Soluble tumor necrosis factor receptors (TNFRs) are important modulators of TNF bioactivity. Proteolytic cleavage of the 28-kDa ectodomain of TNFR1 has been recognized as the mechanism by which soluble TNFR is shed. We now describe the release of exosome-like vesicles as a mechanism for the generation of soluble, full-length 55-kDa TNFR1. We found unexpectedly that the predominant form of soluble TNFR1 in human serum and lung epithelial lining fluid is a full-length 55-kDa protein. Furthermore, supernatants from human vascular endothelial cells contain only full-length 55-kDa TNFR1 that can be sedimented by high-speed centrifugation, floated on sucrose gradients at a density of 1.1 g͞ml, and associated with vesicles that range in diameter from 20 nm to 50 nm. We conclude that the release of TNFR1 exosome-like vesicles represents a previously unrecognized mechanism by which constitutive production of soluble cytokine receptors may be regulated, independent of ectodomain cleavage by receptor sheddases. Binding of tumor necrosis factor (TNF) to the 55-kDa, type I TNF receptor (TNFR1, TNFRSF1A, CD120a, p55) activates signaling pathways that regulate inflammatory, immune, and stress responses, as well as host defense and apoptosis (1). TNF signaling is negatively regulated at two levels to prevent excessive or inappropriate immune or inflammatory responses. First, constitutive TNFR1 signaling is prevented by the binding of silencer of death domains (SODD) to the TNFR1 intracytoplasmic domain (2). TNF binding to TNFR1 releases SODD, thereby allowing the formation of an active TNFR1 signaling complex. The second regulatory mechanism is shedding of cell surface TNF receptors to function as soluble TNF binding proteins that inhibit TNF bioactivity by competing with cell surface TNF receptors for free ligand. Soluble TNF receptors may also reversibly bind to and stabilize trimeric TNF, thereby prolonging its half-life and serving as a slow release reservoir for TNF when levels are low (3).Soluble 27-to 30-kDa TNF-binding proteins, corresponding to the TNFR1 extracellular domain, were originally purified and isolated from human urine and serum (4-8). The demonstration by ELISA of TNFR1 molecules in culture supernatants from Chinese hamster ovary cells transfected with TNFR1 cDNA suggested that the soluble form is generated by proteolytic cleavage of the extracellular domain of cell surface receptors, rather than by alternative splicing (7). Sequence analysis identified the major TNFR1 cleavage site to be in the spacer region adjacent to the transmembrane domain between Asn-172 and Val-173, with a minor site between . Further, the ability of hydroxamic-acid based metalloprotease inhibitors to block TNFR1 shedding suggested that proteolytic cleavage of cell surface TNFR1 receptors is mediated by a zinc metalloprotease (11). Consistent with this, TNF-␣ converting enzyme (TACE, ADAM 17), a member of the metalloproteasedisintegrin (ADAM) family of zinc metalloproteases, was identified as mediating TNFR1 shedding. This conclusio...

Conflict of interest: No conflict of interest has been declared. Nonstandard abbreviations used: TNF receptor (TNFR); TNFRassociated death domain (TRADD); aminopeptidase regulator of TNFR1 shedding (ARTS-1); human umbilical vein endothelial cell (HUVEC); soluble TNFR1 (sTNFR1); TNF-α converting enzyme (TACE); glutathione s-transferase (GST); fastperformance liquid chromatography (FPLC); TNF-α protease inhibitor (TAPI).

Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6R␣), we hypothesized that ARTS-1 might also regulate shedding of IL-6R␣, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6R␣, a size consistent with soluble IL-6R␣ generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6R␣ shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6R␣ shedding, and decreased membrane-associated IL-6R␣ in cell lines overexpressing ARTS-1. The absence of basal IL-6R␣ shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6R␣ shedding. Furthermore, the mechanism of constitutive IL-6R␣ shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6R␣) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6R␣ and TNFR1 shedding.