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

P2X7 Receptor-Stimulated Secretion of MHC ClassII-Containing Exosomes Requires the ASC/NLRP3Inflammasome but Is Independent of Caspase-11

Yan Qu,† Lakshmi Ramachandra,‡ Susanne Mohr,*§ Luigi Franchi,¶ Clifford V. Harding,‡

Gabriel Nunez,¶ and George R. Dubyak2*†‡

We recently reported that P2X7 receptor (P2X7R)-induced activation of caspase-1 inflammasomes is accompanied by release of MHCclass II (MHC-II) protein into extracellular compartments during brief stimulation of murine macrophages with ATP. Here we dem-onstrate that MHC-II containing membranes released from macrophages or dendritic cells (DCs) in response to P2X7R stimulationcomprise two pools of vesicles with distinct biogenesis: one pool comprises 100- to 600-nm microvesicles derived from direct buddingof 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 totalMHC-II pool within 90 min. ATP did not stimulate MHC-II release in macrophages from P2X7R knockout mice. The inflammasomeregulatory 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 theexosome but not the microvesicle MHC-II pool. Treatment of bone marrow-derived macrophages with YVAD-cmk, a peptide inhibitorof caspase-1, also abrogated P2X7R-dependent MHC-II secretion. Surprisingly, however, MHC-II release in response to ATP was intactin caspase-1�/� macrophages. The inhibitory actions of YVAD-cmk were mimicked by the pan-caspase inhibitor zVAD-fmk and theserine protease inhibitor TPCK, but not the caspase-3 inhibitor DEVD-cho. These data suggest that the ASC/NLRP3 inflammasomecomplexes assembled in response to P2X7R activation involve protease effector(s) in addition to caspase-1, and that these proteases mayplay important roles in regulating the membrane trafficking pathways that control biogenesis and release of MHC-II-containingexosomes. The Journal of Immunology, 2009, 182: 5052–5062.

S timulation of P2X7 purinergic receptors (P2X7R)3 triggersthe rapid assembly of inflammasome signaling complexes(1), which include ASC (apoptosis-associated speck-like

protein containing a caspase-recruitment domain) (2) and NLRP3(NLR family, pyrin domain containing 3; formerly Nalp3 or cy-ropyrin) (3); these complexes act as signaling platforms to drivethe proteolytic activation of caspase-1 and maturation of IL-1�. Ina previous study of the mechanisms underlying nonclassical IL-�secretion stimulated by activation of P2X7R in murine macro-phages, we observed that ATP-induced caspase-1 activation and

IL-1� release were temporally correlated with a rapid and robustsecretion of MHC class II (MHC-II) protein (4). That the ATP-triggered MHC-II export was greatly increased by LPS primingand was attenuated by the YVAD caspase-1 inhibitor suggestedthe intriguing possibility that caspase-1 inflammasomes play ad-ditional roles in the biogenesis and trafficking of MHC-II-enrichedmembrane compartments. Although rapid perturbation of ion ho-meostasis is the best characterized cellular response to P2X7R ac-tivation, this ATP-gated nonselective cation channel has also beenassociated with a diverse array of membrane trafficking responsesin immune and inflammatory effector cells (5–8). MHC-II pro-teins, which are selectively expressed in professional APCs,including macrophages, dendritic cells (DCs), and B cells, playcritical roles in mediating the presentation of extracellular patho-gen-derived and other exogenous proteins to CD4� Th cells. No-tably, a previous study by Di Virgilio and colleagues reported thatactivation of P2X7R potentiated Ag presentation by murine DCs(9). However, the mechanisms by which P2X7R signaling regulateMHC-II trafficking and Ag presentation remain unclear.

Altered expression of MHC-II-peptide complexes has been as-sociated with various inflammatory and autoimmune diseases (10,11). Unlike the class I MHC molecules, which bind antigenic pep-tides transported into the endoplasmic reticulum (ER), the forma-tion of foreign Ag-class II complexes occurs in the MHC-II-con-taining compartments (MIICs). These MIICs are specialized lateendosomes/lysosomes that are enriched in MHC-II protein. Othermolecules that are contained within the MIICs include HLA-DM,HLA-DO, IFN-�-inducible lysosomal thiol reductase, and cathe-psins, which are important for exogenous peptide processing, load-ing, and editing (12, 13). The biogenesis and the plasma membrane

*Department of Physiology and Biophysics, †Department of Pharmacology, ‡Depart-ment of Pathology, and §Department of Medicine, Case Western Reserve UniversitySchool of Medicine, Cleveland OH 44120; and ¶Department of Pathology, Universityof Michigan, Ann Arbor, MI 48109

Received for publication September 8, 2008. Accepted for publication January 31,2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health Grants GM36387 (toG.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 ofPhysiology and Biophysics, Case Western Reserve University School of Medicine, 10900Euclid Avenue, Cleveland, OH 44106. E-mail address: [email protected] Abbreviations used in this paper: P2X7R, P2X7 purinergic receptor; ASC, apopto-sis-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, in-traluminal 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.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802968

expression of peptide-MHC-II (pMHC-II) complexes involveseveral membrane trafficking pathways, including: 1) endocytosisof extracellular foreign protein via the recycling endosomes; 2)formation of lysosome-like MIICs through the fusion of late en-dosomes that contain exogenous Ags with the de novo synthesizedMHC-II molecules derived from the Golgi apparatus; and 3) for-mation of multivesicular bodies (MVBs) generated by inward bud-ding from the limiting membrane of the MIIC organelles and ac-cumulation of these inward buds as intraluminal vesicles (ILVs).The pMHC-II complexes are formed via Ag loading onto theMHC-II pools of both ILVs and the limit membrane of these spe-cialized MVBs. This facilitates delivery of pMHC-II complexes tothe cell surface upon exocytotic fusion of the MVBs with theplasma membrane (13–15). Additionally, this exocytosis can resultin the direct secretion of the MHC-II-containing ILVs into theextracellular compartment as so-called exosomes.

Most APCs directly present their Ags to T cells via the cell surfacepool of MHC-II. However, increasing evidence indicates that MVB-derived exosomes containing pMHC-II complexes are also competentto promote T cell activation/proliferation and thereby initiate Ag-spe-cific immune responses in vivo (16, 17). Various studies have indi-cated that released exosomes can activate remote T lymphocytes ei-ther by direct Ag presentation from the pMHC-II displayed on theexosomal surface or by a predominant indirect pathway wherein theseexosomes are taken up by remote naive DCs for DC-mediated Agpresentation (17–19). Recent studies on exosome biology have ex-panded their physiological functions to even broader types of inter-cellular communication, including the exchange of genetic materialsuch as intact mRNAs and microRNAs, transfer of oncogenic growthfactor receptors from tumor cells to remote nontransformed cells,trans-infection of T cells by HIV-1 virions packaged within DC-de-rived exosomes, and transfer of pathogen-associated molecular pat-terns (PAMPs) between macrophages (20–23).

Intracellular signaling cascades that may regulate the varioussteps of exosome biogenesis are poorly defined. The formation andsecretion of MHC-II-containing exosomes in APCs has beenlargely viewed as a constitutive pathway. However, recent studiesperformed in DCs and B cells suggest that both cell surfaceMHC-II levels and the Ag presentation capacities of releasedMHC-II-containing exosomes are dynamically regulated by vari-ous signaling pathways active during particular phases of APCmaturation or stimulation (24–26). In contrast to the slow consti-tutive secretion of exosomes that typically occurs over �24–48 hof media conditioning by cultured APCs, recent studies have dem-onstrated that exosome release can be accelerated by conditions orpharmacological agents that target various signaling pathways(20, 27, 28).

In this study we extend the analysis of P2X7R-induced releaseof MHC-II-containing membranes in murine primary macrophagesand DCs by defining: 1) the basic biophysical and biochemicalproperties of the released MHC-II vesicles; and 2) the role ofparticular inflammasome-associated proteins, including ASC,NLRP3, and caspase-1 in the biogenesis/export of these vesicles.In contrast to the slow constitutive MHC-II secretion previouslydescribed in DCs and B cells, the ATP-triggered MHC-II releaseoccurred in a very rapid and efficient manner (within 15 min afterATP stimulation) and included both larger (100- to 600-nm)plasma membrane-derived microvesicles and smaller (50- to 80-nm) exosome-like vesicles. Moreover, our data indicate that thesignaling cascades required for the secretion of MHC-II mem-branes overlap with, but are distinct from, those required forcaspase-1 activation and IL-1� export. Although maximal P2X7R-dependent MHC-II release requires 1) LPS priming, 2) the inflam-masome adapter proteins ASC and NLRP3, and 3) effector pro-

teins sensitive to the widely used YVAD-cmk inhibitor ofcaspase-1, it is not affected by the absence of caspase-1 expressionper se. The discrepant effects on ATP-triggered MHC-II releaseobserved with ASC�/� or NLRP3�/� bone marrow-derived mac-rophages (BMDMs) vs caspase-1�/� BMDMs reveal an additionalrole for P2X7R-regulated inflammasome adapter proteins otherthan acting as the molecular platform for caspase-1 maturation.

Materials and MethodsReagents

Key reagents and their sources were: Escherichia coli LPS serotypeO1101:B4 (List Biological Laboratories; ATP (Sigma-Aldrich), YVAD-cmk (Bachem), DEVD-cho (BIOMOL), ZVAD-fmk (BIOMOL), and Na-Tosyl-Phe-chloromethylketone (TPCK) (Sigma-Aldrich). The cytotoxicitydetection kit was from Roche. Recombinant murine IFN-� was fromBoehringer Mannheim Biochemica. Anti-caspase-1 p10 rabbit polyclonal,anti-cathepsin B goat polyclonal, anti-actin goat polyclonal, anti-LAMP-1rat monoclonal, and all HRP-conjugated secondary Abs were from SantaCruz Biotechnology. The monoclonal 3ZD anti-IL-1� Ab, which recog-nizes both 33-kDa pro-IL-1� and 17-kDa mature IL-1� in Western blotanalysis, was provided by the Biological Resources Branch, National Can-cer Institute, Frederick Cancer Research and Development Center (Fred-erick, MD). The mouse mAb KL295 against MHC-II �-chain (I-Ab andI-Ad haplotypes) and the rat anti-mouse ASC mAb have been previouslydescribed (29).

Mouse models and methods for isolation and cell culture ofBMDMs and DCs

C57BL/6 mice were purchased from Taconic; P2X7R�/� mice were orig-inally provided by Pfizer Global Research and Development, and thenbackcrossed into a pure C57BL/6 background for �12 generations from aP2X7R�/� mouse strain described previously (30). ASC�/� andNLRP3�/� mice (C57BL/6 background) were generated as described pre-viously in the Nunez Laboratory at the University of Michigan. Caspase-1�/� mice (C57BL/6 background) that were backcrossed for five genera-tions with the C57BL/6J strain were obtained from Richard Flavell (YaleUniversity). All experiments and procedures involving mice were approvedby the Institutional Animal Use and Care Committees of either Case West-ern Reserve University or the University of Michigan.

BMDMs were isolated by previously described protocols (31). Micewere euthanized by CO2 inhalation. Femurs and tibia were removed,briefly sterilized in 70% ethanol, and PBS was used to wash out the marrowcavity plugs. The bone marrow cells were resuspended in DMEM (Sigma-Aldrich) supplemented with 25% L cell-conditioned medium, 15% calfserum (HyClone Laboratories), 100 U/ml penicillin, and 100 �g/ml strep-tomycin (Invitrogen), plated onto 150-mm dishes, and cultured in the pres-ence of 10% CO2. After 5–9 days, the resulting BMDMs were detachedwith PBS containing 5 mM EDTA and 4 mg/ml lidocaine (31), replatedinto 6-well or 12-well plates, and used within 10 days. A similar methodwas used to generate DCs: the bone marrow cells were cultured in DMEMsupplemented with 3–5% J558L cell-conditioned medium, which containsGM-CSF, 15% calf serum, 100 U/ml penicillin, and 100 �g/ml strepto-mycin. Culture medium was changed after 3 days postisolation, and thecells were used after 7 days postisolation.

Western blot analyses for caspase-1 activation and release,IL-1� processing and secretion, exocytosis of secretorylysosomes, and plasma membrane microvesicle shedding

BMDMs were routinely seeded in 6-well plates to a cell density of �2 �106/well and stimulated with 2 ng/ml IFN-� for 16–18 h before LPS prim-ing and ATP stimulation. IFN-� treatment is required to up-regulate ex-pression of MHC-II in BMDMs. After IFN-� priming, the culture mediumwas replaced with fresh medium supplemented with or without 1 �g/mlLPS. In some experiments, cells were pretreated with various pharmaco-logical inhibitors before, and during, LPS priming. Cells were primed withLPS for 4 h at 37°C, followed by washing once with PBS and transfer to1 ml of basal saline solution (BSS) assay medium containing 130 mMsodium gluconate, 5 mM KCl, 20 mM NaHEPES, 1.5 mM CaCl2, and 1.0mM MgCl2 (pH 7.5) supplemented with 5 mM glucose, 5 mM glycine, and0.01% BSA. Experiments testing effects of extracellular Ca2� on MHC-IIrelease utilized BSS with no added CaCl2. Cells were then equilibrated foran additional 5 min at 37°C before stimulation with ATP. To terminate therelease reaction, the entire 1 ml of extracellular medium was transferred toa tube on ice, while the cell monolayer was rapidly washed once with 1 ml

5053The Journal of Immunology

of ice-cold PBS and then lysed by addition of 150 �l of radioimmunopre-cipitation assay (RIPA) extraction buffer (1% Nonidet P-40, 0.5% Na-deoxycholate, 0.1% SDS (pH 7.4) in PBS) containing PMSF, leupeptin,and aprotinin. For processing of extracellular media, the collected 1-mlsamples were centrifuged at 10,000 � g for 10 s to remove any detachedcells, followed by transfer of the supernatant to a fresh tube. The super-natant was concentrated by TCA precipitation using 72 �l of 100% TCAand 15 �l of 10% cholic acid per 1 ml of extracellular media. The precip-itated pellets were washed three times with 1 ml of acetone, dissolved in 10�l of 0.2 M NaOH, diluted with 35 �l of H2O, supplemented with 15 �lof 4� SDS-PAGE sample buffer, and boiled for 5 min. For processing ofcell lysates, the 150-�l RIPA extracts were combined with any detachedcells from the extracellular media, recentrifuged, and supernatants weretransferred to fresh tubes. Cell lysate (45 �l) was supplemented with 15 �lof 4� SDS-PAGE sample buffer and boiled for 5 min. Extracellular mediaand cell lysates were processed by SDS-PAGE, electrophoretic transfer topolyvinylidene difluoride membranes, and standard Western blot protocolsas described previously (32). Primary Abs were used at the following con-centrations: 5 �g/ml for IL-1�, 1 �g/ml for caspase-1, 1 �g/ml for actin,0.04 �g/ml for LAMP-1, 1 �g/ml for cathepsin B, and 0.8 �g/ml forMHC-II. HRP-conjugated secondary Abs were used at a final concentrationof 0.13 �g/ml.

Purification of microvesicles and exosomes

All experiments that characterized the MHC-II-containing membrane ves-icles by centrifugation analysis utilized BMDMs or bone marrow-derivedDCs (BMDCs) grown in 150-mm dishes to a cell density of �10 � 107.The BMDMs were stimulated with 2 ng/ml IFN-� for 16–18 h before LPSpriming and ATP stimulation. Culture medium was replaced with freshmedium supplemented with or without 1 �g/ml LPS. The cells wereprimed with LPS for 4 h at 37°C, followed by washing once with PBS andtransfer to 10 ml of BSS assay medium. Cells were then equilibrated for anadditional 5 min at 37°C before stimulation with 5 mM ATP for indicatedtimes. The collected medium was immediately transferred into a tube con-taining PMSF, leupeptin, and aprotinin on ice, and then followed by se-quential centrifugation at 4°C for 10 min at 300 � g, 20 min at 1,200 � g,30 min at 10,000 � g (Sorvall SS-34 rotor), and 1 h at 100,000 � g(Beckman Coulter Ti 90 rotor). The pellets from 10,000 � g and100,000 � g spins were resuspended in 1 ml of PBS, laid on top of a linearsucrose density gradient (see below), and then centrifuged at 100,000 � gfor 15–16 h (Beckman Coulter SW28 rotor). For Western blot analysis ofthe microvesicles and exosomes, the collected medium was sequentiallycentrifuged at 4°C for 10 min at 300 � g, 20 min at 1,200 � g, 30 min at10,000 � g (Sorvall SS-34 rotor), and 15–16 h at 100,000 � g (BeckmanCoulter Ti 90 rotor). The pellets collected from 10,000 � g and 100,000 �g were resuspended in 1 ml of PBS, precipitated by TCA, and analyzed byWestern blot.

Equilibrium sucrose density gradient centrifugation

Continuous sucrose density gradients were generated by layering 1-ml ali-quots of 12 sucrose solutions (65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10% in 20 mM HEPES/NaOH (pH 7.2)) upon oneanother successively in a 15-ml ultraclear polyallomer centrifuge tube(Beckman Coulter), with the heaviest layer at the bottom and the lightestlayer at the top. The tube was incubated at 4°C for 5–6 h to allow diffu-sional mixing to establish a continuous sucrose density gradient. To per-form sucrose density gradient centrifugation, the collected extracellularmedium from BMDM or BMDC cultures was sequentially centrifuged for10 min at 300 � g and 20 min at 2000 � g to eliminate detached intactcells and large debris. The resulting supernatant was concentrated for�1.5–2 h at 3000 � g using Centriprep (Ultracel YM-3 membrane; Mil-lipore), which decreased the media volume from 10 to �1 ml. The con-centrated extracellular supernatant was then loaded on top of the preequili-brated sucrose density gradient and centrifuged at 100,000 � g for 15 h(Beckman Coulter SW28 rotor). Fractions (1 ml) were taken from the topof the tube and the density of each collected fraction was measured bydirect weighing of the 1-ml aliquot. After weighing, each fraction wasdiluted with 1 ml of PBS and then precipitated with TCA. The precipitatedproteins were analyzed by Western blot for various proteins (MHC-II,caspase-1, IL-1�, LAMP-1, actin) as indicated in particular figures.

Electron microscopy

Purified microvesicles and exosomes were fixed in a mixture of 2.0% glu-taraldehyde and 4% sucrose in a 0.05 M phosphate buffer (pH 7.4) for 2 hand then postfixed in 1% osmium tetroxide for 1 h at room temperature.Samples were then block-stained in 0.5% aqueous uranyl acetate, dehy-drated in ascending concentrations of ethanol, and embedded in Epon 812.

Ultrathin sections (60-nm thicknesses) were cut on a RMC MT6000-XLmicrotome and stained with 2% uranyl acetate in 50% methanol and withlead citrate, and then examined in a JEOL 1200EX electron microscope at80 kV.

Measurement of lactate dehydrogenase (LDH) release

Basal and ATP-stimulated LDH release was measured as described previ-ously using a cytotoxicity detection kit from Roche (33). The results areexpressed as a percentage of total LDH released, which was obtained bydividing the amount of LDH detected extracellularly by the sum total ofLDH detected within the cell and the amount detected in extracellularmedium, and multiplying by 100.

ResultsP2X7R activation stimulates the rapid release of MHC-IImembranes from inflammatory murine macrophages

We previously reported that activation of P2X7R can induce effi-cient intracellular inflammasome assembly and maturation of IL-1�, followed by rapid secretion of the mature IL-1� and activatedcaspase-1 to the extracellular compartment (4). Concurrent withthese responses, we also observed an ATP-induced rapid release ofMHC-II. We compared ATP-induced MHC-II secretion inBMDMs bathed in Na-gluconate balanced salt solution (BSS) (asan optimal experimental test solution for P2X7R activation) (33)vs standard DMEM (as a NaCl-based physiological tissue culturemedium). As indicated in Fig. 1A, under both test conditions arapid secretion of MHC-II was initiated during the first 15 min ofexposure to ATP, and this was followed by a sustained accumu-lation during the next 75 min. Consistent with our previous ob-servations that extracellular Cl� decreases the efficacy of P2X7Ras an activator of caspase-1 inflammasomes (33), we observedattenuated, but still significant, MHC-II release and delayedcaspase-1 activation when BMDMs were stimulated with ATP inDMEM vs Na-gluconate BSS. We used semiquantitative Westernblot analysis (Fig. 1, B and C) to compare the amounts of secretedMHC-II as a percentage of the total intracellular MHC-II inBMDMs pretreated with the different priming stimuli. In cellsprimed with LPS, activation of P2X7R stimulated the release of1.3 � 0.1% (n � 8) of the total MHC-II pool within 15 min afterATP incubation. By 30 min after ATP stimulation, the accumula-tion of secreted MHC-II reached 5.3 � 0.5% (n � 8) of the totalcell content. By 90 min after addition of ATP, 15.3 � 0.1% (n �8) of the total cellular MHC-II pool was transferred to the extra-cellular compartment (Fig. 1D).

In the absence of acute ATP stimulation, the IFN-�- and LPS-primed BMDMs did not release measurable MHC-II during 90-min test incubations (Fig. 1E). We also observed no MHC-II re-lease in BMDMs derived from P2X7R-null mice that were primedwith IFN-� and LPS and stimulated with 5 mM ATP for up to 90min (Fig. 1F). This is notable because murine macrophages ex-press multiple P2 receptors, in addition to P2X7R, that are alsoactivated by extracellular ATP and other nucleotides (34). Similarlevels of total MHC-II were expressed in the P2X7-knockoutBMDMs. As previously described (4, 35, 36), the P2X7R-deficientmacrophages exhibited no caspase-1 activation response toextracellular ATP.

Consistent with our previous findings (33) and those of others(37), sustained activation of P2X7R for 90 min induced significantmacrophage death, but only after priming with LPS. ATP-inducedcell death, as indicated by LDH release, was 38% in IFN-� plusLPS-primed cells vs 5% in cells primed with IFN-� only (supple-mental Fig. 1).4 At 30 min after ATP stimulation, the LDH releasewas proportionately lower (11% in IFN-�/LPS-primed vs 5% in

4 The online version of this article contains supplemental material.

5054 ATP-STIMULATED SECRETION OF MHC-II EXOSOMES

IFN-�-primed cells). ATP-induced cell death was strictly depen-dent on expression of P2X7R, as indicated by the minor LDHrelease (4% at 90 min) in IFN-�/LPS-primed BMDMs fromP2X7R-knockout mice.

Distinct regulatory roles of the inflammasome proteins ASC,NLRP3, and caspase-1 in P2X7R-stimulated MHC-II release

We examined MHC-II release in non-LPS-primed vs LPS-primedmacrophages in response to P2X7R stimulation during a 90-mintest period. When compared with LPS-primed cells, both the ki-netics and extent of ATP-induced MHC-II release were markedlyattenuated in the non-LPS-primed macrophages. At 30 min afterATP stimulation, the nonprimed BMDMs released 1 � 0.3% (n �8) of their MHC-II in contrast to the 5.3 � 0.5% (n � 8) secretedby the LPS-primed cells. Even after 90 min of ATP stimulation,only 3.8 � 1.8% (n � 8) of the MHC-II pool was externalized bythe nonprimed cells relative to the 15.3 � 0.1% (n � 8) releaseobserved in the primed BMDMs (Fig. 2, A and B). This suggests

that LPS-dependent signaling cascades, which are required for ef-ficient coupling of P2X7R to caspase-1 inflammasome assembly,also facilitate ATP-stimulated MHC-II secretion.

Inclusion of YVAD-cmk, an inhibitor of active caspase-1, dur-ing ATP stimulation of the LPS-primed BMDMs significantly al-tered the kinetics and magnitude of MHC-II secretion (Fig. 2, Aand B). Only 0.3 � 0.2% (n � 6) of the MHC-II pool was secretedduring 30 min of ATP stimulation in the presence of YVAD-cmkvs 5.3 � 0.5% (n � 8) release in its absence. Prolongation of theATP stimulation duration to 90 min did not overcome this block-ing effect; at this time point, YVAD-treated cells released 1 �0.5% (n � 6) of their MHC-II relative to the 15.3 � 0.1% (n � 8)release in its absence. These findings suggested a potential role ofactive caspase-1 in regulating the MHC-II trafficking response toP2X7R stimulation. To investigate whether caspase-1 is indeedessential for the P2X7R-stimulated MHC-II release, we comparedthe kinetics and magnitudes of ATP-induced MHC-II release inBMDMs derived from wild-type (WT) vs caspase-1-null mice.

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FIGURE 1. P2X7R-dependent release of MHC-IIfrom inflammatory murine BMDMs. A and B, C57BL/6BMDMs were plated 2 � 106/well and pretreated withIFN-� (2 ng/ml) for 16–18 h before LPS (1 �g/ml)priming for 4 h. Cells were transferred to either BSS orDMEM and stimulated with 5 mM ATP for the indi-cated times. The extracellular media and cell lysateswere separately collected and processed for Westernblot analysis. The blot membrane was first probed withanti-MHC-II Ab, then stripped and sequentially probedwith Abs against caspase-1. These data are representa-tive of similar time course studies from six separate ex-periments with BSS and four separate experiments withDMEM. C, A standard curve was generated from a se-ries of dilutions corresponding to the indicated numberof IFN-� and LPS-primed BMDMs per lane. Densito-metric analysis performed with ImageJ version 1.39uimaging software (59) and was used to correlateMHC-II band intensities with corresponding cell num-ber. D, IFN-� and LPS-primed BMDMs were stimu-lated without or with 5 mM ATP for the indicated timesand the released MHC-II was quantified as the percent-age of total cellular MHC-II content based on standardcurve in C. The data points in the bottom panel representthe means � SE from eight experiments using differentpreparations of C57BL/6 BMDMs; the Western blot inthe upper panel is from a representative experiment. E,IFN-� and LPS-primed BMDMs from WT C57BL/6mice were transferred to BSS and incubated for the in-dicated times in the absence of ATP stimulation. Theextracellular media samples collected at each time pointwere processed for analysis of MHC-II and caspase-1content. Serial dilutions of the cell lysates were pro-cessed in parallel for analysis of total cellular MHC-IIcontent. The Western blot data are representative of ob-servations from three separate experiments. F, IFN-�and LPS-primed BMDMs from P2X7R-knockout(P2X7R�/�) mice were transferred to BSS and stimu-lated for the indicated times in the presence of 5 mM.The extracellular media samples collected at each timepoint were processed for analysis of MHC-II andcaspase-1 content. Serial dilutions of the cell lysateswere processed in parallel for analysis of total cellularMHC-II content. These data are representative of obser-vations from five separate experiments.


Surprisingly, the absence of caspase-1 only modestly affected thekinetics and extent of ATP-triggered MHC-II secretion (Fig. 2, Cand D). A reduction in MHC-II release from the knockout cellswas observed at 30 min (2.6 � 0.3% (n � 5) vs 5.3 � 0.5% (n �8) in the control macrophages). However, with more prolongedATP stimulation periods (90 min), the caspase-1�/� and WTBMDMs released comparable amounts of MHC-II (15.5 � 1.5%(n � 5) vs 15.3 � 0.1% (n � 8) in knockout vs control, respec-tively). Notably, treatment of the caspase-1 knockout macrophageswith YVAD-cmk markedly inhibited ATP-stimulated MHC-II se-cretion at all time points (Fig. 2, C and D); released MHC-II at 90min was 5 � 1% (n � 4) in the presence of the peptide vs 15.5 �1.5% (n � 5) in its absence.

Peptide inhibitors of the various caspases are not completelyselective (38, 39). To test the selectivity of YVAD-cmk as aninhibitor of ATP-stimulated MHC-II release in WT BMDMs, we

also examined the effects of zVAD-fmk (a pan-caspase inhibitor),TPCK (a serine protease inhibitor), or DEVD-cho (a caspase-3inhibitor) (Fig. 2E). The zVAD-fmk and TPCK, but not DEVD-cho, markedly attenuated MHC-II release (measured at 30 min)and completely abrogated caspase-1 activation in response toP2X7R activation. The effects of these protease inhibitors onMHC-II release were not due to nonspecific toxic effects or sup-pression of proximal P2X7R activation because none of the agentsblocked ATP-stimulated exocytosis of secretory lysosomes (as as-sayed by cathepsin B release) (Fig. 2E). We have previously re-ported that P2X7 receptors trigger the mobilization of secretorylysosomes from macrophages via Ca2�-dependent signaling thatcan be dissociated from the parallel pathway that couples thesereceptors to inflammasome activation (4).

To further dissect the possible role of inflammasome complexassembly upstream of caspase-1 in regulating P2X7R-dependent

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FIGURE 2. P2X7R-induced release of MHC-II is potentiated by LPS priming and suppressed by peptide inhibitors of proteases but is independent ofcaspase-1. A and B, BMDMs from WT C57BL/6 mice were pretreated with IFN-� (2 ng/ml) for 16–18 h before priming with or without LPS (1 �g/ml)for 4 h. The primed BMDMs were then stimulated without or with 5 mM ATP for the indicated times in the absence or presence of 50 �M YVAD-cmk.The released MHC-II at each time point was quantified as the percentage of total cellular MHC-II content using the protocol in Fig. 1, C and D. The datapoints in B represent the means � SE from eight experiments that tested the effects of priming and six experiments that tested the effects of the YVAD-cmkinhibitor; the Western blots in A are from representative experiments. C and D, BMDMs from WT C57BL/6 mice (WT) or caspase�/� mice were pretreatedwith IFN-� (2 ng/ml) for 16–18 h before priming with LPS (1 �g/ml) for 4 h. The primed BMDMs were then stimulated without or with 5 mM ATP forthe indicated times in the absence or presence of 50 �M YVAD-cmk. The released MHC-II at each time point was quantified as the percentage of totalcellular MHC-II content using the protocol in Fig. 1, C and D. The data points in D represent the means � SE from eight experiments with WT BMDMs,five experiments with caspase-1�/� BMDMs, and four experiments with caspase-1�/� BMDMs treated with YVAD-cmk; the Western blots in C are fromrepresentative experiments. E, IFN-� and LPS-primed BMDMs from WT C57BL/6 mice were transferred to BSS and incubated with or without 50 �MYVAD-cmk, 50 �M DEVD-cho, 50 �M ZVAD-fmk, or 100 �M TPCK for 30 min before stimulation with 5 mM ATP for an additional 30 min. Theextracellular media and cell lysates were separately collected and processed for Western blot analysis of MHC-II, caspase-1, and cathepsin B. The data arerepresentative of results from three separate experiments.


MHC-II secretion, we used BMDMs derived from WT, ASC-null,and NLRP3-null mice for comparative analyses of MHC-II releasekinetics during ATP stimulation periods ranging from 15 to 60 min(Fig. 3, A and B; the WT and caspase-1-null quantitative data setsfrom Fig. 2D have been replotted for comparison). At all timepoints, P2X7R-dependent MHC-II release was markedly de-creased in ASC�/� and NLRP3�/� macrophages compared withWT cells or caspase-1�/� cells. At 60 min, the extracellular con-tent of MHC-II as a percentage of the total cellular MHC-II poolwas 0.1 � 0.1% (n � 7) for ASC�/� cells and 1.2 � 0.2 (n � 7)for NLRP3�/� macrophages, which contrasted with 7.8 � 1.6%(n � 8) for WT cells. Notably, the release of MHC-II from ASC-null cells measured at 30 min was greater (1 � 0.5% (n � 7)) thanat 60 min; this suggests that previously released MHC-II might bereaccumulated or metabolically cleared in the absence of sustainedexport of MHC-II. As noted previously, the ATP-stimulatedcaspase-1�/� cells released MHC-II at levels that were only mod-estly different from those in WT cells (no difference at 15 min,45% decrease at 30 min, 75% increase at 60 min). We verified thatthe BMDMs derived from these four different mouse strains ex-pressed similar amounts of total MHC-II protein (Fig. 3C). More-over, BMDMs derived from the ASC- or NLRP3-deficient micewere not compromised in ATP-stimulated secretory lysosome exo-cytosis, which is strictly dependent on the influx of extracellularCa2� triggered by P2X7R (4), but were completely deficient in

P2X7R-induced inflammasome activation and release of activecaspase-1 (Fig. 3D).

MHC-II released in response to P2X7R activation is containedin microvesicles and exosome-like vesicles with distinctmorphologies

Previous studies have demonstrated that MHC-II constitutively re-leased from dendritic cells or B cells is predominantly associatedwith membranous exosome vesicles derived from the intraluminalvesicles of MIIC or other MVB-like organelles (40, 41). To furthercharacterize the nature of the MHC-II pool(s) secreted in responseto P2X7R activation, we tested whether the released MHC-II isassociated with membrane compartments that exhibit the estab-lished biophysical properties of exosomes by using equilibriumsucrose density gradient centrifugation. As indicated in Fig. 4A,all MHC-II released in response to ATP was localized to su-crose densities ranging between 1.08 and 1.15 g/ml, consistentwith the buoyant density of exosomes reported in previous stud-ies (16, 20, 41). In contrast, the lysosomal protease cathepsin B,a soluble protein released via secretory lysosome exocytosisstimulated by P2X7R, was exclusively localized at the top ofthe sucrose gradient. Interestingly, we also observed that sub-fractions of LAMP-1, caspase-1 p10 subunit, and mature IL-1�colocalized with MHC-II in the �1.1 g/ml density bands of thesucrose gradient. This further supports our previous hypothesis

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FIGURE 3. The inflammasome proteins ASC andNLRP3, but not caspase-1, are required for maximalP2X7R-induced release of MHC-II. A and B, BMDMsfrom WT, caspase-1�/�, ASC�/�, and NLRP3�/� micewere pretreated with IFN-� (2 ng/ml) for 16–18 h be-fore LPS (1 �g/ml) priming for 4 h. The primedBMDMs were then stimulated without or with 5 mMATP for the indicated times. The extracellular mediaand cell lysates were collected and processed for semi-quantitative Western blot analysis. The releasedMHC-II at each time point was quantified as the per-centage of total cellular MHC-II content using the pro-tocol in Fig. 1, C and D. The data points in B representthe means � SE from eight experiments with WTBMDMs, seven experiments each with ASC�/� andNLRP3�/� BMDMs, and five experiments withcaspase-1�/� BMDMs; the Western blots in A are fromrepresentative experiments. C, Lysate dilutions fromWT, caspase-1�/�, ASC�/�, and NLRP3�/� BMDMswere analyzed for their relative contents of MHC-II,procaspase-1, and ASC. D, BMDMs from WT,ASC�/�, and NLRP3�/� mice were pretreated withIFN-� (2 ng/ml) for 16–18 h before additional incuba-tion without or with LPS (1 �g/ml) for 4 h. The cellswere transferred to either Ca2�-containing or Ca2�-freeBSS and then stimulated with or without 5 mM ATP for15 min. The extracellular media and cell lysates werecollected and processed for Western blot analysis ofMHC-II, caspase-1, and cathepsin B. The data are rep-resentative of observations from five experiments.


that MVB-derived exosomes may comprise a nonclassical se-cretory pathway for release of mature IL-1� and activecaspase-1 from murine macrophages.

Activation of P2X7R is also known to trigger rapid blebbing ofthe plasma membrane (5) and shedding of such blebs as releasedmicrovesicles or microparticles (6–8, 42). To test the contributionof shed microvesicles vs exosomes to the MHC-II fractions ob-served in sucrose gradients, we collected the extracellular mediafrom BMDMs stimulated by ATP (precleared of detached cellsand large debris by successive centrifugation at 300 � g and2000 � g) and then isolated an initial pellet fraction that sedi-mented at 10,000 � g and a secondary pellet that sedimented at100,000 � g. Electron microscopic analysis of these two sediment-able membrane fractions (Fig. 4C) revealed that the 100,000 x gpellet contained homogeneously sized and optically dense vesiclesranging from 50 to 80 nm in diameter, which is consistent with thereported morphological properties of exosomes. In contrast, the10,000 � g pellet contained a more heterogeneous component ofmembrane vesicles that ranged in size between 100 and 600 nm indiameter.

Each resuspended membrane fraction was also loaded on linearsucrose density and recentrifuged at 100,000 � g for 16 h. Asshown in Fig. 4B, MHC-II similarly accumulated in the 1.11–1.14g/ml density fractions in gradients containing either 10,000 � gpellets, which represent the presumed microvesicle pool, or the100,000 � g pellets, which represent the putative exosome pool.Thus, the MHC-II released in response to P2X7R activation ap-pears to be associated with two different membrane compartmentsthat have distinct biogenesis: microvesicles and exosomes. More-

over, although the two membrane compartments have differentmasses, they have similar densities. Hereafter, we refer to the10,000 � g pellet fraction as MHC-II microvesicles and the100,000 � g pellet fraction as MHC-II exosomes. We further char-acterized the ATP-induced MHC-II microvesicles and exosomesfor the presence of LAMP-1 and actin, two other marker proteinspreviously described in some but not all types of released exo-somes (40, 41). Both proteins were observed in MHC-II exosomesand MHC-II microvesicles (Fig. 5A), albeit with markedly lowerlevels of LAMP-1 in the exosome pool at 15 min, but not 30 min,after ATP stimulation.

ASC and NLRP3 are required for the release of MHC-IIexosomes, but not MHC-II microvesicles, in response to P2X7Rstimulation

We compared the distributions of MHC-II microvesicles andMHC-II exosomes released from WT, ASC�/�, and NLRP3�/�

macrophages subjected to identical ATP stimulation periods of30 min. Fig. 5, B and C, show that similar amounts of MHC-IImicrovesicles (also positive for LAMP-1 and actin) were re-leased by ATP-stimulated BMDMs derived from WT, ASC�/�,or NLRP3�/� mice. Conversely, the P2X7R-dependent releaseof MHC-II exosomes (also containing LAMP-1 and actin mark-ers) from ASC�/� and NLRP3�/� macrophages was greatlyreduced relative to that measured in the WT cells. Thus, theASC and NLRP3 inflammasome proteins play critical roles incoupling P2X7R signaling to the biogenesis/export of MVB-derived exosomes, but not the shedding of plasma membranemicrovesicles.

1.04 1.07 1.09 1.11 1.12 1.13 1.15 1.17 1.22 1.24(g/ml)

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FIGURE 4. P2X7R-induced release of MHC-II con-taining membranes includes both microvesicles andexosomes. A, 30–40 � 106 BMDMs were pretreatedwith IFN-� (2 ng/ml) for 16–18 h before LPS (1 �g/ml)priming for 4 h. The cells were transferred to BSS andthen stimulated with 5 mM ATP for 15 min. The col-lected extracellular media were centrifuged successivelyat 300 � g and 2000 � g, concentrated by Centriprepfilters followed by layering on top of linear sucrose den-sity gradients and overnight centrifugation. Each frac-tion from the sucrose gradient was weighed and thensubjected to Western blot analysis. The membrane wassequentially probed with anti-MHC-II, anti-caspase-1,anti-IL-1�, anti-LAMP-1, and anti-cathepsin B. Dataare representative of results from two experiments. B,IFN-� and LPS-primed BMDMs were transferred toBSS and stimulated with 5 mM ATP for 15 min. Theextracellular media was collected, sequentially centri-fuged at 300 � g, 2,000 � g, 10,000 � g, and100,000 � g. The pellets from the 10,000 � g and100,000 � g spins were processed by equilibrium su-crose density gradient centrifugation and the resultingfractions were analyzed by Western blot. The results arerepresentative of two experiments. C, IFN-� and LPS-primed BMDMs were stimulated with 5 mM ATP for 15min. The extracellular media was collected and sequen-tially centrifuged at 300 � g, 2, 000 � g, 10,000 � g,and 100,000 � g. The microvesicles obtained from the10,000 � g pellet and the exosomes obtained from the100,000 � g pellet were analyzed by electron micros-copy. The scale bars are 100 nm and the the images arerepresentative of observations from two experiments.


Immature and mature DCs also rapidly release MHC-IImembrane vesicles in response to extracellular ATP

DCs have been studied extensively to define the mechanismsthat underlie exosome formation/secretion and the intracellulartrafficking of MHC-II molecules (26, 43). We investigatedwhether murine BMDCs might also rapidly release membranesthat contain MHC-II in response to external stimuli such asATP. As illustrated in Fig. 6A, ATP-induced caspase-1 inflam-masome activation and IL-1� maturation were strictly depen-dent on LPS priming of DCs before ATP stimulation. Rapidaccumulation of extracellular LAMP-1 was also triggered byATP in DCs. Both immature DCs (non-LPS primed condition)and mature DCs (24 h LPS primed condition) expressed similarlevels of MHC-II; this high constitutive expression of MHC-IIdistinguishes DCs from macrophages, which express significant

MHC-II only after stimulation with IFN-�. In contrast to mac-rophages (Fig. 1E), both the immature DCs and mature DCsexhibited substantial rates of basal or constitutive secretion ofMHC-II in the absence of ATP stimulation (Fig. 6A). However,ATP induced a marked increase in MHC-II release concurrentlywith caspase-1 activation and IL-1� export.

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FIGURE 5. The ASC and NLRP3 inflammasome proteins are requiredfor P2X7R-induced release of MHC-II exosomes but not MHC-II mi-crovesicles. A, IFN-� and LPS-primed BMDMs were stimulated withoutATP for 30 min or with ATP for 15 or 30 min. The extracellular media wascollected and sequentially centrifuged at 300 � g, 2,000 � g, 10,000 � g,and 100,000 � g. The microvesicles obtained from the 10,000 � g pelletand the exosomes obtained from the 100,000 � g pellet were analyzed byWestern blot analysis. The membrane was probed sequentially with Absagainst MHC-II, caspase-1, LAMP-1, and actin. The results are represen-tative of observations from three experiments. B, IFN-� and LPS-primedBMDM WT, ASC�/�, or NLRP3�/� mice were stimulated with 5 mMATP for 30 min. The extracellular media fractions were collected andsequentially centrifuged at 300 � g, 2,000 � g, and 100,000 � g. Themicrovesicles obtained from the 10,000 � g pellet and the exosomes ob-tained from the 100,000 � g pellet were analyzed by Western blot analysis.The membrane was probed sequentially with Abs against MHC-II,LAMP-1, and actin. The results are representative of data from three ex-periments with WT and two experiments each with ASC�/� or NLRP3�/�

BMDMs. C, The MHC-II bands from the Western blot data in B werequantified by densitometry. The MHC-II band densities from the ASC�/�

and NLRP3�/� sample lanes were normalized to the band densities mea-sured in the WT sample lanes.

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FIGURE 6. Comparison of MHC-II membranes released from imma-ture and mature BMDCs via constitutive or ATP-stimulated mechanisms.A, BMDCs derived from C57BL/6 mice were primed with LPS (1 �g/ml)for 4 h, transferred to BSS, and stimulated with or without 5 mM ATP for15 min. The extracellular media and the cell lysates were processed andanalyzed by Western blot sequentially probed with Abs against MHC-II,caspase-1, IL-1�, and LAMP-1. The results are representative of observa-tions from three experiments. B–D, BMDCs were primed either without(for immature DC) or with 1 �g/ml LPS (for mature DC) for 24 h beforefurther experimentation. B, Immature or mature DCs were incubated inserum-free DMEM media for 24 h before collection and processing of theextracellular fraction. C and D, Mature DCs were transferred to BSS andstimulated either with or without 5 mM ATP for 20 min before collectionand processing of the extracellular fraction. The collected extracellularmedia were sequentially centrifuged to eliminate dead cells and debris,followed by concentration. In B and C, the concentrated samples weredirectly loaded onto linear sucrose gradients. D, The concentrated mediumsamples were supplemented with or without Triton X-100 before loadingonto linear sucrose gradients. The sucrose gradient samples were then cen-trifuged at 100,000 � g for 16 h. Fractions (1 ml) were collected, weighed,precipitated by TCA, and analyzed by Western blot.


We tested whether the ATP-stimulated MHC-II membranes rap-idly released from mature DCs are similar in buoyant density to theexosomes constitutively secreted from both immature and matureDCs. In the absence of external stimuli, both the immature DCs(no LPS priming) and mature DCs (24 h LPS priming) constitu-tively secreted MHC-II during 24 h of medium conditioning, andthis MHC-II was predominantly distributed in the 1.09–1.14 g/mlregions of sucrose gradients (Fig. 6B). Mature DCs also released amodest amount of MHC-II vesicles with similar density charac-teristics even during short-term (20-min) medium conditioning inthe absence of ATP stimulation (Fig. 6C). However, inclusion ofATP during this acute 20-min incubation significantly increasedthis extracellular MHC-II membrane pool. Finally, parallel sam-ples of extracellular medium from ATP-stimulated mature DCswere supplemented with or without Triton X-100 before loadingon the sucrose gradients. Fig. 6D shows that the distribution ofreleased MHC-II was maximal at the 1.14 g/ml region of thesucrose gradient, while the triton-treated MHC-II fraction waslargely shifted to the soluble, low-density (�1.03 g/ml) region.This further indicated that the MHC-II released from DCs in re-sponse to brief ATP stimulation is contained within membranesthat have the same buoyant density as exosomes constitutivelyreleased from immature or mature DCs.

DiscussionThis study provides a detailed characterization of P2X7R-stimu-lated release of MHC-II-containing membranes from murine mac-rophages and DCs in the context of the overall proinflammatoryresponse that involves rapid assembly of the ASC/NLRP3 inflam-masomes that control caspase-1 activation. Our data indicate thatthe rapid MHC-II secretion triggered by P2X7R is associated withtwo types of membrane compartments: microvesicles and exo-somes. Based on biochemical and biophysical analysis, we wereable to distinguish between ATP-induced microvesicles and exo-somes. While both contain MHC-II and exhibit similar densities,each is distinguished by a different morphology, mass, and in-tracellular biogenesis. Electron microscopic images indicatedthat the plasma membrane-derived microvesicles are heteroge-neous in size (100 nm to 600 nm in diameter), while the MVB-derived exosomes are smaller and much more homogeneous insize (50 – 80 nm in diameter). Using both pharmacological andgenetic approaches, we determined that the inflammasome pro-teins ASC and NLRP3 play critical roles in regulating the re-lease of MHC-II exosomes independently of their parallel func-tion as key molecular mediators of the caspase-1 activationresponse to P2X7R signaling.

Plasma membrane-derived microvesicles vs MVB-derivedexosomes as a source of extracellular MHC-II

We (33) and others (37) have reported that sustained P2X7R ac-tivation triggers significant death of LPS-primed macrophages in atime frame similar to that characterizing the release of MHC-II.However, the ATP-triggered microvesicles and exosomes are mor-phologically distinct from apoptotic bodies, which are 1–4 �m indiameter (44). Previous analyses of exosomes constitutively se-creted by DCs and B cells indicated that several MIIC membraneproteins, including LAMP-1, are found mainly on the limitingmembrane of MVBs and are minimally detectable in the secretedexosomes (45). We observed that LAMP-1 and actin were asso-ciated with the ATP-induced exosomes but that the abundance ofthese markers increased with the duration of the ATP stimulus. Incontrast, both LAMP-1 and actin were present in ATP-inducedmicrovesicles, even at the earliest time points of isolation. Thisindicates that the plasma membrane component of LAMP-1, as

well as the underlying actin cytoskeleton, can be incorporatedinto the plasma membrane blebs that rapidly form in response toP2X7R activation and then scission away from the cell surfaceas released microvesicles. The enrichment of LAMP-1 and actinwithin the exosome pool observed with longer ATP stimulationsuggests that plasma membrane-associated proteins are traf-ficked into endosomes during the early phase of P2X7 activa-tion, incorporated into the ILV of MVBs, and then secreted asexosomes following MVB exocytosis. Recent studies have in-dicated that both MHCII and LAMP-1 associated with secretorylysosomes can be rapidly incorporated into the plasma mem-brane of human monocytes in response to Ca2� ionophore treat-ment (46).

Multiple studies have shown that Ag-loaded exosomes can berapidly taken up by naive DCs and macrophages remote from theorigin of exosome generation (17–19). In our experimental cellculture model it is possible that MHC-II membranes released atthe earlier times following the ATP stimulus are resequesteredby viable macrophages at later times. In particular, MHC-IImicrovesicles initially mobilized by inflammasome-indepen-dent mechanisms may be progressively cleared by cells thatlack the ability for sustained production and release of MHC-IIexosomes. This may underlie the reduced levels of extracellularMHC-II observed at 60 or 90 min vs earlier times in someexperiments with ASC-/NLRP3-null macrophages (Fig. 3, Aand B) or YVAD-treated cells (Fig. 2, A and B).

Intracellular signaling cascades that regulate the formation ofmicrovesicles and exosomes

The molecular mechanisms that underlie MHC-II traffickingwithin the endocytic pathway and the sorting signals involved inMVB formation have not been completely defined. Previous stud-ies have demonstrated that the LPS-induced maturation of DCsregulates the cell surface level of MHC-II via a ubiquitination-based mechanism that provides critical signals for both efficientendocytosis from the plasma membrane and cargo protein sortinginto the luminal vesicles of MVBs (24, 25). In murine macro-phages, LPS-dependent signaling is necessary for the P2X7R-cou-pled inflammasome activation that results in processing and re-lease of IL-1� and IL-18 (4, 32). Our new data (Fig. 2) indicatethat LPS-dependent signals are essential for the optimal couplingof P2X7R to MHC-II trafficking and export. That the releaseof LAMP-1-positive MHC-II exosomes, but not MHC-II mi-crovesicles, is abrogated in ASC- and NLRP3-deficient macro-phages supports a model wherein ASC and NLRP3 play specificroles in regulating MVB formation and exosome secretion in re-sponse to P2X7R activation and perhaps other “danger stimuli”that trigger assembly of ASC/NLRP3 inflammasomes. In contrast,P2X7R-dependent induction and release of plasma membrane mi-crovesicles occurs independently of inflammasome assembly.Fang et al. have recently reported that higher order oligomerizationand plasma membrane binding are sufficient to direct the HIV Gagproteins for budding and exosomal sorting (28). This suggests thatprotein aggregates that accumulate at the plasma membrane or atthe limiting membrane of MVBs may comprise one mechanism forcargo sorting into the intraluminal vesicles of the MVBs. Time-lapse confocal analysis of human THP-1 cells stably expressingASC-GFP fusion protein has demonstrated the formation of a largesupramolecular assembly of ASC oligomers, termed the pyropto-some, in response to various proinflammatory stimuli (47). It ispossible that the cytosolic oligomerization of inflammasome com-plex proteins may additionally provide budding/exosomal sortingsignals that accelerate the formation of the MVB-derived exo-somes containing MHC-II.


The surprising finding that deficiency of caspase-1 had nomajor effect on ATP-triggered MHC-II release suggests a reg-ulatory role for ASC/NLRP3 inflammasome complexes in func-tions other than caspase-1 activation. Human macrophages in-fected with Shigella flexneri exhibit a necrosis-like cell deathresponse that is dependent on ASC and NLRP3, but is inde-pendent of caspase-1 and IL-1� (48). Likewise, autophagy re-sulting from Shigella infection in macrophages provides pro-tection against pyroptotic death and is observed in the absenceof caspase-1 and NLRC4, but not of ASC (49). Our observationthat ATP-induced MHC-II secretion was insensitive to geneticdeletion of caspase-1 but sensitive to zVAD-fmk and TPCK,which are widely used pharmacological inhibitors of proteases,indicates that proteases other than caspase-1 may act either up-stream or downstream of ASC/NLRP3 inflammasome assem-bly. The ability of zVAD-fmk and TPCK to mimic the actionsof YVAD-cmk suggests that the reactive fluoro- or chlorometh-ylketone moieties of these inhibitors (38) may underlie theircommon ability to suppress these putative proteases. Notably,destabilization of phagolysosomes, including release of lysoso-mal proteases into the cytosol, has been recently identified as amajor mechanism by which multiple particulate stimuli, includ-ing silica crystals, alum, and amyloid-� aggregates, trigger as-sembly of ASC/NLRP3 inflammasomes (50, 51).

The local composition of lipids, especially cone-shaped lipidssuch as ceramide or lysophosphatidic acid, can rearrange thespatial organization of membrane bilayer leaflets depending onpH and thereby drive vesicle budding by inducing subdomainswith curvature that is different from the adjacent planar mem-brane leaflet (52). In this regard, Trajkovic et al. (53) havereported that cargo proteins in oligodendrocytes can segregateinto distinct subdomains of endosomal membranes and thatsorting of these proteins into a subset of MVBs occurred by aninvagination mechanism independent of the ESCRT (endoso-mal sorting complex required for transport) machinery usuallyassociated with MVB biogenesis. Rather, this ESCRT-indepen-dent process required lipid raft-based microdomains enriched inthe sphingolipid ceramide. It is possible that P2X7R-activatedlipases may mediate similar alterations in local lipid composi-tion, which initiate or accelerate the formation of microvesiclesat the plasma membranes or ILVs/exosomes at endosomalmembranes.

Potential physiological functions of MHC-II exosomes andMHC-II microvesicles mobilized by P2X7R activation

The biological functions of exosomes characterized thus far invarious cell types include Ag presentation, exchange of geneticmaterials such as mRNA and micro-RNA, proinflammatorystimulation, cell differentiation, utilization as biomarkers fordifferent diseases, and transport of alloantigens between donorand recipient DCs during organ transplantation (20 –23, 54 –57).The rapid secretion of MHC-II microvesicles and exosomesfrom P2X7R-activated macrophages may provide an efficientroute not only for releasing proinflammatory cytokines, but alsofor rapid dissemination and presentation of foreign Ags as partof an adaptive immune response that is coordinated with localinflammation. In this regard, it is interesting to note very recentstudies reporting that alum, the classical adjuvant used in vac-cine development, activates the same ASC/NLRP3 inflamma-some targeted by P2X7R (50, 58). The ability of activatedP2X7R to trigger rapid release of a significant mass of MHC-II-containing membranes suggests a link between the well-es-tablished role of this receptor in innate immune responses anda possible role in potentiating adaptive immune responses.

AcknowledgmentsThe authors thank Dr. Hisashi Fujioka for preparation, execution, and in-terpretation of electron microscopy experiments.

DisclosuresThe authors have no financial conflicts of interest.

References1. Di Virgilio, F. 2007. Liaisons dangereuses: P2X7 and the inflammasome. Trends

Pharmacol. Sci. 28: 465–472.2. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee,

M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation ofthe inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218.

3. Mariathasan, S., D. S. Weiss, K. Newton, J. McBride, K. O’Rourke,M. Roose-Girma, W. P. Lee, Y. Weinrauch, D. M. Monack, and V. M. Dixit.2006. Cryopyrin activates the inflammasome in response to toxins and ATP.Nature 440: 228–232.

4. Qu, Y., L. Franchi, G. Nunez, and G. R. Dubyak. 2007. Nonclassical IL-1�secretion stimulated by P2X7 receptors is dependent on inflammasome activationand correlated with exosome release in murine macrophages. J. Immunol. 179:1913–1925.

5. Verhoef, P. A., M. Estacion, W. Schilling, and G. R. Dubyak. 2003. P2X7 re-ceptor-dependent blebbing and the activation of Rho-effector kinases, caspases,and IL-1� release. J. Immunol. 170: 5728–5738.

6. Baroni, M., C. Pizzirani, M. Pinotti, D. Ferrari, E. Adinolfi, S. Calzavarini,P. Caruso, F. Bernardi, and F. Di Virgilio. 2007. Stimulation of P2 (P2X7) re-ceptors in human dendritic cells induces the release of tissue factor-bearing mi-croparticles. FASEB J. 21: 1926–1933.

7. MacKenzie, A., H. L. Wilson, E. Kiss-Toth, S. K. Dower, R. A. North, andA. Surprenant. 2001. Rapid secretion of interleukin-1� by microvesicle shedding.Immunity 15: 825–835.

8. Bianco, F., E. Pravettoni, A. Colombo, U. Schenk, T. Moller, M. Matteoli, andC. Verderio. 2005. Astrocyte-derived ATP induces vesicle shedding and IL-1�release from microglia. J. Immunol. 174: 7268–7277.

9. Mutini, C., S. Falzoni, D. Ferrari, P. Chiozzi, A. Morelli, O. R. Baricordi,G. Collo, P. Ricciardi-Castagnoli, and F. Di Virgilio. 1999. Mouse dendritic cellsexpress the P2X7 purinergic receptor: characterization and possible participationin antigen presentation. J. Immunol. 163: 1958–1965.

10. Swanberg, M., O. Lidman, L. Padyukov, P. Eriksson, E. Akesson, M. Jagodic,A. Lobell, M. Khademi, O. Borjesson, C. M. Lindgren, et al. 2005. MHC2TA isassociated with differential MHC molecule expression and susceptibility to rheu-matoid arthritis, multiple sclerosis and myocardial infarction. Nat. Genet. 37:486–494.

11. Viville, S., J. Neefjes, V. Lotteau, A. Dierich, M. Lemeur, H. Ploegh, C. Benoist,and D. Mathis. 1993. Mice lacking the MHC class II-associated invariant chain.Cell 72: 635–648.

12. Neefjes, J. J., V. Stollorz, P. J. Peters, H. J. Geuze, and H. L. Ploegh. 1990. Thebiosynthetic pathway of MHC class II but not class I molecules intersects theendocytic route. Cell 61: 171–183.

13. Geuze, H. J. 1998. The role of endosomes and lysosomes in MHC class II func-tioning. Immunol. Today 19: 282–287.

14. Kleijmeer, M., G. Ramm, D. Schuurhuis, J. Griffith, M. Rescigno,P. Ricciardi-Castagnoli, A. Y. Rudensky, F. Ossendorp, C. J. Melief,W. Stoorvogel, and H. J. Geuze. 2001. Reorganization of multivesicular bodiesregulates MHC class II antigen presentation by dendritic cells. J. Cell Biol. 155:53–63.

15. Morelli, A. E., A. T. Larregina, W. J. Shufesky, M. L. Sullivan, D. B. Stolz,G. D. Papworth, A. F. Zahorchak, A. J. Logar, Z. Wang, S. C. Watkins, et al.2004. Endocytosis, intracellular sorting, and processing of exosomes by dendriticcells. Blood 104: 3257–3266.

16. Raposo, G., H. W. Nijman, W. Stoorvogel, R. Liejendekker, C. V. Harding,C. J. Melief, and H. J. Geuze. 1996. B lymphocytes secrete antigen-presentingvesicles. J. Exp. Med. 183: 1161–1172.

17. Thery, C., L. Duban, E. Segura, P. Veron, O. Lantz, and S. Amigorena. 2002.Indirect activation of naive CD4� T cells by dendritic cell-derived exosomes.Nat. Immunol. 3: 1156–1162.

18. Quah, B. J., and H. C. O’Neill. 2005. The immunogenicity of dendritic cell-derived exosomes. Blood Cells Mol. Dis. 35: 94–110.

19. Li, X. B., Z. R. Zhang, H. J. Schluesener, and S. Q. Xu. 2006. Role of exosomesin immune regulation. J. Cell. Mol. Med. 10: 364–375.

20. Valadi, H., K. Ekstrom, A. Bossios, M. Sjostrand, J. J. Lee, and J. O. Lotvall.2007. Exosome-mediated transfer of mRNAs and microRNAs is a novel mech-anism of genetic exchange between cells. Nat. Cell Biol. 9: 654–659.

21. Sanderson, M. P., S. Keller, A. Alonso, S. Riedle, P. J. Dempsey, and P. Altevogt.2008. Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exo-some secretion. J. Cell. Biochem. 103: 1783–1797.

22. Izquierdo-Useros, N., M. Naranjo-Gomez, J. Archer, S. C. Hatch, I. Erkizia,J. Blanco, F. E. Borras, M. C. Puertas, J. H. Connor, M. T. Fernandez-Figueras,et al. 2008. Capture and transfer of HIV-1 particles by mature dendritic cellsconverges with the exosome-dissemination pathway. Blood [Epub: Oct. 22, 2008;DOI 10.1182].

23. Bhatnagar, S., K. Shinagawa, F. J. Castellino, and J. S. Schorey. 2007. Exosomesreleased from macrophages infected with intracellular pathogens stimulate aproinflammatory response in vitro and in vivo. Blood 110: 3234–3244.


24. Shin, J. S., M. Ebersold, M. Pypaert, L. Delamarre, A. Hartley, and I. Mellman.2006. Surface expression of MHC class II in dendritic cells is controlled byregulated ubiquitination. Nature 444: 115–118.

25. van Niel, G., R. Wubbolts, T. Ten Broeke, S. I. Buschow, F. A. Ossendorp,C. J. Melief, G. Raposo, B. W. van Balkom, and W. Stoorvogel. 2006. Dendriticcells regulate exposure of MHC class II at their plasma membrane by oligoubiq-uitination. Immunity 25: 885–894.

26. Segura, E., C. Nicco, B. Lombard, P. Veron, G. Raposo, F. Batteux,S. Amigorena, and C. Thery. 2005. ICAM-1 on exosomes from mature dendriticcells is critical for efficient naive T-cell priming. Blood 106: 216–223.

27. Saunderson, S. C., P. C. Schuberth, A. C. Dunn, L. Miller, B. D. Hock,P. A. MacKay, N. Koch, R. W. Jack, and A. D. McLellan. 2008. Induction ofexosome release in primary B cells stimulated via CD40 and the IL-4 receptor.J. Immunol. 180: 8146–8152.

28. Fang, Y., N. Wu, X. Gan, W. Yan, J. C. Morrell, and S. J. Gould. 2007. Higher-order oligomerization targets plasma membrane proteins and HIV gag to exo-somes. PLoS Biol. 5: e158.

29. Dowds, T. A., J. Masumoto, L. Zhu, N. Inohara, and G. Nunez. 2004. Cryopyrin-induced interleukin 1� secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC. J. Biol. Chem. 279: 21924–21928.

30. Myers, A. J., B. Eilertson, S. A. Fulton, J. L. Flynn, and D. H. Canaday. 2005.The purinergic P2X7 receptor is not required for control of pulmonary Myco-bacterium tuberculosis infection. Infect. Immun. 73: 3192–3195.

31. Davies, J. Q., and S. Gordon. 2005. Isolation and culture of murine macrophages.Methods Mol. Biol. 290: 91–103.

32. Kahlenberg, J. M., K. C. Lundberg, S. B. Kertesy, Y. Qu, and G. R. Dubyak.2005. Potentiation of caspase-1 activation by the P2X7 receptor is dependent onTLR signals and requires NF-�B-driven protein synthesis. J. Immunol. 175:7611–7622.

33. Verhoef, P. A., S. B. Kertesy, K. Lundberg, J. M. Kahlenberg, and G. R. Dubyak.2005. Inhibitory effects of chloride on the activation of caspase-1, IL-1� secre-tion, and cytolysis by the P2X7 receptor. J. Immunol. 175: 7623–7634.

34. del Rey, A., V. Renigunta, A. H. Dalpke, J. Leipziger, J. E. Matos, B. Robaye,M. Zuzarte, A. Kavelaars, and P. J. Hanley. 2006. Knock-out mice reveal thecontributions of P2Y and P2X receptors to nucleotide-induced Ca2� signaling inmacrophages. J. Biol. Chem. 281: 35147–35155.

35. Solle, M., J. Labasi, D. G. Perregaux, E. Stam, N. Petrushova, B. H. Koller,R. J. Griffiths, and C. A. Gabel. 2001. Altered cytokine production in mice lack-ing P2X7 receptors. J. Biol. Chem. 276: 125–132.

36. Labasi, J. M., N. Petrushova, C. Donovan, S. McCurdy, P. Lira, M. M. Payette,W. Brissette, J. R. Wicks, L. Audoly, and C. A. Gabel. 2002. Absence of theP2X7 receptor alters leukocyte function and attenuates an inflammatory response.J. Immunol. 168: 6436–6445.

37. Le Feuvre, R. A., D. Brough, Y. Iwakura, K. Takeda, and N. J. Rothwell. 2002.Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated celldeath via a caspase-1-dependent mechanism, independently of cytokine produc-tion. J. Biol. Chem. 277: 3210–3218.

38. Schotte, P., W. Declercq, S. Van Huffel, P. Vandenabeele, and R. Beyaert. 1999.Non-specific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett.442: 117–121.

39. Pereira, N. A., and Z. Song. 2008. Some commonly used caspase substrates andinhibitors lack the specificity required to monitor individual caspase activity.Biochem. Biophys. Res. Commun. 377: 873–877.

40. Thery, C., A. Regnault, J. Garin, J. Wolfers, L. Zitvogel, P. Ricciardi-Castagnoli,G. Raposo, and S. Amigorena. 1999. Molecular characterization of dendriticcell-derived exosomes: selective accumulation of the heat shock protein hsc73.J. Cell Biol. 147: 599–610.

41. Muntasell, A., A. C. Berger, and P. A. Roche. 2007. T cell-induced secretion ofMHC class II-peptide complexes on B cell exosomes. EMBO J. 26: 4263–4272.

42. Pizzirani, C., D. Ferrari, P. Chiozzi, E. Adinolfi, D. Sandona, E. Savaglio, andF. Di Virgilio. 2007. Stimulation of P2 receptors causes release of IL-1�-loadedmicrovesicles from human dendritic cells. Blood 109: 3856–3864.

43. Segura, E., S. Amigorena, and C. Thery. 2005. Mature dendritic cells secreteexosomes with strong ability to induce antigen-specific effector immune re-sponses. Blood Cells Mol. Dis. 35: 89–93.

44. Hristov, M., W. Erl, S. Linder, and P. C. Weber. 2004. Apoptotic bodies fromendothelial cells enhance the number and initiate the differentiation of humanendothelial progenitor cells in vitro. Blood 104: 2761–2766.

45. Escola, J. M., M. J. Kleijmeer, W. Stoorvogel, J. M. Griffith, O. Yoshie, andH. J. Geuze. 1998. Selective enrichment of tetraspan proteins on the internalvesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J. Biol. Chem. 273: 20121–20127.

46. Bunbury, A., I. Potolicchio, R. Maitra, and L. Santambrogio. 2008. Functionalanalysis of monocyte MHC class II compartments. FASEB J. 23: 164–171.

47. Fernandes-Alnemri, T., J. Wu, J. W. Yu, P. Datta, B. Miller, W. Jankowski,S. Rosenberg, J. Zhang, and E. S. Alnemri. 2007. The pyroptosome: a supramo-lecular assembly of ASC dimers mediating inflammatory cell death via caspase-1activation. Cell Death Differ. 14: 1590–1604.

48. Willingham, S. B., D. T. Bergstralh, W. O’Connor, A. C. Morrison,D. J. Taxman, J. A. Duncan, S. Barnoy, M. M. Venkatesan, R. A. Flavell,M. Deshmukh, et al. 2007. Microbial pathogen-induced necrotic cell death me-diated by the inflammasome components CIAS1/cryopyrin/NLRP3 and ASC.Cell Host Microbe 2: 147–159.

49. Suzuki, T., L. Franchi, C. Toma, H. Ashida, M. Ogawa, Y. Yoshikawa,H. Mimuro, N. Inohara, C. Sasakawa, and G. Nunez. 2007. Differential regulationof caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages. PLoS Pathog. 3: e111.

50. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock,K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activatethe NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9:847–856.

51. Halle, A., V. Hornung, G. C. Petzold, C. R. Stewart, B. G. Monks, T. Reinheckel,K. A. Fitzgerald, E. Latz, K. J. Moore, and D. T. Golenbock. 2008. The NALP3inflammasome is involved in the innate immune response to amyloid-�. Nat.Immunol. 9: 857–865.

52. Subra, C., K. Laulagnier, B. Perret, and M. Record. 2007. Exosome lipidomicsunravels lipid sorting at the level of multivesicular bodies. Biochimie 89:205–212.

53. Trajkovic, K., C. Hsu, S. Chiantia, L. Rajendran, D. Wenzel, F. Wieland,P. Schwille, B. Brugger, and M. Simons. 2008. Ceramide triggers budding ofexosome vesicles into multivesicular endosomes. Science 319: 1244–1247.

54. Yu, S., C. Liu, K. Su, J. Wang, Y. Liu, L. Zhang, C. Li, Y. Cong, R. Kimberly,W. E. Grizzle, C. Falkson, and H. G. Zhang. 2007. Tumor exosomes inhibitdifferentiation of bone marrow dendritic cells. J. Immunol. 178: 6867–6875.

55. Vella, L. J., R. A. Sharples, R. M. Nisbet, R. Cappai, and A. F. Hill. 2008. Therole of exosomes in the processing of proteins associated with neurodegenerativediseases. Eur. Biophys. J. 37: 323–332.

56. Pisitkun, T., R. F. Shen, and M. A. Knepper. 2004. Identification and proteomicprofiling of exosomes in human urine. Proc. Natl. Acad. Sci. USA 101:13368–13373.

57. Montecalvo, A., W. J. Shufesky, D. B. Stolz, M. G. Sullivan, Z. Wang,S. J. Divito, G. D. Papworth, S. C. Watkins, P. D. Robbins, A. T. Larregina, andA. E. Morelli. 2008. Exosomes as a short-range mechanism to spread alloantigenbetween dendritic cells during T cell allorecognition. J. Immunol. 180:3081–3090.

58. Li, H., S. B. Willingham, J. P. Ting, and F. Re. 2008. Cutting edge: inflamma-some activation by alum and alum’s adjuvant effect are mediated by NLRP3.J. Immunol. 181: 17–21.

59. Auger, R., I. Motta, K. Benihoud, D. M. Ojcius, and J. M. Kanellopoulos. 2005.A role for mitogen-activated protein kinaseErk1/2 activation and non-selectivepore formation in P2X7 receptor-mediated thymocyte death. J. Biol. Chem. 280:28142–28151.


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