Luteolin exhibits anti-inflammatory effects by blocking the activity of heat shock protein 90 in macrophages

Septic diseases represent the prevalent complications in intensive care units. Luteolin, a plant flavonoid, has potent anti-inflammatory properties; however, the molecular mechanism beneath luteolin mediated immune modulation remains unclear. Here in vitro investigations showed that luteolin dose-dependently inhibited LPS-triggered secretion and relocation of high mobility group B-1 (HMGB1) and LPS-induced production of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) in macrophages. The mechanism analysis demonstrated that luteolin reduced the release of HMGB1 through destabilizing c-Jun and suppressed HMGB1-induced aggravation of inflammatory cascade through reducing Akt protein level. As an inhibitor of Hsp90, luteolin destabilized Hsp90 client protein c-Jun and Akt. In vivo investigations showed that luteolin effectively protected mice from lipopolysaccharide (LPS)-induced lethality. In conclusion, the present study suggested that luteolin may act as a potential therapeutic reagent for treating septic diseases.     

Exercise induces the release of heat shock protein 72 from the human brain in vivo

The present study tested the hypothesis that in response to physical stress the human brain has the capacity to release heat shock protein 72 (Hsp72) in vivo. Therefore, 6 humans (males) cycled for 180 minutes at 60% of their maximal oxygen uptake, and the cerebral Hsp72 response was determined on the basis of the internal jugular venous to arterial difference and global cerebral blood flow. At rest, there was a net balance of Hsp72 across the brain, but after 180 minutes of exercise, we were able to detect the release of Hsp72 from the brain (335 +/- 182 ng/min). However, large individual differences were observed as 3 of the 6 subjects had a marked increase in the release of Hsp72, whereas exercise had little effect on the cerebral Hsp72 balance in the remaining 3 subjects. Given that cerebral blood flow was unchanged during exercise compared with values obtained at rest, it is unlikely that the cerebral Hsp72 release relates to necrosis of specific cells within the brain. These data demonstrate that the human brain is able to release Hsp72 in vivo in response to a physical stressor such as exercise. Further study is required to determine the biological significance of these observations.     

Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans.

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.     

Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria

Mycobacterium tuberculosis (Mtb) survive inside macrophages by manipulating microbicidal functions such as phago-lysosome fusion, production of reactive oxygen species and nitric oxide, and by rendering macrophages non-responsive to IFN-gamma. Mtb-infected lung tissue does however not only contain macrophages, but also significant numbers of infiltrating polymorphonuclear neutrophils (PMN). These are able to phagocytose and kill ingested Mtb, but are short-lived cells that constantly need to be removed from tissues to avoid tissue damage. Phagocytosis of aged or UV-induced apoptotic PMN by macrophages induce an anti-inflammatory response in macrophages. However, in the present study, we show that engulfment of Mtb-induced apoptotic PMN by macrophages initiates secretion of TNF-alpha from the macrophages, reflecting a pro-inflammatory response. Moreover, Mtb-induced apoptotic PMN up-regulate heat shock proteins 60 and 72 (Hsp60, Hsp72) intracellularly and also release Hsp72 extracellularly. We found that both recombinant Hsp72 and released Hsp72 enhanced the pro-inflammatory response to both Mtb-induced apoptotic PMN and Mtb. This stimulatory effect of the supernatant was abrogated by depleting the Hsp72 with immunoprecipitation. These findings indicate that released Hsp72 from Mtb-infected PMN can trigger macrophage activation during the early stage of Mtb infections, thereby creating a link between innate and adaptive immunity.     

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.     

Activation of hepatocytes by extracellular heat shock protein 72

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

Geranylgeranylaceton induces heat shock protein 72 in skeletal muscle cells

Effects of an antiulcer drug, geranylgeranylaceton (GGA), and/or heat-stress on 72 kDa heat shock protein (HSP72) expression and protein content in cultured skeletal muscle cells were studied. Mouse skeletal muscle cells (C(2)C(12)) were subjected to either 1) control (cultured at 37 degrees C without GGA), 2) GGA administration (10(-11) - 10(-8) M), 3) heat-stress at 41 degrees C for 60 min, or 4) GGA administration combined with heat-stress. Expression of HSP72 was up-regulated by GGA administration. Heat-stress further enhanced the GGA-related up-regulation of HSP72. Administration of GGA caused an increase of muscular protein content as a dose-dependent manner. Protein synthesis was also stimulated by heat-stress alone in myotubes. It was suggested that GGA stimulates the differentiation of myoblasts and protein synthesis. These observations may also suggest that the administration of GGA could be one of the useful tools to gain muscular mass not only in athletes, but also in patients during rehabilitation.     

Alternative mechanism by which IFN-gamma enhances tumor recognition: active release of heat shock protein 72

IFN-gamma exhibits differential effects depending on the target and can induce cellular activation and enhance survival or mediate cell death via activation of apoptotic pathways. In this study, we demonstrate an alternative mechanism by which IFN-gamma enhances tumor recognition, mediated by the active release of Hsp72. We demonstrate that stimulation of 4T1 breast adenocarcinoma cells and K562 erythroleukemic cells with IFN-gamma triggers the cellular stress response, which results in the enhanced expression of total Hsp72 expression without a significant increase in cell death. Intracellular expression of Hsp72 was abrogated in cells stably transfected with a mutant hsf-1 gene. IFN-gamma-induced Hsp72 expression correlated with enhanced surface expression and consequent release of Hsp72 into the culture medium. Pretreatment of tumors with compounds known to the block the classical protein transport pathway, including monensin, brefeldin A, tunicamycin, and thapsigargin, did not significantly block Hsp72 release. However, pretreatment with intracellular calcium chelator BAPTA-AM or disruption of lipid rafts using methyl beta-cyclodextrin completely abrogated IFN-gamma-induced Hsp72 release. Biochemical characterization revealed that Hsp72 is released within exosomes and has the ability to up-regulate CD83 expression and stimulate IL-12 release by naive dendritic cells. Pretreatment with neutralizing mAb or depletion of Hsp72 completely abrogated its chaperokine function. Taken together, these findings are indicative of an additional previously unknown mechanism by which IFN-gamma promotes tumor surveillance and furthers our understanding of the central role of extracellular Hsp72 as an endogenous adjuvant and danger signal.     

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-gamma secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.     

The maximal cytoprotective function of the heat shock protein 27 is dependent on heat shock protein 70

Endogenous heat shock proteins (HSPs) 70 and 25/27 are induced in renal cells by injury from energy depletion. Transfected over-expression of HSPs 70 or 27 (human analogue of HSP25), provide protection against renal cell injury from ATP deprivation. This study examines whether over-expressed HSP27 depends on induction of endogenous HSPs, in particular HSP70, to afford protection against cell injury. LLC-PK1 cells transfected with HSP27 (27OE cells) were injured by ATP depletion for 2 h and recovered for 4 h in the presence of HSF decoy, HSP70 specific siRNA (siRNA-70) and their respective controls. Injury in the presence of HSF decoy, a synthetic oligonucleotide identical to the heat shock element, the nuclear binding site of HSF, decreased HSP70 induction by 80% without affecting the over-expression of transfected HSP27. The HSP70 stress response was completely ablated in the presence of siRNA-70. Protection against injury, provided by over-expression of HSP27, was reduced by treatment with HSF decoy and abolished by treatment with siRNA-70. Immunoprecipitation studies demonstrated association of HSP27 with actin that was not affected by either treatment with HSF decoy or siRNA. Therefore, HSP27 is dependent on HSP70 to provide its maximal cytoprotective effect, but not for its interaction with actin. This study suggests that, while it has specific action on the cytoskeleton, HSP 25/27 must have coordinated activity with other HSP classes, especially HSP70, to provide the full extent of resistance to injury from energy depletion.     

Effect of heat acclimation on heat shock protein 72 and interleukin-10 in humans.

Heat acclimation (HA) results in whole body adaptations that increase heat tolerance, and in addition, HA may also result in protective cellular adaptations. We hypothesized that, after HA, basal intracellular heat shock protein (HSP) 72 and extracellular IL-10 levels would increase, while extracellular HSP72 levels decrease. Ten male and two female subjects completed a 10-day exercise/HA protocol (100-min exercise bout at 56% of maximum O(2) uptake in a 42.5 degrees C DB, 27.9% RH environment); subjects exhibited classic adaptations that accompany HA. Peripheral blood mononuclear cells (PBMCs) were isolated before and after each acclimation session on days 1, 6, and 10; plasma and serum were collected before and after exercise on the 1st and 10th day of HA. SDS-PAGE was used to determine PBMC HSP72 levels during HA, and ELISA was used to measure plasma IL-10 and serum HSP72 concentrations. The increase in PBMC HSP72 from pre- to postexercise on the 1st day of HA was not significant (mean +/- SD, 1.0 +/- 0 vs. 1.6 +/- 0.6 density units). Preexercise HSP72 levels on day 1 were significantly lower compared with the pre- and postexercise samples on days 6 and 10 (mean +/- SD, day 6: 2.1 +/- 1.0 and 2.2 +/- 1.0, day 10: 2.0 +/- 1.3 and 2.2 +/- 1.0 density units, respectively, P < 0.05). There were no differences in plasma IL-10 and serum HSP72 postexercise or after 10 days of HA. The sustained elevation of HSP72 from days 6 to 10 may be evidence of a cellular adaptation to HA that contributes to improved heat tolerance and reduced heat illness risk.     

Calcineurin and heat shock protein 72 in functionally overloaded rat plantaris muscle

The involvement of calcineurin (CaN) and heat shock protein (Hsp) 72 in the regulation of fiber size and/or phenotype in response to functional overload (FO) was investigated. In one FO group, the plantaris muscle was overloaded by cutting the distal tendons (5-10 mm length) of the soleus and gastrocnemius of 3-week-old male Wistar rats. Cyclosporin A (CsA), a CaN inhibitor, was injected daily (5 mg/kg body weight, i.p.) in a second group of FO rats (FO+CsA group) for a 2-week period. Compared to age-matched controls (Con), the absolute and relative plantaris weights were increased in both FO groups: the hypertrophic response was attenuated in FO+CsA rats. The mean cross-sectional area of each fiber type was increased (approximately 2.0-fold) in the plantaris of FO rats: CsA treatment attenuated this effect, although the fibers were still larger than in Con rats. The percent composition of myosin heavy chain (MHC) IIb decreased from 54% in Con to 19% in FO rats, whereas types I, IIa, and IIx MHC increased in the FO rats. CsA treatment blunted the shifts in MHC isoforms: the FO+CsA group showed a smaller decrease in type IIb and a smaller increase in type IIx MHC than the FO group. The levels of CaN-A and -B proteins were higher (approximately 2.5-fold) in FO than Con rats, whereas these values were similar in Con and FO+CsA rats. Hsp72 protein levels were higher in FO (3.6-fold) and FO+CsA (5.2-fold) than Con rats, with the values being significantly higher in the FO+CsA than FO rats. CsA treatment in Con rats had no effects on muscle mass, fiber size, MHC composition, and Hsp72 or CaN levels. Combined, these results suggest that CaN levels are related to changes in both fiber size and phenotype, and that Hsp72 levels are more related to the levels of stress added to the muscle rather than to increases in the slow fiber phenotype in functionally overloaded rat plantaris muscles.     

Induction of heat shock protein 72 in the failing heart is attenuated after an exposure to heat shock

Induction of heat shock protein (Hsp) 72 in the right ventricular muscle of the rat with heart failure following acute myocardial infarction (AMI) was examined. AMI was induced by the left coronary artery ligation (CAL). The animals at the 8th, but not 2nd, week after CAL revealed a decrease in cardiac output index (COI), suggesting that heart failure had developed by 8 weeks after CAL. Increases in the right ventricular developed pressure and the ratios of right ventricle/body weight and lung/body weight at the 2nd and 8th weeks showed the development of the right ventricular hypertrophy. After measurement of hemodynamic parameters, the hearts isolated from animals at the 2nd and 8th weeks after CAL (2w- and 8w-CAL hearts, respectively) were perfused and subjected to heat shock (at 42 degrees C, for 15 min) followed by 6-h perfusion. At the end of perfusion, Hsp72 content in the left ventricle without infarct area (viable LV) and the right ventricle (RV) was determined by the Western immunoblotting method. The production of myocardial Hsp72 in the viable LV and RV of the 2w-CAL heart increased after an exposure to heat shock. In contrast, induction of Hsp72 in the viable LV and RV of the 8w-CAL heart was blunted. The results suggest that the development of heart failure following AMI may result in a decrease in the ability for Hsp72 induction not only in the viable LV but also in the RV, leading to contractile dysfunction of the heart.     

Different effect of glutamine on macrophage tumor necrosis factor-alpha release and heat shock protein 72 expression in vitro and in vivo

Macrophage plays a vital role in sepsis. However, the modulatory effect of glutamine （Gin） on macrophage/ monocyte-mediate cytokines release is still controversial. Thus, we investigated the effect of Gin on macro- phage tumor necrosis factor （TNF）-α release and heat shock protein （HSP） 72 expression in vivo and in vitro. Data from our study indicated that the increase of HSP72 expression was significant at 8 mM of Gin 4 h after lipopolysaccharide （LPS） stimulation and became independent of Gin concentrations at 24 h, whereas TNF-α release was dose-and time-dependent on Gin. Heat stress （HS） induced more HSP72 and less TNF-α production compared with the non-HS group. However, the production of TNF-α in cells pretreated with HS was increased with increasing concentrations of Gin. Treatment with various concentrations of Gin for 1 h and then 0.5mM Gin for 4h led to an increase in HSP72 expression, but not in TNF-α production. In sepsis model mice, Gin treatment led to a significantly lower intracellular TNF-α level and an increase in HSP72 expression in mouse peritoneal macrophages. Our results demonstrate that Gin directly increases TNF-α release of LPS-stimulated RAW264.7 macro- phages in a dose-dependent manner, and also decreases mouse peritoneal macrophages TNF-α release in the sepsis model. Taken together, our data suggest that there may be more additional pathways by which Gin modulates cytokine production besides HSP72 expression in macrophage during sepsis.     

Sexual dimorphism of the intracellular heat shock protein 72 response.

The majority of previous work examining stress responses has been done in males. Recently, it has become clear that the impact of stressor exposure is modulated by sex. One stress response that may be affected by sex is the induction of intracellular heat shock protein (HSP) 72, which is a stress- responsive molecular chaperone that refolds denatured proteins and promotes cellular survival. The following study compared HSP72 in males and females and also examined whether the estrous cycle altered HSP72 induction in females. We hypothesized that females compared with males would have a constrained HSP72 response after an acute stressor and that the stress-induced HSP72 response in females would fluctuate with the estrous cycle. Male and female F344 rats were either left in their home cage or exposed to acute tail-shock stress (8-10/group). Immediately following stressor, trunk blood was collected and tissues were flash frozen. Vaginal smear and estrogen enzyme immunoassay were used to categorize the phase of estrous. Results show that female rats had a greater corticosterone response than males, that both males and females exhibit a stress-induced release of progesterone, and that males and females had equal levels of stress-induced circulating norepinephrine. Sexual dimorphism of the HSP72 (ELISA) response existed in pituitary gland, mesenteric lymph nodes, and liver such that female rats had an attenuated HSP72 response compared with males after stress. The adrenal glands, spleen, and heart did not exhibit sexual dimorphism of the HSP72 response. The estrous cycle did not have a significant effect on basal or stress-induced HSP72 in females.     

Nuclear heat shock protein 72 as a negative regulator of oxidative stress (hydrogen peroxide)-induced HMGB1 cytoplasmic translocation and release

In response to inflammatory stimuli (e.g., endotoxin, proinflammatory cytokines) or oxidative stress, macrophages actively release a ubiquitous nuclear protein, high-mobility group box 1 (HMGB1), to sustain an inflammatory response to infection or injury. In this study, we demonstrated mild heat shock (e.g., 42.5 degrees C, 1 h), or enhanced expression of heat shock protein (Hsp) 72 (by gene transfection) similarly rendered macrophages resistant to oxidative stress-induced HMGB1 cytoplasmic translocation and release. In response to oxidative stress, cytoplasmic Hsp72 translocated to the nucleus, where it interacted with nuclear proteins including HMGB1. Genetic deletion of the nuclear localization sequence (NLS) or the peptide binding domain (PBD) from Hsp72 abolished oxidative stress-induced nuclear translocation of Hsp72-DeltaNLS (but not Hsp72-DeltaPBD), and prevented oxidative stress-induced Hsp72-DeltaPBD-HMGB1 interaction in the nucleus. Furthermore, impairment of Hsp72-DeltaNLS nuclear translocation, or Hsp72-DeltaPBD-HMGB1 interaction in the nucleus, abrogated Hsp72-mediated suppression of HMGB1 cytoplasmic translocation and release. Taken together, these experimental data support a novel role for nuclear Hsp72 as a negative regulator of oxidative stress-induced HMGB1 cytoplasmic translocation and release.