Downregulation of heat shock protein 70 protects rheumatoid arthritis fibroblast-like synoviocytes from nitric oxide-induced apoptosis

Introduction  

Heat shock protein 70 (Hsp70) is a well-known anti-apoptotic protein that blocks multiple steps in the stress-induced apoptotic pathway. Enhanced Hsp70 expression has previously been demonstrated in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). The authors investigated the role of Hsp70 in the survival of RA FLSs in a sodium nitroprusside (SNP)-treated environment.     

Intracellular heat shock protein-70 negatively regulates TLR4 signaling in the newborn intestinal epithelium

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal-related mortality in premature infants, and it develops under conditions of exaggerated TLR4 signaling in the newborn intestinal epithelium. Because NEC does not develop spontaneously, despite the presence of seemingly tonic stimulation of intestinal TLR4, we hypothesized that mechanisms must exist to constrain TLR4 signaling that become diminished during NEC pathogenesis and focused on the intracellular stress response protein and chaperone heat shock protein-70 (Hsp70). We demonstrate that the induction of intracellular Hsp70 in enterocytes dramatically reduced TLR4 signaling, as assessed by LPS-induced NF-κB translocation, cytokine expression, and apoptosis. These findings were confirmed in vivo, using mice that either globally lacked Hsp70 or overexpressed Hsp70 within the intestinal epithelium. TLR4 activation itself significantly increased Hsp70 expression in enterocytes, which provided a mechanism of autoinhibition of TLR4 signaling in enterocytes. In seeking to define the mechanisms involved, intracellular Hsp70-mediated inhibition of TLR4 signaling required both its substrate-binding EEVD domain and association with the cochaperone CHIP, resulting in ubiquitination and proteasomal degradation of TLR4. The expression of Hsp70 in the intestinal epithelium was significantly decreased in murine and human NEC compared with healthy controls, suggesting that loss of Hsp70 protection from TLR4 could lead to NEC. In support of this, intestinal Hsp70 overexpression in mice and pharmacologic upregulation of Hsp70 reversed TLR4-induced cytokines and enterocyte apoptosis, as well as prevented and treated experimental NEC. Thus, a novel TLR4 regulatory pathway exists within the newborn gut involving Hsp70 that may be pharmacologically activated to limit NEC severity.     

Critical role of extracellular heat shock cognate protein 70 in the myocardial inflammatory response and cardiac dysfunction after global ischemia-reperfusion

Previous studies showed that Toll-like receptor 4 (TLR4) modulates the myocardial inflammatory response to ischemia-reperfusion injury, and we recently found that cytokines link TLR4 to postischemic cardiac dysfunction. Although TLR4 can be activated in cultured cells by endogenous agents including heat shock protein 70, how it is activated during myocardial ischemia-reperfusion is unknown. In the present study, we examined 1) whether heat shock cognate protein 70 (HSC70), which is constitutively expressed in the myocardium, is released during ischemia-reperfusion; 2) whether extracellular HSC70 induces the myocardial inflammatory response and modulates cardiac function; and 3) whether HSC70 exerts these effects via TLR4. We subjected isolated mouse hearts to global ischemia-reperfusion via the Langendorff technique. Immunoblotting and immunostaining detected the release of HSC70 from the myocardium during reperfusion. Treatment with an antibody specific to HSC70 suppressed myocardial cytokine expression and improved cardiac functional recovery after ischemia-reperfusion. Recombinant HSC70 induced NF-kappaB activation and cytokine expression and depressed myocardial contractility in a TLR4-dependent manner. These effects required the substrate-binding domain of HSC70. Fluorescence resonance energy transfer analysis of isolated macrophages demonstrated that extracellular HSC70 interacts with TLR4. Therefore, this study demonstrates for the first time that 1) the myocardium releases HSC70 during ischemia-reperfusion, 2) extracellular HSC70 contributes to the postischemic myocardial inflammatory response and to cardiac dysfunction, 3) HSC70 exerts these effects through a TLR4-dependent mechanism, and 4) the substrate-binding domain of HSC70 is required to induce these effects. Thus extracellular HSC70 plays a critical role in regulating the myocardial innate immune response and cardiac function after ischemia-reperfusion.     

Flagellin contamination of recombinant heat shock protein 70 is responsible for its activity on T cells

Heat shock proteins (Hsp) 60 and 70 have been intensively studied for their ability to activate innate immunity. Heat shock proteins had been shown to induce the activation of dendritic cells, T cells, and B cells. However, the possible contamination of endotoxin in heat shock protein preparations makes their function as an activator of immune system ambiguous. Here, we examined the ability of bacterial Hsp60 and Hsp70 to activate Jurkat T cells and primary T cells. We found that Burkholderia pseudomallei Hsp70 and Mycobacterium tuberculosis Hsp70 could costimulate Jurkat T cells to make IL-2 and signal through TLR5. This costimulatory activity is not due to endotoxin or contaminants signaling via TLR2 nor TLR4. However, recombinant Hsp70 expressed in Escherichia coli DeltafliC strain completely lost its ability to costimulate T cells. Thus, the activation of T cells by recombinant Hsp70 is ascribed to flagellin contamination.     

The role of heat shock proteins HSP90 in the response of immune cells to centimeter microwaves

The effects of low-level electromagnetic waves (8.15-18 GHz, 1 microW/cm2, 1 h) on the production of heat shock proteins, several cytokines, and nitric oxide in isolated mouse macrophages and lymphocytes were examined both under normal conditions and after the treatment of the cells with geldanamycin (GA), a depressor of activity of the heat shock protein 90 (Hsp90). The irradiation of cells without GA induced the production of Hsp70, nitric oxide (NO), interleukin-1beta (IL-1beta), interleukin-10 (IL-10), and the tumor necrosis factor -alpha (TNF-alpha). No changes in the production of Hsp90 in irradiated cells were observed, but intracellular locations of Hsp25 and Hsp70 altered. The preliminary treatment of cells with GA did not remove the effects of microwaves: in these conditions, the synthesis of all cytokines tested, nitric oxide, as well as total and membrane amount of Hsp70, and the amount of Hsp25 in the cytoplasm and cytoskeleton increased. Moreover, the exposure of cells incubated with GA resulted in the reduction of Hsp90-alpha production.     

Activation of hepatocytes by extracellular heat shock protein 72

Heat shock protein (HSP) 72 is released by cells during stress and injury. HSP-72 also stimulates the release of cytokines in macrophages by binding to Toll-like receptors (TLR) 2 and 4. Circulating levels of HSP-72 increase during hepatic ischemia-reperfusion injury. The role of extracellular HSP-72 (eHSP-72) in the injury response to ischemia-reperfusion is unknown. Therefore, the objective of the present study was to determine whether eHSP-72 has any direct effects on hepatocytes. Primary mouse hepatocytes were treated with purified human recombinant HSP-72. Conditioned media were evaluated by ELISA for the cytokines, TNF-alpha, IL-6, and macrophage inflammatory protein 2 (MIP-2). Stimulation of hepatocytes with eHSP-72 did not induce production of TNFalpha or IL-6 but resulted in dose-dependent increases in MIP-2 production. To evaluate the pathway responsible for this response, expression of TLR2 and TLR4 was confirmed on hepatocytes by immunohistochemistry. Hepatocyte production of MIP-2 was significantly decreased in hepatocytes obtained from TLR2 or TLR4 knockout mice. MIP-2 production was found to be partially dependent on NF-kappaB because inhibition of NF-kappaB with Bay 11-7085 significantly decreased eHSP-72-induced MIP-2 production. Inhibitors of p38 mitogen-activated protein kinase or c-Jun NH(2)-terminal kinase had no effect on production of MIP-2 induced by eHSP-72. The data suggest that eHSP-72 binds to TLR2 and TLR4 on hepatocytes and signals through NF-kappaB to increase MIP-2 production. The fact that eHSP-72 did not increase TNF-alpha or IL-6 production may be indicative of a highly regulated signaling pathway downstream from TLR.     

Selectively increasing inducible heat shock protein 70 via TAT-protein transduction protects neurons from nitrosative stress and excitotoxicity

Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT-Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT-Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre-incubated with 300 nM TAT-Hsp 70 overnight, then exposed to either peroxynitrite (ONOO-) or glutamate. TAT-Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild-type Hsp70, and TAT-beta-galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT-Hsp70 is protective in in vivo models of brain injury.     

Induction of cytokines by heat shock proteins and concanavalin A in murine splenocytes

There has been considerable interest in the effect of heat shock proteins (HSPs) on the innate immune system. Whether HSPs have any direct effects on the activation of lymphocytes is not clear. Using gene expression array, protein array and enzyme-linked immunosorbent assay, we demonstrated that highly purified recombinant murine Hsp60 (rmHsp60), essentially free of lipopolysaccharide contamination, had no effect in the expression of 113 cytokine genes and the release of 22 common cytokines including interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) by murine splenocytes. Likewise, recombinant human Hsp60 (rhHsp60) and rhHsp70 had no effect in the release of IFN-gamma and IL-2. In contrast, concanavalin A induced the expression of a number of cytokine genes and the release of IFN-gamma and IL-2. These results suggest that Hsp60 and Hsp70 do not induce cytokine production by murine splenocytes.     

The anti-inflammatory effects of heat shock protein 72 involve inhibition of high-mobility-group box 1 release and proinflammatory function in macrophages

High-mobility-group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. Anti-inflammatory regulation by the stress response is an effective autoprotective mechanism when the host encounters harmful stimuli, but the mechanism of action remains incompletely delineated. In this study, we demonstrate that increases in levels of a major stress-inducible protein, heat shock protein 72 (Hsp72) by gene transfection attenuated LPS- or TNF-alpha-induced HMGB1 cytoplasmic translocation and release. The mechanisms involved inhibition of the chromosome region maintenance 1 (CRM1)-dependent nuclear export pathway. Overexpression of Hsp72 inhibited CRM1 translocation and interaction between HMGB1 and CRM1 in macrophages post-LPS and TNF-alpha treatment. In addition, overexpression of Hsp72 strongly inhibited HMGB1-induced cytokine (TNF-alpha, IL-1beta) expression and release, which correlated closely with: 1) inhibition of the MAP kinases (p38, JNK, and ERK); and 2) inhibition of the NF-kappaB pathway. Taken together, these experiments suggest that the anti-inflammatory activity of Hsp72 is achieved by interfering with both the release and proinflammatory function of HMGB1. Our experimental data provide important insights into the anti-inflammatory mechanisms of heat shock protein protection.     

Localization of heat shock protein 70 in rat mast cells

Heat shock proteins (Hsp) are intracellular chaperons, as well as extracellular molecules with immunomodulatory and signaling functions engaged in adaptation to stress on the cellular and organism levels. The presence of Hsp in secretory granules of mast cells (MCs) may be correlated with mast cells’ active participation in adaptation to stress. Using immunoelectron microscopy, we showed that Hsp70 was localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat peritoneal mast cells isolated by Percoll density gradient centrifugation was confirmed by immunoblotting. The possible involvement of mast cells in production of extracellular Hsp70, as well as Hsp70 functions inside the mast cells, is discussed.     

Role of scavenger receptors in the binding and internalization of heat shock protein 70

Extracellular heat shock protein 70 (Hsp70) exerts profound effects both in mediating tumor rejection by Hsp70-based vaccines and in autoimmunity. Further progress in this area, however, awaits the identification of the cell surface receptors for extracellular Hsp70 that mediate its immune functions. We have examined a wide range of candidate Hsp70 receptors and find significant binding through two main families of cell surface proteins, including 1) the scavenger receptor (SR) family and 2) C-type lectins of the NK family. In addition, given that the anticancer effects of Hsp70 vaccines have been shown to involve uptake of Ags by APC exposed to Hsp70-tumor Ag complexes, we have examined the ability of the receptors identified here to internalize Hsp70-peptide complexes. Our findings indicate that three members of the SR family (lectin-like oxidized low density lipoprotein receptor 1; fasciclin, epidermal growth factor-like, laminin-type epidermal growth factor-like, and link domain-containing scavenger receptor-1; and SR expressed by endothelial cells-1) are able to bind Hsp70-peptide complexes and mediate its efficient internalization. Indeed, each of the SR was able to mediate efficient uptake of Hsp70 when transfected into Chinese hamster ovary cells previously null for uptake. Curiously, Hsp70 internalization occurs independently of the intracellular domains of the SR, and Hsp70 uptake could be detected when the entire intracellular domain of lectin-like oxidized low density lipoprotein receptor 1 or SR expressed by endothelial cells-1 was truncated. The existence of a wide repertoire of cell surface Hsp70-binding structures may permit intracellular responses to extracellular Hsp70 that are cell specific and discriminate between Hsp70 family members.     

Extracellular heat shock protein 70 mediates heat stress-induced epidermal growth factor receptor transactivation in A431 carcinoma cells

The initial steps of heat stress in A431 cells were previously characterized by ligand-independent EGFR transactivation via an unknown mechanism and concomitant secretion of Hsp70. In this work we demonstrate that the depletion of Hsp70 from the conditioned medium of heated cells abolishes EGFR transactivation indicating that secreted Hsp70 is essential for EGFR transactivation during heat shock. This notion is supported by the findings that purified Hsp70 can induce EGFR transactivation and the activation of EGFR-dependent signaling pathways. Both heat stress and pure Hsp70 stimulate activation of TLR2/4 and their association with EGFR. These results suggest that the secreted Hsp70 mediates the cross-communication of TLR and EGFR signaling systems in A431 cells.     

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.     

Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response

Toll-like receptors (TLRs) play important roles in initiation of innate and adaptive immune responses. Emerging evidence suggests that TLR agonists can serve as potential adjuvant for vaccination. Heat shock proteins (HSPs), functionally serving as TLR4 agonists, have been proposed to act as Th1 adjuvant. We have identified a novel Hsp70 family member, termed Hsp70-like protein 1 (Hsp70L1), shown that Hsp70L1 is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses. However, the underlying mechanism for how Hsp70L1 exerts its Th1 adjuvant activity remains to be elucidated. In this study, we found that Hsp70L1 binds directly to TLR4 on the surface of DCs, activates MAPK and NF-κB pathways, up-regulates I-a(b), CD40, CD80, and CD86 expression and promotes production of TNF-α, IL-1β, and IL-12p70. Hsp70L1 failed to induce such phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating a role for TLR4 in mediating Hsp70L1-induced DC activation. Furthermore, more efficient induction of carcinoembryonic antigen (CEA)-specific Th1 immune response was observed in mice immunized by wild-type DCs pulsed with Hsp70L1-CEA(576-669) fusion protein as compared with TLR4-deficient DCs pulsed with same fusion protein. In addition, TLR4 antagonist impaired induction of CEA-specific human Th1 immune response in a co-culture system of peripheral blood lymphocytes (PBLs) from HLA-A2.1(+) healthy donors and autologous DCs pulsed with Hsp70L1-CEA(576-669) in vitro. Taken together, these results demonstrate that TLR4 is a key receptor mediating the interaction of Hsp70L1 with DCs and subsequently enhancing the induction of Th1 immune response by Hsp70L1/antigen fusion protein.     

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.     

Biological heterogeneity of the peptide-binding motif of the 70-kDa heat shock protein by surface plasmon resonance analysis

70-kDa heat shock protein family is a molecular chaperone that binds to a variety of client proteins and peptides in the cytoplasm. Several studies have revealed binding motifs between 70-kDa heat shock protein family and cytoplasmic proteins by conventional techniques such as phage display library screening. However, little is known about the binding motif based on kinetic parameters determined by surface plasmon resonance analysis. We investigated the major inducible cytosolic 70-kDa heat shock protein (Hsp70)-binding motif with the human leukocyte antigen B*2702-derived peptide Bw4 (RENLRIALRY) by using a Biacore system based on surface plasmon resonance analysis. The K(D) value of Hsp70-Bw4 interaction was 1.8 x 10(-6) m. Analyses with truncated Bw4 variant peptides showed the binding motif of Hsp70 to be seven residues, LRIALRY. To further study the characteristics of this motif, 126 peptides derived from Bw4, each with single amino acid substitution, were synthesized and analyzed for Hsp70 binding affinity. Interestingly, the Hsp70 binding affinity was abrogated when the residues were substituted for by acidic (Asp and Glu) ones at any position. In contrast, if the substitute residue was aromatic (Trp, Tyr, and Phe) or an Arg residue at any position, Hsp70 binding affinity was maintained. Thus, this study presents a new binding motif between Hsp70 and peptides derived from the natural protein human leukocyte antigen B*2702 and may also elucidate some characteristics of the Hsp70 binding characteristic, enhancing our understanding of Hsp70-binding determinants that may influence diverse cellular and physiological processes.     

Immunohistochemical detection of the inducible heat shock protein hsp70: a methodological study.

Stress-inducible Hsp70i and constitutively expressed Hsc70 are highly related heat shock proteins. Aberrant expression levels and intracellular localization of these proteins has been suggested as a potential marker in certain tumors. The aim of our study was to work out a reliable, immunohistochemical detection of the stress-inducible Hsp70i protein and enabling discrimination between Hsp70i and Hsc70 proteins in paraffin-embedded human tissues. We tested the effect of several fixative procedures and antigen retrieval on the effectiveness of the Hsp70i detection in murine cells cultured in vitro and in liver of rats subjected to heat shock. For cells grown in vitro, specific Hsp70i immunoreactivity was obtained with all fixatives used. However, samples fixed in 10% formalin and 4% paraformaldehyde required antigen retrieval. In liver tissue embedded in paraffin, regardless the fixative used, positive Hsp70i staining could be visible only if antigen retrieval was applied. We applied this procedure for detection of Hsp70i in routine sections of breast and lung cancers fixed with 10% formalin and found that the application of thermal antigen retrieval significantly enhanced the SPA810 immunoreactivity and reduced background staining. This procedure enabled also the differential detection of Hsp70i and Hsc70 in routine histopathological preparations.     

The presence and localization of heat shock protein 70 in rat mast cells

Heat shock protein 70 (Hsp70) is considered not only as a cytosolic stress protein, but also as an extracellular molecule with immunomodulatory and signaling functions that play a role in adaptation to stress on cellular and systemic levels. The active involvement of mast cells in adaptation to stress may be associated with the presence of Hsp70 in secretory granules. Using immunoelectron microscopy, we showed that Hsp70 localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat perinoneal mast cells isolated by centrifugation on Percoll was confirmed by immunoblotting. The proposed involvement of mast cells in production of extracellular Hsp70 and possible functions of Hsp70 inside the mast cells granules are discussed.