The nasal mucosa plays an important role in defense of the lung against harmful agents. It has been suggested that this is partly mediated by the production of nitric oxide (NO). We have investigated the localization of the messenger ribonucleic acids (MRNAs) for human endothelial NO synthase (Type III NOS) and inducible NO synthase (Type II NOS) and the immunoreactivities of these enzymes in human nasal mucosa by immunohistochemistry, in situ hybridization, and reduced nicotinamide adenine diphosphate (NADPH) diaphorase histochemistry. Inferior nasal turbinates were obtained from 27 patients at the time of surgery for local disease. Strong immunostaining for Type III NOS was localized to vascular endothelium, surface epithelium, and submucosal glands in all subjects. Moderate immunostaining for Type II NOS was seen in surface epithelium; glandular, inflammatory, and vascular endothelial cells; and smooth-muscle cells in the specimens from patients with chronic rhinitis only. In situ hybridization showed expression of the mRNA for Type III NOS in similar sites to those shown by immunohistochemistry, whereas the mRNA for Type II NOS was predominantly localized to inflammatory cells. The sites of NOS expression were further confirmed by NADPH histochemical staining. These findings demonstrate the cellular expression of NOS in the human nasal mucosa and suggest a possible role for Types II and III NO synthase in the regulation of blood flow, nasal secretion, and ciliary movement in health and disease.
Endothelin-1 (ET-1) is a vasoconstrictor, bronchoconstrictor, and mitogenic peptide which is enzymatically converted from a biologically inactive big ET to mature ET (21 amino acid) by the ET-converting enzyme (ECE). Here, we investigate the expression of ECE-1, big ET-1, and ET-1 in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and compare it to those of normal subjects using immunohistochemistry and in situ hybridization. In normal lungs, focal moderate expression of all three molecules is localized to airway epithelium, pulmonary endothelium, and airway and vascular smooth muscle cells. Serous bronchial glands also expressed ET-1 and ECE-1. In IPF, strong diffuse expression of ECE-1 was seen in airway epithelium, proliferating type II pneumocytes, and in endothelial and inflammatory cells. ECE-1 immunostaining was colocalized to big ET-1 and ET-1 immunostaining, and correlated with disease activity (P < 0.05). To study regulatory mechanisms of ET-1 and ECE-1 expression, human normal bronchial epithelial (NBE) cells were treated with cytokines and analyzed by radioimmunoassay and Northern blot. Incubation of human NBE cells with IL-1alpha and -beta or tumor necrosis factor alpha (TNFalpha) resulted in a significant increase in ET-1 release and mRNA expression. TNFalpha resulted in a significant increase in ECE-1 mRNA expression. These findings demonstrated the colocalization of the precursor and active ET-1, and ECE-1 in the same cell, and that ECE-1 expression is elevated in IPF. In addition, increased expression of ET-1 and ECE-1 in IPF may be mediated by proinflammatory cytokines.
Background25-hydroxycholesterol (25-HC) is one of the oxysterols, which are oxidized derivatives of cholesterol, and has been reported to be involved in the pathogenesis of atherosclerosis and Alzheimer’s disease. In lung, the possible involvement of 25-HC in airway diseases has been revealed. In the present study, we examined whether 25-HC affects the release of cytokines and also modulates the responses of toll-like receptor 3 (TLR3) in airway epithelial cells.MethodsThe effect of 25-HC on the release of cytokines from primary human bronchial epithelial cells after stimulation with or without polyinosine-polycytidylic acid [poly(I:C)], a ligand for TLR3, and the signal transduction were examined.Results25-HC significantly potentiated the release of interleukin-8 (IL-8) and IL-6 from the cells. This effect was more potent compared with that of other oxysterols, 22-HC and 27-HC. GW3965 and TO901317, synthetic agonists of liver X receptors that are receptors for oxysterols, did not augment the IL-8 release. 25-HC enhanced the nuclear factor-kappa B (NF-κB) DNA binding activity and translocation of phosphorylated c-Jun into the nucleus. The release of IL-8 was inhibited by the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), an inhibitor of nuclear factor kappa-B alpha (IκBα) inhibitor, BAY 11–7085, and an inhibitor of nuclear factor kappa-B kinase-2 (IKK-2) inhibitor, SC-514, but not by a c-Jun N-terminal kinase (JNK) inhibitory peptide, L-JNKi1. 25-HC significantly potentiated IL-8 release in poly(I:C)-treated cells and the augmentation was inhibited by CAPE, BAY 11–7085, and SC-514. Furthermore, 25-HC potentiated the translocation of interferon regulatory factor 3 into the nucleus and the release of interferon-beta (IFN-β) in poly(I:C)-treated cells.ConclusionsThese data demonstrated that 25-HC augments the release of IL-8 and IL-6 via NF-κB signalling pathway and enhances the release of IL-8 and IFN-β after stimulation of TLR3 in airway epithelial cells. 25-HC may be involved in the neutrophilic airway inflammation through the stimulant effect of IL-8 and IL-6 release and also potentiate the TLR3-mediated innate immunity in airway diseases.
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