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Extracellular Heat Shock Proteins: A New Location, A New Function

De Maio, Antonio*†‡; Vazquez, Daniel§

doi: 10.1097/SHK.0b013e3182a185ab
Review Article
Editor's Choice

ABSTRACT The expression of heat shock proteins (HSPs) is a basic and well-conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Because these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that HSPs can also be present outside cells where they appear to display a function different than the well-understood chaperone role. Extracellular HSPs act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Because the majority of HSPs do not possess a secretory peptide signal, they are likely to be exported by a nonclassic secretory pathway. Different mechanisms have been proposed to explain the export of HSPs, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular HSPs appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular HSPs suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular HSPs.

*Center for Investigations of Health and Education Disparities; Division of Trauma, Surgical Critical Care and Burns, Department of Surgery; and Department of Neurosciences, School of Medicine University of California San Diego, La Jolla, California; and §Department of Surgery, South Florida University, Tampa, Florida

Received 19 Mar 2013; first review completed 2 Apr 2013; accepted in final form 20 Jun 2013

Address reprint requests to Antonio De Maio, PhD, University of California San Diego, 9500 Gilman Dr, #0739, La Jolla, CA 92093. E-mail:

This study was supported by a grant from the National Institute of General Medical Sciences R01 GM098455.

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When cells are challenged by adverse environmental conditions, they respond by the expression of a family of polypeptides called heat shock or stress proteins (HSPs). These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for insult resolution. Moreover, HSPs confer protection from subsequent insults, which has been coined stress tolerance (1). Thus, the expression of HSPs is a universal response to stress, which is fundamental to ensure survival. The cellular response to stress was discovered over 50 years ago by the Italian scientist Ferruccio Ritossa (2). By serendipity, he found that Drosophila cells exposed to temperatures higher than their normal growing conditions responded with an increase in chromosomal activity. Ritossa was puzzled by the observation, and as a good scientist, he repeated the “accident” several times, included the appropriate controls, and was able to validate his initial unintended observation. Thus, the “heat shock” response was born. Despite the excitement and tremendous importance of his finding, Ritossa had difficulties publishing his study, which was considered “a finding that lacks biological relevance” by the editor of a prestigious scientific journal (2). His findings were ultimately published in the journal Experientia (3), which does not exist anymore. Currently, we all recognize the tremendous importance and impact of the heat shock (HS) response discovery, which plays a major role in current biology (4). Today, we know that these proteins are expressed in response to a large variety of environmental, physiological, and clinical stresses, in addition to temperature increases (1). The transcriptional activity that Ritossa observed was correlated with the expression of HSPs by Tissieres et al. (5), 12 years after the initial observation. Several years after, a Drosophila gene encoding for an inducible HSP was cloned (6–8).

Subsequent studies revealed that polypeptides similar to the inducible HSPs were present in cells during normal physiological conditions, participating in several basic cellular processes, including protein folding, assembly of macromolecule complexes, and signal transduction (1, 9). Overall, HSPs, constitutive and stress-inducible, are classified according to their molecular mass into discrete families. As in many other fields, a variety of names have been given to the HSP family members, creating a vast confusion in the field. Therefore, a consensus nomenclature has been proposed (10). Each subgroup is composed of very similar proteins that differ in their subcellular localization, expression pattern, and minor amino acid sequence (Table 1). Heat shock proteins are better known as molecular chaperones because of their well-recognized ability to fold polypeptides.

Table 1

Table 1

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The main function of HSPs, such as the folding of newly synthesized polypeptides, is carried out in the cytosol. However, these proteins have also been found outside cells, which has become a very puzzling observation. Two major questions have emerged from this unusual observation: How do these proteins get there, and what is their function? The first publication regarding the presence of HSP outside cells was by Tytell et al. (11), who reported a “heat-shock–like protein” as a glia-axon transfer protein of the squid giant axon. Almost simultaneously, Hightower and Guidon (12) described the presence of HSP70 (HSPA1) in the extracellular medium, released by a process that could not be blocked by inhibitors of classic secretory pathways and was independent of the possible release after cell death. They also found that extracellular HSP70 was associated with fatty acids. These publications were against the traditional thinking because the common knowledge at the time indicated that HSPs were exclusively intracellular components. Therefore, Hightower and Guidon’s (12) findings were considered as potential artifacts, similarly to Ritossa’s discovery, and disregarded for many years. It was not until the year 2000 that the interest in extracellular HSPs was regained. Basu et al. (13) showed that extracellular HSP70, assumed to be released after tissue necrosis, modulated the immune system. Similarly, Asea et al. (14) showed that recombinant HSP70 could activate macrophages as measured by an increase in the level of intracellular calcium and the release of cytokines. The potential immune regulatory role of HSP70 was challenged because of the possibility that the effect was due to bacterial lipopolysaccharide contamination (15) or by other bacterial products (16). Several subsequent studies have demonstrated that, indeed, HSP70 is capable of activating cells of the immune system (17–19).

After finding extracellular HSP70 to be an immunomodulator, other HSPs were also detected outside cells. Grp78 (HSPA5), which is an endoplasmic reticulum (ER) resident homologous to HSP70, has been found outside cells (20, 21). Grp75 (HSPA9), also known as mortalin, is a mitochondrial chaperone protein that belongs to the HSP70 family. This protein is released in the extracellular medium upon complement treatment of cells (22). Intracellular HSP90, which is presented in two isoforms: α (HSPC2) and β (HSPC3), differs in its expression pattern, with HSP90β being abundant during normal physiological conditions and HSP90α expressed during stress (23). Initially, HSP90α was detected outside human hybridoma SH-76 cells as a growth-stimulating factor (24). Heat shock protein 90α was also identified as a secreted oxidative stress–induced factor (25). Heat shock protein 90β also has been reported secreted by osteosarcoma cells (26). A homologous form of cytosolic HSP90 is the ER resident Grp94 (HSPC4), which has also been found in the extracellular space (27, 28). The release of Grp94 can be explained because this protein is an ER resident that can be transported through the Golgi complex. As for HSP70, extracellular HSP90 was considered to be a product of cell death. However, further studies have shown that the protein could be secreted by an active mechanism (29, 30). Another HSP, HSP60 (HSPD1), which is located into the mitochondria and cytosol, has been found in plasma after some disease conditions (31, 32). In addition, circulating anti-HSP60 antibodies have been detected in a variety of pathological conditions (33). Small HSPs are also secreted by cells and modulate the immune system (34). In particular, HSP27 (HSPB1) has been noticed in serum of patients during pathological conditions (35, 36). One of the members of the large HSPs family, Grp170 (HSPH4), has also been detected outside cells (37).

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Since the detection of HSPs outside cells or in plasma, a discussion has been generated regarding whether the protein is released secondary to cell death or exported by an active mechanism that is nondependent on cell lysis. The initial studies of Basu et al. (13) proposed that the release of HSP70 was the product of cellular necrosis. On the other hand, Hightower and Guidon (12) demonstrated that the release of HSP70 was not due to cell death, which was later confirmed by Hunter-Lavin et al. (38). Therefore, the question is whether both pathways are possible. The major argument against the secretion of HSPs, particularly HSP70, by an active mechanism is the fact that this protein does not contain a consensus peptide signal for secretion via the classic ER-Golgi pathway. Moreover, typical inhibitors of the ER-Golgi pathway, such as brefeldin A, did not block the release of HSP70 from healthy cells (12). These observations suggest that HSP70 may be exported via an alternative mechanism, which has been named the nonclassic or unconventional secretory pathway (39). Several other proteins, such as interleukin 1α and β, high-mobility group box 1, galectin 1 and 3, and fibroblast growth factor 1, have also been suggested as released by this alternative pathway. However, a unifying mechanism for the release of proteins by the nonclassic secretory pathway has not yet emerged (39). Different scenarios have been proposed for the active export of HSP70 from the cytosol into the extracellular milieu. One suggested mechanism is export via the lysosome-endosome pathway, in which HSP70 is translocated into the lysosome lumen via ATP-binding cassette (ABC) transport-like system and further transported outside cells via the endocytic process (40). Other studies have suggested that HSP70 is released via secretory-like granules (41). However, the most accepted mechanism for the release of HSP70, as well as other HSPs, is via extracellular vesicles (ECVs) (42). These vesicles are derived from the plasma membrane by various processes including membrane blebbing, which is known as ectocytosis, and the derived vesicles coined ectosomes. Alternatively, ECVs could be produced by endocytosis, which causes the formation of multiple vesicle bodies that are released into the extracellular medium, producing vesicles with the same topology as the plasma membrane and are named exosomes (42, 43). Regardless of the origin of the vesicles, there is compelling evidence that HSP can be detected within these vesicles (Table 2). The main difference between these studies is the location of HSPs within the vesicles, which has been proposed to be in the lumen or the membrane of the ECVs. The presence of HSPs in the lumen of ECVs likely results from the protein being trapped into the intracellular space of the vesicle during its formation because these proteins are very abundant in the cytosol (42). The argument against this idea is that proteins in the vesicle lumen may not be able to interact directly with target cells. Thus, it should be assumed that ECVs may burst, releasing the cargo and allowing the HSPs, now free in solution, to interact with cells. Alternatively, it is possible that ECVs fuse with the membranes of cells, releasing the lumen content into the cytosol. A different idea is that HSPs are associated with the membranes of ECVs, which has been shown for several HSPs including HSP70 (18, 44, 45) and HSP60 (46, 47). The presence of HSPs on the membrane (surface) of ECVs is important because it explains the specific interaction with target cells, probable by a process mediated by surface receptors. Moreover, the possibility that multiple copies of the HSP are present within a single ECVs suggests that its biological activity may be enhanced dramatically. In fact, the concentration of HSP70 within ECVs has been calculated in the millimolar range, which is a concentration that is unlikely to be reached by soluble proteins (42). In support of this idea, it has been shown that the specific activity of HSP70 associated with ECVs is more than 250-fold more elevated in the process of activating macrophages than the protein in solution, free of membranes (18). Heat shock protein 90, which also lacks a secretory signal that justifies the transport via the ER-Golgi compartment, has been proposed as being exported via exosomes (48–50) or by direct translocation across the plasma membrane (30).

Table 2

Table 2

We have hypothesized that the interaction of HSP70 with membranes is the first step in the active secretion of this protein (42). The presence of HSP70 on the surface of cells has been well documented by several groups (42, 51). Indeed, the presence of other HSPs on the cell surface has been widely shown by many investigators under different physiological and pathological conditions (42). The constitutive member of the cytosolic HSP70 family, HSC70 (HSPA8), was observed on the bile duct membrane (52). Grp78, also known as BIP, has been detected on the cell surface (53). The mitochondria HSP60 has been reported on the surface of human T-cell lines (54) and endothelial cells (55, 56), as well as in the liver and spleen of mice (57). Heat shock protein 90 was initially detected on the cell surface of tumor cells (58) and subsequently in multipotential mesenchymal precursor cells (59), human neuroblastoma cells (60, 61), and human monocytes (62). Grp94 was observed on the plasma membrane of sarcoma cells (63). The initial observation for the interaction of HSP70 with lipids came from Hightower and Guidon’s (12) work that found extracellular HSP70 associated with fatty acids. Arispe and De Maio (64) showed that the addition of pure HSC70 (HSPA8) to an artificial lipid bilayer resulted in the detection of cationic conductance activity, which was very stable and regulated by nucleotides. These observations were extended to HSP70 (18). Further studies have demonstrated that both HSC70 and HSP70 display a high specificity for phosphatidylserine as compared with many other lipids (65, 66). In addition, HSP70 appeared to interact with phospholipid bis(monoacylglycero)phosphate (67), which is also negatively charged, suggesting that the interaction with lipids is electrostatic. A domain containing a stretch of positively charged amino acids at the C-terminus of HSC70 was found to mediate the interaction of this protein with endosomes, which was confirmed by site-directed mutagenesis (68).

Although the evidence for the active secretion of HSPs from “healthy” cells is notable, it cannot be discarded that, under other circumstances, HSPs are released into the extracellular medium or plasma after cell necrosis. Indeed, the concentration of HSP70 released after necrosis can be potentially very high. Basu et al. (13) have proposed that 1 g of necrotic tissue could release up to 200 µg of HSP70 in the extracellular medium. In this regard, expression of HSP70 has been observed after ischemia/reperfusion (I/R) injury, which resulted in a necrotic focus (69). Assuming that the average amount of HSP70 is in the order of 107 molecules per stressed cell (70), and there is an average of 3 × 108 hepatocytes per rat liver (71), a 1/30 hepatic I/R injury could potentially result in the release of 1014 molecules of HSP70, which is approximately 120 μg. If the total blood volume of a rat is approximately 8 mL, the circulating concentration of HSP70 could be as high as 15 μg/mL, in theory. This massive amount of HSP70 in circulation could have detrimental consequences. For example, HSP70 has been shown to induce cell death (65, 66), which has long been recognized as a secondary effect of HSP70 overexpression in cells (72).

The arguments presented above suggest that HSP70 could be released by two possible mechanisms: passive (necrosis) or active (secretion). For active secretion, HSP70 is likely associated with membranes within ECVs (membrane-bound), whereas for the passive process HSP70 is likely to be in solution or soluble (membrane-free). These possibilities were investigated in various in vivo models. Male rats that underwent regional hepatic I/R by total blockage of the blood supply to the median liver lobe (30 min), whereas blood flow was preserved in the rest of the liver (69), displayed a level of HSP70 in plasma in the order of 60.3 ng/mL, which was more elevated than in sham-operated rats (0.02 ng/mL) and nonmanipulated control animals (nondetectable). The plasma levels correlated with induction of HSP70 in the ischemic liver (detected by Western blotting). In contrast, no extracellular HSP70 was detected in urine samples taken after I/R. The presence of HSP70 in plasma was also investigated after total body thermal stress or HS at 42°C for 10 min as previously described (73). The level of HSP70 in the plasma samples of thermally stressed animals was 99.4 ng/mL as opposed to nonstressed controls (0.04 ng/mL, P = 0.0002, Student t test). Heat shock protein 70 was also detected in bronchoalveolar lavage fluid after HS (2.52 ng/mL), which was significantly higher than that in nonstressed rodents (0.99 ng/mL, P = 0.004, Student t test). After both conditions, I/R or HS, ECVs were isolated from plasma by differential centrifugation, and the content of HSP70 compared with supernatant of the sample corresponding to the membrane-free fraction. Extracellular vesicles derived from thermally stressed rats contained HSP70 (30.9 ng/mL), whereas no detectable HSP70 was found in samples from nonstressed rats (P = 0.01). The content of HSP70 in the ECVs fraction corresponded to 20% of the total concentration in the sample (Table 3). In contrast, all HSP70 detected in plasma samples after I/R was in the membrane-free fraction (Table 3). These observations suggest that extracellular HSP70 could be released by both, cell necrosis (I/R) and active secretion, the latter via ECVs.

Table 3

Table 3

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Clinical relevance of extracellular HSPs

The number of clinical conditions presenting HSP70 in plasma samples is increasing, including the top killers in the Western world: cancer, cardiovascular disease, diabetes, and trauma (42). Major attention has been directed at the effect of extracellular HSPs on the immune system in which they act as signaling molecules. Indeed, HSP70 has been shown to induce the activation of macrophages, monocytes, dendritic cells, and natural killer cells (42). Extracellular HSP70 has also been reported to increase microbicidal capacity and chemotaxis of neutrophils (74, 75) and phagocytosis (76) and modulate the response of monocytes to endotoxin (77). Moreover, extracellular HSP70 has been associated with both immunostimulatory and immunosuppressive activities (78). However, the mechanisms involved in the modulation of the response of cells of the immune system are still unknown. Extracellular HSPs have also been shown to modulate the activity of cells from other biological systems. Thus, extracellular HSP70 has been reported to affect cardiomyocyte contractile dysfunction (79) and increase the growth of tumor cells and resistance to apoptosis (80). Glial cells have also been reported to release HSP70, conferring protection to neighboring neurons (81). Extracellular HSPs have been observed to interact with amyloid β peptides, which are involved in the pathology of Alzheimer disease (82, 83). Moreover, HSP70 and HSP90 inhibited amyloid β aggregation in vitro conditions (84). Extracellular HSP70 has been revealed to protect Schwann from hydrogen peroxide–induced apoptosis (85). Exogenous α-crystallin, a small HSP, has been shown to induce neuroprotection during acute inflammation in mice (86). Extracellular HSP25 (HSPB1) also resulted in reduced cardiotoxicity induced by doxorubicin (87). Extracellular HSP90 has been reported to activate cancer cell motility (88), migration, and metastasis (60, 89). It has also been associated with neuronal motility (90) and wound healing (91, 92). Extracellular HSP90 has been shown to transport antigens from the outside to the cytosol, resulting in cross-presentation (93). In addition, extracellular HSP60 has been demonstrated to modulate the immune system (19, 94). Other studies have proposed that HSP60 induced apoptosis cardiac myocytes (95). Grp170 has also been demonstrated to modulate the innate immune system (37).

The presence of HSP70 in plasma has been identified as a risk predictor for acute coronary syndrome (96). Moreover, HSP70 has been detected in circulation after pregnancy (97), hypertension (98), acute infection (99), brain and spinal cord ischemia (100), and extenuating exercise (101–103). The presence of HSP70 in circulation has also been correlated with improved survival of critically ill patients (104–106). Circulating levels of HSP60 (107) or HSP70 (108, 109) or their antibodies have been proposed as a risk factor for coronary heart disease. In addition, extracellular levels of HSP70 have been correlated with severity and survival after chronic heart failure (110). Heat shock protein 60 has also been detected in saliva and serum of patients with type 2 diabetes mellitus (111), whereas HSP70 has been found during diabetic ketoacidosis (112). Both HSP70 and HSP60 have been detected in circulation after soft tissue trauma (113). The levels of HSP27 were increased in serum of patients with chronic pancreatitis and pancreatic carcinoma (35, 36).

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The presence of HSPs in the extracellular medium has been a very controversial topic, probably due to the reduced understanding of the mechanism for the release of these proteins that lack a consensus secretory peptide signal. However, evidence accumulated during the last several years has demonstrated that the presence of extracellular HSPs is not an artifact, but rather a novel biological event. Therefore, the role of extracellular HSPs appears to be at the level of signaling or cellular communication rather than at the traditional chaperone activity. Indeed, there is no evidence that extracellular HSPs could carry out their chaperone activity outside cells. Moreover, the requirement of cochaperones and other factors for their intracellular function is unlikely to be necessary for their extracellular role as signaling molecules. Certainty, it is possible that we will encounter multiple roles for extracellular HSPs, which will likely depend on the cellular milieu or type of target cell. A particularly interesting finding is the modulation of the immune system, which has been envisioned as a priming event or a novel form of cellular communication in the case of stress conditions. Indeed, the modulation of the immune system associated with ECVs bearing HSPs has been coined the Stress Observation System, which has the primary function of activating the immune system to avoid the propagation of the insults (42).

Another variant to add flavor to the role of extracellular HSPs is the observation that they could be derived from both healthy and necrotic cells. Moreover, extracellular HSPs can be detected in two “flavors”: membrane-bound or membrane-free, depending on their origin. It is likely that these two variants of extracellular HSPs could play alternative roles in the modulation of the immune system or other systems. Finally, the detection of extracellular HSPs, which is a very improbable observation that is against the conventional wisdom, is changing the biology of the stress response just as it was challenged 50 years ago by Ritossa’s discovery.

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The authors thank Molly Wofford for her impeccable editorial assistance and Dr. Lawrence Hightower for important historical information.

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1. De Maio A: Heat shock proteins: facts, thoughts, and dreams. Shock 11: 1–12, 1996.
2. Ritossa F: Discovery of the heat shock response. Cell Stress Chaperones 1: 97–98, 1996.
3. Ritossa F: A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18: 571–573, 1962.
4. De Maio A, Santoro MG, Tanguay RM, Hightower LE: Ferruccio Ritossa’s scientific legacy 50 years after his discovery of the heat shock response: a new view of biology, a new society, and a new journal. Cell Stress Chaperones 17: 139–143, 2012.
5. Tissieres A, Mitchell HK, Tracy UM: Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs. J Mol Biol 84: 389–398, 1974.
6. Schedl P, Artavanis-Tsakonas S, Steward R, Gehring WJ, Mirault ME, Goldschmidt-Clermont M, Moran L, Tissieres A: Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein. Cell 14: 921–929, 1978.
7. Livak KJ, Freund R, Schweber M, Wensink PC, Meselson M: Sequence organization and transcription at two heat shock loci in Drosophila. Proc Natl Acad Sci U S A 75: 5613–5617, 1978.
8. Craig EA, McCarthy BJ, Wadsworth SC: Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster. Cell 16: 575–588, 1979.
9. Hartl FU, Hayer-Hartl M: Converging concepts of protein folding in vitro and in vivo. Nature Structural Mol Biol 16: 574–581, 2009.
10. Kampinga HH, Hageman J, Vos MJ, Kubot H, Tanguay RM, Bruford EA, Cheetham ME, Chen B, Hightower LE: Guidelines for the nomenclature of the human heat shock proteins. Cell Stress Chaperones 14: 105–111, 2009.
11. Tytell M, Greenberg SG, Lasek RJ: Heat shock-like protein is transferred from glia to axon. Brain Res 363: 161–164, 1986.
12. Hightower LE, Guidon PT: Selective release from cultured mammalian cells of heat-shock (stress) proteins that resemble glia-axon transfer proteins. J Cell Physiol 138: 257–266, 1989.
13. Basu S, Binder RJ, Suto R, Anderson KM, Srivastava PK: Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Intl Immunol 12: 1539–1546, 2000.
14. Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nature Med 6: 435–442, 2000.
15. Gao B, Tsan MF: Endotoxin contamination in recombinant human heat shock protein 70 (HSP70) preparation is responsible for the induction of tumor necrosis factor alpha release by murine macrophages. J Biol Chem 278: 174–179, 2003.
16. Bendz H, Marincek BC, Momburg F, Ellwart JW, Issels RD, Nelson PJ, Noessner E: Calcium signaling in dendritic cells by human or mycobacterial HSP70 is caused by contamination and is not required for HSP70-mediated enhancement of cross-presentation. J Biol Chem 283: 26477–26483, 2008.
17. Zheng H, Nagaraja GM, Kaur P, Asea EE, Asea A: Chaperokine function of recombinant HSP72 produced in insect cells using a baculovirus expression system is retained. J Biol Chem 285: 349–356, 2010.
18. Vega VL, Rodriguez-Silva M, Frey T, Gehrmann M, Diaz JC, Steinem C, Multhoff G, Arispe N, De Maio A: HSP70 translocates into the plasma membrane after stress and is released into the extracellular environment in a membrane-associated form that activates macrophages. J Immunol 180: 4299–4307, 2008.
19. Henderson B, Pockley AG: Molecular chaperones and protein-folding catalysts as intercellular signaling regulators in immunity and inflammation. J Leukoc Biol 88: 445–462, 2010.
20. Delpino A, Castelli M: The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation. Biosci Rep 22: 407–420, 2002.
21. Kern J, Untergasser G, Zenzmaier C, Sarg B, Gastl B, Gunsilius E, Steurer M: GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib. Blood 114: 3960–3967, 2009.
22. Pilzer D, Fishelson Z: Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis. Int Immunol 17: 1239–1248, 2005.
23. De Maio A: HSP90. In: Creighton TE, (ed.): Wiley Encyclopedia of Molecular Medicine. Vol. 5. John Wiley & Sons, Inc, pp 1675–1676, 2002.
24. Kuroita T, Tachibana H, Ohashi H, Shirahata S, Murakami H: Growth stimulating activity of heat shock protein 90 alpha to lymphoid cell lines in serum-free medium. Cytotechnology 8: 109–117, 1992.
25. Liao DF, Jin ZG, Baas AS, Daum G, Gygi SP, Aebersold R, Berk BC: Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells. J Biol Chem 275: 189–196, 2000.
26. Suzuki S, Kulkarni AB: Extracellular heat shock protein HSP90beta secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-beta1. Biochem Biophys Res Commun 398: 525–531, 2010.
27. Booth C, Koch GL: Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell 59: 729–737, 1989.
28. Evdokimovskaya Y, Skarga Y, Vrublevskaya V, Morenkov O: Release of the glucose-regulated protein 94 by baby hamster kidney cells. Cell Biochem Funct 30: 558–562, 2012.
29. Tsutsumi S, Neckers L: Extracellular heat shock protein 90: a role for a molecular chaperone in cell motility and cancer metastasis. Cancer Sci 98: 1536–1539, 2007.
30. Li W, Sahu D, Tsen F: Secreted heat shock protein-90 (HSP90) in wound healing and cancer. Biochim Biophys Acta 1823: 730–741, 2012.
31. Pockley AG, Wu R, Lemne C, Kiessling R, de Faire U, Frostegard J: Circulating heat shock protein 60 is associated with early cardiovascular disease. Hypertension 36: 303–307, 2000.
32. Lewthwaite J, Owen N, Coates A, Henderson B, Steptoe A: Circulating human heat shock protein 60 in the plasma of British civil servants: relationship to physiological and psychosocial stress. Circulation 106: 196–201, 2002.
33. Henderson B, Pockley AG: Proteotoxic stress and circulating cell stress proteins in the cardiovascular diseases. Cell Stress Chaperones 17: 303–311, 2012.
34. van Noort JM, Bsibsi M, Nacken P, Gerritsen WH, Amor S: The link between small heat shock proteins and the immune system. Intl J Biochem Cell Biol 44: 1670–1679, 2012.
35. Liao WC, Wu MS, Wang HP, Tien YW, Lin JT: Serum heat shock protein 27 is increased in chronic pancreatitis and pancreatic carcinoma. Pancreas 38: 422–426, 2009.
36. Melle C, Ernst G, Escher N, Hartmann D, Schimmel B, Bleul A, Thieme H, Kaufmann R, Felix K, Friess HM, et al.: Protein profiling of microdissected pancreas carcinoma and identification of HSP27 as a potential serum marker. Clin Chem 53: 629–635, 2007.
37. Zuo D, Yu X, Guo C, Yi H, Chien X, Conrad DH, Guo TL, Chen Z, Fisher PB, Subjeck JR, et al.: Molecular chaperoning by glucose-regulated protein 170 in the extracellular milieu promotes macrophage-mediated pathogen sensing and innate immunity. FASEB J 26: 1493–1505, 2012.
38. Hunter-Lavin C, Davies EL, Bacelar MM, Marshall MJ, Andrew SM, Williams JH: HSP70 release from peripheral blood mononuclear cells. Biochem Biophys Res Comm 324: 511–517, 2004.
39. Nickel W, Seedorf M: Unconventional mechanisms of protein transport to the cell surface of eukaryotic cells. Ann Rev Cell Dev Biol 24: 287–308, 2008.
40. Mambula SS, Calderwood SK: Heat shock protein 70 is secreted from tumor cells by a nonclassical pathway involving lysosomal endosomes. J Immunol 177: 7849–7857, 2006.
41. Evdonin AL, Martynova MG, Bystrova OA, Guzhova I, Margulis B, Medvedeva N: The release of HSP70 from A431 carcinoma cells is mediated by secretory-like granules. Eur J Cell Biol 85: 443–455, 2006.
42. De Maio A: Extracellular heat shock proteins, cellular export vesicles, and the Stress Observation System: a form of communication during injury, infection, and cell damage. Cell Stress Chaperones 16: 235–249, 2011.
43. Thery C, Ostrowski M, Segura E: Membrane vesicles as conveyors of immune responses. Nature Rev 9: 581–593, 2009.
44. Gastpar R, Gehrmann M, Bausero MA, Asea A, Gross C, Schroeder JA, Multhoff G: Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells. Cancer Res 65: 5238–5247, 2005.
45. Chalmin F, Ladoire S, Mignot G, Vincent J, Bruchard M, Remy-Martin J, Boireau W, Rouleau A, Simon B, Lanneau D, et al.: Membrane-associated HSP72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells. J Clin Invest 120: 457–471, 2010.
46. Gupta S, Knowlton AA: HSP60 trafficking in adult cardiac myocytes: role of the exosomal pathway. Am J Physiol Heart Circ Physiol 292: H3052–H3056, 2007.
47. Merendino AM, Bucchieri F, Campanella C, Marciano V, Ribbene A, David S, Zummo G, Burgio G, Corona DF, Conway de Macario E, et al.: HSP60 is actively secreted by human tumor cells. PLoS One 5: e9247, 2010.
48. Clayton A, Turkes A, Navabi H, Mason MD, Tabi Z: Induction of heat shock proteins in B-cell exosomes. J Cell Sci 118: 3631–3638, 2005.
49. Cheng CF, Fan J, Fedesco M, Guan S, Li Y, Bandyopadhyay B, Bright AM, Yerushalmi D, Liang M, Chen M, et al.: Transforming growth factor alpha (TGFalpha)–stimulated secretion of HSP90alpha: using the receptor LRP-1/CD91 to promote human skin cell migration against a TGFbeta-rich environment during wound healing. Mol Cell Biol 28: 3344–3348, 2008.
50. McCready J, Sims JD, Chan D, Jay DG: Secretion of extracellular HSP90alpha via exosomes increases cancer cell motility: a role for plasminogen activation. BMC Cancer 10: 294, 2010.
51. Multhoff G, Hightower LE: Cell surface expression of heat shock proteins and the immune response. Cell Stress Chaperones 1: 167–176, 1996.
52. Mills DR, Haskell MD, Callanan HM, Flanagan DL, Brilliant KE, Yang D, Hixson DC: Monoclonal antibody to novel cell surface epitope on HSC70 promotes morphogenesis of bile ducts in newborn rat liver. Cell Stress Chaperones 15: 39–53, 2010.
53. Zhang Y, Liu R, Ni M, Gill P, Lee AS: Cell surface relocalization of the endoplasmic reticulum chaperone and unfolded protein response regulator GRP78/BiP. J Biol Chem 285: 15065–15075, 2010.
54. Soltys BJ, Gupta RS: Cell surface localization of the 60 kDa heat shock chaperonin protein (HSP60) in mammalian cells. Cell Biol Intl 21: 315–320, 1997.
55. Xu Q, Schett G, Seitz CS, Hu Y, Gupta RS, Wick G: Surface staining and cytotoxic activity of heat-shock protein 60. Circ Res 75: 1078–1085, 1994.
56. Pfister G, Stroh CM, Perschinka H, Kind M, Knoflach M, Hinterdorfer P, Wick G: Detection of HSP60 on the membrane surface of stressed human endothelial cells by atomic force and confocal microscopy. J Cell Sci 118: 1587–1594, 2005.
57. Belles C, Kuhl A, Nosheny R, Carding SR: Plasma membrane expression of heat shock protein 60 in vivo in response to infection. Infect Immun 67: 419–4200, 1999.
58. Ferrarini M, Heltai S, Zocchi MR, Rugarli C: Unusual expression and localization of heat-shock proteins in human tumor cells. Intl J Cancer 51: 613–619, 1992.
59. Gronthos S, Zannettino AC, Graves SE, Ohta S, Hay SJ, Simmons PJ: Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res 14: 47–56, 1999.
60. Tsutsumi S, Neckers L: Extracellular heat shock protein 90: a role for a molecular chaperone in cell motility and cancer metastasis. Cancer Sci 98: 1536–1539, 2007.
61. Cid C, Regidor I, Poveda PD, Alcazar A: Expression of heat shock protein 90 at the cell surface in human neuroblastoma cells. Cell Stress Chaperones 14: 321–327, 2009.
62. Cecchini P, Tavano R, Polverino de Laureto P, Franzoso S, Mazzon C, Montanarri P, Papini E: The soluble recombinant Neisseria meningitidis adhesin NadA(Delta351-405) stimulates human monocytes by binding to extracellular HSP90. PLoS One 6: e25089, 2011.
63. Altmeyer A, Maki RG, Feldweg AM, Heike M, Protopopov VP, Masur SK, Srivastava PK: Tumor-specific cell surface expression of the-KDEL containing, endoplasmic reticular heat shock protein gp96. Int J Cancer 69: 340–349, 1996.
64. Arispe N, De Maio A: ATP and ADP modulate a cation channel formed by HSC70 in acidic phospholipid membranes. J Biol Chem 275: 30839–30843, 2000.
65. Arispe N, Doh M, Simakova O, Kurganov B, De Maio A: HSC70 and HSP70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability. FASEB J 18: 1636–1645, 2004.
66. Schilling D, Gehrmann M, Steinem C, De Maio A, Pockley AG, Abend M, Molls M, Multhoff G: Binding of heat shock protein 70 to extracellular phosphatidylserine promotes killing of normoxic and hypoxic tumor cells. FASEB J 23: 2467–2477, 2009.
67. Kirkegaard T, Roth AG, Petersen NH, Mahalka AK, Olsen OD, Moilanen I, Zylicz A, Knudsen J, Sandhoff K, Arenz C, et al.: HSP70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology. Nature 463: 549–553, 2010.
68. Sahu D, Zhao Z, Ulmer TS, Conti P, Woodley DT, Li W: A potentially common peptide target in secreted heat shock protein-90alpha for hypoxia-inducible factor-1alpha–positive tumors. Mol Biol Cell 23: 602–613, 2011.
69. Gingalewski C, Theodorakis NG, Yang J, Beck SC, De Maio A: Distinct expression of heat shock and acute phase genes during regional hepatic ischemia-reperfusion. Am J Physiol 271: R634–R640, 1996.
70. Cornivelli L, Zeidan Q, De Maio A: HSP70 interacts with ribosomal subunits of thermotolerant cells. Shock 20: 320–325, 2003.
71. Michalopoulos GK, DeFrances MC: Liver regeneration. Science 276: 60–66, 1997.
72. Feder JH, Rossi JM, Solomon J, Solomon N, Lindquist S: The consequences of expressing HSP70 in Drosophila cells at normal temperatures. Genes Dev 6: 1402–1413, 1992.
73. De Maio A, Beck SC, Buchman TG: Induction of translational thermotolerance in livers of thermally stressed rats. Eur J Biochem 218: 413–420, 1993.
74. Ortega E, Giraldo E, Hinchado MD, Martinez M, Ibanez S, Cidoncha A, Collazos ME, Garcia JJ: Role of HSP72 and norepinephrine in the moderate exercise-induced stimulation of neutrophils’ microbicide capacity. Eur J Appl Physiol 98: 250–255, 2006.
75. Ortega E, Hinchado MD, Martin-Cordero L, Asea A: The effect of stress-inducible extracellular HSP72 on human neutrophil chemotaxis: a role during acute intense exercise. Stress 12: 240–249, 2009.
76. Wang R, Kovalchin JT, Muhlenkamp P, Chandawarkar RY: Exogenous heat shock protein 70 binds macrophage lipid raft microdomain and stimulates phagocytosis, processing, and MHC-II presentation of antigens. Blood 107: 1636–1642, 2006.
77. Abboud PA, Lahni PM, Page K, Giuliano JS Jr, Harmon K, Dunsmore KE, Wong HR, Wheeler DS: The role of endogenously produced extracellular HSP72 in mononuclear cell reprogramming. Shock 30: 285–292, 2008.
78. Pockley AG, Muthana M, Calderwood SK: The dual immunoregulatory roles of stress proteins. Trends Biochem Sci 33: 71–79, 2008.
79. Mathur S, Walley KR, Wang Y, Indrambarya T, Boyd JH: Extracellular heat shock protein 70 induces cardiomyocyte inflammation and contractile dysfunction via TLR2. Circ J 75: 2445–2452, 2011.
80. Wu FH, Yuan Y, Li D, Liao SJ, Yan B, Wei JJ, Zhou YH, Zhu JH, Zhang GM, Feng ZH: Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance. Cancer Lett 317: 157–164, 2012.
81. Guzhova I, Kislyakova K, Moskaliova O, Fridlanskaya I, Tytell M, Cheetham M, Margulis B: In vitro studies show that HSP70 can be released by glia and that exogenous HSP70 can enhance neuronal stress tolerance. Brain Res 914: 66–73, 2001.
82. Carnini A, Scott LO, Ahrendt E, Proft J, Winkfein RJ, Kim SW, Colicos MA, Braun JE: Cell line specific modulation of extracellular abeta42 by HSP40. PLoS One 7: e37755, 2012.
83. Bruinsma IB, de Jager M, Carrano A, Versleijen AA, Veerhuis R, Boelens W, Rosemuller AJ, de Waal RM, Verbeek MM: Small heat shock proteins induce a cerebral inflammatory reaction. J Neurosci 31: 11992–12000, 2011.
84. Evans CG, Wisen S, Gestwicki JE: Heat shock proteins 70 and 90 inhibit early stages of amyloid beta-(1-42) aggregation in vitro. J Biol Chem 281: 33182–33191, 2006.
85. Luo X, Tao L, Lin P, Mo X, Chen H: Extracellular heat shock protein 72 protects Schwann cells from hydrogen peroxide-induced apoptosis. J Neurosci Res 90: 1261–1269, 2012.
86. Masilamoni JG, Vignesh S, Kirubagaran R, Jesudason EP, Jayakumar R: The neuroprotective efficacy of alpha-crystallin against acute inflammation in mice. Brain Res Bull 67: 235–241, 2005.
87. Krishnamurthy K, Kanagasabai R, Druhan LJ, Ilangovan G: Heat shock protein 25-enriched plasma transfusion preconditions the heart against doxorubicin-induced dilated cardiomyopathy in mice. J Pharmacol Exp Ther 341: 829–839, 2012.
88. McCready J, Sims JD, Chan D, Jay DG: Secretion of extracellular HSP90alpha via exosomes increases cancer cell motility: a role for plasminogen activation. BMC Cancer 10: 294, 2010.
89. Becker B, Multhoff G, Farkas B, Wild PJ, Landthaler M, Stolz W, Vogt T: Induction of HSP90 protein expression in malignant melanomas and melanoma metastases. Exp Dermatol 13: 27–32, 2004.
90. Sidera K, Samiotaki M, Yfanti E, Panayotou G, Patsavoudi E: Involvement of cell surface HSP90 in cell migration reveals a novel role in the developing nervous system. J Biol Chem 279: 45379–45388, 2004.
91. Li W, Li Y, Guan S, Fan J, Cheng CF, Bright AM, Chinn C, Chen M, Woodley DT: Extracellular heat shock protein-90alpha: linking hypoxia to skin cell motility and wound healing. EMBO J 26: 1221–1233, 2007.
92. Song X, Luo Y: The regulatory mechanism of HSP90alpha secretion from endothelial cells and its role in angiogenesis during wound healing. Biochem. Biophys. Res Commun 398: 111–117, 2010.
93. Oura J, Tamura Y, Kamiguchi K, Kutomi G, Sahara H, Torigoe T, Himi T, Sato N: Extracellular heat shock protein 90 plays a role in translocating chaperoned antigen from endosome to proteasome for generating antigenic peptide to be cross-presented by dendritic cells. Int Immunol 23: 223–237, 2011.
94. Habich C, Burkart V: Heat shock protein 60: regulatory role on innate immune cells. Cell Mol Life Sci 64: 742–751, 2007.
95. Kim SC, Stice JP, Chen L, Jung JS, Gupta S, Wang Y, Baumgarten G, Trial J, Knowlton AA: Extracellular heat shock protein 60, cardiac myocytes, and apoptosis. Circ Res 105: 1186–1195, 2009.
96. Zhang X, Xu Z, Zhou L, Chen Y, He M, Cheng L, Hu FB, Tanguay RM, Wu T: Plasma levels of HSP70 and anti-HSP70 antibody predict risk of acute coronary syndrome. Cell Stress Chaperones 15: 675–686, 2010.
97. Molvarec A, Tamasi L, Losonczy G, Madach K, Prohaszka Z, Rigo J Jr: Circulating heat shock protein 70 (HSPA1A) in normal and pathological pregnancies. Cell Stress Chaperones 15: 237–247, 2010.
98. Molvarec A, Prohaszka Z, Nagy B, Szaley J, Fust G, Karadi I, Rigo J Jr: Association of elevated serum heat-shock protein 70 concentration with transient hypertension of pregnancy, preeclampsia and superimposed preeclampsia: a case-control study. J Hum Hypertens 20: 780–786, 2006.
99. Njemini R, Lambert M, Demanet C, Mets T: Elevated serum heat-shock protein 70 levels in patients with acute infection: use of an optimized enzyme-linked immunosorbent assay. Scan. J Immunol 58: 664–669, 2003.
100. Hecker JG, McGarvey M: Heat shock proteins as biomarkers for the rapid detection of brain and spinal cord ischemia: a review and comparison to other methods of detection in thoracic aneurysm repair. Cell Stress Chaperones 16: 119–131, 2011.
101. Walsh RC, Koukoulas I, Garnham A, Moseley PL, Hargreaves M, Febbraio MA: Exercise increases serum HSP72 in humans. Cell Stress Chaperones 6: 386–393, 2001.
102. Febbraio MA, Ott P, Nielsen HB, Steensberg A, Keller C, Krustup P, Secher NH, Pedersen BK: Exercise induces hepatosplanchnic release of heat shock protein 72 in humans. J Physiol 544: 957–962, 2002.
103. Periard JD, Ruell P, Caillaud C, Thompson MW: Plasma HSP72 (HSPA1A) and HSP27 (HSPB1) expression under heat stress: influence of exercise intensity. Cell Stress Chaperones 17: 375–383, 2012.
104. Pittet JF, Lee H, Morabito D, Howard M, Welch W, Mackersie R: Serum levels of HSP 72 measured early after trauma correlate with survival. J Trauma 52: 611–617, 2002.
105. Ziegler TR, Ogden LG, Singleton KD, Luo M, Fernandez-Esstivariz C, Griffith D, Galloway J, Wischmeyer P: Parenteral glutamine increases serum heat shock protein 70 in critically ill patients. Intensive Care Med 31: 1079–1086, 2005.
106. Ganter MT, Ware LB, Howard M, Roux J, Gartland B, Matthay MA, Fleshner M, Pittet JF: Extracellular heat shock protein 72 is a marker of the stress protein response in acute lung injury. Am J Physiol Lung Cell Mol Physiol 291: L354–L631, 2006.
107. Zhang X, He M, Cheng L, Chen Y, Zhou L, Zeng H, Pockley AG, Hu FB, Wu T: Elevated heat shock protein 60 levels are associated with higher risk of coronary heart disease in Chinese. Circulation 118: 2687–2693, 2008.
108. Zhang X, Xu Z, Zhou L, Chen Y, He M, Cheng L, Hu FB, Tanguay RM, Wu T: Plasma levels of HSP70 and anti-HSP70 antibody predict risk of acute coronary syndrome. Cell Stress Chaperones 15: 675–686, 2010.
109. Zhu J, Quyyumi AA, Wu H, Csako G, Rott D, Zalles-Ganley A, Ogunmakinwa J, Halcox J, Epstein SE: Increased serum levels of heat shock protein 70 are associated with low risk of coronary artery disease. Arterioscler Thromb Vasc Biol 23: 1055–1059, 2003.
110. Genth-Zotz S, Bolger AP, Kalra PR, von Haehling S, Doehner W, Coats A, Volk HD, Anker SD: Heat shock protein 70 in patients with chronic heart failure: relation to disease severity and survival. Int J Cardiol 96: 397–401, 2004.
111. Yuan J, Dunn P, Martinus RD: Detection of HSP60 in saliva and serum from type 2 diabetic and non-diabetic control subjects. Cell Stress Chaperones 16: 689–693, 2011.
112. Oglesbee MJ, Herdman AV, Passmore GG, Hoffman WH: Diabetic ketoacidosis increases extracellular levels of the major inducible 70-kDa heat shock protein. Clin Biochem 38: 900–904, 2005.
113. Flohé SB, Bangen JM, Flohé S, Agrawal H, Bergmann K, Schade FU: Origin of immunomodulation after soft tissue trauma: potential involvement of extracellular heat-shock proteins. Shock 27: 494–502, 2007.

Ectosomes; exosomes; immune cell activation; nonclassic secretory pathway; stress; vesicles; cellular communication

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