Release Of Heat Shock Protein Research Articles

HSP60 mediates the neuroprotective effects of curcumin by suppressing microglial activation.

Curcumin has anti-inflammatory and antioxidant properties and has been widely used to treat or prevent neurodegenerative diseases. However, the mechanisms underlying the neuroprotective effects of curcumin are not well known. In the present study, the effect of curcumin on lipopolysaccharide (LPS)-stimulated BV2 mouse microglia cells was investigated using enzyme-linked immunosorbent assays of the culture medium and western blotting of cell lysates. The results showed that curcumin significantly inhibited the LPS-induced expression and release of heat shock protein 60 (HSP60) in the BV2 cells. The level of heat shock factor (HSF)-1 was upregulated in LPS-activated BV2 microglia, indicating that the increased expression of HSP60 was driven by HSF-1 activation. However, the increased HSF-1 level was downregulated by curcumin. Extracellular HSP60 is a ligand of Toll-like receptor 4 (TLR-4), and the level of the latter was increased in the LPS-activated BV2 microglia and inhibited by curcumin. The activation of TLR-4 is known to be associated with the activation of myeloid differentiation primary response 88 (MyD88) and nuclear factor (NF)-κB, with the subsequent production of proinflammatory and neurotoxic factors. In the present study, curcumin demonstrated marked suppression of the LPS-induced expression of MyD88, NF-κB, caspase-3, inducible nitric oxide synthase, tumor necrosis factor-α, interleukin (IL)-1β and IL-6 in the microglia. These results indicate that curcumin may exert its neuroprotective and anti-inflammatory effects by inhibiting microglial activation through the HSP60/TLR-4/MyD88/NF-κB signaling wpathway. Therefore, curcumin may be useful for the treatment of neurodegenerative diseases that are associated with microglial activation.

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

Autoimmunity--promoting and stabilizing innate immunity 'UNWUCHT'.

Membrane-bound heat shock proteins facilitate the uptake of dying cells and cross-presentation of cellular antigen.

Heat shock proteins (HSPs) were originally identified as stress-responsive proteins and serve as molecular chaperones in different intracellular compartments. Translocation of HSPs to the cell surface and release of HSPs into the extracellular space have been observed during the apoptotic process and in response to a variety of cellular stress. Here, we report that UV irradiation and cisplatin treatment rapidly induce the expression of membrane-bound Hsp60, Hsp70, and Hsp90 upstream the phosphatidylserine exposure. Membrane-bound Hsp60, Hsp70 and Hsp90 could promote the release of IL-6 and IL-1β as well as DC maturation by the evaluation of CD80 and CD86 expression. On the other hand, Hsp60, Hsp70 and Hsp90 on cells could facilitate the uptake of dying cells by bone marrow-derived dendritic cells. Lectin-like oxidized LDL receptor-1 (LOX-1), as a common receptor for Hsp60, Hsp70, and Hsp90, is response for their recognition and mediates the uptake of dying cells. Furthermore, membrane-bound Hsp60, Hsp70 and Hsp90 could promote the cross-presentation of OVA antigen from E.G7 cells and inhibition of the uptake of dying cells by LOX-1 decreases the cross-presentation of cellular antigen. Therefore, the rapid exposure of HSPs on dying cells at the early stage allows for the recognition by and confers an activation signal to the immune system.

An in vitro model to consider the effect of 2 mM glutamine and KNK437 on endotoxin-stimulated release of heat shock protein 70 and inflammatory mediators

ObjectiveGlutamine has been shown to promote the release of heat shock protein 70 (HSP70) both within experimental in vitro models of sepsis and in adults with septic shock. This study aimed to investigate the effects of 2 mM glutamine and an inhibitor of HSP70 (KNK437) on the release of HSP70 and inflammatory mediators in healthy adult volunteers. MethodsAn in vitro whole blood endotoxin stimulation assay was used. ResultsThe addition of 2 mM glutamine significantly increased HSP70 levels over time (P < 0.05). HSP70 release had a positive correlation at 4 h with IL-1 β (r = 0.51, P = 0.03) and an inverse correlation with TNF-α (r = −0.56, P = 0.02) and IL-8 levels (r = −0.52, P = 0.03), and there were no significant correlations between HSP70 and IL6 or IL-10 or glutamine. Glutamine supplementation significantly (P < 0.05) attenuated the release of IL-10 at 4 h and IL-8 at 24 h, compared with conditions without glutamine. In endotoxin-stimulated blood there were no significant differences in the release of IL-6, TNF-α, and IL-1 β with glutamine supplementation at 4 and 24 h. However, glutamine supplementation (2 mM) appeared to attenuate the release of inflammatory mediators (IL-1 β, IL-6, TNF-α), although this effect was not statistically significant. The addition of KNK437, a HSP70 inhibitor, significantly diminished HSP70 release, which resulted in lower levels of inflammatory mediators (P < 0.05). ConclusionGlutamine supplementation promotes HSP70 release in an experimental model of sepsis. After the addition of KNK437, the effects of glutamine on HSP70 and inflammatory mediator release appear to be lost, suggesting that HSP70 in part orchestrates the inflammatory mediator response to sepsis. The clinical implications require further investigation.

Abstract 3609: In vitro comparison of conventional hyperthermia and electro-hyperthemia

Abstract Purpose: Deep heating hypertermia methods are a promising cancer treatment modalities used to enhance conventional treatment. This could be achieved through radiofrequency field en-bloc heating by Thermotron RF-8 and selective tumor heating by Oncotherm-Labehy, respectively. This is the first study to evaluate the different cytotoxic effect and action mechanism on cancer cells after mild hyperthermia (MHT) by Thermotron RF-8 or Oncothermia-Labehy. Experimental Design: Hepatoma HepG2 cells were used for MHT treatment (42°C for 30minute). Either by Oncotherm-Labehy or Thermotron RF-8 or water bath treatment were compared. Apoptosis evaluated 24h after MTH as measured by Annexin-V assay and cell cycle analysis. Mitochondria-targeted damage as measured by ROS production with or without Mito-TEMPO treatment and membrane-targeted damage as measured by caspase-8 expression, adhesion molecule expression, heat shock protein (HSP) release and calcium channel disturbance were evaluated and compared. Results: MHT resulted in similar cell cycle arrest on G2/M. Oncothermia treatment resulted in higher apoptosis rate (38.87±6.58) than RF-8 (25.13±2.26), water bath (18.77±0.35) and control (14.93±0.76). The expression of Caspase-8 is higher by oncothermia treatment, while caspase-3 and 9 levels were similar between 3 types of treatment. HSPs were upregulated by MHT, however a marked HSP release outside cell and E-cadherin overexpression on the membrane were only observed after oncothermia treatment. Conclusion: In this report, we found that different hyperthermia devices may induce different mechanisms on their cytotoxic effects and cell responses to heat. Oncothermia may selectively target on cell membrane before targeting on mitochondria, while RF-8 has no membrane targeting effect. These results may provide rational basis for future clinical application of hyperthermia treatment. Citation Format: Kwan-Hwa Chi, Yu-Shan Samuel Wang, Cheng-Chung Huang, Chao-Chun Chang, Mau-Shin Chi, Kai-Lin Yang, Yuk-Wah Tsang, Gabor Andocs. In vitro comparison of conventional hyperthermia and electro-hyperthemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3609. doi:10.1158/1538-7445.AM2015-3609

A hyposensitive anticancer drug induces higher surface expression and release of heat shock proteins in a human hepatocellular carcinoma cell line.

Heat shock proteins (HSPs) respond to multiple stresses and have been implicated as essential immune chaperones that regulate innate and adaptive immunity. The exposure of HSPs containing tumour peptide complex to immune surveillance elements may elicit a specific anti-tumour response. The present study examined the potential of anticancer drugs to induce apoptosis of HepG2 cells and elicit the expression of HSP proteins, including HSP70 and gp96, on the membrane or their release to the extracellular environment, leading to HSP exposure. In the present study, etoposide and carboplatin were classified by an adenosine triphosphate assay as representatives of hypersensitive and hyposensitive anticancer drugs, respectively. Flow cytometry, immunofluorescence, ELIZA and reverse transcription quantitative polymerase chain reaction were all used to detect changes in the HSPs. The results demonstrated that etoposide and carboplatin induced apoptosis of HepG2 cells. In addition, following treatment with etoposide or carboplatin, HSP70/gp96 expression increased, demonstrating a 'transfer expression' pattern: The cytosol expression decreased while the surface expression increased. These alterations progressed steadily with notable alterations following treatment with etoposide for 24 h or carboplatin for 72 h. Additionally, at the end of treatment, release of HSP70/gp96 to the extracellular environment increased. Notably, following treatment with the hyposensitive anticancer drug carboplatin for 72 h, the surface expression of gp96 in HepG2 cells was significantly increased. These results suggest that when combined with cancer cell apoptosis, anticancer drugs induce the membrane expression and release of HSP70/gp96 in hepatocellular carcinoma (HCC) cells, which may represent a crucial event in the immune anti-tumour response. Notably, treatment with the hyposensitive anticancer drug for a longer time period resulted in greater surface expression and release of gp96, which suggests a potential use for hyposensitive anticancer drugs in HSP-based dendritic cell vaccine preparation and chemoimmunotherapy for HCC patients.

Pim1 kinase protects airway epithelial cells from cigarette smoke-induced damage and airway inflammation.

Exposure to cigarette smoke (CS) is the main risk factor for developing chronic obstructive pulmonary disease and can induce airway epithelial cell damage, innate immune responses, and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation, and levels of keratinocyte-derived cytokine (KC) and several damage-associated molecular patterns in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in the presence or absence of Pim1 inhibitor and assessed for loss of mitochondrial membrane potential, induction of cell death, and release of heat shock protein 70 (HSP70). We observed increased expression of Pim1, but not of Pim2 and Pim3, in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared with wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, whereas release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of double-strand DNA or HSP70 in Pim1-deficient mice compared with wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC.

The bradykinin-forming cascade: a historical perspective.

The formation of bradykinin in plasma requires interaction of three proteins, namely coagulation factor XII (Hageman factor), prekallikrein and high-molecular-weight kininogen (HK). Prekallikrein and HK circulate as a bimolecular complex. Initiation of the cascade upon binding to negatively charged surfaces (or macromolecules) is dependent on factor XII autoactivation, conversion of prekallikrein to kallikrein, and a feedback activation of factor XII by kallikrein. The latter reaction is extremely rapid relative to factor XII autoactivation. The kallikrein then digests HK to liberate bradykinin. The natural surface appears to be vascular endothelial cells which express binding proteins for factor XII and HK, and activation can proceed along the cell surface. Recent findings demonstrate that prekallikrein has enzymatic activity separate from that of kallikrein such that it can stoichiometrically bind and cleave HK to liberate bradykinin. It is normally prevented from doing so by the plasma C1 inhibitor. Release of heat shock protein 90 (HSP-90) from endothelial cells can convert prekallikrein to kallikrein (stoichiometrically) within the prekallikrein-HK complex, even in the absence of factor XII, and the prekallikrein-HK complex can autoactivate to generate kallikrein if phosphate is the buffering ion. The effects of phosphate ion and HSP-90 are additive. Thus, an active site appears to be induced in prekallikrein by binding to HK and any of the aforementioned reactions can generate kallikrein prior to factor XII activation by autoactivation of the HK-PK complex. This brief review highlights the major discoveries made over the past 50 years which have led to our current concepts regarding the constituents and mechanisms of activation of the plasma bradykinin-forming cascade.

In vivo modification of Abeta plaque toxicity as a novel neuroprotective lithium-mediated therapy for Alzheimer’s disease pathology

BackgroundAlzheimer’s disease (AD) is characterized by the abnormal accumulation of extracellular beta-amyloid (Abeta) plaques, intracellular hyperphosphorylated tau, progressive synaptic alterations, axonal dystrophies, neuronal loss and the deterioration of cognitive capabilities of patients. However, no effective disease-modifying treatment has been yet developed. In this work we have evaluated whether chronic lithium treatment could ameliorate the neuropathology evolution of our well characterized PS1M146LxAPPSwe-London mice model.ResultsThough beneficial effects of lithium have been previously described in different AD models, here we report a novel in vivo action of this compound that efficiently ameliorated AD-like pathology progression and rescued memory impairments by reducing the toxicity of Abeta plaques. Transgenic PS1M146LxAPPSwe-London mice, treated before the pathology onset, developed smaller plaques characterized by higher Abeta compaction, reduced oligomeric-positive halo and therefore with attenuated capacity to induce neuronal damage. Importantly, neuronal loss in hippocampus and entorhinal cortex was fully prevented. Our data also demonstrated that the axonal dystrophic area associated with lithium-modified plaques was highly reduced. Moreover, a significant lower accumulation of phospho-tau, LC3-II and ubiquitinated proteins was detected in treated mice. Our study highlights that this switch of plaque quality by lithium could be mediated by astrocyte activation and the release of heat shock proteins, which concentrate in the core of the plaques.ConclusionsOur data demonstrate that the pharmacological in vivo modulation of the extracellular Abeta plaque compaction/toxicity is indeed possible and, in addition, might constitute a novel promising and innovative approach to develop a disease-modifying therapeutic intervention against AD.

New Paradigm for Intrinsic Function of Heat Shock Proteins as Endogenous Ligands in Inflammation and Innate Immunity

Recently, growing evidences that extracellular heat shock protein (HSP) functions as endogenous immunomodulator for innate and adaptive immune responses have been demonstrated. Because HSPs inherently act as chaperones within the cells, passive release such as cell necrosis and active release including secretion in the form of exosome have been suggested for HSP release into extracellular milieu. Such extracellular HSPs have been shown to be activators for innate immune responses through Toll-like receptors (TLRs). However, it has also been suggested that HSPs augmented the ability of associated innate ligands such as LPS to stimulate cytokine production and dendritic cell (DC) maturation. More interestingly, recent study demonstrated that innate immune responses elicited by both endogenous and exogenous danger signals were spatially and temporally regulated and this can be manipulated using Hsp90 or oxygen-regulated protein 150 (ORP150), thereby controlling the immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects the antigen cross-presentation and innate immune responses. Precise analysis of HSP biology can lead us to establish outstanding HSPbased immunotherapy.

Can we assess an acute myocardial infarction in patients with acute coronary syndrome according to diagnostic accuracy of heat shock proteins?

Heat shock proteins (HSPs) have changed very little with evolution, suggesting that they play important role(s) in cellular survival. Specifically, HSPs protect cells from induced cell death. Their expression is triggered by heat or other stress, such as ischemia. HSPs provide protection against protein denaturation, although they slightly differ with respect to group affiliation. Release of HSPs from necrotic and ischemic cardiomyocytes into the intercellular space and plasma may correlate with the intensity of the pro-inflammatory response observed during and immediately after myocardial infarction. We hypothesized that the plasma concentration of particularly inducible forms of HSPs from different groups (HSP 90, HSP 70, HSP 60 and/or HSP 20) can be used as early specific markers for diagnosing myocardial infarction in patients with acute coronary syndrome. Our hypothesis is supported by the following data: (I) HSP expression occurs very early after acute coronary events; (II) HSP concentrations increase rapidly in the peripheral blood; (III) HSP concentrations correlate with markers of myocardial necrosis and pro-inflammatory biochemical parameters. The magnitude of the increase in plasma HSP concentrations over initial concentrations during the period of highest sensitivity and specificity of the assay could be important for early detection of myocardial infarction and distinguishing it from unstable angina. We suggest that these parameters, along with close observation of patients with chest pain, will assist providers who must differentiate between acute myocardial damage and other organ diseases.

Heat-Shock Proteins as Endogenous Ligands Building a Bridge Between Innate and Adaptive Immunity

There has been growing evidence that heat-shock protein (HSP) functions as an endogenous immunomodulator for innate and adaptive immune responses. Since HSPs inherently act as chaperones within cells, passive release (e.g., by cell necrosis) and active release (including release by secretion in the form of an exosome) have been suggested as mechanisms of HSP release into the extracellular milieu. Such extracellular HSPs have been shown to be activators of innate immune responses through Toll-like receptors. However, it has also been suggested that HSPs augment the ability of associated innate ligands such as lipopolysaccharides to stimulate cytokine production and dendritic cell maturation. More interestingly, a recent study has demonstrated that innate immune responses elicited by danger signals were regulated spatiotemporally and that can be manipulated by HSPs, thereby controlling immune responses. We will discuss how spatiotemporal regulation of HSP-chaperoned molecules within antigen-presenting cells affects adaptive immunity via antigen cross-presentation and innate immune responses. Precise analysis of HSP biology should lead to the establishment of effective HSP-based immunotherapy.

Stress-induced facilitation of host response to bacterial challenge in F344 rats is dependent on extracellular heat shock protein 72 and independent of alpha beta T cells

Activation of the in vivo stress response can facilitate antibacterial host defenses. One possible mechanism for this effect is stress-induced release of heat shock protein 72 (Hsp72) into the extracellular environment. Hsp72 is a ubiquitous cellular protein that is up-regulated in response to cellular stress, and modulates various aspects of immune function including macrophage inflammatory/bactericidal responses and T-cell function when found in the extracellular environment. The current study tested the hypothesis that in vivo extracellular Hsp72 (eHsp72) at the site of inflammation contributes to stress-induced restricted development of bacteria, and facilitated recovery from bacteria-induced inflammation, and that this effect is independent of alpha beta (αβ) T cells. Male F344 rats were exposed to either inescapable electrical tail-shocks or no stress, and subcutaneously injected with Escherichia coli (ATCC 15746). The role of eHsp72 was investigated by Hsp72-immunoneutralization at the inflammatory site. The potential contribution of T cells was examined by testing male athymic (rnu/rnu) nude rats lacking mature αβ T cells and heterozygous thymic intact control (rnu/+) rats. The results were that stressor exposure increased plasma concentrations of eHsp72 and facilitated recovery from bacterial inflammation. Immunoneutralization of eHsp72 at the inflammatory site attenuated this effect. Stressor exposure impacted bacterial inflammation and eHsp72 equally in both athymic and intact control rats. These results support the hypothesis that eHsp72 at the site of inflammation, and not αβ T cells, contributes to the effect of stressor exposure on subcutaneous bacterial inflammation.

Radiation combined with hyperthermia induces HSP70-dependent maturation of dendritic cells and release of pro-inflammatory cytokines by dendritic cells and macrophages

PurposeHyperthermia (HT) treatment of cancer patients was revived over the last years and has been proven to be beneficiary for many cancer entities when applied temperature controlled in multimodal treatments. We examined whether a combination of ionizing irradiation (X-ray) and HT (41.5°C; 1h) can induce the release of heat shock protein (HSP) 70 by tumor cells and thereby lead to the activation of dendritic cells and macrophages. Material and methodsExtracellular HSP70 was detected in supernatants (SN) of treated colorectal tumor cells by ELISA. Maturation of dendritic cells (DC) after contact with the SN was measured by flow-cytometry. Phagocytosis assays were conducted to get hints about the immune stimulating potential of the tumor cells after the respective treatments. ResultsAn increased surface expression of HSP70 was observed after X-ray or X-ray plus HT while the amount of extracellular HSP70 was only increased when HT was given additionally. A high up-regulation of the co-stimulation molecule CD80 and the chemokine receptor CCR7 on DC was measured after contact with SN of X-ray plus HT treated cells. This was dependent on extracellular HSP70. Combined treatments further led to significantly increased phagocytosis rates of macrophages and DC and increased pro-inflammatory cytokine (IL-8 and IL-12) secretion. ConclusionX-ray combined with HT induces HSP70 dependent activation of immune cells and might generate a tumor microenvironment beneficial for cure.

혈관평활근세포에서 HSP90에 의한 CXCL10 증가에 관여하는 인자에 대한 연구

Heat shock protein (HSP)은 외부적인 자극에 반응하여 세포를 보호하는 역할을 한다. 또한 HSP90은 혈관질환에서 처럼 세포가 사멸되거나 손상을 입는 경우 세포 밖으로 유리된다. 그러나 지금까지 세포 밖 HSP90이 혈관평활근세포에 어떠한 영향을 주는지에 대한 연구는 미약하다. 따라서 본 연구는 혈관평활근세포에서 HSP90이 CXCL10 발현에 대한 영향과 그 기전을 규명하였다. HSP90에 노출된 혈관평활근세포에서 CXCL10 transcript가 증가하고, CXCL10 단백질의 분비가 증가되었다. HSP90에 의한 CXCL10 분비는 Toll-like receptor (TLR)-2/-4 억제제인 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine (OxPAPC)과 NADPH oxidase 억제제인 diphenyleneiodium 그리고 Akt 경로를 억제하는 LY294002와 Akti IV에 의하여 크게 감소되었다. 또한 TLR-4의 dimerization을 저해하는 curcumin 역시 HSP90에 의한 CXCL10의 분비를 억제하였다. 전사인자인 nuclear factor kappa B(NF-<TEX>${\kappa}$</TEX>B)의 생물학적 억제제인 inhibitory kappa B (I<TEX>${\kappa}$</TEX>B)와 NF-<TEX>${\kappa}$</TEX>B 억제작용이 있는 rasveratrol은 HSP90에 의한 CXCL10 분비를 억제하였다. 이러한 결과는 혈관평활근세포에서 HSP90에 의한 CXCL10의 발현에 TLR-4와 Akt 및 NF-<TEX>${\kappa}$</TEX>B가 관여함을 의미한다. Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). We investigated whether extracellular HSP90 predisposed VSMCs to pro-inflammatory phenotype. Exposure of human aortic smooth muscle cells to HSP90 not only significantly enhanced CXCL10 secretion but also increased CXCL10 transcription. HSP90-mediated CXCL10 secretion was attenuated by OxPAPC, a TLR-2/4 inhibitor, and curcumin, a TLR-4 dimerization inhibitor. Inhibitors of diphenyleneiodium chloride and the Akt pathway also attenuated CXCL10 secretion in response to HSP90. The gene delivery of I<TEX>${\kappa}$</TEX>B using recombinant adenoviruses and treatment with resveratrol, which inhibit NF-<TEX>${\kappa}$</TEX>B activity, significantly attenuated HSP90-induced CXCL10 secretion from VSMCs. We propose that extracellular HSP90 contributes to an inflammatory reaction in the stressed vasculature by inducing CXCL10 expression of VSMCs, and that TLR-4, Akt, and NF-<TEX>${\kappa}$</TEX>B play active roles in the process.

Elevated serum levels of heat shock protein 70 can be detected after radiofrequency ablation

Due to their adjuvant effect and their ability to chaperone tumor-associated peptides, heat shock proteins constitute a potent alarm signal for the immune system and can lead to activation of anti-tumor T-cell immunity. Radiofrequency ablation has been reported to induce heat shock protein expression especially that of heat shock protein 70 in sublethally damaged tumor cells. In this study, we evaluated the release of heat shock protein 70 into the serum of cancer-bearing patients directly after radiofrequency ablation. Sera of 22 patients undergoing radiofrequency ablation for the treatment of primary and secondary malignancies of the liver, kidney, and lung, as well as control sera of 20 patients undergoing diagnostic liver biopsy were analyzed using a manufactured heat shock protein 70 ELISA. A significant increase in serum levels of heat shock protein 70 was detectable in the patient cohort 1day after radiofrequency ablation. More than a twofold increase was observed in nine out of 22 patients, which tended to correlate with favorable clinical outcome. No patient of the control group revealed a comparable increase. Radiofrequency ablation can lead to a release of heat shock protein 70 into the serum, which is transiently detectable 1day after treatment. Elevated heat shock protein 70 serum levels may constitute a biomarker for favorable clinical outcome.

Extracellular heat shock protein 90 induces interleukin-8 in vascular smooth muscle cells

Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). The aim of this article is to investigate whether extracellular HSP90 predisposes VSMCs to pro-inflammatory phenotype. Exposure of aortic smooth muscle cells to HSP90 elevated IL-8 release and IL-8 transcript via promoter activation. HSP90-induced IL-8 promoter activation was suppressed by dominant-negative forms of Toll-like receptor (TLR)-4 and MyD88, but not by dominant-negative-forms of TLR-3, TLR-2, and TRIF. IL-8 up-regulation in response to HSP90 was also attenuated by IκB, rasveratrol, curcumin, diphenyleneiodium, N-acetylcystein, U0126, and SB202190. Mutation at the NF-κB- or C/EBP-binding site, but not at the AP-1-binding site, in the IL-8 promoter region suppressed the promoter activation by HSP90. This study proposes that extracellular HSP90 would contribute to IL-8 elevation in the stressed vasculature, and that TLR-4, mitogen-activated protein kinases, NF-κB, and reactive oxygen species are involved in that process.

Release of heat shock protein 70 and the effects of extracellular heat shock protein 70 on the production of IL-10 in fibroblast-like synoviocytes

It has recently been suggested that heat shock protein (Hsp) 70, an intracellular protein, can be released into the extracellular compartment and exert important immunomodulatory functions. Although elevated Hsp70 has been found in synovial fluid from patients with rheumatoid arthritis (RA), its sources and extracellular functions remain unclear. In this study, we explored whether stress response such as heat stress or exposure to tumor necrosis factor-alpha (TNF-alpha) could induce Hsp70 release from RA fibroblast-like synoviocytes (FLSs) and whether extracellular Hsp70 would stimulate cytokine production in RA FLSs. Cultured FLSs were obtained from patients with RA. The expression of intracellular Hsp70 was studied by Western blot. Hsp70 release and the production of interleukin (IL)-6, IL-8, and IL-10 by RA FLSs were studied by specific enzyme-linked immunosorbent assays. The levels of Toll-like receptor (TLR) 2 and 4 mRNA and protein in FLSs were analyzed using reverse transcription-polymerase chain reaction and Western blotting. Treatment with sublethal heat shock or TNF-alpha results in the up-regulation of intracellular Hsp70 in FLSs and Hsp70 release from RA FLSs. In vitro studies show that extracellular Hsp70 can induce anti-inflammatory cytokine IL-10 production in FLSs. The mRNA and protein expression of TLR2 and TLR4 was demonstrated in FLSs, and TLR4 blocking abrogated the up-regulatory effects of Hsp70 on IL-10 production. Thus, these results lend support to the hypothesis that Hsp70 is actively released from FLSs in response to heat shock or TNF-alpha and Hsp70 may be a major paracrine/autocrine inducer of IL-10 production in FLSs via TLR4.

A Vicious Cycle Involving Release of Heat Shock Protein 60 from Injured Cells and Activation of Toll-Like Receptor 4 Mediates Neurodegeneration in the CNS

Infection, ischemia, trauma, and neoplasia elicit a similar inflammatory response in the CNS characterized by activation of microglia, the resident CNS monocyte. The molecular events leading from CNS injury to the activation of innate immunity is not well understood. We show here that the intracellular chaperone heat shock protein 60 (HSP60) serves as a signal of CNS injury by activating microglia through a toll-like receptor 4 (TLR4)-dependent and myeloid differentiation factor 88 (MyD88)-dependent pathway. HSP60 is released from CNS cells undergoing necrotic or apoptotic cell death and specifically binds to microglia. HSP60-induced synthesis of neurotoxic nitric oxide by microglia is dependent on TLR4. HSP60 induces extensive axonal loss and neuronal death in CNS cultures from wild-type but not TLR4 or MyD88 loss-of-function mutant mice. This is the first evidence of an endogenous molecular pathway common to many forms of neuronal injury that bidirectionally links CNS inflammation with neurodegeneration.

Release of heat shock proteins from human neuroblastoma cells exposed to acrylamide

When human neuroblastoma cells (SH-SY5Y) were exposed to 0.5 - 5 mM acrylamide for 18 hr, the levels of heat shock proteins (HSPs) of 90, 70 and 27 kDa (Hsp90, Hsp70, and Hsp27, respectively) were elevated in the incubation media depending on the dose of acrylamide whereas only the Hsp70 level increased within cells. U0126, a specific inhibitor of extracellular signal-regulated protein kinase kinase and a potent suppressor of the cytotoxicity of acrylamide, suppressed the increase in the levels of all HSPs in the incubation media but not their expression within cells. Total protein concentrations in the incubation media increased depending on the dose of acrylamide, and this increase was associated with the increasing number of bands detected by silver staining after SDS-polyacrylamide gel electrophoresis. One of the clearest bands was identified as Hsp90 by peptide mass fingerprinting. Thus, acrylamide causes release of proteins, including that of HSPs, from SH-SY5Y cells. HSP in extracellular fluid may be a good indicator of cytotoxicity of acrylamide.