P2X7 Receptor-Stimulated Secretion of MHC Class II-Containing Exosomes Requires the ASC/NLRP3 Inflammasome but Is Independent of Caspase-1

• Published in: The Journal of immunology (1950) Vol. 182; no. 8; pp. 5052 - 5062
• Main Authors: Qu, Yan, Ramachandra, Lakshmi, Mohr, Susanne, Franchi, Luigi, Harding, Clifford V, Nunez, Gabriel, Dubyak, George R
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15-04-2009
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Apoptosis Regulatory Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins - genetics; Carrier Proteins - metabolism; Caspase 1 - metabolism; Cell Differentiation - drug effects; Cell Differentiation - immunology; Cells, Cultured; Cytoskeletal Proteins - metabolism; Dendritic Cells - cytology; Dendritic Cells - drug effects; Dendritic Cells - immunology; Exosomes - immunology; Exosomes - metabolism; Exosomes - secretion; Histocompatibility Antigens Class II - immunology; Macrophages - immunology; Macrophages - metabolism; Mice; Mice, Inbred C57BL; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Receptors, Purinergic P2 - deficiency; Receptors, Purinergic P2 - genetics; Receptors, Purinergic P2 - metabolism; Receptors, Purinergic P2X7
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.0802968

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 approximately 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.