The effect of stress-inducible extracellular Hsp72 on human neutrophil chemotaxis: a role during acute intense exercise.
ABSTRACT We studied the physiological role of the 72 kDa extracellular heat shock protein (Hsp72, a stress-inducible protein) in modulating neutrophil chemotaxis during a single bout of intense exercise performed by sedentary women, together with various cell mechanisms potentially involved in the modulation. For each volunteer, we evaluated neutrophil chemotaxis and serum Hsp72 concentration before and immediately after a single bout of exercise (1 h on a cycle ergometer at 70% VO(2) max), and 24 h later. Both parameters were found to be stimulated by the exercise, and had returned to basal values 24 h later. In vitro, there was a dose-dependent increase in chemotaxis when neutrophils were incubated both with physiological Hsp72 concentrations and with a 100 x greater concentration. The chemotaxis was greater when the neutrophils were incubated with the post-exercise Hsp72 concentration than with the basal concentration, suggesting a physiological role for this protein in the context of the stimulation of neutrophil chemotaxis by intense exercise. The 100 x Hsp72 concentration stimulated chemotaxis even more strongly. In addition, Hsp72 was found to have chemoattractant and chemokinetic effects on the neutrophils at physiological concentrations, with these effects being significantly greater with the post-exercise than with the basal Hsp72 concentration. The Hsp72-induced stimulation of neutrophil chemotaxis disappeared when the toll-like receptor 2 (TLR-2) was blocked, and phosphatidylinositol-3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and nuclear transcription factor kappa B (NF-kappaB) were also found to be involved in the signaling process. No changes were observed, however, in neutrophil intracellular calcium levels in response to Hsp72. In conclusion, physiological concentrations of the stress protein Hsp72 stimulate human neutrophil chemotaxis through TLR-2 with its cofactor CD14, involving ERK, NF-kappaB, and PI3K, but not iCa(2 + ), as intracellular messengers. In addition, Hsp72 seems to participate in the stimulation of chemotaxis induced by a single bout of intense exercise performed by sedentary women.
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ABSTRACT: Expression of intracellular HSP70 is associated with cytoprotective effects against a wide range of stressful stimuli, such as inflammation, oxidative stress, hypoxia, endotoxins, infections, and fever. This cytoprotective effect is mainly attributed to their ability to stabilize protein structures through chaperone-like reversible interactions. HSP70 was recently detected in the extracellular medium, and its presence in serum is commonly associated with pathological situations, where it exerts modulatory effects on cells of the immune system. Previously, we have described the relationship between serum HSP70 levels, oxidant status, and clinical outcome of septic patients; the group of patients with higher prooxidant status and higher serum HSP70 had also higher mortality. To investigate the possible association between oxidized HSP70 and cytoprotection or cell death, we incubated RAW 264.7 macrophages with oxidized HSP70 and evaluated nitrite production, cell proliferation, cell viability, TNF-α release, and phagocytic activity. We also evaluated structural modifications caused by oxidation in purified HSP70. Oxidation of HSP70 altered its protein structure; besides, the modulatory effect of oxidized HSP70 on RAW264.7 cells was different from that of native HSP70. Macrophages treated with oxidized HSP70 presented lower proliferation and viability, lower phagocytic activity, and lower TNF-α release. These results indicate that oxidation of extracellular HSP70 modified its signaling properties, causing alterations on its modulatory effects on macrophage function and viability.Cell Stress and Chaperones 05/2014; · 2.48 Impact Factor
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ABSTRACT: The expression of heat shock proteins (hsp) is a basic and well conserved cellular response to an array of stresses. These proteins are involved in the repair of cellular damage induced by the stress, which is necessary for the salutary resolution from the insult. Moreover, they confer protection from subsequent insults, which has been coined stress tolerance. Since these proteins are expressed in subcellular compartments, it was thought that their function during stress conditions was circumscribed to the intracellular environment. However, it is now well established that hsp can also be present outside cells where they appear to display a function different than the well understood chaperone role. Extracellular hsp act as alert stress signals priming other cells, particularly of the immune system, to avoid the propagation of the insult and favor resolution. Since the majority of hsp do not possess a secretory peptide signal, they are likely be exported by a non-classical secretory pathway. Different mechanisms have been proposed to explain the export of hsp, including translocation across the plasma membrane and release associated with lipid vesicles, as well as the passive release after cell death by necrosis. Extracellular hsp appear in various flavors, including membrane-bound and membrane-free forms. All of these variants of extracellular hsp suggest that their interactions with cells may be quite diverse, both in target cell types and the activation signaling pathways. This review addresses some of our current knowledge about the release and relevance of extracellular hsp.Shock (Augusta, Ga.) 06/2013; · 2.87 Impact Factor
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ABSTRACT: Background/aims: Extracellular heat shock protein 72 (eHSP72) is increased in the plasma of both types of diabetes and is positively correlated with inflammatory markers. Since aging is associated with a low-grade inflammation and insulin resistance (IR) we aimed to: i) analyse the concentration of eHSP72 in elderly people and determine correlation with insulin resistance and ii) determine the effects of eHSP72 on β-cell function and viability in human and rodent pancreatic β-cells. Methods: Fasting blood samples were collected from fifty older people (27♀, 23♂; 63.4±4.4 years old; BMI=25.5±2.7 kg/m2). Plasma samples were analysed for eHSP72, insulin, TNF-α, leptin, adiponectin, cortisol, glycemic and lipid profile. In vitro studies were conducted using rodent islets and clonal rat and human pancreatic β-cell lines (BRIN-BD11 and 1.1B4, respectively). Cells/Islets were incubated for 24h with eHSP72 (0, 0.2, 4, 8 and 40ng/mL). Cell viability was measured using three different methods. The impact of HSP72 on β-cell metabolic status was determined using Seahorse Bioscience XFe96 technology. To assess if the effects of eHSP72 was mediated by Toll-like receptors (TLR-2/TLR4), we co-incubated rodent islets with eHSP72 and the TLR2/4 inhibitor OxPAPC (30mg/mL).Results:We found a positive correlation between plasma eHSP72 and HOMA-IR (r=0.528, p,<0.001), TNF-α (r=0.389, p<0.014), cortisol (r=0.348, p<0.03) and leptin/adiponectin (r=0.334, p<0.03). In vitro studies: Insulin secretion was decreased in an eHSP72 dose-dependent manner in BRIN-BD11 cells (from 257.7±33 to 84.1±10.2mg/mg protein/24h with 40ng/mL of eHSP72), and in islets in the presence of 40ng/mL (from 0.48±0.07 to 0.33±0.009mg/20islets/24h). Similarly, eHSP72 reduced b-cell viability (at least 30% for BRIN-BD11 and 10% for 1.1B4). Bioenergetic studies revealed that eHSP72 altered pancreatic β-cell metabolism. OxPAPC restored insulin secretion in islets incubated with 40ng/mL of eHSP72. Conclusions: We demonstrated a positive correlation between eHSP72 and IR. In addition, we suggest that chronic eHSP72 exposure may mediate β-cell failure.Clinical Science 12/2013; · 4.86 Impact Factor