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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jan 4, 2007.
Published in final edited form as:
PMCID: PMC1762097
NIHMSID: NIHMS15242

Alternative Mechanism by which IFN-γ Enhances Tumor Recognition: Active Release of Heat Shock Protein 721

Abstract

IFN-γ exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-γ enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-γ triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-γ-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl β-cyclodextrin completely abrogated IFN-γ-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-γ promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.

Heat shock proteins (Hsps)3 are highly conserved proteins found in all prokaryotes and eukaryotes. In response to a wide variety of stressful stimuli, there is a marked increase in total Hsp synthesis (1), known as the cellular stress response, which is designed to enhance the ability of the cell to cope with increasing concentrations of unfolded or denatured proteins. Of all Hsps, the Hsp70 family constitute the most conserved and best studied class. This family consists of the constitutively expressed Hsp70 (Hsc70; 73 kDa), the stress-inducible Hsp70 (Hsp70; 72 kDa), the mitochondrial Hsp70 (Hsp75; 75 kDa), and the endoplasmic reticulum (ER) Hsp70 (Grp78; 78 kDa). From its original description as an intracellular molecular chaperone, increasing evidence suggests additional functions of Hsp72 that is dependent on the localization and type of cells/tissues this unique protein comes in contact.

The “danger theory” postulates that the host releases endogenous signals capable of stimulating immunity and that immune activation involves danger/nondanger molecular recognition schemas and suggests that innate immune cells are activated by danger signals that are derived from stressed or damaged self-proteins (2, 3). Accumulating evidence indicates that extracellular Hsp72 fulfils the criteria of an endogenous danger signal; when admixed with APCs, Hsp72 possesses potent cytokine activity, with the ability to bind with high affinity to the plasma membrane, elicit a rapid intracellular Ca2+ ([Ca2+]i) flux, activate NF-κB nuclear translocation, augment the expression and release of proinflammatory cytokines (4-7), induce NO release (8), up-regulate costimulatory molecule expression (9), and induce dendritic cell (DC) maturation (9-11). On a molecular level, Hsp72-induced proinflammatory cytokine production is mediated via the MyD88/IL-1R-associated kinase/NF-κB signal transduction pathway and uses both TLR2 (receptor for Gram-positive bacteria) and TLR4 (receptor for Gram-negative bacteria) to transduce its proinflammatory signal in a CD14-dependent fashion (9, 12, 13). On a cellular level, human glioma cells export Hsp70 into culture medium under normal and stressed conditions (14). In addition, neuroblastoma cells take up biotinylated Hsc/Hsp70 and exhibit a thermo-tolerance phenotype when exposed to lethal heat stress (44°C) and to staurosporine-induced apoptosis, suggesting a mechanism by which extracellular Hsp70 might affect neuronal function (14). On a tissue/organ level, recent studies have begun to address the postulate that intracellular Hsp72 is actively released into the circulation to signal impending danger (15-17).

IFNs are a family of natural glycoproteins that share antiviral, immunomodulatory, and antiproliferative effector functions. IFN-γ is produced predominately by Th cells, and CTL and NK cells play an important role in proinflammatory responses (18). IFN-γ exhibits differential effects depending on the target and can induce cellular activation and enhances survival or mediates cell death via activation of apoptotic pathways. In the clinic, IFN-γ exhibits antitumor effects against a variety of cancers, including lymphomas, melanomas, and multiple myelomas (19). In patients with breast cancer, encouraging results have been demonstrated when IFN-γ is used in combination with ILs (20). Treatment of tumor cells with IFN-γ in vitro induces cell cycle arrest at low doses or apoptosis at higher doses. Indeed, a low concentration of IFN-γ sensitizes tumor apoptosis induced by CD95L (Fas ligand) (21, 22), via the elevation of caspase-8 (23, 24). However, tumors have developed numerous ways in which to evade recognition by immune effector cells, including the down-regulation of surface recognition structures such as MHC molecules, costimulatory molecules, adhesion molecules, and/or release of immunosuppressive mediators, including IL-10, TGF-β, and PGE2, which inhibit T lymphocyte, NK cell, and DC functions (25).

In this study, we demonstrate a hitherto unknown mechanism by which that IFN-γ induces tumor recognition. We show that treatment of tumors with IFN-γ at concentrations that do not significantly induce cell death activates the cellular stress response, which in turn results in an increase in intracellular Hsp72 expression and its consequent release into the extracellular milieu. Furthermore, we show that IFN-γ-induced Hsp72 release occurs via a mechanism independent of the classical protein transport pathway and requires intact lipid raft formation. In addition, we show that Hsp72 is released within exosomes and that the released Hsp72-exosomes enhance cytokine release and up-regulate costimulatory molecule expression by DCs. The biological significance of these findings is that here we present an additional mechanism by which IFN-γ enhances tumor surveillance that is dependent on the active release of Hsp72 from tumors.

Materials and Methods

Reagents

Recombinant human and mouse IFN-γ, IL-10, TGF-β1, and neutralizing anti-IFN-γ mAb were purchased from R&D Systems. Samples are routinely monitored for endotoxin contamination, and endotoxin levels are reported as <0.1 EU/1 μg protein. Anti-Hsp72 mAb (StressGen Biotechnologies). Monensin, brefeldin A, tunicamycin, thapsigargin, BAPTA-AM, EGTA, and methyl β-cyclodextrin (MβD) (Sigma-Aldrich).

Cell lines and culture conditions

The K562 cell line, derived from a human erythroleukemia patient, was purchased from American Type Cell Culture and maintained in culture with RPMI 1640 culture medium (Invitrogen Life Technologies) supplemented with 5% FCS (BioWhittaker), 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml amphotericin B (ICN), 5.6 mM HEPES (Sigma-Aldrich), and 23.8 mM NaHCO3 referred to further as a complete medium. To avoid stress induced by cells overgrowth, cultures were maintained in a density of 2 × 105 cells/ml and passaged with fresh complete medium every 48 h. Cell viability was assessed using trypan blue exclusion test and routinely found to contain <5% dead cells. 4T1 breast carcinoma cells (a kind gift from Dr. C. Nicchitta, Duke University Medical Center, Durham, NC) are a 6-thioguanine resistant cell line selected from the 410.4 tumor without mutagen treatment. 4T1 cells were maintained on RPMI 1640 medium with 2 mM l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10% FBS at 37°C in a humidified incubator with 5% CO2 atmosphere.

Stable transfection

The C-terminal truncation mutant DN-HSF1 (mut) and empty vector (ev) was a gift from Dr. S. K. Calderwood (Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA) as described previously (26). Stable transfection was conducted using liposomal transfection reagent DOTAP® (Roche Diagnostics). Briefly, cells were seeded in 6-well tissue culture plates at 2–5 × 105/well and transfected according to the manufacturer's instructions. Cells were maintained in RPMI 1640 medium containing 10% FCS for 24 h. Neomycin (G418) was added to a concentration of 300 ng/ml until surviving cells reached confluence. Surviving clones were then selected and amplified.

Measurement of lactate dehydrogenase (LDH) release

LDH is a stable cytosolic enzyme that is released upon cell lysis. LDH released into cell culture medium by dead cells and total LDH contained in living cells was measured using the CytoTox 96 Nonradioactive Cytotoxicity Assay, according to the manufacturer's instructions (Promega) and as described previously (27). Briefly, after various treatment protocols, culture medium (500 μl) was removed, and the remaining cells lysed by adding 500 μl of 5% Triton X-100 solution. After 30 min at room temperature, cell lysate was recovered and incubated for an additional 30 min in the dark with a buffer containing NAD+, lactate, and tetrazolium. LDH converts lactate to pyruvate, generating NADH, which reduces tetrazolium (yellow) to formazan (red), which is detected by fluorescence (490 nm). LDH release, a marker for cell death, was expressed as a percentage of the LDH in the medium over the total LDH (lysate).

Protein separation and Western blot analysis

Following various treatment protocols, cells were washed once with complete medium, centrifuged, and pellets were lysed with 100 μl of lysing buffer containing a mixture of proteases inhibitors (antipain, bestain, chymostatin, E-64, pepstatin, phosphoramidon, pefabloc, EDTA, and aprotinin; Complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics). Cells were then incubated for 30 min on ice and sonicated (Brandson 1510) for 15 min. The cells suspension was passed through a 26-gauge needle, and protein quantification was performed using the Bradford method. Proteins were separated in a 10% SDS-PAGE by carefully placing 3 μg of protein in each lane. Nitrocellulose membrane (Invitrogen Life Technologies) was used to transfer the proteins, and the membrane was blocked with 5% skim milk (in TBS 1% (pH 7.4) and 0.01% Tween 20) and incubated for 1 h at room temperature with the appropriate primary Ab (anti-Hsp70, anti-Hsc70, calnexin (StressGen Biotechnologies), or anti-tubulin (Oncogene Research Products)). Blots were incubated 50 min at room temperature with 0.5 μg of appropriate species-matched anti-peroxidase, and the reaction was detected using the Luminol reagent for chemiluminescence (Santa Cruz Biotechnology). The intensity of the bands were analyzed by densitometry with a video densitometer (Chemilmager 5500; Alpha Innotech) using the AAB software (American Applied Biology).

Flow cytometric analysis of tumor cells

After treatment, 2 × 105 cells were pelleted and stained with 0.5 μg of mAb anti-Hsp70 (StressGen Biotechnologies) or anti-CD83 (Beckman Coulter) for 30 min on ice. Cells were then washed and incubated with 0.6 μg of the F(ab′)2 anti-rat IgG-FITC (Caltag Laboratories) or 0.5 μg of the F(ab′)2 anti-mouse IgG-FITC (Caltag Laboratories) and analyzed by flow cytometry. Samples were acquired in a FACSCalibur cytometer and analyzed using the CellQuest software (BD Biosciences). A total of 20,000 cells/condition was recorded, and viable cells were defined according to the forward light scatter (FSC) and side scatter (SSC) pattern.

Coupling of exosomes to latex beads and flow cytometric analysis

Phenotypic analysis of exosomes was performed as described previously (28). Briefly, recovered exosomes (30 μg) were incubated with 4-μm diameter aldehyde/sulfate latex beads (Interfacial Dynamics) for 15 min at room temperature. Two hours later, the reaction was stopped by addition of 100 mM glycine. Exosome-coated beads were then washed three times in PBS (containing 10% FCS) and stained with specific Abs. Samples were washed twice with PBS (containing 2% FCS) and analyzed on a BD Biosciences FACSCalibur using CellQuest Software. Beads were assessed from the dot plot representation of FSC vs SSC. Single beads were gated for fluorescence analysis. A “bead-only” control, as well as an isotype-matched Ab control, were prepared and the fluorescence intensity was normalized to 1 × 101 fluorescence units for each Ab.

Hsp72 depletion

Hsp72 was depleted from recovered supernatant by a affinity column chromatography as described previously (29). Briefly, because Hsp72 binds to ATP-agarose, recovered supernatant was mixed with a buffer containing 0.5 M KC1 plus 10 mM EDTA and slowly passed over a 25-ml column of ATP-agarose. The column flow-through (50 ml) was attached to an Amicon filter and dialyzed overnight against two changes of HEPES buffer plus 100 mM KC1 and 5 mM DTT and was then concentrated by Centricon C-10 ultrafiltration to one-half the original volume (0.5 ml). The Hsp72 retained by the ATP-agarose column was eluted with wash buffer containing 5 mM ATP and then concentrated and treated as above and used as a positive control.

Hsp72 ELISA

After various treatment protocols, cell culture medium was centrifuged to discard floating cells and cellular debris, and the total protein content was determined by Bradford analysis using BSA as a standard. The supernatant was aliquoted and treated with or without 1% Triton X-100 or 1% Lubrol WX or 0.5% Brij 98 for 10 min at 4°C with gentle rocking, and Hsp70 content was measured by standard sandwich ELISA as previously described (30) with minor modifications. Briefly, 96-well microtiter plates (Nunc Immunoplate Maxisorp; Invitrogen Life Technologies) were coated with murine monoclonal anti-human Hsp70 (clone C92F3A-5; StressGen Biotechnologies) in carbonate buffer (pH 9.5) (2 μg/ml) overnight at 4°C. Plates were then washed with PBS containing 1% Tween 20 (PBS-T) and blocked by incubation with 1% BSA in PBS-T. Supernatant was added, and bound Hsp72 was detected by the addition of rabbit polyclonal anti-Hsp70 Ab (SPA-812; StressGen Biotechnologies). Bound polyclonal Ab was detected with alkaline phosphatase-conjugated murine mAb to rabbit immunoglobulins (Sigma-Aldrich), followed by p-nitrophenyl phosphate substrate (Sigma-Aldrich). The resultant absorbance was measured at 405 nm with a Bio-Rad Benmark Plus plate reader. Standard dose-response curves were generated in parallel with Hsp70 (0–20,000 ng/ml; StressGen Biotechnologies), and the concentrations of Hsp70 were determined by reference to these standard curves with ASSAYZAP data analysis software (Biosoft). The interassay variability of the Hsp70 immunoassays was <10%.

Sucrose density gradient centrifugation and acetylcholinesterase (AChE) activity

Exosomes were carefully layered onto a sucrose density gradient (ranging from 1.08 to 1.24 g sucrose/ml and ultracentrifuged at 150,000 × g for 12 h at 4°C as described previously (31). Gradient fractions were collected and analyzed by for the presence of Hsp72 by Western blot analysis. AChE activity of various fractions was measured using the protocol described previously (32). Briefly, fractions (50 μl) were suspended in 1.25 mM AChE (supplemented with 0.1 mM 5.5-dithio-bis (2-nitrobenzoic acid)), and changes in absorption were monitored at 412 nm over a 10-min period at 37°C.

Statistical analysis

Data obtained from Western blot analysis were analyzed according to the relationship between the signal intensity and the area and presented as OD. Data from flow cytometry are shown as histograms and fluorescent arbitrary units, and the mean fluorescent intensity was obtained to compare between groups. Data are shown as percentages, mean and SD comparison was done using a Student's t test, and p < 0.05 was considered statistically significant.

Results

Intracellular and phenotypic changes induced by cytokine treatment on tumor cells

Recently, we demonstrated that certain cytokines found in high concentrations within the zone of inflammation, including IFN-γ and IL-10, induce the active release of Hsc70 from K562 erythroleukemic cells (33). In the present study, we investigate the mechanism of cytokine-induced Hsp72 release. To answer this question, we used the murine 4T1 breast adenocarcinoma and human K562 erythroleukemic cell lines either stably transfected with mutant heat shock factor-1 (HSF-1) (mut), which interferes with the cell's ability to produce Hsps in response to a variety of stressful stimuli (34) or empty vector control (ev). Measurement of total Hsp72 expression under normal culture conditions demonstrated that 4T1 cells have normal expression of the constitutive Hsc70 (data not shown) but extremely low expression of inducible Hsp72 (Fig. 1A). In response to nonlethal HS (43°C, 30 min), 4T1 cells respond in a normal fashion, triggering the cellular stress response that results in the up-regulation of the inducible Hsp72 (Fig. 1A). Treatment of 4T1 cells with IFN-γ at a concentration that does not induce significant cell death resulted in an increase in total Hsp72 expression (Fig. 1A). To our surprise, IL-10, a cytokine known to abrogate the proinflammatory effects of IFN-γ, was equally efficient in enhancing total Hsp72 expression (Fig. 1A). Wild-type K562 cells (data not shown) and K562 cells transfected with empty vector (K562ev) were found to express comparatively high baseline concentration of total Hsp72 and respond to nonlethal HS (43°C, 30 min) by up-regulating the inducible Hsp72 by ~4-fold (Fig. 1B). IFN-γ and HS treatment significantly increased total Hsp72 expression above baseline but did not significantly increase cell death (Fig. 1B). Treatment of K562ev cells with IL-10 increased total Hsp72 expression slightly above baseline values but had no significant effect on cell death, as compared with controls (Fig. 1B). K562 cells stably transfected with mutant HSF-1 (K562mut) did not significantly increase total Hsp72 expression above baseline when exposed to IFN-γ, IL-10, or nonlethal HS (Fig. 1B). Phenotypic analysis of Hsp72 surface expression revealed that transfection of K562 and 4T1 cells with mutant HSF-1 (K562mut and 4T1mut cells) completely abrogated the surface expression of Hsp72, as compared with K562ev and 4T1ev cells, respectively (Table I). Treatment of K562ev and 4T1ev cells but not K562mut or 4T1mut cells with IFN-γ or nonlethal HS treatment (43°C, 30 min) resulted in significant up-regulation in Hsp72 surface expression (Table I). However, TGF-β1 treatment of all cell lines at a concentration known to significantly inhibit LPS-induced NK-κB activation by the murine microglial cell line N9 (35) did not significantly increase Hsp72 surface expression (Table I).

FIGURE 1
Cellular expression of Hsp72 induced by stress. 4T1 breast adenocarcinoma cells (106)(A) or K562 erythroleukemia cells (106)(B); K562ev (empty vector; left panel) or K562mut (mutant; right panel) were stimulated with PBS (lane 1), 750 U/ml IFN-γ ...
Table I
Surface expression of Hsp72 induced by stress

Active release of Hsp72 by tumor cells

To determine whether stressful stimuli indeed augments the active release of Hsp72, cells were treated with IFN-γ, IL-10, or TGF-β1 or exposed to nonlethal HS. The supernatant was recovered and measured for released Hsp72 using a modified Hsp72 ELISA. Treatment of K562ev cells with IFN-γ and IL-10 dose dependently increased the release of Hsp72 into the supernatant, without significant increase in cell death (Fig. 2A and B). However, TGF-β1 used over a range of concentrations (5–50 ng/ml) that have been shown previously to significantly inhibit LPS-induced NK-κB activation (35) did not significantly increase Hsp72 release or induce cell death (Fig. 2C). Cells treated with protein controls (BSA) or OVA (data not shown) did not significantly increase Hsp72 release above baseline concentrations. To demonstrate that the released Hsp72 was indeed the inducible Hsp72, we repeated the same experiment in K562mut cells. Because they carry a mutation in the hsf-1 gene, which interferes with the cells ability to produce Hsp70 in response to nonlethal HS (34). Treatment of K562mut cells with IFN-γ, IL-10, or TGF-β1 over the range of concentrations did not significantly increase Hsp72 release as compared with cells treated with PBS alone (Fig. 2, A–C). Exposure of K562ev cells to a range of temperatures between 37°C and 45°C resulted in a proportional increase in Hsp72 release that did not reach a plateau (Fig. 2D). A 2°C increase in temperature above baseline temperature (37°C, 30 min) to 39°C for 30 min resulted in significant increase in Hsp72 release without significant cell death (Fig. 2D). This was the same between the temperature range of 39°C to 43°C; however, a 1°C increase to 44°C resulted in significant cell death (Fig. 2D). K562mut cells already showed significant cell death when exposed to 43°C for 30 min. (Fig. 2D). Thus, reflecting an essential requirement for an efficient heat shock response to inhibit the effects of HS-induced cell death.

FIGURE 2
Stress-induced release of Hsp72 from K562 tumor cells. K562 erythroleukemic cells (106); mutant (K562mut; right panels) or empty vector control (K562ev) was treated with increasing concentrations of IFN-γ (●) or protein control (BSA; ○) ...

Recent studies suggest that tumor cells can actively release exosomes in response to exposure to nonlethal HS (36). We hypothesized that the cytokines IFN-γ and IL-10 shown to induce Hsp72 release by tumors could induce Hsp72 release by a mechanism involving exosomes. To test this hypothesis, we admixed human monocytes with supernatant recovered from K562ev cells treated with either IFN-γ, IL-10, or nonlethal HS (43°C, 30 min). After various treatment protocols, the recovered supernatant was exposed to 1% Triton X-100 before admixing it with human monocytes overnight at 37°C. We consistently demonstrated a significant increase in Hsp72 concentration after exposure to 1% Triton X-100 as compared with supernatant exposed to PBS alone (Fig. 3). Supernatant recovered from K562mut cells contained significantly less Hsp72 as compared with supernatant from K562ev cells, indicating that there is significant spontaneous release of Hsp72 from cells. Treatment of K562mut supernatant with 1% Triton X-100 significantly increased the concentration of Hsp72 as compared with PBS-treated supernatant (Fig. 3). Triton X-100, a mild detergent, has been shown to solubilize exosomes and release its internal contents (36). Our studies indicate that some of the internal contents of stress-induced exosomes is Hsp72. Further evidence that Hsp72 is released within detergent-sensitive vesicles was demonstrated in experiments using two additional detergents; treatment of recovered supernatant with 1% Lubrol WX (Serva-Crescent Chemical) and 0.5% Brij 98 (polyoxyethylene 20 oleyl ether; Sigma-Aldrich) resulted in significant elevated Hsp72 concentrations as compared with treatment with 1% Triton X-100 (Fig. 3). Taken together, these results suggest that Hsp72 is spontaneously and actively released from tumors into the extracellular milieu within detergent sensitive structures, possibly exosomes.

FIGURE 3
Enhancement of Hsp72 concentration by mild detergent treatment. K562 erythroleukemic cells; empty vector (K562ev; top panel) or mutant (K562mut; bottom panel) was treated with 10 ng/ml IL-10 or 750 U/ml IFN-γ or exposed to nonlethal HS (43°C, ...

Characterization of actively released Hsp72

To prove that Hsp72 is actively released within exosomes, supernatant was collected and treated according to the protocol designed to isolate and enrich exosomes (see Materials and Methods). Exosomes from K562ev cells exposed to nonlethal HS (43°C, 30 min) contained significantly higher expression of Hsp72 than exosomes from K562mut cells (Fig. 4A). AChE activity (a typical exosomes enzyme) of isolated exosomes recovered from nontreated K562ev and K562mut cells showed maximal activity at a density of 1.17 g/ml (Fig. 4B, lower panel), a known characteristic of exosomal vesicles (37). Western blot analysis of all the fractions recovered after sucrose density ultracentrifugation revealed that the fraction with highest AChE activity corresponds to the maximal amount of Hsp72 (Fig. 4B, upper panel). The surface profile of exosomes analyzed using exosomes-latex-bead technique revealed that exosomes recovered from K562ev express Hsp72 and MHC class I molecules at a higher intensity than K562mut cells (Fig. 4C). Analysis of the internal content of the Hsp72 containing exosomes revealed that both K562ev and K562mut-derived exosomes contained cytosolic proteins, including Hsp72, Hsc70, and tubulin. However, the Hsp72 containing exosomes was negative for the expression of the ER-residing protein, calnexin (Fig. 4D). Taken together, these results suggest that the Hsp72 containing vesicles are exosomes that originate from cytosol as opposed to ER-Golgi source.

FIGURE 4
Biochemical characterization of stress-induced Hsp72. A, Measurement of total Hsp72 content of exosomes. Exosomes isolated from 107 K562ev or K562mut cells were analyzed by Western blot using Hsp72-specific Ab (top panel). The intensity of the bands were ...

Mechanism of active release of Hsp72

To determine the mechanism of active release of Hsp72 within exosomes, cells were treated with various inhibitors of the classical protein transport pathway or inhibitors of cellular lipid rafts. A modified Hsp72 ELISA was used to measure the concentration of Hsp72 in the supernatant. Pretreatment of cells with inhibitors of the classical protein transport pathways, including brefeldin A, monensin, tunicamycin, and thapsigargin, did not significantly inhibit HS-induced or IFN-γ-induced Hsp72 release (Table II). However, pretreatment of cells with MβD (Sigma-Aldrich), a compound known to disrupt lipid rafts integrity by removing cholesterol, and BAPTA-AM, an inhibitor of [Ca2+]i, but not EGTA, an inhibitor of extracellular Ca2+, significantly inhibited HS- and IFN-γ-induced Hsp72 release (Table II). We demonstrate that pre-treatment of 4T1 cells with 0.63–2.50 mM MβD alone did not significantly enhance total Hsp72 expression (Fig. 5A). However, pretreatment of cells with MβD, followed by exposure to HS, dose dependently increased total Hsp72 expression, as judged by Western blot analysis (Fig. 5A). Similarly, immunofluorescent microscopy demonstrates that pre-exposure of tumors to MβD, followed by HS, induces increased expression of Hsp72 within cells at comparable levels to HS alone. However, exposure to MβD did not induce significant expression of Hsp72 (Fig. 5B). Significantly higher Hsp72 expression is demonstrated in cells pre-exposed to MβD, followed by IFN-γ or IL-10 treatment or HS, as compared with cells treated with cytokines or HS alone (Fig. 5C). We interpreted that MβD-mediated increase in stress-induced increase of total Hsp72 expression to be due to the inability of Hsp72 to leave the cell because the lipid raft integrity of the plasma membrane has been destroyed. This was confirmed by experiments in which supernatant recovered after various treatments were probed for Hsp72 expression by Western blot analysis. We demonstrate that pretreatment of 4T1 cells with MβD completely abrogates IFN-γ-or IL-10- or HS-induced Hsp72 release (Fig. 5D). Taken together, these results suggest that Hsp72 is released within exosomes via a nonclassical protein transport pathway that requires intact lipid raft integrity.

FIGURE 5
Dose-response curve for MβD-mediated inhibition of Hsp72 release. A, K562 erythroleukemic empty vector (106) cells were pretreated with MβD (2.5 mM, lanes 1 and 5; 1.25 mM, lanes 2 and 6; 0.625 mM, lanes 3 and 7; 0.0 mM, lanes 4 and 8 ...
Table II
Inhibition of HS- and IFN-γ-induced Hsp72 release: role of nonclassical protein transport pathway

Biological activity of released Hsp72 is found within stress-induced exosomes

Novel immunostimulatory functions have been ascribed to Hsp72, including its ability to stimulate the release of proinflammatory cytokines (4-7) and induce NO release (8) by APCs and up-regulate costimulatory molecule expression (9) and induce DC maturation (9-11). To determine whether indeed the biological activity of released Hsp72 is found within the exosomes fraction, we used the following schema (Fig. 6A); tumors were pretreated with brefeldin A or MβD before treatment with either IFN-γ or exposure to nonlethal HS. After which, exosomes or supernatant was recovered and depleted of either IFN-γ (using neutralizing anti-IFN-γ mAb) or Hsp72 (using anti-Hsp72 column). The recovered samples were admixed with immature DCs and IL-12 release (Fig. 6B), or CD83 surface expression (Table III) was measured (Fig. 6A). Pre-exposure of K562 cells to MβD completely abrogated the ability of exosomes and supernatant recovered after IFN-γ or HS to stimulate IL-12 release by DCs (Fig. 6B). The depletion of IFN-γ or Hsp72 from exosomes or supernatant had no significant effect on IL-12 release. However, exosomes or supernatant recovered from IFN-γ- or HS-treated K562ev cells after pretreatment with brefeldin A effectively stimulated IL-12 release (Fig. 6B). The depletion of Hsp72 from the supernatant recovered after any treatment completely abrogated its ability to stimulate IL-12 release (Fig. 6B). As an additional control, exposure of exosomes or supernatant to extreme heat (100°C, 1 h) or proteinase K (10 μg/ml, 3 h at 37°C) denaturation completely abrogated its ability to stimulate IL-12 release by immature DCs (data not shown).

FIGURE 6
A, Experimental flow chart; K562 cells were pretreated with brefeldin A, MβD, or control (culture medium), then incubated with IFN-γ or exposed to nonlethal HS (heat shock; 43°C, 30 min) treatment. Exosomes or supernatant was recovered ...
Table III
The released Hsp70 stimulates the maturation of naive DC

In a separate experiment, K562ev cells were pretreated with IFN-γ or TGF-β1 or HS. After incubation, supernatant was recovered and passed through a polymyxin B column five times to remove any residual endotoxin contamination. Supernatant was then mixed with either anti-IFN-γ-neutralizing mAb or passed through an anti-Hsp72 column five times to deplete Hsp72. Supernatant from cells either exposed to HS or treated with IFN-γ, but not TGF-β1, significantly increased CD83 expression (Table III). Neutralization of IFN-γ using anti-IFN-γ did not significantly inhibit the ability of supernatant from cells either exposed to HS or treated with IFN-γ to enhance CD83 expression (Table III). However, supernatant from cells either exposed to HS or treated with IFN-γ that were passed through an anti-Hsp72 column completely lost its ability to augment CD83 expression (Table III). Taken together, these data suggest that the biological activity of released Hsp72 is found within the exosomes fraction, and disruption of lipid raft integrity suppresses Hsp72-mediated functions such as IL-12 release and DC maturation.

Discussion

Our study presents an additional mechanism by which IFN-γ enhances tumor immunogenicity, which is dependent on the active release of inducible Hsp72. The working hypothesis of this study is that certain cytokines normally found in high concentrations within inflammatory foci mediate the active release of Hsp72. The released Hsp72 acts as a chaperokine, thereby augmenting the effect of the cytokine. The cytokine-mediated release of Hsp72 is accomplished by activating the cellular stress response, which results in the synthesis and expression and release of Hsp72 within exosomes. We have demonstrated previously that IFN-γ and IL-10 induce the active release of Hsc70 from K562 erythroleukemic cells (33). However, the mechanism by which this active release occurs was not addressed. In the present study, we show that IFN-γ and IL-10 activates the cellular stress response in 4T1 murine breast adenocarcinoma (Fig. 1) and K562 erythroleukemic (Fig. 2) cells, as judged by increased expression of the stress-inducible Hsp72. We did not expect IL-10 to exhibit similar effects as IFN-γ on tumor cells. However, although generally considered an immunosuppressive cytokine, IL-10 possesses anti-inflammatory properties in several in vitro and in vivo models, depending on a number of variables (for review, see Ref. 38). Our results demonstrate that IL-10 exhibits immunostimulatory instead of immunosuppressive characteristics. Therefore, we searched for a cytokine with consistent immunosuppressive characteristics irrespective of the variables. Treatment of tumors with TGF-β1 at a concentration demonstrated to significantly inhibit LPS-induced NF-κB by murine microglial cells line N9 (35) did not induce Hsp72 surface expression (Table I), Hsp72 release, or cell death (Fig. 2) or stimulate CD83 expression (Table III), at the concentration range used, indicating that the observed IFN-γ- and IL-10-induced increase in Hsp72 surface expression and release is a specific physiological event. The fact that elevated levels of serum IL-10 have been demonstrated in patients with advanced gastrointestinal malignancies compared with healthy controls and patients with metastatic disease showing significantly higher levels than patients with undisseminated disease (39) and that serum IFN-γ concentrations are elevated in a patients with cervical cancer have also been demonstrated (40). This indicates that our model is biologically significant. Although the concentration of IL-10 and IFN-γ used in our studies are higher than those found in the serum of patients with certain cancers, it is possible that higher concentrations of cytokine exist within inflammatory foci caused by tumors.

If Hsp72 is actively released in response to IFN-γ, disrupting its expression should abrogate Hsp72 release. Indeed, treatment of K562mut and 4T1mut cells that are stably transfected with mutation in the hsf-1 gene, which interferes with the cells ability to produce Hsp72 (34), failed to activate Hsp72 synthesis (Fig. 1), surface expression (Table I), and release (Fig. 2) in response to IFN-γ treatment. The slight, albeit insignificant, increase in Hsp72 expression in K562mut and 4T1mut cells is in agreement with recent findings that HSF-1 (the transcription factor that regulates Hsp70 synthesis) is not the only regulator of Hsp70 synthesis (see review by Calderwood (41)). Most proteins are transported to the extracellular milieu via the classical protein transport pathways dependent on the ER-Golgi transit (42). However, monensin, a compound known to block transport out of the Golgi apparatus, brefeldin A, a known inhibitor of anterograde transport from ER to Golgi, thapsigargin, an ATPase inhibitor that specifically depletes calcium from the ER, and tunicamycin, a known inhibitor of N-glycosylation, were unable to suppress IFN-γ and HS-induced Hsp72 release (Table II). An important requirement for efficient secretion is the rise in [Ca2+]i. As expected, depletion of [Ca2+]i using BAPTA-AM, a [Ca2+]i chelator, but not extracellular Ca2+, using EGTA, an extracellular Ca2+ chelator, resulted in inhibition of IFN-γ-, IL-10-, and HS-mediated Hsp72 release (Table II). Taken together, these results suggest that IFN-γ-induced release of Hsp72 is mediated via a nonclassical protein transport pathway that is dependent on [Ca2+]i (Table II). These results are in agreement with the recent studies showing that inhibitors of the classical protein transport pathway does not block HS-induced Hsp72 release (43).

Our results showing that treatment of the supernatant recovered after cytokine or HS treatment with the nonionic detergent Triton X-100 significantly enhanced the detection/measurement of released Hsp72 (Fig. 3). The insolubility in Triton X-100 is a hallmark for the presence of exosomes. This suggests that a portion of the Hsp72 is released in the context of exosomes. Exosomes are internal vesicles of multivesicular bodies that are released into the extracellular milieu upon fusion of multivesicular bodies with the cell surface (44-46). Recently, a microdomain that appears to play a role as a building unit for various plasma membrane protrusions has been shown to be soluble in Triton X-100 and resistance to Lubrol WX (47). However, other studies have begun to ask whether there is more than one kind of raft (48). Our data shows that pretreatment of supernatant recovered after cytokine or HS treatment with the nonionic detergents Lubrol WX and Brij 98 increases the detection/measurement of released Hsp72 ~4- to 5-fold greater than pretreatment with Triton X-100 alone (Fig. 3). It is well accepted that, even under control culture conditions, the cells are under a certain amount of stress. This stress results in baseline concentrations of spontaneously released Hsp72 into the culture medium. This explains the results demonstrating that, after exposure of supernatant to the mild detergents Triton X-100, Brij 98, or Lubrol WX, there is consistently more detectable Hsp72, as judged by standard sandwich ELISA (Fig. 3).

Biochemical and phenotypic analyses of the released Hsp72 confirmed that Hsp72 is released in vesicles, known as exosomes (Fig. 4, A–D). We demonstrate that maximum AChE activity, an enzyme characteristic for exosomal vesicles (32), peaked at the same density of 1.17 g/ml, which corresponds to maximum Hsp70 content. This is in agreement with data that show tumor-derived exosomes float at a density of 1.17–1.24 g/ml (28, 46, 49). Phenotypic analysis using latex beads revealed that the exosomes express Hsp72 and MHC class I (Fig. 4C). Analysis of the protein content of the exosomes show the presence of Hsp72, Hsc70, and the cytoplasmic protein tubulin but absence of the ER protein, calnexin (Fig. 4D). Work from the Multhoff laboratory shows that exosomes additionally express Bag-4, known as the silencer of death domain and a member of the antiapoptotic Bcl-2-associated athanogene family (50). Rab-4, a GTPase protein associated with early endosome to plasma membrane transport but neither Rab-7, associated with late endosomes to lysosomes, nor Rab-9, associated with late endosomes to trans-Golgi, nor Rab-11, associated with trans-Golgi to plasma membrane transport, was found to be expressed in the exosomes (50).

The plasma membrane of cells is partially composed of arranged domains known as lipid rafts. These lipid rafts are composed of sphingolipids, sphingomyelin, saturated phospholipids, and cholesterol and contain a definite set of proteins (51). Evidence that the active release of Hsp72 requires intact lipid raft formation is demonstrated in experiments in which pre-exposure of tumors with MβD, a compound known to disturb lipid raft organization by specifically depleting cholesterol, dose dependently inhibited, HS-induced Hsp72 release (Fig. 5). MβD at the concentrations used in these experiments did not alone induce significant Hsp72 expression or release (Fig. 5). However, cells pre-exposed to MβD and subsequently treated with IFN-γ or HS expressed more total Hsp72 than cells treated with IFN-γ or HS alone (Fig. 5). However, this seems counterintuitive because measurement of super-natant from these experiments demonstrated that pre-exposure of cells to MβD abrogates IFN-γ- and HS-induced Hsp72 release (Fig. 5D). The observed increase in expression of Hsp72 within the cell is because MβD has disrupted the lipid raft integrity in the plasma membrane, thereby resulting in an accumulation of Hsp72 within the cell. In agreement with these findings, depletion of cholesterol using MβD has been shown to inhibit HS-mediated release of Hsp70 from Caco-2 and human intestinal cells and induce the redistribution of Hsp70 to detergent-resistant microdomains (43). Studies showing that Hsp70 family members, including Hsp72 and Hsc70, interact directly with lipids (52) might suggest that depletion of cholesterol using MβD can interfere with the interaction of Hsp72 with lipids rather than lipid rafts. However, other studies showing that receptor molecules, including CD14, Hsp90, GDF5, and TLR4, are involved in LPS-induced activation and are present in microdomains after stimulation of monocytes with LPS (53) suggest an essential role for lipid raft integrity in LPS-induced TNF-α secretion (53). Taken together, these studies indicate an important role for lipid rafts in the transport and release of Hsp72.

Chaperokine is a recent term coined to better describe the unique function of extracellular Hsp72 as both chaperone and cytokine (for review, see Refs. 54 and55). To determine whether the released Hsp72 retained the chaperokine activity of extracellular Hsp72, K562 cells were pretreated with brefeldin A or MβD and incubated with IFN-γ or exposed to nonlethal HS (Fig. 6A). Exosomes or supernatant was recovered and depleted of either IFN-γ (using anti-IFN-γ mAb) or Hsp72 (passed through an anti-Hsp72 column). The samples were admixed with naive DCs for the measurement of IL-12 release or CD83 surface expression. Exosomes from control or brefeldin A pretreated but not cell pre-exposed to MβD were more efficient at stimulating IL-12 secretion (Fig. 6B) and inducing DC maturation (Table III). The failure of neutralizing Abs to IFN-γ to have any significant effect on up-regulation of IL-12 release demonstrate that the biological effects are due to Hsp72 (Fig. 6B). This is confirmed in experiments in which supernatant is passed through an anti-Hsp72 column to deplete Hsp72. Admixing DCs with supernatant depleted of Hsp72 failed to induce IL-12 release (Fig. 6B) or deliver the maturation signal to immature DCs (Table III), confirming that the biological activity of the supernatant is contained within the Hsp72 fraction. Although the possibility exists that some other component of the supernatant was inadvertently depleted in the anti-Hsp72 column. This is not very plausible because this technique has been routinely used to deplete, isolate, enrich, and purify Hsp72 from a number of complex protein lysates with excellent results (29, 56). At the time of the submission of this report, we were unable to get sufficient quantities of exosomes to pass through an anti-Hsp72 column to perform a complete experiment. However, because previous results have shown that the chaperokine activity of recovered supernatant closely correlates with recovered exosomes, we postulate that we would obtain similar results. The high background activation of exosomes compared with IL-12 secretion by exosomes from untreated cells is possibly due to the spontaneous release of Hsp72 from other cell lines (50) and the previously described ability of exosomes to potently stimulate the immune system (57). Indeed, supernatant recovered from human pancreas (Colo/Colo+) and colon (CX/CX+) carcinoma sublines activate CD94+ NK cell-mediated lysis and act as chemoattractants for NK cells (50). In addition, these authors demonstrate that depletion of exosomes completely abrogates CD94+ NK cell migration (50). Taken together, these results demonstrate that the Hsp72 containing exosomes exhibits potent biological activity associated with the chaperokine activity of Hsp72.

Although we have not used rHsp72 in these studies, we were careful to monitor for endotoxin contamination that might confound our studies. Some recent studies have reported that endotoxin contamination of certain commercial preparations of rHsp70 was responsible for their observed immunostimulatory properties (58, 59). This is a serious problem for researchers interested in studying the mechanism of Hsp70-induced functions in vitro and others involved in designing Hsp-based immunotherapeutics. However, this concern was recently addressed by MacAry et al. (60) using physical and functional assays to ensure the “cleanliness” of the Hsp70 protein preparations. Physical assays include adding an additional microdialysis step to remove unbound peptide and other contaminants, passing all Hsp70 protein preparations through polymyxin B column, and only using preparations with <1.0 endotoxin units/20 μg of protein. Functional assays include boiling Hsp70 protein preparations at 100°C at 60 min, which denatures Hsp70 but not LPS, pretreatment of Hsp70 preparations with proteinase K, which inhibit Hsp70-induced but not LPS-induced cytokine release by APCs, addition of soluble CD14 to macrophages enhances LPS-induced but not Hsp70-induced cytokine release, pretreatment of macrophages with BAPTA-AM, an intracellular calcium chelator, inhibits efficient Hsp70-induced but not LPS induced-cytokine release, and measurement of intracellular calcium ([Ca2+]i) flux, which is only induced by Hsp70 not LPS (5, 60). When similar measures were used to control for LPS contamination of rHsp70 preparations, Hsp70 augments DC effector functions and, when admixed with specific Ags, triggers autoimmune diseases in vivo (61), and DCs pulsed with peptide-loaded rHsp70 generated potent Ag-specific CTL responses (60). However, mutation of the peptide binding domain of Hsp70 rendered the mutants incapable of generating Ag-specific CTL responses (60). During the revision of this article, a study was published that identified stimulating and inhibitory epitopes within microbial Hsp70 that modulate cytokine and chemokine release and the maturation of DCs (62). Because the results were based on recombinant technology and yet both preparations elicit disparate effects, this seems to exclude the possibility that endotoxin contamination of Hsp70 preparations is responsible for their observed effects. Finally, it must be noted that although the most up-to-date techniques to eliminate endotoxin contamination may be used, it is virtually impossible to completely eliminate this possibility. However, all these studies strongly suggest that when special care is taken to control for LPS contamination, clear effects of Hsp70 can be demonstrated.

In summary, this study tests the postulate that treatment of tumors with a sublethal dose of IFN-γ activates the cellular stress response, which results in the release of Hsp72 into the extracellular milieu within exosomes (Fig. 7). IFN-γ-induced increase in total Hsp72 expression is abrogated in cells transfected with mutant HSF-1, known to interfere with the trimerization and nuclear translocation of HSF-1 to the heat shock element and subsequent transcription of Hsp72 but not by pretreatment of wild-type cells with neutralizing IFN-γ mAb (Fig. 7). Pretreatment of cells with inhibitors of the classical protein transport pathway does not block intracellular expression of Hsp72 or its release (Fig. 7). However, release of Hsp72 can be blocked by pretreatment with a [Ca2+]i chelator, BAPTA-AM or MβD, a compound known to disrupt lipid rafts. Biochemical characterization of the released Hsp72 demonstrates that it is released within exosomes, although some Hsp72 is expressed on the surface of the exosomes as well. Thus, the released Hsp72 can be measured by the classical sandwich ELISA. Treatment of Hsp72-exosome supernatant with Brij 98, Lubrol WX, or Triton X-100 augments the amount of Hsp72, indicating that Hsp72 is found within detergent soluble vesicles. In this context, Hsp72-exosomes act as a powerful endogenous adjuvant according to the concepts of immunogenicity proposed by Chaput et al. (57, 63). In our model, Hsp72 not bound within exosomes delivers signal 0, i.e., trigger signaling of pattern recognition receptors and activation of innate immune cells to release cytokines/chemokines, acts as a danger molecule by inducing APCs to up-regulate costimulatory molecules, and delivers signal 2 through cytokines and costimulation as natural adjuvants. The rest of the contents of the exosome, including MHC class I, class II, and CD86, delivers signal 2. Our working hypothesis is that antigenic peptides carried by Hsp72 bound within the exosome (Hsp72-exosome) will be more efficiently taken up, processed, and presented in the context of MHC class I than unbound Hsp72 for the activation of specific CD8+ CTL-mediated tumor killing by APCs (Fig. 7). Previous studies demonstrate that expression of Hsp72 on the surface of tumors stimulates NK-specific tumor killing (64, 65) and results in tumor regression (66). Gastpar et al. (50) demonstrate that the released Hsp72 stimulate migratory and cytolytic activity in NK cells. Our studies indicate an additional previously unknown mechanism by which IFN-γ promotes tumor surveillance and helps to further our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.

FIGURE 7
Schematic representation of the working hypothesis for this study. Sublethal dose of IFN-γ (lightning) activates trimerization and nuclear translocation of cytoplasmic HSF-1 (red rods) to the heat shock element (HSE) and subsequent transcription ...

Acknowledgments

We thank Dr. Stuart K. Calderwood for the gift of the C-terminal truncation mutant DN-HSF1 and empty vector (Beth Israel Deaconess Medical Center, Harvard Medical School) and Dr. Susana Fiorentino (Institut National de la Santé et de la Recherche Médicale Unite 462, Hôpital Saint-Louis, Paris, France) for helpful discussions; and Diana T. Page and Edwina Asea (Boston University School of Medicine, Boston, MA) and Lydia Rossbacher (University Hospital Regensberg, Regensberg, Germany) for expert technical assistance.

Footnotes

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported in part by National Institute of Health Grant RO1CA91889, grants from the Joint Center for Radiation Therapy Foundation and Harvard Medical School, institutional support from the Department of Medicine, Boston University School of Medicine, and the Scott & White Clinic, an endowment from the Cain Foundation (to A.A.), a BioChance grant (0312338) from the Bundesministerium für Forschung und Tecnologie, the European Union Project Trans-Europe (QLRT 2001 01936), the Deutsche Forschungsgemenischaft (MU 1238 7/2), and a grant from Multimmune (to G.M.).

3Abbreviations used in this paper: Hsp, heat shock protein; AChE, acetylcholinesterase; [Ca2+]i, intracellular Ca2+; DC, dendritic cell; ER, endoplasmic reticulum; FSC, forward light scatter; HS, heat stress; Hsc70, constitutively expressed Hsp70; HSF-1, heat shock factor-1; Hsp72, stress-inducible 70-kDa Hsp; LDH, lactate dehydrogenase; MβD, methyl β-cyclodextrin; SSC, side scatter.

DisclosuresThe authors have no financial conflict of interest.

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