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It was hypothesized that cell-to-cell interaction between human alveolar macrophages (AM) and alveolar epithelium, might be an important factor leading to nitric oxide synthase-2 (NOS2) messenger ribonucleic acid (mRNA) and protein expression by constituent cells of the alveolar wall and/or AM.NOS2 mRNA and the protein expression patterns of human AM and alveolar epithelial cells type II (AEC-II) isolated from normal parts of lung resections of patients with pulmonary malignancies were determined. In addition, NOS2 mRNA expression in human AM co-cultured with autologous AEC-II in the presence of pro-inflammatory cytokines interleukin (IL)-1b, tumour necrosis factor (TNF)-a, interferon (IFN)-c or lipopolysaccharide (LPS) was investigated. The effect of human surfactant protein-A (SP-A) on IFN-c-mediated NOS2 mRNA expression in human AM was also studied.Neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated AEC-II. In contrast, freshly isolated AM from bronchoalveolar lavage or lung tissue samples expressed immunoreactivity for NOS2 protein, but no NOS2 mRNA could be detected by reverse transcriptase polymerase chain reaction. All stimuli tested failed to induce NOS2 mRNA expression in human AM in vitro. Only AM-AEC-II co-culture in the presence of IFN-c led to NOS2 mRNA and protein expression. In situ hybridization of NOS2 mRNA on lung tissue explants and immunohistochemical staining of cytospin preparations of AM-AEC-II co-cultures demonstrated that NOS2 is expressed in AM but not in AEC-II. This co-culture effect could not be reproduced by substitution of AEC-II with SP-A.These data give evidence of a regulatory network controlling human nitric oxide synthase-2 expression in the lower respiratory tract.

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-␥, interleukin-1␤, and tumor necrosis factor-␣, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24-or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.nitric oxide synthase; mRNA; RT-PCR; interferon-␥; tumor necrosis factor-␣; interleukin-1␤; type II human alveolar epithelial cells; A549 cell line NITRIC OXIDE SYNTHASES (NOS) constitute a family with at least three distinct isoforms; neuronal (nNOS or NOS1), inducible (iNOS or NOS2) and endothelial (eNOS or NOS3) (20, 21). NOS1 and NOS3 are constitutively active and produce small amounts of nitric oxide (NO), whereas NOS2 is induced by inflammatory cytokines and produces a larger amount of NO. All three types of NOS are expressed in human lung, and NOS mRNA expression and enzyme activity have been described in human bronchial epithelial cells, macrophages, endothelial cells, and vascular smooth muscle cells (18,34). However, relatively little is known regarding the expression of NOS2 and NOS3 in type II human alveolar epithelial cells (AEC II) and how these enzymes are regulated. Although several laboratories have reported that rat AEC II express NOS2 mRNA and produce NO in vitro (12,26) and that human alveolar epithelium exhibits an NOS-like enzymatic activity in vivo (18), there has been neither molecular nor biochemical confirmation of the expression of NOS2 and NOS3 in human primary cultured AEC II. Asano et al. (4) and Kwon and George (19) have demonstrated that the human type II alveolar epithelial cell line (A549) expresses NOS2 and produces NO in response to proinflammatory cytokines such as interferon-␥ (IFN-␥), interleukin-1␤ (IL-1␤), and tumor necrosis factor-␣ (TNF-␣). Therefore, the aims of the present study were 1) to determine whether human AEC II express NOS2 and NOS3 mRNA in primary culture, 2) to characterize the regulation of mRNA expression of NOS isoforms in these cells by proinflammatory cytokines, and 3) to compare these...

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This study compared airway responsiveness in vitro, as measured in isolated bronchi, with responsiveness in vivo in patients with COPD and smokers with normal lung function. In 9 patients with COPD (mean (range) FEV, 55 (30-78) %predicted) and 8 smokers with normal lung function (FEV1 101 (89-117) %predicted), who underwent surgery for lung cancer, responses to inhaled histamine and salbutamol were assessed before surgery. Bronchial specimens of 1-4 mm internal diameter were studied in the organ bath and histamine concentration-response curves assessed. All patients with COPD and none of the control individuals were hyperresponsive to inhaled histamine. Five patients with COPD and no control patient showed a bronchodilator response to salbutamol. Opposite to these findings, bronchial rings in the organ bath demonstrated a rightward shift of histamine concentration-response curves in COPD compared to controls, (p < 0.005). Accordingly, pED50 but not Emax differed statistically (p = 0.0016) between groups, mean+/-SEM values of pED50 in COPD (controls) being 4.67+/-0.08 (5.29+/-0.15) and of Emax 672+/-86 (772+/-120) mg. Patients with COPD showing hyperresponsiveness to inhaled histamine demonstrated lower responsiveness of their isolated bronchi compared to smokers with normal lung function. This suggests that in vivo hyperresponsiveness is based on other mechanisms than alterations in smooth muscle physiology.