Activated macrophages express high levels of Nrf2, a transcription factor that positively regulates the gene expression of antioxidant and detoxication enzymes. In this study, we examined how Nrf2 contributes to the anti-inflammatory process. As a model system of acute inflammation, we administered carrageenan to induce pleurisy and found that in Nrf2-deficient mice, tissue invasion by neutrophils persisted during inflammation and the recruitment of macrophages was delayed. Using an antibody against 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), it was observed that macrophages from pleural lavage accumulate 15d-PGJ 2 . We show that in mouse peritoneal macrophages 15d-PGJ 2 can activate Nrf2 by forming adducts with Keap1, resulting in an Nrf2-dependent induction of heme oxygenase 1 and peroxiredoxin I (PrxI) gene expression. Administration of the cyclooxygenase 2 inhibitor NS-398 to mice with carrageenan-induced pleurisy caused persistence of neutrophil recruitment and, in macrophages, attenuated the 15d-PGJ 2 accumulation and PrxI expression. Administration of 15d-PGJ 2 into the pleural space of NS-398-treated wild-type mice largely counteracted both the decrease in PrxI and persistence of neutrophil recruitment. In contrast, these changes did not occur in the Nrf2-deficient mice. These results demonstrate that Nrf2 regulates the inflammation process downstream of 15d-PGJ 2 by orchestrating the recruitment of inflammatory cells and regulating the gene expression within those cells.

Myofibroblasts have been thought to participate in subepithelial fibrosis in asthma, but the mechanism of myofibroblast induction has not been fully understood. In this study we investigated injury-related myofibroblast induction in a coculture system of guinea-pig epithelial cells and fibroblasts cocultured in a human amnion chamber. After pseudostratified epithelial cells were mechanically scraped, migrated flat epithelial cells differentiated into cuboidal appearances on Day 4 and then returned to their original shapes on Day 8. During the course of the epithelial redifferentiation, it was found by Northern blot analysis, immunohistochemistry for alpha-smooth muscle actin, and electron microscopic observation that the myofibroblasts were transiently induced on Day 4. The myofibroblast induction was inhibited by the blocking of transforming growth factor (TGF)-beta1 and thrombospondin (TSP)-1, indicating that the activation of TGF-beta1 by TSP-1 would induce myofibroblasts. This finding was also supported by a transient upregulation of TSP immunoreactivity and TSP-1 messenger RNA (mRNA) in fibroblasts. Interestingly, epithelial injury reduced TGF-beta1 immunoreactivity in the amnion membrane but did not affect TGF-beta1 mRNA in epithelial cells and fibroblasts, indicating that TGF-beta1 supplied from the extracellular matrix can participate in myofibroblast induction. Concurrently with myofibroblast induction, procollagen type I and III mRNAs were upregulated in fibroblasts, and obvious collagen deposition was observed ultrastructurally around the myofibroblasts compared with the fibroblasts. These results indicate that induced myofibroblasts can be functionally more active in producing collagen than are resting fibroblasts. The present study suggests that epithelial injury stimulates TGF-beta1 release from the extracellular matrix and its activation via TSP-1 production, causing collagen synthesis through myofibroblast induction.

These results demonstrated in vivo that 15d-PGJ2 plays a protective role against ALI by exploiting the Nrf2-mediated transcriptional pathway.

Given the high frequency of AITD in patients with type 1 diabetes, these data suggest the possibility that the association of CTLA4 with type 1 diabetes in previous studies may have been secondary to AITD, suggesting the importance of subclassification of type 1 diabetes relative to AITD in genetic studies.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture.3 We confirmed that supplementation with 0.5 mm CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mm CR significantly suppressed the endothelial permeability induced by serotonin and H 2 O 2 . 5 In cell adhesion experiments, supplementation with 5 mm CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mm CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A 2A receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells. British Journal of Pharmacology (2003) 139, 715 -720. doi:10.1038/sj.bjp.0705316 Keywords: Creatine;endothelial cell; H 2 O 2 ; permeability; phosphocreatine; ATP; serotonin; adhesion; ICAM-1; E-selectin Abbreviations: CR, creatine; Dil-Ac-LDL, LDL labeled with 1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindo-carbocyanine perchlorate; EBM, endothelial cell basal medium; FITC-dex, fluorescein isothiocyanate-dextran; FMLP, n-formyl-MetLeu-Phe; GPA, b-guanidinopropionic acid; HBSS, Hank's balanced saline solution; LDL, low-density lipoprotein; b-NAD þ , b-nicotinamide adenine dinucleotide; PC, phosphocreatine IntroductionCreatine (CR) is tightly linked to the intracellular energy under the form of ATP (Wyss & Kaddurah-Daouk, 2000). Cells take in CR from the blood stream through a CR transporter and store it as CR and its phosphorylated form, phosphocreatine (PC) (Snow & Murphy, 2001). CR receives a phosphoryl group from ATP to generate PC by using the CR kinase inside cells. Intracellular ATP has such a short half-life that cells are equipped with two major channels of ATP supply. One is glycolysis and oxidative phosphorylation; the other is the PC energy system. While the former constantly provides ATP, the latter works on acute demand as an energy buffer to maintain an intracellular ATP level. Because PC transfers the phosphoryl group to ADP to synthesize ATP quickly, the total amount of ATP and PC represents an intracellular energy pool. The PC energy system is well characterized in the case of skeletal muscle cells because about 94% of CR is distributed therein (Wyss & Kaddurah-Daouk, 2000). It is known that supplementation with high-dose CR further increases intracellular energy pools of muscle tissue as well as serum CR levels in vivo. ...

Pulmonary fibrosis is an end-stage disorder for which efficacious therapeutic options are not readily available. Although its pathogenesis is poorly understood, pulmonary fibrosis occurs as a result of various inflammations. NKT cells modulate inflammation because of their ability to produce large amounts of cytokines by stimulation with their glycolipid ligand. In the present study, we investigated the effects of α-galactosylceramide (α-GalCer), a selective NKT cell ligand, on the development of bleomycin-induced pulmonary fibrosis. Treatment of mice with α-GalCer prolonged their survival under bleomycin administration by attenuating the development of pulmonary fibrosis. The protective effects of α-GalCer were associated with an increase in the pulmonary level of IFN-γ and a decrease in the pulmonary level of fibrogenic cytokines such as TGF-β and connective tissue growth factor. The initial pulmonary inflammation caused by bleomycin was also attenuated by α-GalCer with the reduction of the macrophage inflammatory protein-2 level. The protective effects of α-GalCer were markedly reduced in mice lacking NKT cells or as a result of treatment with anti-IFN-γ Ab. These results suggest that α-GalCer suppresses bleomycin-induced acute pulmonary inflammation and thus attenuates the development of pulmonary fibrosis possibly by regulating several cytokine levels.

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OBJECTIVE -To clarify the role of the T-lymphocyte-associated-4 (CTLA-4) polymorphism in the susceptibility to child-onset type 1 diabetes with regard to its clinical characteristics and complications with autoimmune thyroid disease (AITD) in the Japanese population.RESEARCH DESIGN AND METHODS -The CTLA-4 49 A/G polymorphism was detected by the PCR-restriction fragment-length polymorphism (RFLP) method in 97 type 1 diabetic subjects and 20 patients with Graves' disease, a cohort which included 4 patients who also had type 1 diabetes.RESULTS -The genotypes and allele frequencies of this polymorphism did not differ between the type 1 diabetic subjects and the control subjects. The G allele frequency was 63.9% in the type 1 diabetic subjects. The G allele frequency in the subgroup of patients with a high titer of autoantibodies to the GAD antibody (Ab) was 72.9% (P ϭ 0.0499 vs. control subjects); in the subgroup of patients without HLA DRB1*0405, it was 72.6% (P ϭ 0.0271 vs. control subjects); and in the subgroup of patients with a residual ␤-cell function, it was 78.6% (P ϭ 0.0391 vs. control subjects). The G allele frequency in the patients with Graves' disease was also significantly higher at 78.1% (P ϭ 0.0405 vs. control subjects). Furthermore, the frequency in our diabetic subjects complicated with Graves' disease was even higher (87.5%).CONCLUSIONS -We have demonstrated that a distinct association exists between the G allele of CTLA-4 and high values of GAD Ab, residual ␤-cell function, and the absence of HLA-DRB1*0405.