Activation of P2X7R in macrophages triggers the caspase-1 mediated proteolytic maturation and release of the proinflammatory cytokines IL-1β and IL-18. Aberrant activation and secretion of these key cytokines has been associated with a variety of inflammatory or auotimmune diseases in humans such as arthritis, pulmonary fibrosis, Alzheimer's disease, diabetes, and atherosclerosis.
Several mechanistically distinct models have been proposed to explain the rapid export of caspase-1 processed IL-1β from monocyte/macrophages in response to activation of P2X7R by extracellular ATP. Our experiments revealed: 1) a novel correlation between IL-1β secretion and the release of the MHC-II membrane protein which is a marker of plasma membranes, recycling endosomes, multivesicular bodies (MVB), and released exosomes; 2) mechanistic dissociation of IL-1β export from either secretory lysosome exocytosis or plasma membrane microvesicle shedding on the basis of different requirements for extracellular Ca2+ and differential sensitivity to pharmacological agents that block activation of caspase-1 inflammasomes.
Regardless of its mechanistic relationship to IL-1β export, the P2X7R-induced release of exosomes represents a further addition to the remarkably diverse array of membrane trafficking responses that have already been associated with P2X7R activation in macrophages. Our data indicate that the released MHC-II containing membranes comprise two pools of vesicles with distinct biogenesis: one comprises the plasma membrane derived microvesicles while the second pool is composed of the MVB-derived exosomes. The two isolated membrane compartments have distinct morphology, dissimilar intraluminal contents, and different masses, but exhibit similar density distributions when analyzed by equilibrium sucrose gradient centrifugation. Inflammasome component proteins, ASC and NLRP3, which are essential for the caspase-1 activation, were also required for the P2X7R-regulated release of the exosomes but not the microvesicles. However, the P2X7R-stimulated MHC-II release was completely intact in caspase-1-/- macrophages. These data suggest that the ASC/NLRP3 inflammasome complexes assembled in response to P2X7R activation may recruit additional YVAD-sensitive effector proteins other than caspase-1 and that these effectors play unique roles in regulating the biogenesis and release of MHCII-containing exosomes. Data obtained from in vitro antigen presentation assays indicated that both the microvesicles and exosomes released during P2X7R activation have the capacity to present antigens to T cell hybridomas.