Identification and characterization of TNFR1 exosome‐like vesicles in human plasma

Abstract

Extracellular TNFR1 (TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and release of full-length TNFR1 exosome-like vesicles. The goal of this study was to identify and characterize TNFR1 exosome-like vesicles in human plasma. Western blots demonstrated a 48-kDa TNFR1 in plasma from healthy volunteers. Fractionation by gel exclusion chromatography revealed that the 48-kDa TNFR1 co-segregated with LDL particles. TNFR1 exosome-like vesicles in human serum were confirmed by immunoelectron microscopy. The 48-kDa TNFR1 segregated independently from LDL particles by peak density (1.1 g/ml vs. 1.03 g/ml), which demonstrates that TNFR1 exosome-like vesicles are distinct from LDL particles. ICAM-1, LAMP-1, and LAMP-2, known exosome-associated proteins, co-segregated with the HDL fraction, which suggests that TNFR1 exosome-like vesicles are distinct from typical exosomes. The reduced size of the 48-kDa exosome-associated TNFR1 reflected reduced N-linked carbohydrate content. Thus, we propose human plasma contains TNFR1 exosome-like vesicles that may regulate TNF-mediated inflammatory events. This research is supported by DIR, NHLBI, NIH.