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Division of Molecular and Cellular Biology, Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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Abstract |
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. Our studies show that HSP70 release involves transit though an endolysosomal compartment and is inhibited by lysosomotropic compounds. In addition, the rate of HSP70 secretion correlates well with the appearance of the lysosomal marker LAMP1 on the cell surface, further suggesting the role for endolysosomes. The entry of HSP70 into this secretory compartment appears to involve the ABC family transporter proteins and ABC transporter inhibitor glibenclamide antagonizes secretion. Although the cell signals involved in triggering stress induced HSP70 release though this lysosomal pathway are largely unknown, our experiments suggest a regulatory role for extracellular ATP. These mechanisms appear to be shared by IL-1 secretion. Following release, we observed the binding of extracellular HSP70 to the cell surface of the prostate carcinoma cells. These findings suggest that secreted HSP70 can take part in paracrine or autocrine interactions with adjacent cell surfaces. Our experiments therefore suggest a mechanism for HSP70 secretion and binding to the surface of other cells that may be involved in recognition of the tumor cells by the immune system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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and IL-18 were used by HSP70 (12, 13). It has been shown that exposing cells to heat shock causes the release of a number of proteins that lack secretion leaders, including basic fibroblast growth factor 1 (FGF-1) and IL-1
release and this release appears to be unaffected by inhibitors of common secretory pathways such as colchicine and brefeldin A (14, 15, 16) (S. K. Calderwood, unpublished data). Although the pathways of secretion of leaderless proteins are not fully defined, a number of contrasting mechanisms have been elucidated. Most notable among these are the pathway of lysosomal exocytosis used by IL-1 release and a plasma membrane translocation mechanism for FGF-1 release after heat shock (12, 15, 17). IL-1 release follows a complex pathway that includes transport of IL-1 to the cell surface in lysosomal endosomes and passage across the plasma membrane by a mechanism involving ABC family transmembrane transporters (12). Secretory lysosomes are acidic organelles sharing similar traits with conventional lysosomes, including a requirement for the maintenance of low pH, that are involved in the secretion of many leaderless proteins (18). ABC family transmembrane-transport proteins are ubiquitous polypeptides found in all cellular organisms, and in unicellular organisms mediate secretion of most leaderless secretory proteins (17, 19). ABC transporter molecules include proteins of fairly large Mr such as toxins secreted by Gram-negative bacteria (20). In mammalian cells, secretion of IL-1 by macrophages also involves an ABC family transporter and is stimulated though the binding of extracellular ATP to cell surface purinergic receptors (21, 22).
Our current experiments show that HSP70 is released from prostate carcinoma cells though an active mechanism independently of either the rate of HSP70 synthesis or cell death. Active HSP70 secretion involves translocation though a lysosomal compartment and can be inhibited by lysomotropic agents. HSP70 release correlates well with the appearance of the lysosomal-associated membrane protein 1 (LAMP1) on the cell surface, suggesting that HSP70-containing lysosomal vesicles may lyse on exit from cells and deposit LAMP1 on the cell surface, whereas HSP70 is released into the external milieu. The factors required for HSP70 release further resemble those involved in IL-1 secretion and glibenclamide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), potent inhibitors of ABC family transporter activity, inhibit HSP70 secretion. We also examined signals that may be involved in HSP70 release and our experiments suggest that HSP70 release requires extracellular ATP. HSP70 secretion therefore appears to share mechanisms with other leaderless cell proteins and may play a part in cell regulation by interaction with receptors on the cell surface.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The prostate carcinoma cell lines PC-3 and LNCaP were cultured respectively in complete Ham’s F-12 and RPMI 1640 medium supplemented with 10% FCS (Mediatech), glutamine (Invitrogen Life Technologies), penicillin, and streptomycin (Invitrogen Life Technologies).
Hyperthermia treatments and HSP70 release
Five million cells at a concentration of 1 x 106 cells/ml (PC-3 or LNCaP) were cultured at 37°C overnight and fed with fresh medium before treatment at 37°C and 43°C for 30 min or 37°C and 40°C for 6 h representing the untreated control group and the heat shock group, respectively. Heat shock was conducted in a circulating water bath and temperature monitored to a resolution of 0.1°C using a high-resolution mercury-in-glass thermometer. Aliquots of medium were harvested at the indicated times in experiments and cleared by spinning at 900 x g for 15 min. These aliquots of medium were replaced by replicate volumes of fresh medium to preserve a constant extracellular volume. HSP70 release was corrected for the resultant dilution.
Detection of extracellular HSP70 by ELISA
Sandwich ELISA was used for detection of HSP70 in supernatants described previously (23). Briefly, ultra-high-binding 96-well microtiter ELISA plates (ThermoLabsystems) were coated overnight with HSP70 mAb (StressGen Biotechnologies) in carbonate buffer followed by three washes in PBS plus 0.1% Tween 20 (PBST; Sigma-Aldrich). The plates were blocked with 1% BSA (Sigma-Aldrich) in PBST followed by three washes in PBST. Samples and recombinant human HSP70 protein (StressGen Biotechnologies) were added to the wells and incubated for 1 h followed by three washes in PBST. Rabbit polyclonal anti-HSP70 (StressGen Biotechnologies) was added to each well and incubated for 1 h followed by three washes with PBST. Anti-rabbit IgG alkaline phosphatase-conjugated mAb (
) was added to the 96-well plates followed by three washes with PBST. Finally, the substrate p-nitrophenyl phosphate (Sigma-Aldrich) was added, and absorbance was read by spectrophotometer (Bio-Rad) at absorbances of 405 and 650 nm (reference wavelength).
Western analysis of intracellular HSP70 expression
Aliquots of 1 x 106 cells were washed three times in ice-cold PBS, solubilized in reducing sample buffer, and resolved by 10% SDS-PAGE under reducing conditions. Proteins were then electrophoretically transferred to nitrocellulose membranes (Amersham Biosciences) and blocked for 1 h with 10% nonfat dry milk (Bio-Rad) in PBST buffer (Boston BioProducts). Filters were hybridized sequentially with the following: HSP70 mAb (StressGen Biotechnologies), anti-mouse IgG hp-conjugate (Amersham Biosciences) and Ab-Ag complexes developed by ECL-plus luminescence-based detection agent (Amersham Biosciences) according to the manufacturer’s instructions.
Western analysis of ABCA-1 expression
A total of 1 x 106 cells was grown until growth was confluent, and cells were harvested and washed three times in PBS. Total cell lysates were prepared by solubilizing cells in reducing sample buffer, and samples were run on 6% SDS-PAGE under reducing conditions. Transfer of proteins was conducted as described above, and detection was confirm by using ABCA-1 Abs (Novus Biologicals). Actin controls were run on each lysate.
Isolation of cell lysosomal fraction
PC3 and LNCaP cells were grown until there was packed cell volume of 3 ml, which was resuspended in an equal volume of packed cells with hypotonic buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT) and allowed to swell for 15 min on ice. Cells were lysed by rapidly pushing cells through a 1 ml syringe with a 25-gauge needle five times while keeping the cells on ice. Crude nuclear pellet was pelleted by centrifugation for 30 s at 12,000 x g. Supernatants were stored and lysosomal fraction isolated as described by Andrei et al. (12). Briefly, supernatants were treated with proteinase K for 30 min on ice and inhibited with protease inhibitors. The supernatant was diluted 10-fold in homogenizing buffer and centrifuged at 50,000 x g for 20 min. The pellet was washed once in homogenizing buffer and solubilized in reducing sample buffer, and HSP70 was detected by Western blots as described previously. Cathepsin D was detected as the control for the lysosomal compartment by Western blot.
Immunofluorescence microscopy
Cells were plated onto fibronectin-coated 8-well chamber glass slides with cover (Nalge Nunc International) at 105 cells per well. Twenty-four hours later, the culture medium was removed and replaced with fresh medium before the cells were hyperthermia treated as described above. The cells were immediately fixed with 3.7% paraformaldehyde. After fixation for 15 min, the cells were washed once with PBS and blocked with blocking buffer (PBS containing 3% BSA) for 30 min. For intracellular staining, the cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min at room temperature after the fixing step. The cells were next washed once with PBS, incubated for 1 h anti-HSP70 (StressGen Biotechnologies) or LAMP1 (CD107a; BD Pharmingen) mAbs followed by five washes in wash buffer (PBS containing 0.05% Tween 20). The cells were incubated with fluorescein-conjugated anti-mouse/rabbit IgG Ab (Alexa 594 and Alexa 488; Invitrogen Life Technologies), respectively. Slides were then washed and mounted in aqueous mounting solution using cover glass (Fisher Scientific) before fluorescent microscopy. Images were acquired using a Nikon Eclipse E600 microscope fitted with a RT SPOT digital camera and processed using SPOT software (Diagnostic Instruments).
Statistical analysis
Data are shown as mean ± SD. Statistical significance of differences between experimental groups was analyzed by using a Student’s t test. Statistical significance was defined as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Results |
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We first investigated whether the rate of HSP70 release is related to the overall intracellular levels of the protein using PC-3 and LNCaP cells. These cell lines were chosen for their contrasting levels of intracellular HSP70. We have shown previously that although LNCaP express minimal levels of intracellular HSP70, PC-3 express HSP70 abundantly even at nonstress temperatures (24). As a stimulus for HSP70 release, we used heat shock which has previously been shown to cause release of a number of leaderless proteins including FGF-1 and IL-1 (25). We used two heat shock conditions for the experiments, including 40°C, which is in the fever range and minimally toxic in tissue culture and stimulates tumor immunity in mice (26, 27) and 43°C, which strongly induces transcriptional activation of hsp70 genes (28). As mentioned, PC-3 cells have high endogenous HSP70 levels which increase significantly after heat shock at 43°C although with comparatively minor increases at 40°C. Similar trends were observed in LNCaP cells that have almost undetectable levels of HSP70 in unstressed cells but undergo significant HSP70 induction at 40°C and 43°C (Fig. 1, A and B). These heat shock treatments resulted in minimal (<1%) levels of necrosis and apoptosis at the 40°C or 43°C condition in both cell lines at the period investigated (data not shown). This is in line with many previous studies of the kinetics of heat-induced cell death that show minimal effects on cell survival of 40°C heating or heating at 43°C up to 30 min reviewed by Hahn and Li (28). Low, but significant levels of necrosis and apoptosis (5–10%) did develop in the prostate cancer cells at later times (24–48 h) (29, 30). Our recent studies indicate that increasing temperatures above 43°C when necrosis increases actually decreases HSP70 secretion, presumably by denaturing components in the secretion pathway (30). HSP70 release at the time period examined in this study is apparently due to active secretion (30).
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The experiments in Fig. 2 suggested that HSP70 release is independent of de novo synthesis. We have directly examined the requirement for translation using protein synthesis-inhibitor cycloheximide (Fig. 3, A and B). Cycloheximide did not inhibit the rate of HSP70 release in either cell line further indicating that HSP70 translation is not required for release (Fig. 3, A and B). In addition, we found that the rate of HSP70 release was temperature dependent and minimal release was seen at 4°C, suggesting a requirement for metabolic activity in HSP70 secretion (Fig. 3, A and B). There were significant differences observed in HSP70 release at both 43°C and 40°C compared with 37°C controls. These experiments thus suggest an active process of HSP70 release that can be stimulated by heat shock. Interestingly, we observed that HSP70 release was stimulated only in full medium and was not observed in cells incubated in balanced salt solutions (Tables I and II). This effect did not appear to involve energy depletion or the absence of the stimulatory effects of serum, as HSP70 release was not observed in PBS supplemented with either 5 mM glucose or 10% FCS (Tables I and II). As the active secretion of some intracellular proteins may require Ca2+, we therefore tested the potential role of extracellular Ca2+ in HSP70 release (Tables I and II). In cells incubated at various concentrations of extracellular Ca2+ (40–400 mM) in PBS (Tables I and II), we did not see restoration of stress-induced HSP70 release. In addition, we did not observe differences in HSP70 release when cells in full medium were pretreated with the intracellular Ca2+ chelator BAPTA-AM or the extracellular Ca2+ chelator EGTA before stress (Table I and II). We did, however, observe a significantly higher rate of HSP70 release in cells treated with the calcium ionophore ionomycin alone compared with nontreated cells, whereas there was a significant additive increase when the cells were treated with ionomycin and heat compared with heat treatment alone (Table I and II).
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; addition of Mg2+ decreases secretion due ATP sequestration, whereas addition of Mg2+ chelator EDTA can increase secretion though its chelation of Mg2+ and release of ATP in unbound form (12). We incubated the prostate carcinoma cells in growth medium supplemented with 20 mM MgCl2 before heat shock. Indeed, the addition of MgCl2 had a profound significant effect on the rate of HSP70 release and almost completely inhibited both the basal release and heat-induced HSP70 release in both cell lines (Fig. 4, A and B). This suggests that extracellular ATP is present in a free form and that addition of MgCl2 sequesters free ATP and blocks HSP70 release. EDTA significantly enhanced HSP70 release but only under the 6 h at 40°C condition. The rationale behind these findings is not clear but may be related to the longer incubation time at 40°C (Fig. 4, C and D) (21). However, addition of ATP alone does not cause release of HSP70 in the absence of heat shock, suggesting that it is necessary but not sufficient for HSP70 release (data not shown). Similarly, findings are observed with IL-1 in which extracellular ATP is required for secretion, but not sufficient in itself (21).
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The ability of HSP70 to exit cells under nontoxic conditions suggests a pathway for traversing cell membranes and active secretion. Therefore, we next examined the pathway of HSP70 release, exploring the hypothesis that HSP70 release may follow mechanisms comparable to IL-1 secretion. We first explored a role for secretory lysosomes in HSP70 release and investigated whether increasing pH in these compartments using lysosomotropic agents inhibits HSP70 secretion. Both methylamine and ammonium chloride, which increase intralysosomal pH, decreased the rate of HSP70 release in PC-3 and LNCaP cells (Fig. 5). Incubation with methylamine significantly caused a >50% reduction in HSP70 release in both cell lines exposed to either heat shock condition (Fig. 5, A and B). The effects of NH4Cl were significantly more marked, with abolition of both basal and heat shock-induced HSP70 release (Fig. 5, C and D). Thus, lysomotropic drugs inhibit HSP70 secretion, and these experiments indicate a key role for the endolysosme pathway in HSP70 release.
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Because previous studies have implicated a role for ABC family transporters in the secretion of IL-1, we examined whether this mechanism might also relate to HSP70 secretion (12). Cells were pretreated with the ABC inhibitor glibenclamide before examination of basal and heat shock-induced HSP70 secretion. Glibenclamide significantly inhibited heat-induced HSP70 release in LNCaP and PC-3 cells although it had a less-pronounced effect on basal release, suggesting a role for ABC family transporters in HSP70 secretion (Fig. 7, A and B). Previous studies have implicated the ABC family member ABCA-1 in IL-1 secretion (17). The role for ABCA-1 can be examined using another chemical, inhibitor DIDS. DIDS inhibits ABCA-1 by blocking the chloride current necessary for its function (21). DIDS proved to significantly inhibit HSP70 secretion at both basal and heat shock-stimulated PC-3 and LNCaP cells (Fig. 7, C and D). Fig. 7E indicates that ABCA-1 is expressed in both cell lines both under nonheat shock and heat shock conditions. ABCA-1 may be involved in the multidrug resistance phenotype exhibited by both of the tumor cells. These experiments therefore indicate that HSP70 secretion requires the ABCA-1 isoform ABC transporters.
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We next examine whether a fraction of the HSP70 secreted from tumor cells can bind to the cell surface (Fig. 8). Such a mechanism was suggested by kinetic studies of extracellular HSP70 accumulation as in Fig. 2. In each case, we observed that after initial accumulation, extracellular HSP70 levels declined. This decrease in extracellular HSP70 may be due to binding and internalization of secreted HSP70. Indeed, cell surface HSP70 was minimal in unstressed cells but was readily detectable after 43°C for 30 min or 6 h at 40°C in both cell lines (Fig. 8, A and C). This increase in surface expression of HSP70 correlates with the increases observed in LAMP1 surface expression (Fig. 6). We also examined the behavior of intracellular HSP70 after heat shock (Fig. 8B). HSP70 appears to be evenly distributed within the cell at 37°C but was translocated to the nucleus at 43°C, where it was found in granular structures (31). However, 40°C heating did not appear to markedly alter the intracellular HSP70 distribution (Fig. 8B). This experiment therefore indicates a profound and complex redistribution of HSP70 in cells after heat shock. For instance, at 43°C, HSP70 may be translocated to the nucleus (Fig. 8B) or associated with the cell surface (Fig. 8A). We observed similar findings with the intracellular staining of LNCaP cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The mechanisms of secretion of leaderless proteins such as HSP70 are complex and incompletely understood. However, two main pathways have been deduced. The first (i) used by proteins such as FGF-1 and IL-1, appears to translocate across the plasma membrane on partial denaturation to a molten globule form (15). It has been conjectured that heat shock stimulates this pathway in part by permitting the proteins to partially unfold, a requirement for membrane translocation of many proteins (33, 34). More common (ii) appears to be the pathway used by IL-1, which involves entry of the secreted protein into endolysosomes and release from the cell by a vesiculation mechanism (35). Our largely pharmacological dissection indicates that HSP70 release belongs to this second class of leaderless secretion, and its mechanisms of release further resemble IL-1 in requiring the activity of ABC family transmembrane transporters and the likely participation of purinergic receptors (21). Other secreted proteins that are largely of cytoplasmic/nuclear distribution in addition to HSP70, such as high-mobility protein b1 (HMGB1) and engrailed-2, appear to be secreted along this pathway under specialized conditions that promote secretion (36, 37). Curiously, heat shock has been shown to cause the secretion of two proteins, FGF-1 and IL-1, through the alternative pathway (i) involving heat-induced membrane translocation (15). HSP70 secretion by heat shock may therefore use a hybrid pathway involving mechanisms found in pathways (i) and (ii). For instance, binding of FGF-1 and IL-1 to phosphatidylserine on the inner leaflet of the plasma membrane before translocation may be involved in a flipping mechanism in which phosphatidylserine is externalized to the outer leaflet of the plasma membrane accompanied by associated proteins (38). Intriguingly, HSP70 has been shown to bind phosphatidylserine, although the significance of this interaction for HSP70 release has not been determined (39). There is previous evidence that heat shock stimulates lysosomal exocytosis in vivo in liver cells and peritoneal macrophages (40, 41). Our studies do not indicate the sorting mechanism required for a fraction of the HSP70 to translocate to the lysosomal endosome secretion pathway during heat shock. However, because the majority of intracellular HSP70 is either retained in the cytoplasm or translocates to the nucleus on stress (Fig. 8B), it seems likely that a signal such as posttranslational modification may be required for its sorting to the secretion pathway (Fig. 8A). HSP70 can be phosphorylated under some circumstances and may be ubiquitinated on association with the ubiquitin ligase CHIP (42, 43). Translocation of a fraction of HMGB1 and engrailed-2 to the secretory pathway involves hyperacetylation and dephosphorylation, respectively (44, 45).
Several other schemes for HSP70 release have been proposed and indeed may be operable under some circumstances. It has been proposed that HSP70 may be released from cells in lipid-bounded endosomes (46). Such a scheme is not supported by the experiments in our study of prostate carcinoma cells in which we could only detect HSP70 in the extracellular medium in free solution. However, it is possible that a small fraction of HSP70 may be released from other cell types by such a mechanism (47, 48). Secretion mechanisms may vary between cell types and, for instance, IL-1 secretion involves quite distinct mechanisms in different cell types (12, 21). In addition, pretreatment of prostate cancer cells with glibenclamide prevented heat-induced HSP70 release but not basal release of HSP70, indicating that heat-induced release is regulated by ABC transporters, whereas basal release likely involves another mechanism (Fig. 7, C and D). In either case, it seems likely that HSP70 transits though a lipid-bounded vesicular structure along the pathways to secretion, which in the case of this study is a lysosomal compartment, and finally crosses the plasma membrane to be secreted (49).
Our experiments also suggest that HSP70 released from cells exists in free form or may bind to adjacent cells though an autocrine or paracrine mechanism (Fig. 8). Secreted HSP70 may bind to receptors on tumor cells as shown above and mark such cells for destruction by NK cells, or may become associated with APCs such as DC and induce DC maturation and Ag cross-presentation (50, 51). NK cells have been shown to bind directly to HSP70 on the surfaces of tumor cells and lead to direct cell killing (52). The role of HSP70 release in tumor cell killing by T lymphocytes has been examined by Vile and colleagues (7) who showed that HSP70 overexpression in tumors and its induced release can lead to profound immunostimulation and tumor rejection (11). However, it is not clear what the significance of low-level release of HSP70-peptide complexes in untreated tumor cells (Fig. 2) might be. Tumor cell killing by NK cells or alternatively tolerance to tumor Ags could occur. Such tolerance can, however, be broken by increasing intracellular HSP70 levels in tumors and maximizing release (7). The extracellular properties of HSP70 resemble those of HMGB1, another mammalian danger signal, in that it can be released both after cell necrosis or by active secretion mechanisms both in immune and nonimmune cells (53). In addition, stimulation of HSP70 secretion by heat shock may have direct physiological significance and may underlie some of the known effects of febrile heat on the immune response. Febrile heat facilitates some aspects of the innate immunity and causes lymphocyte infiltration and enhanced NK cell-dependent tumor cell killing, some of which changes may involve secreted HSP70 (26). Febrile heat also promotes maturation and migration of APC (27, 54).
Our experiments, therefore suggest that HSP70 under some circumstances may acquire a novel, extracellular role. The modification required in order for HSP70 to be secreted is not known, although the pathway involved has remarkable similarities to IL-1 secretion in macrophages and glial cells (12, 21). As HSP70 has potent immunological properties, this pathway may play a role in tumor immune response.
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants CA047407, CA094397, and CA094397-05S1 (to S.S.M.). 
2 Address correspondence and reprint requests to Dr. Stuart K. Calderwood, Beth Israel Deaconess Medical Center, 21-27 Burlington Avenue, Room 553, Boston, MA 02215. E-mail address: scalderw{at}bidmc.harvard.edu
3 Abbreviations used in this paper: HSP, heat shock protein; DC, dendritic cells, FGF-1, fibroblast growth factor 1; LAMP1, lysosomal-associated membrane protein 1; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; HMGB1, high-mobility protein b1.
Received for publication January 1, 2006. Accepted for publication September 11, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B pathway. Int. Immunol. 12: 1539-1546. involves exocytosis of endolysosome-related vesicles. Mol. Biol. Cell 10: 1463-1475. blocks the release of FGF1 in response to heat shock. J. Biol. Chem. 276: 5147-5151. point the way. J. Cell Sci. 116: 4871-4881. secretion is impaired by inhibitors of the Atp binding cassette transporter, ABC1. Blood 90: 2911-2915. by P2X7-stimulated and lipopolysaccharide-primed mouse Schwann cells. Glia 49: 511-519. [Medline] release by activating the purinergic P2Z receptor of human macrophages. J. Immunol. 159: 1451-1458. [Abstract] secretion: implications for inflammatory processes. Proc. Natl. Acad. Sci. USA 101: 9745-9750. This article has been cited by other articles:
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H. Jensen, L. Andresen, K. A. Hansen, and S. Skov Cell-surface expression of Hsp70 on hematopoietic cancer cells after inhibition of HDAC activity J. Leukoc. Biol., October 1, 2009; 86(4): 923 - 932. [Abstract] [Full Text] [PDF]
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V. L. Vega, M. Rodriguez-Silva, T. Frey, M. Gehrmann, J. C. Diaz, C. Steinem, G. Multhoff, N. Arispe, and A. De Maio 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., March 15, 2008; 180(6): 4299 - 4307. [Abstract] [Full Text] [PDF]
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J. H. H. Williams and H. E. Ireland Sensing danger--Hsp72 and HMGB1 as candidate signals J. Leukoc. Biol., March 1, 2008; 83(3): 489 - 492. [Abstract] [Full Text] [PDF]
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R. M. Srivastava, C. Varalakshmi, and A. Khar The Ischemia-Responsive Protein 94 (Irp94) Activates Dendritic Cells through NK Cell Receptor Protein-2/NK Group 2 Member D (NKR-P2/NKG2D) Leading to Their Maturation J. Immunol., January 15, 2008; 180(2): 1117 - 1130. [Abstract] [Full Text] [PDF]
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M. A. Chase, D. S. Wheeler, K. M. Lierl, V. S. Hughes, H. R. Wong, and K. Page Hsp72 Induces Inflammation and Regulates Cytokine Production in Airway Epithelium through a TLR4- and NF-{kappa}B-Dependent Mechanism J. Immunol., November 1, 2007; 179(9): 6318 - 6324. [Abstract] [Full Text] [PDF]
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), 43°C for 30 min (
), and 37°C control group (
) or nonheat shocked cells in tissue culture dishes containing 5 x 106 cells. Following heat treatments, the dishes were incubated at 37°C for recovery. Supernatants were taken at 60-min intervals (A and B) and 10-min intervals (C and D) and analyzed for the presence of HSP70 by ELISA. Data represent the mean ± SD of three independent experiments. Statistical analyses compared 0 time point and 60 in A and B and 0 min vs 30 min in C and D. **, p < 0.01; comparing 0 vs 360 min in A and B and 0 vs 360 in C and D (***, p < 0.001).
) and control group without cyclohexamide treatment (CHX; 
