Exosome-dependent Trafficking of HSP70

The heat shock proteins (HSPs) are a family of intracellular proteins found in all eukaryotes and prokaryotes. Their functions are well characterized and are central to maintaining cellular homeostasis and in promoting cell survival in response to stressful cellular conditions. However, several studies provide evidence that specific members of the HSP family might be secreted via an unidentified exocytotic pathway. Here we show that exosomes, small membrane vesicles that are secreted by numerous cell types, contribute to the release of HSP70 from human peripheral blood mononuclear cells (PBMCs) in both basal and stress-induced (heat shock at 40 or 43 °C for 1 h) states. HSP70 release from PBMCs is independent of the common secretory pathway because Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP70 release. Furthermore, we show that HSP70 release from PBMCs does not occur via a lipid raft-dependent pathway, because treatment with methyl-β-cyclodextrin, a raft-disrupting drug, had no affect on HSP70 release. To examine whether exosomes contributed to HSP70 release from PBMCs, exosomes were purified from PBMC cultures, and exosomal number and HSP70 content were determined. We demonstrate that although heat shock does not influence the exosomal secretory rate, the HSP70 content of exosomes isolated from heat shocked PBMCs is significantly higher than control. These data identify a novel secretory pathway by which HSP70 can be actively released from cells in both the basal and stress-induced state. The heat shock proteins (HSPs) are a family of intracellular proteins found in all eukaryotes and prokaryotes. Their functions are well characterized and are central to maintaining cellular homeostasis and in promoting cell survival in response to stressful cellular conditions. However, several studies provide evidence that specific members of the HSP family might be secreted via an unidentified exocytotic pathway. Here we show that exosomes, small membrane vesicles that are secreted by numerous cell types, contribute to the release of HSP70 from human peripheral blood mononuclear cells (PBMCs) in both basal and stress-induced (heat shock at 40 or 43 °C for 1 h) states. HSP70 release from PBMCs is independent of the common secretory pathway because Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP70 release. Furthermore, we show that HSP70 release from PBMCs does not occur via a lipid raft-dependent pathway, because treatment with methyl-β-cyclodextrin, a raft-disrupting drug, had no affect on HSP70 release. To examine whether exosomes contributed to HSP70 release from PBMCs, exosomes were purified from PBMC cultures, and exosomal number and HSP70 content were determined. We demonstrate that although heat shock does not influence the exosomal secretory rate, the HSP70 content of exosomes isolated from heat shocked PBMCs is significantly higher than control. These data identify a novel secretory pathway by which HSP70 can be actively released from cells in both the basal and stress-induced state. The heat shock proteins (HSP 1The abbreviations used are: HSP, heat shock protein; PBMC, peripheral blood mononuclear cell; MVBs, multivesicular bodies; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BFA, Brefeldin A; MBC, methyl-β-cyclodextrin; LDH, lactate dehydrogenase; RM ANOVA, repeated measures analysis of variance; MHC, major histocompatibility complex. (s)) are a family of quintes-sentially intracellular proteins found in all eukaryotes and prokaryotes. HSP families include both constitutive and stress-inducible members whose primary function is to interact with naïve and denatured proteins to prevent the aggregation of aberrantly folded proteins, facilitate the folding of naïve proteins, facilitate the refolding of denatured proteins, and to aid intracellular protein trafficking (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar). Although these functions underscore the importance of intracellular HSPs in the maintenance of cellular homeostasis and in promoting cell survival in response to stressful cellular conditions, accumulating evidence suggests that HSPs are actively secreted and have important extracellular functions. The concept that specific HSPs can be actively secreted was suggested ∼15 years ago following the demonstration that heat-shocked rat embryo cells rapidly released HSP70 and HSP110 (2Hightower L.E. Guidon Jr., P.T. J. Cell. Physiol. 1989; 138: 257-266Crossref PubMed Scopus (312) Google Scholar). Importantly, several studies have confirmed these earlier findings, demonstrating that HSP70 is secreted from a variety of cell types in response to cellular stress (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 4Guzhova I. Kislyakova K. Moskaliova O. Fridlanskaya I. Tytell M. Cheetham M. Margulis B. Brain Res. 2001; 914: 66-73Crossref PubMed Scopus (257) Google Scholar, 5Hunter-Lavin C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google Scholar, 6Febbraio M.A. Ott P. Nielsen H.B. Steensberg A. Keller C. Krustrup P. Secher N.H. Pedersen B.K. J. Physiol. 2002; 544: 957-962Crossref PubMed Scopus (143) Google Scholar, 7Fleshner M. Campisi J. Amiri L. Diamond D.M. Psychoneuroendocrinology. 2004; 29: 1142-1152Crossref PubMed Scopus (110) Google Scholar). The HSP70 protein lacks a secretory signal sequence, and, concordantly, cellular HSP70 release is unaffected by inhibitors of the common secretory pathway (2Hightower L.E. Guidon Jr., P.T. J. Cell. Physiol. 1989; 138: 257-266Crossref PubMed Scopus (312) Google Scholar, 3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Although it has been suggested that cellular HSP70 release may be the result of nonspecific processes, such as cell lysis, several lines of evidence argue against this notion (2Hightower L.E. Guidon Jr., P.T. J. Cell. Physiol. 1989; 138: 257-266Crossref PubMed Scopus (312) Google Scholar, 3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 6Febbraio M.A. Ott P. Nielsen H.B. Steensberg A. Keller C. Krustrup P. Secher N.H. Pedersen B.K. J. Physiol. 2002; 544: 957-962Crossref PubMed Scopus (143) Google Scholar). Interestingly, two recent studies have provided evidence that specialized membrane microdomains, termed lipid rafts, may play a role in HSP70 exocytosis (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 5Hunter-Lavin C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google Scholar). Given the critical intracellular roles played by HSPs it may appear counterintuitive that a secretory pathway exists allowing cells to release HSPs both basally and in response to cellular stress. However, a growing body of evidence suggests that specific members of the HSP family possess potent immunoregulatory functions (8Vabulas R.M. Ahmad-Nejad P. da Costa C. Miethke T. Kirschning C.J. Hacker H. Wagner H. J. Biol. Chem. 2001; 276: 31332-31339Abstract Full Text Full Text PDF PubMed Scopus (685) Google Scholar, 9Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (801) Google Scholar, 10Vabulas R.M. Braedel S. Hilf N. Singh-Jasuja H. Herter S. Ahmad-Nejad P. Kirschning C.J. Da Costa C. Rammensee H.G. Wagner H. Schild H. J. Biol. Chem. 2002; 277: 20847-20853Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, 11Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar, 12Kol A. Lichtman A.H. Finberg R.W. Libby P. Kurt-Jones E.A. J. Immunol. 2000; 164: 13-17Crossref PubMed Scopus (473) Google Scholar). Recombinant human HSP70 stimulates the production of interleukin-1β, tumor necrosis factor-α, interleukin-6, and interleukin-12 and promotes the up-regulation of co-stimulatory (CD86) and antigen presenting major histocompatibility complex (MHC) II molecules in monocytes and dendritic cells (11Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar, 13Asea A. Kraeft S.K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1371) Google Scholar). Furthermore, cell surface-bound HSP70 renders tumor cells more sensitive to natural killer cell-mediated cytolytic attack (14Gross C. Koelch W. DeMaio A. Arispe N. Multhoff G. J. Biol. Chem. 2003; 278: 41173-41181Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 15Multhoff G. Botzler C. Jennen L. Schmidt J. Ellwart J. Issels R. J. Immunol. 1997; 158: 4341-4350PubMed Google Scholar). These findings have led to the hypothesis that extracellular HSPs, released either as a result of cellular necrosis or via a stress-induced exocytotic pathway, may act as a potent danger signal to the immune system (9Vabulas R.M. Ahmad-Nejad P. Ghose S. Kirschning C.J. Issels R.D. Wagner H. J. Biol. Chem. 2002; 277: 15107-15112Abstract Full Text Full Text PDF PubMed Scopus (801) Google Scholar, 16Campisi J.L. Leem T.H. Fleshner M. Cell Stress Chaperones. 2003; 8: 272-286Crossref PubMed Scopus (173) Google Scholar). Our initial experiments were aimed at determining whether heat shock at physiological/pathophysiological temperatures, i.e. during febrile states, stimulated the release of HSPs. Interestingly, in the course of conducting these studies, we were unable to confirm a role for lipid rafts in either heat shock-induced or basal HSP70 release from human peripheral blood mononuclear cells (PBMCs). Almost years Multhoff and G. L.E. Cell Stress Chaperones. PubMed Google that exosomes, vesicles secreted following the of multivesicular with the may provide a secretory pathway allowing cells to actively release specific HSPs. The concept of the was following the that Although have a well role in the of proteins via with the studies in that small vesicles be secreted the extracellular via of with the membrane B. G. Cell Biol. 2004; PubMed Scopus Google Scholar). recent has proteins with exosomes, several molecules with roles in the of immune L. A. A. J. C. P. G. S. Nat. Med. PubMed Scopus Google Scholar). the we identify exosomes as of basal and stress-induced HSP70 from These data identify a novel secretory pathway by which PBMCs are to actively release and cell fetal bovine and phosphate-buffered were from Brefeldin and were from The lactate was from were the from was from protein were from The used were as and The HSP70 were from Cell were isolated from peripheral blood from the blood was in and of at for the was and in at for the was and the cell was and in a at the was and cells were in of cell FBS, were on a and at 1 to either or and were to in were in with fetal and at in were in with fetal and were with the of and for by a in and which cells had lipid was for which cells were for were to heat either 40 or 43 or for in were to at for to for the and release of HSPs. bovine to experiments in which exosomes were to be cell FBS, was at to were isolated to the of A. M. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). exosomes were from of PBMC The were on and at for to the cells and at for to cellular were from the by at for The exosomal was in and in of the of exosomes released we the of an that is specific to these vesicles A. M. J. Cell 2002; PubMed Google Scholar). was as by A. M. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). 40 of the was in of of this was to on a and were to in a of and the in at was The data of cell and were following treatment conditions. were in and and inhibitor were at for and protein were determined. 40 of was to and for at and the proteins were by with a were to with and with an against HSP70 to by to were a and the protein were and on a system with To the intracellular and extracellular and cell of HSP70 from and heat-shocked a was used as by the were to the in were with for 1 which were to heat shock or by a i.e. cells were to for the of the The of the treatment in protein via the classical protein transport pathway was confirmed by the of tumor necrosis and in from PBMCs stimulated with either or and cellular cells were with or for 1 at were for at and in for the of the The cell were and the intracellular were a to the was on a at a as by the are as were the or repeated measures analysis of a was found were with was Stress and HSP70 in Cell examine the cellular of HSP70 we whether heat shock or 43 °C for 1 by at °C for h) the release of HSP70 with °C for heat shock in a of intracellular HSP70 with in and PBMCs response to conditions, HSP70 was in the extracellular from and PBMCs with (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google we a in the extracellular of HSP70 following heat shock in of the cell types and Furthermore, heat shock at 40 °C during febrile the release of HSP70 from PBMCs Interestingly, this was no in the intracellular of HSP70 To whether heat shock had the in the cell was determined. heat shock or 43 had no on release not with we Cell was than and we did not heat shock or 43 and conditions. These data demonstrate that heat shock the release of HSP70 from a variety of cell types and that this release is independent of cell HSP70 by of the or the of examine the of the common secretory pathway in HSP70 release PBMCs were for 1 with that protein transport from the to the and to either heat shock or as in the cell for the of the with had no affect on the intracellular HSP70 Furthermore, HSP70 release was unaffected by treatment following or heat shock or 43 treatment had no on release not These data findings (2Hightower L.E. Guidon Jr., P.T. J. Cell. Physiol. 1989; 138: 257-266Crossref PubMed Scopus (312) Google Scholar, 3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google and demonstrate that in PBMCs the common secretory pathway does not play a role in either basal or heat shock-induced HSP70 release. has been that lipid rafts play a role in heat shock-induced HSP70 release from cells (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). is the major lipid of lipid rafts and is for maintaining is a that cellular and a to E. L. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). to examine whether lipid rafts were in HSP70 release from PBMCs, PBMCs were with or for 1 h) and to either heat shock or as 1 of PBMCs were in cell for the of the treatment had affect on intracellular HSP70 at or 40 PBMCs to 43 °C for 1 in the of either or had a significantly intracellular HSP70 response Interestingly, we no affect of treatment on the release of HSP70 following either or heat shock Furthermore, the of intracellular HSP70 at 43 °C by no affect of was on HSP70 release following heat shock at 43 °C To confirm the of treatment we the cellular in PBMCs with either or for 1 or that were treatment was in with and a in cellular content by and with cells These data not a role for lipid rafts in either basal or heat shock-induced HSP70 release from Interestingly, treatment of PBMCs with for 1 in an in release with cells the by (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google cells were with for To examine the that the of treatment on HSP70 release by (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google may be the result of PBMCs were with for 1 and to either heat shock or at or 43 °C by at °C for in the by (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google cells were with for the of the of PBMCs to for in a in the release of with that this treatment was with heat shock in an in both the intracellular and extracellular HSP70 with °C and Interestingly, although treatment of PBMCs with for in a of intracellular the extracellular HSP70 was to PBMCs, in both and 43 °C These data demonstrate that of PBMCs to of in an in the extracellular HSP70 because of the in membrane and of HSP70 the extracellular to the of HSP70 from PBMCs in and has been that exosomes may provide a by which cells can actively release HSP70 G. L.E. Cell Stress Chaperones. PubMed Google Scholar). to examine whether exosomes play a role in HSP70 in both the basal and stress-induced we PBMCs to heat shock at 43 °C by at or at conditions. were purified to the of A. M. M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google and exosomal HSP70 content was by To confirm the of the a of were was in cell to of and were isolated as and the number of exosomes purified from the was via the of exosomal an specific to exosomes are released in the basal to we purified exosomes from PBMCs for or the the of the PBMC the the we i.e. the the number of exosomes secreted number of proteins are found in exosomes A. M.J. L. J. 2004; 164: Full Text Full Text PDF PubMed Scopus Google several HSPs, i.e. and To examine whether HSP70 is in exosomes, purified exosomes from PBMCs for or were with an HSP70 was in exosomes isolated at To examine whether exosomes play a role in the release of HSP70 from PBMCs were to heat shock or 43 °C for 1 by a at or °C for h) and exosomes were purified as We that heat shock may influence HSP70 release via exosomes in heat shock may the secretory rate, heat shock may HSP70 content and heat shock may HSP70 release via a of these Although we no in the number of exosomes secreted in response to heat shock the of HSP70 isolated exosomes was significantly in heat-shocked PBMCs with the of experiments we have a novel secretory pathway for HSP70 cellular Our data show that HSP70 is exosomes and that exosomes contribute to the release of HSP70 from PBMCs in both the basal and stress-induced state. Although heat shock had no on the secretory rate, exosomal HSP70 content was in heat-shocked data not a role for either the common cellular secretory pathway or lipid rafts in the of stress protein from PBMCs the extracellular with data (2Hightower L.E. Guidon Jr., P.T. J. Cell. Physiol. 1989; 138: 257-266Crossref PubMed Scopus (312) Google Scholar, 3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 5Hunter-Lavin C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google we found that the release of HSP70 from PBMCs by a than the common secretory pathway, because BFA, an inhibitor of protein transport via the did not influence HSP70 release. However, and in to (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google not a role for lipid rafts in HSP70 release from studies (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 5Hunter-Lavin C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google the of lipid rafts in HSP70 has been by cells with the lipid raft-disrupting The of in lipid is on to cellular an of lipid rafts to the maintenance of treatment of PBMCs with MBC, at that cellular had no on HSP70 release in either the basal or stress-induced state. of PBMCs with at for and to that used in the by (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google was we that the of on HSP70 release (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google may be a of However, demonstrate that HSP70 release from PBMCs with for was than in is that this in HSP70 release is as a of the in membrane in the of HSP70 from the the extracellular Interestingly, it was that the of HSP70 from PBMCs, and, it was suggested that lipid rafts are in this C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google Scholar). The of used to lipid i.e. cellular are in we have that 1 treatment in a small in PBMC Given that the lipid raft-disrupting of are as a of the of cellular and that the of used in the by C. Davies E.L. Bacelar M.M. Marshall M.J. Andrew S.M. Williams J.H. Biochem. Biophys. Res. Commun. 2004; 324: 511-517Crossref PubMed Scopus (199) Google was were not in this the that lipid rafts play a role in HSP70 release from PBMCs on these on findings, and of (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google it appear that exocytotic in cell types to facilitate the release of it is that exosomes are secreted from several and dendritic and K. M.J. W. J. Cell 2000; PubMed Google Scholar). Given the cellular of HSP70 PBMCs, cells (3Broquet A.H. Thomas G. Masliah J. Trugnan G. Bachelet M. J. Biol. Chem. 2003; 278: 21601-21606Abstract Full Text Full Text PDF PubMed Scopus (250) Google and I. Kislyakova K. Moskaliova O. Fridlanskaya I. Tytell M. Cheetham M. Margulis B. Brain Res. 2001; 914: 66-73Crossref PubMed Scopus (257) Google have all been to release studies be to confirm the of specific exocytotic in HSP70 release from cell Although heat shock at 40 °C for 1 in a in HSP70 heat shock at 43 °C for 1 in a in HSP70 with Interestingly, although we a in exosomal HSP70 content at 40 with heat shock at 43 °C in a small in exosomal HSP70 content with heat shock at 40 °C these data that although exosomes contribute to the release of HSP70 in the basal and stress-induced and 43 an is to play a role in HSP70 release at 43 the of studies we have release to the of cell Our data show that heat shock does not influence release. However, it is important to that cell does occur in a of cells the of the We that the in HSP70 release from PBMCs to 43 °C for 1 may be in to the in the intracellular HSP70 and the of HSP70 the extracellular via a studies be to examine this Our studies not identify the of HSP70 release via However, recent studies an important role for exosomes in the of immune exosomes purified from G. W. R. C.J. J. Med. PubMed Scopus Google and dendritic cells L. A. A. J. C. P. G. S. Nat. Med. PubMed Scopus Google are in and molecules and co-stimulatory molecules and These data have led to the that secreted exosomes may play a role in the of immune in C. L. S. Nat. Immunol. 2002; PubMed Scopus Google Scholar). Interestingly, the treatment of dendritic cells with HSP70 the of and co-stimulatory molecules (11Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1265) Google and H. A. E. 2003; PubMed Scopus Google Scholar). Given that HSP70 and purified exosomes have a of and HSP70 is secreted exosomes, we that the of secreted exosomes in be because of the of of the is is HSP70 to MVBs, the from which exosomes are are from the of and have a well role in the of proteins from the cell via with B. G. Cell Biol. 2004; PubMed Scopus Google Scholar). However, in to with are to exocytotic with the membrane and release for exosomes are the K. M.J. W. J. Cell 2000; PubMed Google Scholar). Interestingly, HSP70 is on the cell (14Gross C. Koelch W. DeMaio A. Arispe N. Multhoff G. J. Biol. Chem. 2003; 278: 41173-41181Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, G. L.E. Cell Stress Chaperones. PubMed Google the that cell surface-bound HSP70 is via and secreted via the of with the the of findings demonstrating that exosomes tumor L. A. A. J. C. P. G. S. Nat. Med. PubMed Scopus Google exosomes isolated from dendritic cells have been for the of C. L. S. Nat. Immunol. 2002; PubMed Scopus Google Scholar). proteins to the of immune and and and, as the of HSP70 exosomes may contribute to Given the of and data demonstrating that the HSP70 content of exosomes is significantly following a heat it is an that the of exosomes for in may be by cells to heat we show for the that exosomes contribute to HSP70 release from PBMCs in both the basal and stress-induced or 43 °C for 1 h) state. Furthermore, although lipid rafts contribute to the release of HSP70 from heat-shocked that lipid rafts not contribute to HSP70 release from Given the functions of both HSP70 and purified exosomes and that HSP70 is exosomes, we that exosomal HSP70 may for of the of We Andrew for with and