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,
* Department of Physiology and Biophysics,
Department of Pharmacology,
Department of Pathology, and
Department of Medicine, Case Western Reserve University School of Medicine, Cleveland OH 44120; and
¶ Department of Pathology, University of Michigan, Ann Arbor, MI 48109
We recently reported that P2X7 receptor (P2X7R)-induced activation of caspase-1 inflammasomes is accompanied by release of MHC class II (MHC-II) protein into extracellular compartments during brief stimulation of murine macrophages with ATP. Here we demonstrate that MHC-II containing membranes released from macrophages or dendritic cells (DCs) in response to P2X7R stimulation comprise two pools of vesicles with distinct biogenesis: one pool comprises 100- to 600-nm microvesicles derived from direct budding of the plasma membrane, while the second pool is composed of 50- to 80-nm exosomes released from multivesicular bodies. ATP-stimulated release of MHC-II in these membrane fractions is observed within 15 min and results in the export of 
15% of the total MHC-II pool within 90 min. ATP did not stimulate MHC-II release in macrophages from P2X7R knockout mice. The inflammasome regulatory proteins, ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) and NLRP3 (NLR family, pyrin domain containing 3), which are essential for caspase-1 activation, were also required for the P2X7R-regulated release of the exosome but not the microvesicle MHC-II pool. Treatment of bone marrow-derived macrophages with YVAD-cmk, a peptide inhibitor of caspase-1, also abrogated P2X7R-dependent MHC-II secretion. Surprisingly, however, MHC-II release in response to ATP was intact in caspase-1–/– macrophages. The inhibitory actions of YVAD-cmk were mimicked by the pan-caspase inhibitor zVAD-fmk and the serine protease inhibitor TPCK, but not the caspase-3 inhibitor DEVD-cho. These data suggest that the ASC/NLRP3 inflammasome complexes assembled in response to P2X7R activation involve protease effector(s) in addition to caspase-1, and that these proteases may play important roles in regulating the membrane trafficking pathways that control biogenesis and release of MHC-II-containing exosomes.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants GM36387 (to G.R.D.), AI035726/AI069085 (to C.V.H.), and AI063331/AI064748 (to G.N.).
2 Address correspondence and reprint requests to Dr. George R. Dubyak, Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106. E-mail address: george.dubyak{at}case.edu
3 Abbreviations used in this paper: P2X7R, P2X7 purinergic receptor; ASC, apoptosis-associated speck-like protein containing a caspase-recruitment domain; BMDC, bone marrow-derived dendritic cell; BMDM, bone marrow-derived macrophage; BSS, basal saline solution; DC, dendritic cell; ER, endoplasmic reticulum; ILV, intraluminal vesicle; MHC-II, MHC class II; MIIC, MHC-II-containing compartment; MVB, multivesicular body; NLRP3, NLR family, pyrin domain containing 3; pMHC-II, peptide-MHC class II; TPCK, Na-Tosyl-Phe-chloromethylketone; WT, wild type.
4 The online version of this article contains supplemental material.
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  S. F. Moore and A. B. MacKenzie NADPH Oxidase NOX2 Mediates Rapid Cellular Oxidation following ATP Stimulation of Endotoxin-Primed Macrophages J. Immunol., September 1, 2009; 183(5): 3302 - 3308. [Abstract] [Full Text] [PDF]
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