Circulating Heat Shock Protein 60 Levels Are Elevated in HIV Patients and Are Reduced by Anti-Retroviral Therapy

Circulating heat shock protein 60 (Hsp60) and heat shock protein 10 (Hsp10) have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART). We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients.     

Expression of 60 kDa heat shock protein (Hsp60) on plasma membrane of Daudi cells

In Daudi cells, a fraction of the 60 kDa heat shock protein (Hsp60), which is typically a mitochondrial protein, is located on cell membrane. This was demonstrated by the recovery of biotinylated Hsp60 in the anti-Hsp60 immunoprecipitate obtained from cells in which surface-exposed proteins were selectively labeled with biotin. In further experiments, isolated membrane proteins (obtained by two different biochemical methods) were probed in Western blot with two antibodies (N-20 and K-19) directed against different epitopes located, respectively, at the amino- and at the carboxyl-terminus of the Hsp60. Both these antibodies recognized, among the isolated membrane proteins, a unique band with an electrophoretic mobility identical to that of the cytoplasmic Hsp60, thus demonstrating that Hsp60 is present on cell surface as an intact, full-length, protein. FACS analysis, performed with the same two highly specific anti-Hsp60 antibodies, confirmed that both the N-terminus and the C-terminus of the Hsp60 are exposed outside the cell and are accessible for recognition by the corresponding antibody. Moreover, quantitative analysis of the data showed that constitutive cell surface expression of the Hsp60 is limited to a small fraction (about 10%) of the whole cell population.     

Heat shock protein 10 inhibits lipopolysaccharide-induced inflammatory mediator production

Heat shock protein 10 (Hsp10) and heat shock protein 60 (Hsp60) were originally described as essential mitochondrial proteins involved in protein folding. However, both proteins have also been shown to have a number of extracellular immunomodulatory activities. Here we show that purified recombinant human Hsp10 incubated with cells in vitro reduced lipopolysaccharide (LPS)-induced nuclear factor-kappaB activation and secretion of several inflammatory mediators from RAW264.7 cells, murine macrophages, and human peripheral blood mononuclear cells. Induction of tolerance by contaminating LPS was formally excluded as being responsible for Hsp10 activity. Treatment of mice with Hsp10 before endotoxin challenge resulted in the reduction of serum tumor necrosis factor-alpha and RANTES (regulated upon activation, normal T cell expressed and secreted) levels and an elevation of serum interleukin-10 levels. Hsp10 treatment also delayed mortality in a murine graft-versus-host disease model, where gut-derived LPS contributes to pathology. We were unable to confirm previous reports that Hsp10 has tumor growth factor properties and suggest that Hsp10 exerts anti-inflammatory activity by inhibiting Toll-like receptor signaling possibly by interacting with extracellular Hsp60.     

Neuronal differentiation in PC12 cells is accompanied by diminished inducibility of Hsp 70 and Hsp60 in response to heat and ethanol

The stress response of PC12 cells was characterized by evaluating the production of heat shock proteins of the 70 kDa (Hsp70), 60 kDa (Hsp60) and 90 kDa (Hsp90) families by western blot analysis. Induction of Hsp synthesis was elicited by brief exposure to elevated temperatures or by addition of ethanol to the cultures. Normal PC12 cells responded to stress with rapid up-regulation of Hsp70 and Hsp60 production. However, fully differentiated PC12 cells (induced by nerve growth factor, NGF) failed to produce Hsp70 or Hsp60 in response to heat or ethanol treatment. The disappearance of the heat shock response of the cells was directly related to the extent of neuronal differentiation. The cellular levels of the constitutive proteins, Hsc70 and Hsp90, were not altered by differentiation of the cells. Production of Hsps was restored in the differentiated cells by removal of NGF which coincided with the loss of neurite expression and retraction of processes.     

Circulating anti-heat-shock-protein antibodies in normal pregnancy and preeclampsia

It has been previously reported that circulating anti-heat-shock-protein (Hsp) antibody levels are elevated in cardiovascular disorders. The aim of the present study was to determine circulating antihuman Hsp60, antimycobacterial Hsp65, and antihuman Hsp70 antibody levels in healthy pregnant women and preeclamptic patients and to investigate their relationship to the clinical characteristics of the study subjects, as well as to the markers of inflammation (C-reactive protein (CRP)), endothelial activation (von Willebrand factor antigen), or endothelial injury (fibronectin), oxidative stress (malondialdehyde) and to serum Hsp70 levels. Ninety-three preeclamptic patients and 127 normotensive healthy pregnant women were involved in this case control study. Serum anti-Hsp60, anti-Hsp65, anti-Hsp70, and Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Serum CRP levels were determined by an autoanalyzer using the manufacturer’s kit. Plasma von Willebrand factor antigen levels were quantified by ELISA, while plasma fibronectin concentration by nephelometry. Plasma malondialdehyde levels were measured by the thiobarbituric-acid-based colorimetric assay. For statistical analyses, nonparametric methods were applied. Anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibodies were detected in all of our serum samples. There were no significant differences in serum anti-Hsp60, anti-Hsp65, and anti-Hsp70 antibody levels between the control and preeclamptic groups. Serum levels of Hsp70 and CRP, as well as plasma levels of VWF antigen, fibronectin, and malondialdehyde, were significantly higher in preeclamptic patients than in normotensive healthy pregnant women. Serum anti-Hsp60 antibody levels showed significant correlations with serum anti-Hsp65 antibody levels both in the control and the preeclamptic groups (Spearman R = 0.55 and 0.59; p < 0.001, respectively). However, no other relationship was found between clinical features (maternal age, smoking status, parity, body mass index, gestational age at blood draw, systolic and diastolic blood pressure, gestational age at delivery, and fetal birth weight) and measured laboratory parameters of the study subjects and serum anti-Hsp antibody levels in either study group. In conclusion, anti-Hsp60 and anti-Hsp70 antibodies as naturally occurring autoantibodies are present in the peripheral circulation of healthy pregnant women. Nevertheless, humoral immunity against heat shock proteins was not associated with preeclampsia. Further studies are warranted to explore the role of heat shock proteins and immune reactivity to them in the immunobiology of normal pregnancy and preeclampsia.     

Extracellular heat shock protein 70 (HSPA1A) and classical vascular risk factors in a general population

Atherosclerosis is a chronic inflammatory and autoimmune disease. Candidate molecules/autoantigens include heat shock proteins (HSPs); Hsp70 (HSPA1A) is one of the best studied HSPs. Various studies have shown a correlation between extracellular Hsp70 (eHsp70) and anti-Hsp70/anti-Hsp60 antibody concentration and development of atherosclerosis. A random sample of 456 people aged 40–60 (218 males, 234 females) was studied to investigate the prevalence of traditional vascular risk factors and eHsp70 and anti-Hsp70/anti-Hsp60 antibodies levels, according to the risk of vascular disease. Task Force Chart was applied for classification. Subjects were divided into three groups: G0 (with no vascular risk factor or a risk lower than 5%), n = 239; G1 (moderated 10–20% risk, who do not have established disease) n = 161; and G2 (established atherosclerosis disease) n = 52. eHsp70 and anti-Hsp70 were significantly lower in the atherosclerosis group (group 2) with respect to the other groups. Disease-free people showed the highest anti-Hsp60 concentration compared with the other two groups. A correlation has not been demonstrated between the concentrations of circulating Hsp70 (HSPA1A), anti-Hsp70, and anti-Hsp60 and classical vascular risk factors and C-reactive protein. Low levels of eHsp70 and anti-Hsp70 antibodies should be considered as candidate FRV. Simultaneous decrease of eHsp70 and anti-Hsp70 antibodies would be explained by circulating immune complex formation, and both could be proposed as biomarkers for the progression of atherosclerotic disease. Levels of circulating anti-Hsp60 antibodies may constitute a marker of inflammation in atherosclerosis.     

Autoantibodies to heat shock protein 60, 70, and 90 are not altered in the anti-SARS-CoV-2 IgG-seropositive humans without or with mild symptoms

Highly conserved heat shock proteins (Hsps) are localized in the cytoplasm and cellular organelles, and act as molecular chaperones or proteases. Members of Hsp families are released into the extracellular milieu under both normal and stress conditions. It is hypothesized that the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) has the potential to elicit autoimmunity due to molecular mimicry between human extracellular Hsps and immunogenic proteins of the virus. To confirm the above hypothesis, levels of circulating autoantibodies directed to the key human chaperones i.e., Hsp60, Hsp70, and Hsp90 in the anti-SARS-CoV-2 IgG-seropositive participants have been evaluated. Twenty-six healthy volunteers who got two doses of the mRNA vaccine encoding the viral spike protein, anti-SARS-CoV-2 IgG-positive participants (n = 15), and healthy naïve (anti-SARS-CoV-2 IgG-negative) volunteers (n = 51) have been included in this study. We found that the serum levels of anti-Hsp60, anti-Hsp70, and anti-Hsp90 autoantibodies of the IgG, IgM, or IgA isotype remained unchanged in either the anti-COVID-19-immunized humans or the anti-SARS-CoV-2 IgG-positive participants when compared to healthy naïve volunteers, as measured by enzyme-linked immunosorbent assay. Our results showing that the humoral immune response to SARS-CoV-2 did not include the production of anti-SARS-CoV-2 antibodies that also recognized extracellular heat shock protein 60, 70, and 90 represent a partial evaluation of the autoimmunity hypothesis stated above. Further testing for cell-based immunity will be necessary to fully evaluate this hypothesis.     

The atheroprotective properties of Hsp70: a role for Hsp70-endothelial interactions?

Although heat shock (stress) proteins are typically regarded as being exclusively intracellular molecules, it is now apparent that they can be released from cells in the absence of cellular necrosis. We and others have reported the presence of Hsp60 (HSPD1) and Hsp70 (HSPA1A) in the circulation of normal individuals and our finding that increases in carotid intima-media thicknesses (a measure of atherosclerosis) in subjects with hypertension at a 4-year follow-up are less prevalent in those having high serum Hsp70 (HSPA1A) levels at baseline suggests that circulating Hsp70 (HSPA1A) has atheroprotective effects. Given that circulating Hsp70 (HSPA1A) levels can be in the range which has been shown to elicit a number of biological effects in vitro, and our preliminary findings that Hsp70 (HSPA1A) binds to and is internalised by human endothelial cell populations, we speculate on the mechanisms that might be involved in the apparent atheroprotective properties of this protein.     

Heat shock protein synthesis over time in infective Trichinella spiralis larvae raised in suboptimal culture conditions

Changes in the viability, infectivity and heat shock protein (Hsp) levels are reported in Trichinella spiralis first stage larvae (L1) stored in 199 medium for up to seven days at 37 degrees C. These conditions induce stress that the larvae, eventually, cannot overcome. After three days of storage, the infectivity and viability were unchanged, although higher Hsp70 levels were observed. After this time, larvae gradually lost viability and infectivity, coinciding with a decrease in Hsp70 and Hsp90 and an increase in actin (a housekeeping protein). In addition, a possibly inducible heat shock protein, Hsp90i, appeared as constitutive Hsp90 disappeared. No significant changes in Hsp60 levels were detected at any time. These results suggest that heat shock proteins initially try to maintain homeostasis, but on failing, may be involved in cell death.     

Impact of aging on heat shock protein expression in the substantia nigra and striatum of the female rat

Many heat shock proteins are chaperones that help refold or degrade misfolded proteins and battle apoptosis. Because of their capacity to protect against protein misfolding, they may help keep diseases of aging at bay. A few reports have examined heat shock proteins (eg. Hsp25, Hsp60, Hsp70, and heat shock cognate 70 or Hsc70) as a function of age in the striatum and nigra. In the present study, we examined the impact of aging on Hsp25, heme oxygenase 1 (HO1 or Hsp32), Hsp40, Hsp60, Hsc70, Hsc/Hsp70 interacting protein (Hip), 78 kDa glucose-regulated protein (GRP78), Hsp90, and ubiquitinated proteins in the nigra and striatum of the female rat by infrared immunoblotting. Female animals are not typically examined in aging studies, adding further to the novelty of our study. Striatal HO1 and Hsp40 were both higher in middle-aged females than in the oldest group. Hsp60 levels were also highest in middle age in the nigra, but were highest in the oldest animals in the striatum. Striatal levels of Hsc70 and the co-chaperone Hip were lower in the oldest group relative to the youngest animals. In contrast, Hsp25 rose with advancing age in both regions. Hsp25 was also colocalized with tyrosine hydroxylase in nigral neurons. Ubiquitinated proteins exhibited a trend to rise in the oldest animals in both regions, and K48 linkage-specific ubiquitin rose significantly from 4–6 to 16–19 months in the striatum. Our study reveals a complex array of age-related changes in heat shock proteins. Furthermore, the age-related rises in some proteins, such as Hsp25, may reflect endogenous adaptations to cellular stress.     

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37°C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.     

Kinetics of thermally induced heat shock protein 27 and 70 expression by bone marrow-derived mesenchymal stem cells

Although bone marrow-derived mesenchymal stem cells (MSCs) are an attractive cell therapy candidate, their potential is limited by poor survival following transplantation. Over-expression of anti-apoptotic heat shock proteins using viral vectors can improve the survival of these cells under stressful conditions in vitro and in vivo. It is also possible to induce heat shock protein expression in many cell types by simply exposing them to a transient, nonlethal elevation in temperature. The response profile of MSCs to such a thermal stress has not yet been reported. Therefore, this study sought to determine the kinetics of thermally induced heat shock protein expression by MSCs in vitro. To determine if heat shock protein expression was a function of thermal stress exposure time, MSCs were exposed to 42°C for 15, 30, 45, and 60 min and were harvested 24 h later. To establish the time-course of heat shock protein expression, MSCs were heat shocked for 60 min and harvested 2, 24, 48, 72, 96, and 120 h later. The cells were then analyzed for Hsp27 and Hsp70 expression by Western blot. Densitometric analysis revealed that exposure to a thermal stress induced expression of both Hsp27 and Hsp70 and that the level of expression was dependant on stress exposure time. Following 60 min of heat stress, both Hsp27 and Hsp70 accumulated maximal expression after 48 h with both proteins returning to constitutive expression levels by 120 h. This study demonstrates that heat shock protein expression can be induced in MSCs by a simple thermal stress.     

Glucose ingestion attenuates the exercise-induced increase in circulating heat shock protein 72 and heat shock protein 60 in humans

Heat shock protein (Hsp) 72 is a cytosolic stress protein that is highly inducible by several factors including exercise. Hsp60 is primarily mitochondrial in cellular location, plays a key role in the intracellular protein translocation and cytoprotection, is increased in skeletal muscle by exercise, and is found in the peripheral circulation of healthy humans. Glucose deprivation increases Hsp72 in cultured cells, whereas reduced glycogen availability elevates Hsp72 in contracting human skeletal muscle. To determine whether maintained blood glucose during exercise attenuates the exercise-induced increase in intramuscular and circulating Hsp72 and Hsp60, 6 males performed 120 minutes of semirecumbent cycling at approximately 65% maximal oxygen uptake on 2 occasions while ingesting either a 6.4% glucose (GLU) or sweet placebo (CON) beverage throughout exercise. Muscle biopsies, obtained before and immediately after exercise, were analyzed for Hsp72 and Hsp60 protein expression. Blood samples were simultaneously obtained from a brachial artery, a femoral vein, and the hepatic vein before and during exercise for the analysis of serum Hsp72 and Hsp60. Leg and hepatosplanchnic blood flow were measured to determine Hsp72-Hsp60 flux across these tissue beds. Neither exercise nor glucose ingestion affected the Hsp72 or Hsp60 protein expression in, or their release from, contracting skeletal muscle. Arterial serum Hsp72 increased (P < 0.05) throughout exercise in both trials but was attenuated (P < 0.05) in GLU. This may have been in part because of the increased (P < 0.05) hepatosplanchnic Hsp72 release in CON, being totally abolished (P < 0.05) in GLU. Serum Hsp60 increased (P < 0.05) after 60 minutes of exercise in CON before returning to resting levels at 120 minutes. In contrast, no exercise-induced increase in serum Hsp60 was observed in GLU. We detected neither hepatosplanchnic nor contracting limb Hsp60 release in either trial. In conclusion, maintaining glucose availability during exercise attenuates the circulating Hsp response in healthy humans.     

Specific incorporation of heat shock protein 70 family members into primate lentiviral virions

To determine if any heat shock proteins are incorporated into human immunodeficiency virus type 1 (HIV-1) virions in a manner similar to that of the peptidyl-prolyl isomerase cyclophilin A, we probed purified virions with antibodies against heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70, Hsc70, and Hsp90. Of these proteins, Hsp60, Hsp70, and Hsc70 associated with virions purified based on either particle density or size and were shown to be incorporated within the virion membrane, where they were protected from digestion by exogenous protease. Virion incorporation of Hsp70 was also observed with HIV-2 and with simian immunodeficiency viruses SIV(MAC) and SIV(AGM), but it appears to be specific for primate lentiviruses, since Hsp70 was not detected in association with Moloney murine leukemia virus virions. Of the HIV-1 genes, gag was found to be sufficient for Hsp70 incorporation, though Hsp70 was roughly equimolar with pol-encoded proteins in virions.     

The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.     

Heat shock protein 60 and adipocytes: Characterization of a ligand-receptor interaction

Adipocyte-derived mediators contribute to chronic, diabetes-associated inflammation. We recently demonstrated, that heat shock protein 60 (Hsp60) is an effective inductor of inflammatory adipocyte activities. In the present study, we characterized the initial Hsp60 binding to adipocyte receptor structures. Analyses with preadipocytes and adipocytes from the murine 3T3-L1 line and with primary cultures from the New Zealand obese mouse, a model of human obesity, revealed comparable specific, dose-dependent and saturable Hsp60 binding, confirming the characteristics of a ligand-receptor interaction. Furthermore, we identified the N-terminal regions aa1-50 and aa91-110 of the Hsp60 molecule as relevant epitopes involved in binding to receptor structures on these cells. Our results demonstrate differentiation-independent conserved Hsp60 reactivity in permanent and primary adipocytes, strongly indicating that Hsp60 is an important regulator of inflammatory adipocyte activities.     

Hsp60D is essential for caspase-mediated induced apoptosis in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Apart from their roles as chaperones, heat shock proteins are involved in other vital activities including apoptosis with mammalian Hsp60 being ascribed proapoptotic as well as antiapoptotic roles. Using conditional RNAi or overexpression of Hsp60D, a member of the Hsp60 family in Drosophila melanogaster, we show that the downregulation of this protein blocks caspase-dependent induced apoptosis. GMR-Gal4-driven RNAi for Hsp60D in developing eyes dominantly suppressed cell death caused by expression of Reaper, Hid, or Grim (RHG), the key activators of canonical cell death pathway. Likewise, Hsp60D-RNAi rescued cell death induced by GMR-Gal4-directed expression of full-length and activated DRONC. Overexpression of Hsp60D enhanced cell death induced either by directed expression of RHG or DRONC. However, the downregulation of Hsp60D failed to suppress apoptosis caused by unguarded caspases in DIAP1-RNAi flies. Furthermore, in DIAP1-RNAi background, Hsp60D-RNAi also failed to inhibit apoptosis induced by RHG expression. The Hsp60 and DIAP1 show diffuse and distinct granular overlapping distributions in the photoreceptor cells with the bulk of both proteins being outside the mitochondria. Depletion of either of these proteins disrupts the granular distribution of the other. We suggest that in the absence of Hsp60D, DIAP1 is unable to dissociate from effecter and executioner caspases, which thus remain inactive.     

Induced thermotolerance and associated expression of the heat-shock protein Hsp70 in adult Drosophila melanogaster

1. Inducible heat-shock proteins are synthesized when temperatures are increased to levels substantially above normal. The functional role of these proteins is well known at the cellular level. Today increasing interest has been directed towards the importance of heat-shock proteins for resistance of whole organisms to high-temperature stress and other environmental stressors.
2. Here the functional relationship between the heat-shock protein, Hsp70, and thermal resistance in adult Drosophila melanogaster was examined by comparing thermal resistance, i.e. survival at 39 °C for 85 min, and levels of Hsp70 at various times elapsed (2, 4, 8, 16, 32 and 64 h) after thermotolerance was induced by short-term acclimation/heat hardening at 37 °C for 55 min.
3. Levels of Hsp70 in both males and females were highest 2 h after heat hardening and declined with longer times elapsed. The rate of decrease initially was very fast but diminished with increasing time. After 32 h the level of Hsp70 approached the level in flies that were not hardened. Levels of Hsp70 in males exceeded that of females during the entire period.
4. Survival of both sexes increased with increasing time after heat hardening and reached an optimum between 8 and 32 h. Thereafter resistance decreased with longer times elapsed. Survival of females generally exceeded that of males except after 16 and 64 h.
5. Regression analysis applied to the data on Hsp70 levels revealed that the model describing these data could not explain the data for survival. Also, higher levels of Hsp70 in males compared with females were not associated with greater survival in males. However, statistical analysis on paired measurements of Hsp70 and survival revealed a positive association between Hsp70 level and survival at each time elapsed after induction of thermotolerance.