Studies on the fate of receptor-bound 125I-interleukin 1 beta in porcine synovial fibroblasts.

Binding of porcine interleukin 1, radiolabeled with Bolton-Hunter reagent (125I IL 1), to monolayers of porcine synovial fibroblasts (PSF) was found to be a temperature-dependent process. The rate of uptake and the amount of cell-associated ligand was higher at 37 degrees C than at 4 degrees C or 19 degrees C, and exceeded the apparent equilibrium binding capacity. The amount of bound 125I IL 1 that was removed by brief treatment with acidic buffers decreased from 80% at 4 degrees C to 35% for PSF incubated at 37 degrees C; this procedure was used to distinguish surface-bound from internalized ligand. In untreated PSF, surface binding was maximal at 1 hr and was maintained for at least 5 hr during which time the internal pool continued to increase. The lysosomotropic agent methylamine (20 mM) decreased surface binding by 50%; monensin (20 microM) decreased the rate and extent of internalization. Cycloheximide (10 micrograms/ml) did not affect ligand uptake, hence, continual expression of surface receptors could not be ascribed to their de novo synthesis. 40% of the radioactivity taken up by PSF during incubation at 37 degrees C subsequently appeared in the culture medium upon prolonged postincubation (5 hr) in the absence of added 125I IL 1: 60% of this fraction was trichloroacetic acid-soluble in untreated cultures, but the extent of degradation was halved by treatment with methylamine or monensin. Direct measurement of the rate of internalization of prebound 125I IL 1 was obtained by monitoring the formation of covalently cross-linked ligand-receptor complexes after warming PSF monolayers to 37 degrees C. By using gel electrophoresis we observed a decrease (t1/2 = 9 to 11 min) in labeling of the major cross-linkable species.