TUMOR-NECROSIS-FACTOR - RECEPTOR-BINDING AND EXPRESSION OF RECEPTORS IN CULTURED MOUSE HEPATOCYTES

Recombinant murine tumor necrosis factor (TNF-alpha) was labeled with I-125 and used to determine the binding characteristics, internalizati on and intracellular degradation in cultured mouse hepatocytes. [I-125 ]TNF-alpha bound specifically to hepatocytes and Scatchard analysis of the data indicated binding to both a low-affinity (K-d = 20 nM) high capacity (51225 sites/cell) component and high-affinity component (K-d = 4 pM), with low capacity (290 sites/cell). The extent of TNF-alpha binding to hepatocytes correlated closely with its biological activity in hepatocytes, as indexed by depletion of intracellular ATP. At conc entrations lower than 0.06 nM there was minimal binding and no effect on cellular ATP, whereas maximal binding at concentrations greater tha n 45 nM caused 80% depletion (in comparison to controls) of hepatocyte ATP. Incubation at 37 degrees C resulted in rapid uptake, internaliza tion and degradation of [I-125]TNF-alpha. This was followed by release of degraded material from hepatocytes. Examination, by reverse transc riptase/polymerase chain reaction technology, of hepatocyte RNA extrac ted after the 4-hr adherence period revealed that mouse hepatocytes ex pressed mRNA for both TNF-alpha receptor 1 and TNF-alpha receptor 2, a nd that the relative abundance of TNF-alpha receptor 1 was approximate ly 7-fold greater than that for TNF-alpha receptor 2. Because it has b een shown that these receptors have different affinities for TNF-alpha , this may explain the high- and low-affinity binding sites present on cultured mouse hepatocytes.