Activation of Macrophages by P2X7-Induced Microvesicles from Myeloid Cells Is Mediated by Phospholipids and Is Partially Dependent on TLR4

Abstract

ATP-mediated activation of the purinergic receptor P2X(7) elicits morphological changes and proinflammatory responses in macrophages. These changes include rapid shedding of microvesicles (MV) and the nonconventional secretion of cytokines, such as IL-1beta and IL-18 following priming. In this study, we demonstrate the activation potential of P2X(7)-induced MV isolated from nonprimed murine macrophages. Cotreatment of nonprimed macrophages with ATP and calcium ionophore induced a rapid release of MV that were predominantly 0.5-1 microm in size. Exposure of primary murine bone marrow-derived macrophages to these MV resulted in costimulatory receptor upregulation and TNF-alpha secretion. Cell homogenates or supernatants cleared of MV did not activate macrophages. MV-mediated activation was p38 MAPK and NF-kappaB dependent, and partially dependent on TLR4 activity, but was high-mobility group box 1 independent. Biochemical fractionation of the MV demonstrated that the phospholipid fraction, not the protein fraction, mediated macrophage activation through a TLR4-dependent process. P2X(7) activation is known to induce calcium-independent phospholipase A(2), calcium-dependent phospholipase A(2), and phospholipase D activities, but inhibition of these enzymes did not inhibit MV generation or shedding. However, blocking phospholipase D activity resulted in release of MV incapable of activating recipient macrophages. These data demonstrate a novel mechanism of macrophage activation resulting from exposure to MV from nonprimed macrophages, and identifies phospholipids in these MV as the biologically active component. We suggest that phospholipids delivered by MV may be mediators of sterile inflammation in a number of diseases.