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...

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferatoractivated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasmaderived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

Rationale: Although lipids, apolipoproteins, and lipoprotein particles are important modulators of inflammation, varying relationships exist between these parameters and asthma.Objectives: To determine whether serum lipids and apolipoproteins correlate with the severity of airflow obstruction in subjects with atopy and asthma.Methods: Serum samples were obtained from 154 atopic and nonatopic subjects without asthma, and 159 subjects with atopy and asthma. Serum lipid and lipoprotein levels were quantified using standard diagnostic assays and nuclear magnetic resonance (NMR) spectroscopy. Airflow obstruction was assessed by FEV 1 % predicted.Measurements and Main Results: Serum lipid levels correlated with FEV 1 only in the subjects with atopy and asthma. Serum levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) were positively correlated with FEV 1 in subjects with atopy and asthma, whereas a negative correlation existed between FEV 1 and serum levels of triglycerides, low-density lipoprotein (LDL) cholesterol, apolipoprotein B (apoB), and the apoB/apoA-I ratio. NMR spectroscopy identified a positive correlation between FEV 1 and HDL NMR particle size, as well as the concentrations of large HDL NMR particles and total IDL NMR (intermediate-density lipoprotein) particles in subjects with atopy and asthma. In contrast, LDL NMR particle size and concentrations of LDL NMR and VLDL NMR (very-low-density lipoprotein) particles were negatively correlated with FEV 1 in subjects with atopy and asthma.Conclusions: In subjects with atopy and asthma, serum levels of apoA-I and large HDL NMR particles are positively correlated with FEV 1 , whereas serum triglycerides, LDL cholesterol, and apoB are associated with more severe airflow obstruction. These results may facilitate future studies to assess whether apoA-I and large HDL NMR particles can reduce airflow obstruction and disease severity in asthma.