Shigekiyo Matsumoto

Changes in cell culture temperature alter release of inflammatory mediators in murine macrophagic RAW264.7 cells

Abstract.Objectives:To examine whether moderate changes in cell culture temperature influence the production of various cytokines and associated mediators of inflammation.Methods:We performed lipopolysaccharide (LPS) stimulation of the murine macrophagic RAW264.7 cell line under hyperthermic (40 °C), normothermic (37 °C) and hypothermic (34 °C) conditions. We then measured the levels of heat shock protein 70 (HSP70), heat shock factor protein (HSF) and nuclear factor–kB (NF-kB) dimers (p50 and p65) in the cells, and the levels of high mobility group box 1 (HMGB1) and the cytokines tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in the culture supernatants.Results:Levels of HMGB1, IL-1β, IL-6, and TNF-α, as well as NF-kB dimers (p50 and p65), were all reduced following LPS stimulation at 40 °C and 34 °C compared with those at 37 °C. Levels of HSP70 and HSF increased at 40 °C and 34 °C.Conclusions:The application of moderate hyperthermia and hypothermia after LPS-induced cell activation attenuated the inflammatory response and reduced the likelihood of cell damage. These findings suggest that moderate temperature changes modulate the inflammatory response and could be a useful therapy against sepsis.

In vivo and in vitro effects of the anticoagulant, thrombomodulin, on the inflammatory response in rodent models.

Sepsis remains a major health threat in intensive care medicine. The physiological functions of the coagulation cascade extend beyond blood coagulation and play a pivotal role in inflammation. We investigated whether the use of recombinant thrombomodulin (rTM), which has activity comparable with antithrombin, tissue factor pathway inhibitor, and activated protein C, could inhibit secretion of cytokines and high-mobility group box 1 (HMGB1) protein, thus reducing lung damage in a rat model of LPS-induced systemic inflammation. Rats treated with an intravenous injection of either rTM or saline were injected concurrently with intravenous LPS. In addition, mouse macrophage RAW264.7 cells were stimulated with LPS, with or without simultaneous rTM treatment. Histological examination revealed marked reductions of interstitial congestion, edema, inflammation, and hemorrhage in lung tissue harvested 12 h after treatment with both agents compared with LPS administration alone. LPS-induced secretion of proinflammatory cytokines and HMGB1 protein was inhibited by treatment with rTM. The presence of HMGB1 protein in the lung was examined by immunohistochemistry; the number of HMGB1-positive cells was significantly lower in LPS-treated animals that also received rTM. In the in vitro studies, rTM administration inhibited the activation of nuclear factor-kappa B by inhibiting I kappa B phosphorylation. The anticoagulant rTM blocked the LPS-induced inflammatory response and protected against acute lung injury normally associated with endotoxemia in this rat sepsis model. Given these results, rTM is a strong candidate as a therapeutic agent for various systemic inflammatory diseases.

Association Between Heat Stress Protein 70 Induction and Decreased Pulmonary Fibrosis in an Animal Model of Acute Lung Injury

The hyperthermia-induced activation of the stress protein response allows cells to withstand metabolic insults that would otherwise be lethal. This phenomenon is referred to as thermotolerance. Heat shock protein 70 (HSP70) has been shown to play an important role in this hyperthermia-related cell protection. HSP70 confers protection against cellular and tissue injury. Our objective was to determine the effect of heat stress on the histopathology of pulmonary fibrosis caused by the administration of lipopolysaccharide (LPS) in Wistar rats. The rats were randomly divided into three groups. In the control group, rats were heated to 42°C for 15 min. In the LPS group, rats were given LPS in 0.9% NaCl solution (10 mg/kg body weight). In the WH (whole-body hyperthermia) +LPS group, rats were heated to 42°C for 15 min, and 48 h later they were injected with LPS dissolved in a 0.9% NaCl solution (10 mg/kg body weight). We investigated lung histopathology and performed a Northern blot analysis daily. Hyperthermia was shown to reduce tissue injury caused by the administration of LPS. Pulmonary tissue HSP70 mRNA was found to be elevated at 3 h after heating. HSP70 protein levels in the serum increased after whole-body hyperthermia. However, neither the expression of HSP47 mRNA nor the expression of type I or type III collagen mRNA was induced by the administration of LPS after whole-body hyperthermia. These data indicate that thermal pretreatment is associated with the induction of HSP70 protein synthesis, which subsequently attenuates tissue damage in experimental lung fibrosis.

Antisense oligonucleotide inhibition of heat shock protein (HSP) 47 improves bleomycin-induced pulmonary fibrosis in rats.

BackgroundThe most common pathologic form of pulmonary fibrosis arises from excessive deposition of extracellular matrix proteins such as collagen. The 47 kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen.ObjectivesTo determine whether inhibition of HSP47 could have beneficial effects in mitigating bleomycin-induced pulmonary fibrosis in rats.MethodsAll experiments were performed with 250–300 g male Wistar rats. Animals were randomly divided into five experimental groups that were administered: 1) saline alone, 2) bleomycin alone, 3) antisense HSP47 oligonucleotides alone, 4) bleomycin + antisense HSP47 oligonucleotides, and 5) bleomycin + sense control oligonucleotides. We investigated lung histopathology and performed immunoblot and immunohistochemistry analyses.ResultsIn rats treated with HSP47 antisense oligonucleotides, pulmonary fibrosis was significantly reduced. In addition, treatment with HSP47 antisense oligonucleotides significantly improved bleomycin-induced morphological changes. Treatment with HSP47 antisense oligonucleotides alone did not produce any significant changes to lung morphology. Immunoblot analyses of lung homogenates confirmed the inhibition of HSP47 protein by antisense oligonucleotides. The bleo + sense group, however, did not exhibit any improvement in lung pathology compared to bleomycin alone groups, and also had no effect on HSP47 expression.ConclusionThese findings suggest that HSP47 antisense oligonucleotide inhibition of HSP47 improves bleomycin-induced pulmonary fibrosis pathology in rats.

Coexpression of HSP47 Gene and Type I and Type III Collagen Genes in LPS-Induced Pulmonary Fibrosis in Rats

Diffuse alveolar damage is the histopathologic hallmark of acute respiratory distress syndrome (ARDS). A significant proportion of ARDS survivors have residual pulmonary fibrosis and compromised pulmonary function. On the other hand, heat shock protein 47 (HSP47) is a collagen-binding stress protein that is assumed to act as a collagen-specific molecular chaperone during the biosynthesis and secretion of procollagen in living cells. The synthesis of HSP47 has been reported to correlate with that of collagen in several cell lines. We examined the expression of HSP47 mRNA and protein during the progression of lipopolysaccharide (LPS)-induced ARDS in rat lung. Male Wistar rats were randomly divided into two groups: a control group with instillation of 0.9% NaCl solution alone, and a LPS group with instillation of LPS dissolved in 0.9% NaCl solution (10 mg/kg). Histologic changes thereafter appeared in the LPS-treated rats. Northern blot analysis revealed the expression of HSP47 mRNA to be markedly induced during the progression of lung damage in parallel with type I and type III collagen mRNA. These results suggest that the upregulation of HSP47 and collagen may play an important role in the fibrotic process of LPS-induced ARDS lung.

Human Atrial Natriuretic Peptide Attenuates Renal Ischemia-Reperfusion Injury

BACKGROUND Acute kidney injury (AKI) is common in the intensive care unit, and one of its primary causes is renal ischemia-reperfusion (I/R) injury. Human atrial natriuretic peptide (hANP) exerts various pharmacologic effects, including renal protection. In the present study, we evaluated the renal protective effect of hANP in a rat model of renal I/R. MATERIALS AND METHODS Male Wistar rats were divided into three groups that received the following treatments: induction of renal I/R (I/R group); continuous intravenous injection of hANP followed 30 min later by induction of renal I/R (hANP+I/R group); and sham treatment (control group). Rats were sacrificed after 60 min of ischemia and 24 h of reperfusion or sham treatment. To evaluate the renal protective effects if hANP, serum blood urea nitrogen (BUN) and creatinine (Cre) concentrations were determined, kidneys were histologically assessed, and serum biomarkers of oxidative stress were evaluated. In addition, antimycin A (AMA)-stimulated RAW264.7 cells were treated with hANP to assess its antioxidant effects. RESULTS Serum BUN and Cre levels were elevated in the I/R group; however, these increases were significantly inhibited in the hANP + I/R group. Similarly, kidney tissue damage observed in the I/R group was attenuated in the hANP + I/R group. In vitro, AMA-stimulated cells treated with hANP showed reduced reactive oxygen species activity compared to cells treated with AMA alone. CONCLUSIONS Our findings indicate that hANP may be effective in the treatment of various types of I/R injuries.

Antithrombin III prevents cerulein-induced acute pancreatitis in rats.

Objectives: Systemic inflammatory mediators, including the protein high-mobility group box 1 (HMGB1), play an important role in the development of acute pancreatitis. Anticoagulants, such as antithrombin III (AT III), inhibit inflammation resulting from various causes, but their mechanism of action is not well understood. Because acute pancreatitis is a severe inflammatory disease, we hypothesized that AT III would inhibit inflammation and prevent cerulein-induced acute pancreatitis. Methods: Experimental animals received or were saline injected with a bolus of 250 IU/kg of AT III followed by intraperitoneal injections of 50 mg/kg of cerulein. Levels of cytokines (interleukin 6 and tumor necrosis factor &agr;), nitric oxide (NO), and HMGB1 were measured in serum and pancreatic tissue at regular intervals for 12 hours after the cerulein injection. Results: Pancreas histopathology and wet-dry ratio significantly improved in the AT III-injected (250 IU/kg) animals compared with the saline-injected rats. Serum and pancreas HMGB1 levels decreased over time in AT III-treated animals. Antithrombin III also decreased cytokine, NO, and HMGB1 levels during cerulein-induced inflammation. As a result, AT III ameliorated the pathologic pancreas in the rat model of cerulein-induced acute pancreatitis. Conclusions: Antithrombin III treatment inhibited the secretion of cytokines, NO, and HMGB1 and prevented cerulein-induced acute pancreatitis in the rat model.

Heat shock protein 72 protects insulin-secreting beta cells from lipopolysaccharide-induced endoplasmic reticulum stress.

Purpose: Hyperthermia-induced activation of stress response proteins allows cells to withstand metabolic insults. In this study we set out to determine whether insulin secretion by pancreatic beta cells was affected by the acute inflammatory response, systemic inflammation-induced hyperglycaemia, and whole-body hyperthermia. Given that systemic-inflammation induces ER stress, we further examined whether hyperthermia can attenuate the extent of LPS-induced ER stress. Materials and methods: Rats were randomised and divided into three treatment groups. Control rats received a 0.9% NaCl solution. Rats in the lipopolysaccharide (LPS) group received 7.5 mg of LPS/kg. Rats in the whole-body hyperthermia (WBH) + LPS group were exposed to 42 °C for 15 min, followed by injection with 7.5 mg of LPS/kg after 48 h. Glucose-potentiated insulin release and extent of ER stress were measured in beta cells. Results: LPS inhibited glucose-induced insulin release from islet cells and induced the expression of Bip/GRP78, XBP-1, and CHOP transcripts. The inhibition of glucose-induced insulin release and induction of ER stress proteins by LPS was attenuated by WBH. Conclusions: Our findings suggest that LPS-induced systemic inflammation decreased insulin release due to the effects of ER stress proteins on insulin secretion. Furthermore, the induction of ER stress proteins was prevented by pretreating rats with WBH. This may suggest that inhibiting the induction of ER stress proteins through WBH can restore insulin release in various disease states.

Retraction Note to: Coexpression of HSP47 Gene and Type I and Type III Collagen Genes in LPS-Induced Pulmonary Fibrosis in Rats

Diffuse alveolar damage is the histopathologic hallmark of acute respiratory distress syndrome (ARDS). A significant proportion of ARDS survivors have residual pulmonary fibrosis and compromised pulmonary function. On the other hand, heat shock protein 47 (HSP47) is a collagen-binding stress protein that is assumed to act as a collagen-specific molecular chaperone during the biosynthesis and secretion of procollagen in living cells. The synthesis of HSP47 has been reported to correlate with that of collagen in several cell lines. We examined the expression of HSP47 mRNA and protein during the progression of lipopolysaccharide (LPS)-induced ARDS in rat lung. Male Wistar rats were randomly divided into two groups: a control group with instillation of 0.9% NaCl solution alone, and a LPS group with instillation of LPS dissolved in 0.9% NaCl solution (10 mg/kg). Histologic changes thereafter appeared in the LPS-treated rats. Northern blot analysis revealed the expression of HSP47 mRNA to be markedly induced during the progression of lung damage in parallel with type I and type III collagen mRNA. These results suggest that the upregulation of HSP47 and collagen may play an important role in the fibrotic process of LPS-induced ARDS lung.

Uncoupling of peripheral and master clock gene rhythms by reversed feeding leads to an exacerbated inflammatory response after polymicrobial sepsis in mice.

Abstract Reversed feeding uncouples peripheral and master clock gene rhythms and leads to an increased risk of disease development. The aim of this study was to determine the effects of clock gene uncoupling on sepsis-induced inflammation using a mouse cecal ligation and puncture (CLP) model. C57BL/6N mice were entrained to a 12-h light-dark cycle (lights on at 7 AM). Mice were permitted ad libitum feeding either during the night (7 PM–7 AM) or the nonphysiological light phase (7 AM–7 PM) for a week before CLP. In daytime-fed mice, phase inversion of clock gene expression was observed in the liver, but not in the suprachiasmatic nucleus. Daytime-fed mice also had decreased body weight and food intake. Survival rate was significantly lower in daytime-fed mice (29%) compared with nighttime-fed mice (54%) 72 h after CLP (P = 0.03). Serum levels of interleukin 6 (IL-6), tumor necrosis factor &agr;, high mobility group box 1, IL-1&agr;, IL-9, eotaxin, and granulocyte colony-stimulating factor increased in daytime-fed mice compared with nighttime-fed mice after CLP. Baseline expression levels of sirtuin peroxisome 1 and proliferator-activated receptor &ggr; coactivator 1&agr; in the liver decreased in daytime-fed mice compared with nighttime-fed mice. Thus, daytime feeding induces clock gene uncoupling, which leads to decreased expression of longevity-related and energy metabolism proteins. Daytime feeding may also increase the levels of inflammatory cytokines, thereby increasing mortality in a mouse sepsis model. Our findings suggest that uncoupling of peripheral and master clock gene rhythms by reversed feeding exacerbates inflammatory responses.