Takashi Furuno

The Role of Neutrophil Elastase in Human Pulmonary Artery Endothelial Cell Injury

Neutrophils are thought to play a key role in tissue injury. We investigated the role of human neutrophil-derived elastase in the induction of injury to human pulmonary artery endothelial cells. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity, thrombomodulin, and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. Attachment alone of neutrophils to endothelial cells appeared to induce activation because elastase release and N-formyl-mentionyl-leucyl-phenylalanine (fMLP)-induced superoxide (O2) production from neutrophils incubated with endothelial cells were greater than from neutrophils only. When endothelial cell were incubated with neutrophils stimulated by fMLP or phorbol myristate acetate, the amount of elastase in the medium and endothelial cell damage was further enhanced. However, when neutrophils were blocked from direct attachment to endothelial cells using a membrane filter, endothelial cell damage was ameliorated, while exogenous neutrophil elastase and medium containing neutrophil-released elastase did not induce endothelial cell injury. An inhibitor of neutrophil elastase, ONO-5046 Na, as well as erythromycin, which reduces neutrophil-derived elastase, dramatically inhibited neutrophil-induced endothelial cell injury. Superoxide dismutase (SOD) partially inhibited injury. Injury was completely inhibited by treatment with a combination of ONO-5046 Na and SOD. These results suggest that attachment of neutrophils to endothelial cells is important for endothelial cell damage and that neutrophil-derived elastase plays an important role in endothelial cell injury in combination with O2. In addition, ONO-5046 Na and erythromycin may be useful in treating diseases worsened by excessive neutrophil activity.

Neutrophil-induced Endothelial Cell Damage: Inhibition by a 14-Membered Ring Macrolide Through the Action of Nitric Oxide

Macrolide antibiotics have been used worldwide for about 40 years. The clinical effectiveness of oral erythromycin for diffuse panbronchiolitis has been established and erythromycin seems to act not only as an antibacterial but also as an anti-inflammatory agent. We investigated the effect of 14-membered ring macrolides, erythromycin and clarithromycin, on human neutrophil functions and endothelial cell damage induced by neutrophils. The superoxide production of neutrophils and Ca2+ influx into neutrophils induced by N-formyl-methionyl-leucyl-phenylalanine was inhibited by treatment with erythromycin but not by treatment with clarithromycin. When endothelial cells were cocultured with neutrophils, nitric oxide (NO) presumably released from endothelial cells were enhanced by treatment with erythromycin but not by treatment with clarithromycin and endothelial cell injury induced by neutrophils was ameliorated by addition of erythromycin but not by clarithromycin. The reduction of neutrophil-induced endothelial cell injury by erythromycin was abolished by treatment with carboxy-PTIO which traps NO in the medium. Moreover, nitrite in the medium in which endothelial cells were incubated with neutrophils was enhanced by treatment with erythromycin and the enhancement of nitrite by erythromycin was partially cancelled by addition of H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). Erythromycin seems to ameliorate neutrophil-induced endothelial cell injury by affecting not only neutrophil functions but the release of NO from endothelial cells through the action of PKA. The usefulness for the treatment of diseases worsened by the interaction between neutrophils and endothelium might be different among 14-membered ring macrolides.

RELEASE OF NITRIC OXIDE AND EXPRESSION OF CONSTITUTIVE NITRIC OXIDE SYNTHASE OF HUMAN ENDOTHELIAL CELLS : ENHANCEMENT BY A 14-MEMBERED RING MACROLIDE

A 14-membered ring macrolide, erythromycin, acts not only as an antibacterial but also as an anti-inflammatory agent. We have previously reported that erythromycin modulates neutrophil functions and ameliorates neutrophil-induced endothelial cell damage through the action of cyclic AMP-dependent protein kinase (PKA) and nitric oxide (NO). We investigated the effect of erythromycin on human endothelial cell functions. Erythromycin enhanced intracellular calcium ion concentration ([Ca2+]i) of endothelial cells and NO release from endothelial cells. The enhancement of NO release from endothelial cells by erythromycin was abolished by addition of EGTA in the medium and was partially reduced by addition of H-89, an inhibitor of PKA. These results suggest that erythromycin enhances NO release from endothelial cells through the action of PKA and [Ca2+]i. In addition, constitutive NO synthase (cNOS) protein expression of endothelial cells was dose-dependently enhanced by treatment with erythromycin, which might also contribute to the enhancement of NO release from endothelial cells by erythromycin. The effect of erythromycin as an anti-inflammatory agent might be partially mediated through the enhancement of NO release from endothelial cells and the drug might be a useful tool for the investigation of cNOS of endothelial cells.

An inhibitor of cyclic AMP-dependent protein kinase enhances the superoxide production of human neutrophils stimulated by N-formyl- methionyl-leucyl-phenylalanine

Intact human neutrophils produced superoxide (O2−) by the stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) even when the extracellular Ca2+ was absent (0.56±0.13 nmol/min per 106 cells). The production by fMLP was enhanced more than twice in the presence of the extracellular Ca2+. Moreover, the O2− production by fMLP in the presence of extracellular Ca2+ was enhanced nearly three times by the treatment of cells with H-89, an inhibitor of cyclic AMP-dependent protein kinase (PKA). The enhancement was not observed when the extracellular Ca2+ was depleted from the reaction mixture. In addition, H-89 did not enhance fMLP-induced O2− production of electropermeabilized neutrophils in which the intracellular Ca2+ concentration was fixed to about 100 nM. These observations suggest that not only Ca2+ influx but the inhibition of PKA is necessary for the maximum O2− production by fMLP and that the O2− production is partially suppressed by the activation of PKA induced by fMLP.

The Role of Nitric Oxide in Human Pulmonary Artery Endothelial Cell Injury Mediated by Neutrophils

Human endothelial cells are injured by the action of leukocytes. We investigated the role of nitric oxide (NO) in the induction of injury to human pulmonary artery endothelial cells. NO has been a putative source of cytotoxic reactive oxygen species in some settings. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. This effect was augmented by treatment with carboxy-PTIO, which traps NO in the medium, or with L-NAME, an inhibitor of NO synthase. When endothelial cells were incubated with neutrophils stimulated by phorbol myristate acetate, an activator of protein kinase C, endothelial cell damage was further enhanced and the amount of NO in the medium was decreased. Dibutyryl cyclic AMP, a cell-permeable analogue of cyclic AMP, protected against neutrophil-induced endothelial cell injury and increased NO release into the medium. The effects of dibutyryl cyclic AMP were abrogated by treatment with H-89, a potent inhibitor of cyclic AMP-dependent protein kinase. The protective effect on neutrophil-induced endothelial cell injury by dibutyryl cyclic AMP was abolished by addition of carboxy-PTIO or L-NAME. Thus, our studies suggest that NO, presumably released from endothelial cells, protects against endothelial injury by activated neutrophils and the protective effect by cyclic AMP during coculture with activated neutrophils is mediated through the action of NO. However, when monocytes activated by lipopolysaccharide and IFN-gamma were used instead of neutrophils, endothelial cells were likewise injured, but a much higher level of NO was detected and injury was diminished by addition of carboxy-PTIO to the medium. These observations suggest that the high levels of NO released by activated monocytes contribute to endothelial injury, whereas low levels of NO protect endothelial cells against injury by neutrophils.

Case report: the first report of idiopathic hypereosinophilic syndrome involved with lung and middle ear.

It has been reported that various organs are involved in idiopathic hypereosinophilic syndrome. Frequently, the heart, lung, skin, and nervous system are involved. Involvement of the middle ear, however, has not yet been reported. In this article, the authors describe the first case of hypereosinophilic syndrome involving the lung and middle ear. A 39-year-old woman had a 4-month history of low grade fever, non-productive cough, and a feeling of fullness and hearing loss in both ears. Peripheral blood cell count showed eosinophilia. Bilateral tympanic cavities were obstructed with granulation tissue, and she was diagnosed as obliterative otitis media. The granulation tissue consisted of foamy histiocytes and eosinophils. Chest X-ray film and computed tomography showed patchy infiltrative shadow in the lung. Histologic examination of the open lung biopsied specimen showed alveolar spaces infiltrated by eosinophils. After treatment with 30 mg oral prednisolone daily, there was a rapid improvement in her clinical condition. Based on the clinical course and the histologic findings of this case, obliterative otitis media may be caused by eosinophilic infiltration and eosinophilic pneumonia.

Modulatory effect of roxithromycin on human neutrophil function

Neutrophils are thought to play a key role in tissue injury. We investigated the effect of roxithromycin, a 14-membered ring macrolide, on human neutrophil functions. The drug inhibitedN-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide (O2−) production and Ca2+ influx of human neutrophils. The inhibition was overcome by adding an inhibitor of cyclic AMP-dependent protein kinase (PKA), H-89. These results suggest that the drug affects O2− production and intracellular Ca2+ concentration of neutrophils via the action of PKA. Moreover, roxithromycin ameliorated endothelial cell injury induced by neutrophils, which may be, in part, due to the effect of the drug on neutrophils. Thus, roxithromycin may contribute to the treatment of diseases worsened by the excessive action of neutrophils.

Inhibition by Erythromycin of Human Pulmonary Artery Endothelial Cell Injury Induced by Human Neutrophils

Neutrophils are thought to play a key role in tissue injury. We investigated the role of human neutrophils in the induction of injury to the human pulmonary artery endothelial cells and the effect of erythromycin on neutrophil-induced endothelial cell damage. Incubation of unstimulated neutrophils with endothelial cells increased the release of lactate dehydrogenase (LDH) activity and preloaded fura-2 from endothelial cells. When neutrophils were activated by phorbol myristate acetate, the release of LDH and fura-2 was enhanced further. Superoxide dismutase partially inhibited the release of LDH and fura-2 induced by neutrophils, whereas erythromycin markedly inhibited the release of endothelial cell LDH and fura-2 induced by neutrophils. These results suggest that endothelial cell injury is, at least in part, mediated by the action of superoxide and that erythromycin protects against neutrophil-induced endothelial cell injury.

Tyrosine-Specific Protein Kinase Participates in the Pathogenesis of Acute Immune Complex Alveolitis in Rats

Rabbit IgG antibodies against ovalbumin (OA) was injected intravenously into Wistar rats. When the animals were challenged with OA aerosolized by ultrasonic nebulization, acute lung injury occurred as reflected by increased recovery of bronchoalveolar cells, especially polymorphonuclear leukocytes (PMN) in bronchoalveolar lavage fluid (BALF). Lung morphology demonstrated cellular infiltration in the alveolar septa and intra-alveolar hemorrhage. When the rats were administered ST-638, a novel tyrosine kinase inhibitor, intraperitoneally prior to nebulization, the number of PMN in BALF decreased in a dose-dependent manner and superoxide anion (O2-)-producing activity in peripheral leukocytes was significantly suppressed. Furthermore, the reagent inhibited migration of human peripheral blood neutrophils induced by the chemotactic peptide f-met-leu-phe in vitro. These studies strongly indicate that tyrosine kinase plays an important role in immune complex-triggered neutrophil-related lung disorders, and the novel tyrosine kinase inhibitor ST-638 attenuates lung injury by preventing superoxide production and neutrophil migration.