Itaru Ishida

Hypoxia Diminishes Toll-Like Receptor 4 Expression Through Reactive Oxygen Species Generated by Mitochondria in Endothelial Cells

Hypoxia and inflammation often occur simultaneously due to prevention of adequate gas exchange. Understanding the influence of hypoxia on the inflammatory response is important because hypoxia directly regulates expression of many genes, including those regulating inflammation, and plays a role in modulating the resolution of an inflammatory response. LPS is a major mediator of cellular injury and inflammation that induces its effects through Toll-like receptor 4 (TLR4). The aim of this study was to evaluate the effect of hypoxia on TLR4 expression. Hypoxia decreased TLR4 expression on cultured endothelial cells. Furthermore, LPS-induced ICAM-1 up-regulation was decreased by hypoxia. Because reactive oxygen species (ROS) generated from mitochondria are one of the signaling molecules induced by hypoxia, the role of ROS in hypoxia-induced TLR4 down-regulation was evaluated. Our data showed that hypoxia increased ROS generation and that hypoxia-induced TLR4 down-regulation was inhibited by myxothiazol, a mitochondrial site III electron transport inhibitor. Hypoxia also inhibited AP-1 translocation. Since the TLR4 promoter has a binding site for AP-1, hypoxia-induced TLR4 down-regulation may be due to an ROS-mediated decrease in AP-1-binding activity. We conclude that hypoxia decreases TLR4 expression in endothelial cells and that this change is mediated by mitochondrial ROS leading to attenuation of AP-1 transcriptional activity.

Effects of rewarming on nuclear factor-κb and interleukin 8 expression in cold-preserved alveolar epithelial cells

Background. Nuclear factor-&kgr;B (NF-&kgr;B) and interleukin (IL)-8 play important roles in the pathophysiology of acute lung injury after lung transplantation. Because alveolar epithelium is one of the most important sites at which IL-8 production takes place after reperfusion of donor lungs, we examined the effects of cold/rewarming on NF-&kgr;B and IL-8 expression in alveolar epithelial cells. Methods. A549 cells were preserved at 4°C for 5 hr and then rewarmed for up to 20 hr. NF-&kgr;B was analyzed by electrophoretic mobility shift assay. IL-8 mRNA expression was examined by reverse transcription-polymerase chain reaction. IL-8 concentration in the cell culture medium after rewarming was measured by enzyme-linked immunosorbent assay. Results. NF-&kgr;B was increased in the nuclear extracts as early as 30 min after rewarming. There was a marked increase in the IL-8 mRNA expression at 1 and 3 hr after rewarming. IL-8 concentration in the cell culture medium was progressively increased during 20 hr following rewarming. The cell culture medium inhibited apoptosis of neutrophils significantly. The cold/rewarming-induced IL-8 production was reduced to approximately 50% by introducing an antisense oligonucleotide for the p65 subunit of NF-&kgr;B and by treatment with N-acetyl-leucinyl-leucinyl-norleucinal and pyrrolidine dithiocarbamate. The effect of dexamethasone treatment was dose dependent (reduced to approximately 30% at 10−5 M dexamethasone). Conclusions. Our results indicate that rewarming of cold-preserved alveolar epithelial cells itself may be an important initiator of the inflammatory cascades, including NF-&kgr;B activation and IL-8 release. Inhibition of NF-&kgr;B would be worth trying to control unnecessary IL-8 production and the inflammatory response in the donor lungs.

Roentgenographically occult bronchogenic squamous cell carcinoma involving mediastinal lymph nodes after removal of initial lesion by the diagnostic examination

A 69-year-old male was suspected of having lung cancer by sputum cytology and diagnosed as roentgenographically occult squamous cell carcinoma (ROSCC) at the spur of left B(1+2)/B(3). However, after the first bronchoscopy, no suspicious lesion was detected by any examinations. Therefore, we considered that cancer cells had been removed completely by the initial examination, and the patient was followed up by sputum cytology, chest roentgenogram, and bronchoscopy. Sixteen months later from the initial examination, bronchoscopy was performed for follow-up. The bronchoscopic findings showed the elevation of the surface of left B(1+2) a+b, but the cytologic specimen by brushing toward B(1+2) a+b showed negative findings. However, the lesion had developed to polypoid-shaped tumor and obstructed B(1+2) a+b after the next 6 months. The tumor was diagnosed as squamous cell carcinoma, and hilar and mediastinal nodal involvement was suspected on chest computed tomography. The standard thoracotomy was performed and the pathological results showed positive for nodal involvement on hilus and mediastinum. The tumor is considered to arise from the residual cancer cells of initially detected ROSCC. In conclusion, although some ROSCCs regress by the diagnostic examinations, it is important to detect the recurrence of residual cancer cells as early as possible by intensive follow-up.