Mobilizable intracellular pool of p55 (type I) tumor necrosis factor receptors in human neutrophils

The distribution of type I (p55) and type II (p75) tumor necrosis factor receptors (TNF-Rs) in human polymorphonuclear neutrophils (PMNs) was analyzed by Western blotting of subcellular fractions obtained by centrifugation of PMN cavitates on Percoll density gradients. In resting PMNs, the p55 receptor was associated with both gamma and beta fractions, enriched in plasma membranes and specific granules, respectively, whereas the p75 TNF-R was located only in the gamma fraction. Intracellular p55 TNF-R bound 125I-labeled TNF in a ligand blot assay and migrated as a diffuse band of 46-60 kd, similar to the plasma membrane receptor. Activation of PMNs with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), in conditions that induced the selective release of the tertiary granule marker gelatinase, caused the shedding of most of membrane p55 TNF-R as a 28-kd soluble form but had little effect on the distribution of the receptors in beta fractions. By contrast, when specific granule secretion was induced in the presence of cytochalasin B, a marked decrease in reactivity of beta fractions with anti-p55 TNF-R antibody was observed. At the same time, the amount of soluble 28-kd fragment found in the supernatant was increased. Thus, the intracellular pool of p55 TNF-R is associated with functional secretory granules and is redistributed in response to PMN activation.