted curves passing through the data represent the calculated dissociation kinetics based on the dissociation rate constants obtained from the experiments shown in Fig. 1.

Figure 3

TNF binding kinetics on U937 cells, coexpressing TNF-R1 and TNF-R2. U937 cells (1 × 10⁶ cells) were preincubated with receptor-specific antibodies for 1 hr on ice (A and C) or left untreated (B and D). ¹²⁵I-TNF dissociation and association kinetics of cells with TNF-R1 (▪), TNF-R2 (□), or both receptors (◊) present were performed at 37°C as described in Fig. 1. The continuous curves passing through the data were calculated from best-fit parameter values by using single-exponential time-dependency curves (A and C) or two-phase-exponential time-dependency curves (B) and the resulting rate constants are given in the graph. The dotted curve passing through the data in D represents the calculated two-phase-exponential association kinetics based on the individual net association rates of TNF-R1 and TNF-R2, respectively, determined in parallel (C). Shown is a typical experiment (n = 3).

Figure 4

Comparison of TNF and LTα bioactivities and dissociation rates on human and mouse cells. TNF (•) and LTα (○) were titrated on human HeLa cells (A) and mouse MethA cells (B) in a standard cytotoxicity assay. Note that the full cytotoxic effect of TNF cannot be reached with 2 nM LTα. The dissociation rates of ¹²⁵I-TNF (•) and ¹²⁵I-LT (○) from HeLa cells (C) and MethA cells (D) were determined as described in Fig. 1. Shown are typical experiments (HeLa, n = 6; MethA, n = 3).