Frequency-specific association of antibodies against heat shock proteins 60 and 70 with noise-induced hearing loss in Chinese workers

Noise exposure may result in production of auto-antibodies against heat shock proteins (Hsps), which might be of significance in the pathogenesis or prognosis (or both) of auto-immune ear diseases. However, it is not known whether these antibodies are associated with noise-induced hearing loss (NIHL) in workers exposed to noise in occupational settings. Using immunoblotting with human recombinant Hsps, audiological assessment, and multivariate logistic regression models, we investigated the presence of antibodies against Hsp60 and Hsp70 and hearing levels, and analyzed their associations with NIHL in 399 workers exposed to noise between 75 and 115 dB. Our findings showed that the prevalence of positive anti-Hsp70 was significantly higher in the workers with slight and moderate high-frequency hearing loss than in normal workers (P < 0.05). Furthermore, the prevalence of positive anti-Hsp60 in workers with moderate low-frequency NIHL was significantly higher than in the normal (P < 0.01). The levels of anti-Hsp70 and anti-Hsp60 seemed correlated, and the level of anti-Hsp70 better predicted the level of anti-Hsp60. An elevated plasma level of anti-Hsp70 was associated with a nonsignificantly increased risk of high-frequency NIHL (adjusted OR = 1.45; 95% CI = 0.89-2.36) and an elevated plasma level of anti-Hsp60 was associated with a nonsignificantly increased risk of the low-frequency NIHL (adjusted OR = 2.25; 95% CI = 0.85-5.96). These results suggest that the production of anti-Hsp60 and anti-Hsp70 may play a role in the pathogenesis of NIHL, and that anti-Hsps may be a risk factor. The precise mechanisms for the elevation of antibodies against Hsps caused by noise exposure and their possible role in the development of NIHL warrant further investigations.     

Plasma antibodies against heat shock protein 70 correlate with the incidence and severity of asthma in a Chinese population

Background

The heat shock proteins (Hsps) are induced by stresses such as allergic factors and inflammatory responses in bronchi epithelial cells and therefore may be detectable in patients with asthma. However, the etiologic link between anti-Hsps and asthma (its severity and related inflammatory responses such as interleukin-4 and immunoglobulin E) has not been established. We determined whether antibodies against Hsp60 and Hsp70 were present in patients with asthma and evaluated their associations with risk and severity of asthma.

Methods

We determined the levels of anti-Hsp60 and anti-Hsp70 by immunoblot and their associations with risk and symptom severity of asthma in 95 patients with asthma and 99 matched non-symptomatic controls using multivariate logistic regression analysis.

Results

Compared to the controls, asthma patients were more likely to have detectable anti-Hsp60 (17.2% vs 5.1%) and anti-Hsp70 (33.7% vs 8.1%) (p ≤ 0.001). In particular, the presence of anti-Hsp70 was associated with a greater than 2 fold risk for asthma (adjusted OR = 2.21; 95% CI = 1.35~3.59). Furthermore, both anti-Hsp60 and anti-Hsp70 levels were positively correlated with symptom severity (p < 0.05) as well as interleukin-4 and immunoglobulin E (p < 0.05). Individuals with antibodies against anti-Hsp60 and anti-Hsp70 were more likely to have a family history of asthma (p < 0.001) and higher plasma concentrations of total immunoglobulin E (p = 0.001) and interleukin-4 (p < 0.05) than those without antibodies.

Conclusions

These data suggest that anti-Hsp60 and especially anti-Hsp70 correlate with the attacks and severity of asthma. The underlying molecular mechanisms linking antibodies to heat shock proteins and asthma remain to be investigated.     

Birth defects and anti-heat shock protein 70 antibodies in early pregnancy

Abstract It has been suggested that induction of the heat shock response in the mammalian embryo during the critical period of organogenesis can result in anatomical malformation. We measured serum heat shock protein 70 (Hsp70), anti-Hsp70, and anti-Hsp60 in samples taken from expectant mothers at 16 weeks gestation. Samples from women whose babies were born with a birth defect (n = 30) were compared with controls who gave birth to healthy babies (n = 46). Anti-Hsp70 levels were significantly elevated in patients who later gave birth to babies with cleft lip or palate or neurological abnormalities (n = 10): 260 (223-406) microg/mL compared to 150 (88-207) microg/mL in controls (P < 0.001). No significant differences were found in serum Hsp70 and anti-Hsp60 levels between cases and controls. This finding of increased maternal anti-Hsp70 in patients who later gave birth to babies with these abnormalities suggests a previous stressful event may have contributed to the pathogenesis. Further work is required to determine whether Hsp70 has a direct or indirect role in this pathogenesis or whether anti-Hsp70 is simply a marker of a prior increase in Hsp70 due to a physiological stress that itself resulted in the damage. This work is consistent with previous studies showing a buffering role for Hsps in evolution.     

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.     

Association of increased heat shock protein 70 levels in the lymphocyte with high risk of adverse pregnancy outcomes in early pregnancy: a nested case-control study

Studies suggest that heat shock proteins (Hsps), Hsp70 in particular, may play a role in embryogenesis and reproduction. As the first trimester is the critical period of human fetal development, we tested whether there is an association between Hsp70 expression in lymphocytes and adverse pregnancy outcomes (APOs) during that period. We measured lymphocyte Hsp70 levels by immunoblot in 55 pregnant women with APOs and 110 well-matched controls selected from 778 pregnant women in a nested case-control study. Multivariate logistic regression models were used to investigate the association between lymphocyte Hsp70 levels and risk of developing APOs. Our data showed that Hsp70 levels in women with APOs, especially those younger than 29 years old, were significantly higher than controls (193 vs 135 units, P < 0.001) and that the elevated Hsp70 levels were associated with a significantly increased risk of APOs (adjusted OR = 1.014; 95% CI = 1.008-1.020, P < 0.001). Our results also showed that the sensitivity, specificity, and positive and negative predictive values were 78%, 60%, 50%, and 85%, respectively, among these pregnant women. Adjusted ORs and 95% CI for the association between a Hsp70 value > 153 IOD and APOs were statistically significant (OR = 8.78, 95% CI = 2.79-27.64, P < 0.001). These results suggest that Hsp70 may play a role in the etiology of APOs. However, the underlying mechanisms for the elevation of Hsp70 in women with APOs and whether Hsp70 can be applied as a clinical indicator of APOs warrant further investigations.     

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.     

Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = -3.708, P < 0.0001) but not with sex (r = -10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).     

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

The role of heat shock proteins in inflammatory injury induced by cold stress in chicken hearts

The aim of this study was to investigate the effects of cold stress on the expression levels of heat shock proteins (Hsps90, 70, 60, 40, and 27) and inflammatory factors (iNOS, COX-2, NF-κB, TNF-α, and PTGEs) and oxidative indexes in hearts of chickens. Two hundred forty 15-day-old male chickens were randomly divided into 12 groups and kept at the temperature of 12 ± 1 °C for acute and chronic cold stress. There were one control group and five treatment groups for acute cold stress, three control groups, and three treatment groups for chronic cold stress. After cold stress, malondialdehyde level increased in chicken heart; the activity of superoxide dismutase and glutathione peroxidase in the heart first increased and then decreased. The inflammatory factors mRNA levels were increased in cold stress groups relative to control groups. The histopathological analysis showed that heart tissues were seriously injured in the cold stress group. Additionally, the mRNA levels of Hsps (70, 60, 40, and 27) increased significantly (P < 0.05) in the cold stress groups relative to the corresponding control group. Meanwhile, the mRNA level and protein expression of Hsp90 decreased significantly (P < 0.05) in the stress group, and showed a gradually decreasing tendency. These results suggested that the levels of inflammatory factors and Hsps expression levels in heart tissues can be influenced by cold stress. Hsps commonly played an important role in the protection of the heart after cold stress.     

Plasma levels of Hsp70 and anti-Hsp70 antibody predict risk of acute coronary syndrome

Although immune reactions against heat shock proteins have been implicated in the pathogenesis of atherosclerosis, conflicting associations between Hsp70, anti-Hsp70 antibody and coronary heart disease (CHD) have been reported. This study assessed whether there is a significant association between extracellular human Hsp70, anti-Hsp70 antibody and acute coronary syndrome (ACS) and stable angina (SA), and examined dynamic changes in Hsp70 and anti-Hsp70 antibody levels induced by acute myocardial infarction (AMI). Plasma Hsp70 and anti-Hsp70 antibody levels in 291 patients with ACS (179 AMI, 112 unstable angina), 126 patients with SA and 417 age and sex-matched healthy subjects, and in 40 patients after admission for AMI, and on day 2, 3, and 7 after the onset of AMI were determined using enzyme-linked immunosorbent assays. Hsp70 levels were significantly higher in ACS and SA and anti-Hsp70 antibody levels were only markedly lower in ACS than controls. After adjustment for traditional CHD risk factors, increasing levels of Hsp70 were significantly associated with an increased risk and severity of ACS (P for trend < 0.001), whereas increasing levels of anti-Hsp70 antibody were associated with a decreased risk of ACS (P for trend = 0.0003). High levels of Hsp70 combined with low levels of anti-Hsp70 antibody had a joint effect on the risk of ACS (OR, 5.14, 95% CI, 3.00-8.79; P < 0.0001). In patients with AMI, Hsp70 levels decreased rapidly from days 1-7 after onset, whereas anti-Hsp70 antibody levels increased in patients with AMI. These findings suggest that higher Hsp70 levels or lower anti-Hsp70 antibody levels are independently associated with a higher risk of ACS. Higher Hsp70 levels and lower anti-Hsp70 antibody levels combine to further increase this risk.     

Circulating anti-Hsp70 levels in nascent metabolic syndrome: the Casale Monferrato Study

The metabolic syndrome (MetS) confers an increased risk of both type 2 diabetes and cardiovascular diseases (CVD). Heat shock protein 70 (Hsp70), an intracellular polypeptide, can be exposed on the plasma membrane and/or released into the circulation, eliciting both native and immune responses that may contribute to vascular damage. Our aim was to assess if serum anti-Hsp70 antibody levels were cross-sectionally associated with uncomplicated MetS. A cross-sectional case–control study from the nondiabetic cohort of the Casale Monferrato Study was performed. Subjects with established CVD and/or abnormal renal function were excluded. Case subjects (n?=?180) were defined as those fulfilling the criteria for the diagnosis of MetS. Control subjects (n?=?136) were completely free of any component of the MetS. Serum anti-Hsp70 levels were measured by immunoenzymatic assay. We found that anti-Hsp70 antibody levels were significantly higher in cases than in control subjects [122.6 (89.5–155.6) vs 107.1 (77.3–152.4) μg/ml, p?=?0.04], even after age and sex adjustment. In logistic regression analysis, higher levels of log-anti-Hsp70 conferred greater odds ratio (OR) for MetS, independently of age and sex. There was a statistically significant trend of ORs across quartiles of anti-Hsp70 and values greater than 108.0 μg/ml conferred a 77 % increased OR of MetS as compared with values in the lower quartiles. The strength of the association slightly decreased after further adjustment for apolipoprotein B, smoking, and albumin excretion rate. In conclusion, our results show that serum anti-Hsp70 antibody levels are independently associated with nascent MetS.     

Establishment of thermotolerance in maize by exposure to stresses other than a heat shock does not require heat shock protein synthesis

Maize (Zea mays) seedlings were pretreated prior to heat shock with either a progressive water stress of −0.25 megapascal PEG/hour from 0 to −1.25 megapascal over a 6-hour time period, or various concentrations of copper, cadmium, or zinc for 4 days. When the subsequent heat shock of 40 or 45°C was administered for 3 hours, the seedlings showed an induced thermotolerance to these temperatures, which were otherwise lethal to control (water grown) seedlings. Thermotolerance was exhibited by both the root and the shoot of pretreated seedlings, even though the water and heavy metal stresses were applied only to the roots. Neither of these pretreatments had induced the synthesis of detectable levels of heat shock proteins (Hsps) at the time of heat shock. Pretreatment of seedlings with a progressive heat shock of 2°C/hour from 26 to 36°C, which did induce Hsps 18, 70, and 84, resulted in tolerance of a severe water stress of −1.5, −1.75, or −2.0 megapascal for 24 hours. But these seedlings producing Hsps were no better protected against water stress than those pretreated with a progressive water stress which did not produce Hsps. Hsps appear not to act as general stress proteins and their presence is not always required for the establishment of thermotolerance.     

Mobile phones, heat shock proteins and cancer

There are several reports which indicate that electromagnetic radiation (such as from mobile phones) at non-thermal levels may elicit a biological effect in target cells or tissues. Whether or not these biological effects lead to adverse health effects, including cancer, is unclear. To date there is limited scientific evidence of health issues, and no mechanism by which mobile phone radiation could influence cancer development. In this paper, we develop a theoretical mechanism by which radiofrequency radiation from mobile phones could induce cancer, via the chronic activation of the heat shock response. Upregulation of heat shock proteins (Hsps) is a normal defence response to a cellular stress. However, chronic expression of Hsps is known to induce or promote oncogenesis, metastasis and/or resistance to anticancer drugs. We propose that repeated exposure to mobile phone radiation acts as a repetitive stress leading to continuous expression of Hsps in exposed cells and tissues, which in turn affects their normal regulation, and cancer results. This hypothesis provides the possibility of a direct association between mobile phone use and cancer, and thus provides an important focus for future experimentation.     

Overexpression of heat shock proteins differentially modulates protein kinase C expression in rat neonatal cardiomyocytes

Previous studies have suggested that protein kinase C (PKC) is involved in heat shock protein (Hsp)-mediated cardioprotection. Therefore, we wanted to determine whether overexpression of Hsps modulates PKC expression, which will give us further insight into understanding the mechanism by which Hsps and PKC interact to protect cells from stress-induced injury. Specifically, we overexpressed the inducible form of Hsp70 (Hsp70i) or Hsp90 in rat neonatal cardiomyocytes and evaluated PKCdelta or PKCepsilon expression by immunoblotting and immunofluorescent confocal microscopy. Western analysis showed that overexpression of Hsp70i or Hsp90 decreased PKCepsilon expression. However, overexpression of Hsp70i or Hsp90 did not modify PKCdelta expression over control levels. Overexpression of constitutively active PKCdelta or PKCepsilon increased Hsp70i expression over control levels. The data suggest that overexpression of Hsps differentially modulates expression of PKC isoforms in rat neonatal cardiomyocytes. Furthermore, PKC may directly play a role in Hsp-mediated cardioprotection by upregulating Hsp70i expression.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

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.