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Biomedical subjects

D N Watkins

Publications and source records attributed to D N Watkins.

At least 19 recordsLinked to original sources
PubMed2004

Hedgehog signalling in foregut malignancy.

Hedgehog (Hh) signalling mediates axial patterning and stem cell fate in development. This is mediated by Sonic, Desert and Indian Hedgehogs whose morphogen gradients determine the level of signalling in recipient tissues. Aberrant, cell autonomous, ligand-dependent Hh signalling has recently been demonstrated in small cell lung cancer (SCLC), as well as in upper gastrointestinal malignancies arising from pancreas, esophagus and stomach. These tumors lack mutations in the Hh receptor PATCHED, identifying a mechanism of pathway activation distinct from Gorlin's syndrome associated neural and skin tumors. We believe that this phenomenon represents a conserved mechanism for establishing niche-independent stem cell fates in cancer which is essential for malignant transformation and metastasis. Specific inhibition of Hh signalling by the naturally occurring plant alkaloid cyclopamine provides the opportunity for pharmacologic assessment of the role of Hh signalling in these tumors. Cyclopamine inhibits growth of SCLC and a wide range of foregut derived malignancies both in vitro and in vivo. This demonstrates an ongoing requirement for Hh signalling in these highly lethal and aggressive tumors. A novel therapeutic strategy is proposed using pharmacologic targeting of Hh dependent tumors with high potency pathway antagonists.

Animals↗
PubMed2002

Oxidant stress induces gamma-glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI-H292 cells.

BACKGROUND: The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by gamma-glutamylcysteine synthetase (gamma-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known. OBJECTIVE: To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells. METHODS: NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS: Exposure to menadione (MQ, 10-200 microm, 30-120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6-12 h after withdrawal of the oxidant stress (MQ, 50 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-GCS activity measured 6 h (21.7 +/- 3.4 nmol/min/mg, SD, n = 5, P < 0.01) or 12 h (23.2 +/- 4.6, P < 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 +/- 1.0). Similarly, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-GCS mRNA expression was completely inhibited by actinomycin D. CONCLUSIONS: Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased gamma-GCS mRNA transcription and enzyme activity.

Bronchi↗
PubMed2001

Mammalian Scratch participates in neuronal differentiation in P19 embryonal carcinoma cells.

Mammalian Scratch (Scrt) is a Snail family zinc finger transcription factor that is specifically expressed in newly differentiating, post-mitotic central nervous system neurons. While Scrt-related genes appear essential for invertebrate neurogenesis, the role of Scrt in mammalian neural development is unknown. In this study, we found that neural differentiation of multipotent mouse P19 embryonal carcinoma cells by retinoic acid led to the appearance of Scrt together with neuron-specific class III beta-tubulin (Tuj1), following the earlier elaboration of Mash1. Transient co-transfection in P19 cells with either Mash1 or NeuroD2 plus E12 also induced Scrt gene expression. Moreover, overexpression of Scrt alone was sufficient to confer Tuj1 immunoreactivity and neuronal morphology in a subset of P19 cells. Scrt thus appears to function downstream of proneural bHLH proteins in promoting mammalian neural differentiation in this model system.

Animals↗
PubMed2001

Cell surface tumor endothelial markers are conserved in mice and humans.

We recently identified genes encoding tumor endothelial markers (TEMs) that displayed elevated expression during tumor angiogenesis. From both biological and clinical points of view, TEMs associated with the cell surface membrane are of particular interest. Accordingly, we have further characterized four such genes, TEM1, TEM5, TEM7, and TEM8, all of which contain putative transmembrane domains. TEM5 appears to be a seven-pass transmembrane receptor, whereas TEM1, TEM7, and TEM8 span the membrane once. We identified mouse counterparts of each of these genes, designated mTEM1, mTEM5, mTEM7, and mTEM8. Examination of these mTEMs in mouse tumors, embryos, and adult tissues demonstrated that three of them (mTEM1, mTEM5, and mTEM8) were abundantly expressed in tumor vessels as well as in the vasculature of the developing embryo. Importantly, expression of these mTEMs in normal adult mouse tissues was either undetectable or detected only in a small fraction of the vessels. These results demonstrate conservation of human and mouse tumor angiogenesis at the molecular level and support the idea that tumor angiogenesis largely reflects normal physiological neovasculaturization. The coordinate expression of TEM1, TEM5, and TEM8 on tumor endothelium in humans and mice makes these genes attractive targets for the development of antiangiogenic therapies.

Animals↗
PubMed2001

p14ARF silencing by promoter hypermethylation mediates abnormal intracellular localization of MDM2.

The INK4a/ARF locus encodes two distinct tumor suppressors, p16INK4a and p14ARF. Although the contribution of p16INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation has been widely documented, little is known about specific p14ARF lesions and their consequences. Recent data indicate that p14ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14ARF methylation. Taken together, these data support that epigenetic silencing of p14ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.

DNA Methylation↗
PubMed2001

Mammalian Scratch: a neural-specific Snail family transcriptional repressor.

Members of the Snail family of zinc finger transcription factors are known to play critical roles in neurogenesis in invertebrates, but none of these factors has been linked to vertebrate neuronal differentiation. We report the isolation of a gene encoding a mammalian Snail family member that is restricted to the nervous system. Human and murine Scratch (Scrt) share 81% and 69% identity to Drosophila Scrt and the Caenorhabditis elegans neuronal antiapoptotic protein, CES-1, respectively, across the five zinc finger domain. Expression of mammalian Scrt is predominantly confined to the brain and spinal cord, appearing in newly differentiating, postmitotic neurons and persisting into postnatal life. Additional expression is seen in the retina and, significantly, in neuroendocrine (NE) cells of the lung. In a parallel fashion, we detect hScrt expression in lung cancers with NE features, especially small cell lung cancer. hScrt shares the capacity of other Snail family members to bind to E-box enhancer motifs, which are targets of basic helix--loop--helix (bHLH) transcription factors. We show that hScrt directly antagonizes the function of heterodimers of the proneural bHLH protein achaete-scute homolog-1 and E12, leading to active transcriptional repression at E-box motifs. Thus, Scrt has the potential to function in newly differentiating, postmitotic neurons and in cancers with NE features by modulating the action of bHLH transcription factors critical for neuronal differentiation.

Amino Acid Motifs↗
PubMed2000

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.

Adenine↗
PubMed2000

Nitrite generation and antioxidant effects during neutrophil apoptosis.

Neutrophil apoptosis is important for the resolution of airway inflammation in a number of lung diseases. Inflammatory mediators, endogenous reactive oxygen and nitrogen species, and intracellular and extracellular antioxidants may all influence neutrophil apoptosis. This study investigated the involvement of these factors during apoptosis of neutrophils cultured in vitro. Neutrophils undergoing spontaneous apoptosis in culture as assessed by annexin V binding generated significant amounts of nitrite. Incubation with agonistic anti-Fas monoclonal antibody or tumor necrosis factor-alpha (TNF-alpha) enhanced neutrophil apoptosis at 6 h, although it decreased nitrite accumulation. Although granulocyte-macrophage colony-stimulating factor significantly reduced neutrophil apoptosis, this was also associated with decreased nitrite accumulation. In contrast, inhibition of apoptosis at 16 h by dibutyryl cyclic adenosine monophosphate was associated with increased nitrite accumulation. Exogenous glutathione (GSH) or N-acetylcysteine significantly enhanced neutrophil apoptosis at 6 h and stimulated the production of H(2)O(2), which may mediate apoptosis through intracellular hydroxyl radical production. Intracellular GSH concentrations decreased in neutrophils undergoing apoptosis, and this was more marked in neutrophils treated with anti-Fas or TNF-alpha. These results suggest a causal association between reduced endogenous nitric oxide production, reduced intracellular GSH, and Fas- and TNF-alpha-mediated neutrophil apoptosis, whereas enhanced neutrophil survival mediated by dibutyryl cyclic adenosine monophosphate is associated with increased nitrite generation and maintenance of intracellular GSH. The interaction of endogenous reactive oxygen species with extracellular antioxidants such as GSH could also contribute to the complex processes regulating neutrophil apoptosis and hence the resolution of inflammation in the lung.

Acetylcysteine↗
PubMed2000

Oncostatin M synergises with house dust mite proteases to induce the production of PGE(2) from cultured lung epithelial cells.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE(2) production (22 fold, P<0.05). OSM also synergistically enhanced PGE(2) production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE(2) or NO production, although it did induce the release of GM-CSF. These observations suggest that OSM is an important co-factor in the release of PGE(2) and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

Animals↗
PubMed1999

PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro.

BACKGROUND: Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. OBJECTIVE: We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE2, TNF-alpha, and nitric oxide. METHODS: Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 micromol/L), TNF-alpha (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 micromol/L), and 2,2;-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 micromol/L). Control cultures consisted of untreated, IL5-treated (100 U/mL), and anti-Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. RESULTS: PGE2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP2 receptor agonist, 11-deoxy PGE1, at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-alpha delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2, 2;-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE2, cyclic AMP, and TNF-alpha. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. CONCLUSION: Inhibition of spontaneous eosinophil apoptosis by PGE2 appears to be mediated by EP2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE2 may increase the proinflammatory potential of these cells in chronic asthma.

Adult↗
PubMed1999

Expression and localization of COX-2 in human airways and cultured airway epithelial cells.

Cyclo-oxygenase is the rate-limiting enzyme in the prostanoid pathway. Although expression of the inducible isoform of cyclo-oxygenase (COX-2) is associated with cytokine-mediated inflammation, recent evidence suggests a homeostatic role for epithelial COX-2 in the gastrointestinal tract. The aim of this study was to examine the expression and localization of COX-2 in human airway epithelium both in vivo and in vitro. Human airway specimens from patients undergoing lung resection surgery for primary lung tumours (n=10) or nasal mucosal resection for non-inflammatory nasal obstruction (n=5) were examined for COX-2 expression by in situ hybridization and immunohistochemistry. COX-2 expression was also studied in two human airway epithelial cell lines (BEAS-2B and A549) using reverse transcription polymerase chain reaction and Northern and Western blot analysis. COX-2 messenger ribonucleic acid (mRNA) and protein were localized to individual columnar epithelial cells and to airway resident inflammatory cells in 9/10 lower and 5/5 upper airway specimens. Expression of COX-2 did not correlate with evidence of airway inflammation. Focal expression of COX-2 mRNA and protein was observed in bronchus-associated lymphoid tissue. Both COX-2 mRNA and protein were detected in BEAS-2B and A549 cells cultured under standard conditions. In conclusion, expression of COX-2 in human airway epithelium occurs in the upper and lower airways, is widespread in airway epithelial and airway resident inflammatory cells in the absence of overt airway inflammation, and is detectable in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. These data suggest a potentially important homeostatic role for COX-2 in the regulation of human airway contractility, inflammation and immune responses.

Adult↗
PubMed1999

Expression and localization of cyclo-oxygenase isoforms in non-small cell lung cancer.

The beneficial effects of cyclo-oxygenase (COX) inhibitors in both colon cancer and adenomatous polyps suggest a role for the prostanoid pathway in epithelial malignancy. Although variable prostanoid synthesis in non-small cell lung cancer (NSCLC) has been demonstrated in freshly obtained tissue, COX messenger ribonucleic acid (mRNA) and protein localization in such tumours had not been investigated ex vivo. Thirty-four cases of primary NSCLC were examined for both constitutive (COX-1) and inducible COX (COX-2) by means of in situ hybridization and immunohistochemistry. COX-1 mRNA expression was absent or below the level of detection via in situ hybridization. COX-1 immunohistochemistry demonstrated uniform faint cytoplasmic staining in tumour cells and stromal inflammatory cells. Semiquantitative analysis of COX-2 expression in NSCLC demonstrated the highest levels of both mRNA and protein in adenocarcinoma cells (n=10, p<0.005 compared with large cell and squamous cell carcinoma), intermediate and variable expression in large cell carcinoma (n=11) and low or absent expression in squamous cell tumours (n=13). Levels of COX-2 expression in infiltrating inflammatory cells was the same in all tumour types. In conclusion, tumour cell cyclo-oxygenase-2 rather than cyclo-oxygenase-1 expression may account for the variable prostanoid production seen in non-small cell lung cancer, and primary lung adenocarcinoma expresses the highest levels of cyclooxygenase-2. Assessment of cyclo-oxygenase-2 expression ex vivo should be performed in studies examining the potential therapeutic effects of cyclo-oxygenase inhibitors in non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed1998

Glutathione peroxidase activity and mRNA expression in eosinophils and neutrophils of asthmatic and non-asthmatic subjects.

Asthma has been reported to be associated with a reduction in the activity of glutathione peroxidase (GSH-Px), an important antioxidant enzyme. However, the expression of GSH-Px enzyme activity has not previously been investigated in human eosinophils, which are important inflammatory cells involved in asthma. Reverse transcriptase-polymerase chain reaction and Southern blotting demonstrated that eosinophils express GSH-Px mRNA and the relative expression of GSH-Px was greater in eosinophils than in neutrophils for both asthmatic and non-asthmatic subjects. The presence of GSH-Px protein in eosinophil and neutrophil lysates was confirmed by size exclusion chromatography and by Western blotting. GSH-Px enzyme activity as measured by a spectrophotometric assay was greater in eosinophil (48.4+/-1.6 micromol NADPH oxidized x min(-1) x g(-1) protein) than in neutrophil lysates (18.1+/-0.4, n = 24, P < 0.0001). GSH-Px activities of eosinophils and neutrophils from asthmatic subjects did not differ from those of non-asthmatic subjects. Eosinophil GSH-Px activity was correlated with peripheral blood eosinophil count only in asthmatic subjects (rs = 0.59, n = 12, P = 0.04). Increased GSH-Px expression in eosinophils compared with neutrophils of asthmatic patients may provide antioxidant protection against the greater amounts of reactive oxygen species generated by these cells and may enhance the survival of eosinophils at sites of inflammation in asthma.

Adult↗
PubMed1998

The effect of aspirin on thrombin stimulated platelet adhesion receptor expression and the role of neutrophils.

AIMS: Aspirin has proven clinical efficacy in limiting the thrombotic complications of atherosclerotic vascular disease but its mechanism of action remains unclear. Recent evidence suggests the anti-platelet action of aspirin may be partly mediated by neutrophil derived nitric oxide (NO). The aim of the study was to determine the effects of aspirin on thrombin-induced platelet expression of the alpha-granule membrane protein, P-selectin, and the platelet surface glycoprotein required for aggregation, GPIIb-IIIa, and to assess whether this was enhanced by the presence of neutrophils. METHODS: Platelet P-selectin and GPIIb-IIIa receptor expression were assessed by flow cytometric analysis of washed platelets stimulated with thrombin (0.025 iu ml(-1), sub aggregatory concentration) alone or after pre-incubation with aspirin (0.05, 0.1, 0.5, 1.0 mg m1(-1) either in the presence or absence of neutrophils (100 platelets per neutrophil). NO release was determined by assay of nitrite in the supernatants from parallel samples. RESULTS: In preliminary aggregation studies, aspirin at all concentrations inhibited arachidonic acid but not thrombin-induced platelet aggregation. Similarly, aspirin at all concentrations failed to inhibit thrombin-induced platelet P-selectin or GPIIb-IIIa expression and this was not influenced by the presence of neutrophils. A reduction in P-selectin and GPIIb-IIIa receptor density on non-activated platelets co-incubated with unstimulated neutrophils was associated with NO release from neutrophils, but this was not enhanced by the addition of aspirin. CONCLUSIONS: These results confirm that thrombin-induced platelet alpha-granule release, with consequent P-selectin expression, and platelet GPIIb-IIIa expression, are not affected by aspirin inhibition of cyclo-oxygenase and suggest that the anti-thrombotic efficacy of aspirin in vivo may partly depend on other mechanisms. This study did not demonstrate an effect of neutrophils or neutrophil derived NO on aspirin inhibition of platelet adhesion receptor expression.

Adult↗
PubMed1998

Expression and localization of the inducible isoform of nitric oxide synthase in nasal polyp epithelium.

BACKGROUND: The pathogenesis of nasal polyp disease is poorly understood. Recent evidence has suggested that nitric oxide (NO), an endogenous soluble gas vasodilator and inflammatory mediator, may be synthesised within the nasal cavity. Three nitric oxide synthase isoforms have been identified in humans, with the inducible isoform (iNOS) generally expressed in the setting of inflammation. OBJECTIVE: The aim of this study was to detect and localize iNOS expression in nasal polyp tissue, and compare these findings with normal nasal turbinate tissue. METHODS: We examined the expression and localisation of inducible nitric oxide synthase (iNOS) in human nasal airway specimens from patients undergoing elective nasal turbinectomy (n = 5) or nasal polypectomy (n = 5). iNOS mRNA expression was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis and localised by in situ hybridization. Densitometric data were analysed using Student's unpaired t-test. Adjacent sections were also examined for iNOS protein expression by immunohistochemistry. RESULTS: Semi-quantitative RT-PCR/Southern analysis of RNA obtained from the 10 surgical specimens demonstrated that iNOS mRNA expression was significantly increased in the five nasal polyps (P < 0.05). In situ hybridization studies revealed strong iNOS mRNA signal localized to the respiratory epithelium of nasal polyps, but not nasal turbinates. This pattern was confirmed by immunohistochemistry. Localization to inflammatory cells or other subepithelial structures was not seen. CONCLUSIONS: We conclude that iNOS expression is upregulated in nasal polyp disease, and is localized to the polyp epithelial layer. These data reinforce the concept that the epithelial layer may be important in the pathogenesis of nasal disease, and suggest a potential role for NO in the formation of nasal polyps.

Epithelium↗
PubMed1997

Regulation of the inducible cyclo-oxygenase pathway in human cultured airway epithelial (A549) cells by nitric oxide.

1. In airway epithelium, nitric oxide (NO) is synthesized in the setting of inflammation by inducible nitric oxide synthase (iNOS). Although the role of epithelial derived NO in the regulation of human airways is unknown, prostaglandin E2 (PGE2) is recognised as an important inhibitory mediator in human airways. Cyclo-oxygenase (COX) is the rate limiting enzyme in the production of prostanoids and since inflammatory pathways enhance the expression of an inducible COX (COX-2), both COX-2 and iNOS may be co-expressed in response to an inflammatory stimulus. Although regulation of the COX-2 pathway by NO has been demonstrated in animal models, its potential importance in human airway epithelium has not been investigated. 2. The effect of endogenous and exogenous NO on the COX-2 pathway was investigated in the A549 human airway epithelial cell culture model. Activity of the COX-2 pathway was assessed by PGE2 EIA, and iNOS pathway activity by nitrite assay. A combination cytokine stimulus of interferon gamma (IFNgamma) 100 u ml(-1), interleukin-1beta (IL-1beta) 1 u ml(-1) and lipopolysaccharide (LPS) 10 microg ml(-1) induced nitrite formation which could be inhibited by the competitive NOS inhibitor N(G)-nitro-L-arginine-methyl-ester (L-NAME). IL-1beta alone (1-50 u ml(-1) induced PGE2 formation without significant nitrite formation, a response which was inhibited by the COX-2 specific inhibitor nimesulide. Submaximal stimuli used for further experiments were IFNgamma 100 u ml(-1), IL-1beta 1 u ml(-1) and LPS 10 microg ml(-1) to induce both the iNOS and COX-2 pathways, and IL-1beta 3 u ml(-1) to induce COX-2 without iNOS activity. 3. Cells treated with IFNgamma 100 u ml(-1), IL-1beta I u ml(-1) and LPS 10 microg ml(-1) for 48 h either alone, or with the addition of L-NAME (0 to 10(-2) M), demonstrated inhibition by L-NAME of PGE2 (3.61 +/- 0.55 to 0.51 +/- 0.04 pg/l0(4) cells; P<0.001) and nitrite (34.33 +/- 8.07 to 0 pmol/10(4) cells; P<0.001) production. Restoration of the PGE2 response (0.187 +/- 0.053 to 15.46 +/- 2.59 pg/10(4) cells; P<0.001) was observed after treating cells with the same cytokine stimulus and L-NAME 10(-6) M, but with the addition of the NOS substrate L-arginine (0 to 10(-5) M). 4. Cells incubated with IL-1beta 3 u ml(-1) for 6 h, either alone or with addition of the NO donor S-nitroso-acetyl-penicillamine (SNAP) (0 to 10(-4) M), demonstrated increased PGE2 formation (1.23 +/- 0.03 to 2.92 +/- 0.19 pg/10(4) cells; P< 0.05). No increase in PGE2 formation was seen when the experiment was repeated in the presence of the guanylate cyclase inhibitor methylene blue (50 microM). Cells treated with SNAP alone did not demonstrate an increased PGE2 formation. Cells incubated with IL-1beta 3 u ml(-1) for 6 h in the presence of dibutyryl cyclic guanylate monophosphate (0 to 10(-3) M) also demonstrated an increased PGE2 response (2.56 +/- 0.21 to 4.53 +/- 0.64 pg/10(4) cells; P<0.05). 5. These data demonstrate that in a human airway epithelial cell culture system, both exogenous and endogenous NO increase the activity of the COX-2 pathway in the setting of inflammatory cytokine stimulation, and that this effect is likely to be mediated by guanylate cyclase. This suggests a role for NO in the regulation of human airway inflammation.

Cytokines↗
PubMed1997

Expression and activity of nitric oxide synthases in human airway epithelium.

Nitric oxide (NO) synthesized by airway epithelium may be important in the regulation of airway inflammation and reactivity. As such, the expression and localization of nitric oxide synthase (NOS) isoforms was assessed in human airway tissue obtained following thoracotomy, and in cultured human airway epithelial (BEAS-2B) cells. NOS expression was determined by reverse transcription-polymerase chain reaction (RT-PCR)/Southern blot analysis, and localized by in situ hybridization and immunohistochemistry. No synthesis by cell cultures was detected by nitrite assay. Endothelial and neuronal constitutive NOS mRNAs were not detected in airways or cell cultures. Inducible NOS (iNOS) mRNA was detected in 5 of 6 airway specimens, and in situ hybridization demonstrated iNOS mRNA expression in columnar epithelial cells. This was confirmed by immunohistochemistry using an iNOS specific antibody. BEAS-2B cell cultures were stimulated with (I) combinations of tumor necrosis factor alpha (TNF alpha) (50-2,000 U/ml)/interferon gamma (IFN gamma) (20-500 U/ml)/lipopolysaccharide (LPS) (10 micrograms/ml) or (2) histamine (10(-3) M-10(-5) M). Cell cultures treated with TNF alpha/IFN gamma/LPS in combination expressed iNOS mRNA, and this was associated with increases in supernatant nitrite concentrations over 24 h; however, this response diminished with successive passage of cells. Histamine treatment did not result in iNOS mRNA expression or detectable NO synthesis. We conclude that iNOS in human airway tissue is localized to the airway epithelium. Cytokine/ LPS stimulation, but not histamine, results in iNOS mRNA expression and NO synthesis in BEAS-2B cells. BEAS-2B cells may not be a suitable model for the study of NO biology in airway epithelium.

Blotting, Northern↗