Modulation of neutrophil and mononuclear cell adherence to bronchial epithelial cells.

Neutrophils and mononuclear cells have been associated with the lower respiratory tract inflammation observed in both acute and chronic bronchitis. In order to transit into and remain within the airways, neutrophils and mononuclear cells would likely need to adhere to bronchial epithelium. To test this hypothesis, bovine bronchial epithelial cells (BBECs) were isolated and cultured on a round coverslip. After 7 to 10 days, 51Cr-labeled neutrophils and mononuclear cells were evaluated for their capacity to adhere to the BBEC monolayer. Both neutrophils and mononuclear cells readily bound to the BBEC monolayer (10.8 +/- 1.2% bound neutrophils; 40.5 +/- 2.8% bound mononuclear cells). Stimulation of the neutrophils and mononuclear cells with phorbol 12-myristate 13-acetate (PMA) increased the adherence (45.8 +/- 10.6% bound neutrophils, P less than 0.01 compared with unstimulated cells; 58.7 +/- 6.2% bound mononuclear cells, P less than 0.01 compared with unstimulated cells). Importantly, stimulating the BBEC monolayer with PMA, bacterial lipopolysaccharide, or a cigarette smoke extract for 4 to 72 h also increased the adherence of both cell types (P less than 0.01, all comparisons at 24 h). The adherence was not decreased by exposure of either the BBEC monolayer, the neutrophils, or the mononuclear cells to cycloheximide or to the anti-CD11/CD18 monoclonal antibody 60.3 (P greater than 0.05). However, exposure of the BBEC monolayer to trypsin before addition of the neutrophils significantly decreased adherence (P less than 0.05). Because neutrophils and mononuclear cells are thought to mediate cell cytotoxicity by adhering to the target cells, BBECs were labeled with 51Cr, and 51Cr release was measured as an index of cytotoxicity. There was a modest increase in 51Cr release by the addition of unstimulated neutrophils and mononuclear cells, and culturing the BBEC monolayer with PMA before the addition of the neutrophils or mononuclear cells resulted in a further modest enhancement of 51Cr release (P less than 0.05). Similar results were obtained using lactate dehydrogenase release as a measure of cytotoxicity. These results demonstrate that inflammatory cells can adhere to BBECs and may be capable of mediating cytotoxicity and adherence and cytotoxicity can be increased by stimulating BBECs.     

Effect of IL-17 on ICAM-1 expression of human bronchial epithelial cells, NCI-H 292]

Bronchial asthma is characterized as a chronic inflammation of the airway infiltrated by eosinophils, lymphocytes, and neutrophils. ICAM-1 expression on airway epithelium facilitates adhesion between these inflammatory cells and bronchial epithelial cells, and induces the activation of inflammatory cells. ICAM-1 expression was affected by various cytokines, such as IL-17. IL-17 is a novel cytokine released by CD4+ activated memory T cells. In this study, we examined the effect of IL-17 on ICAM-1 expression by RT-PCR and flow cytometry. Human bronchial epithelial cells, NCI-H 292 cells, were stimulated with IL-17 (100 ng/ml) and/or IFN-gamma (100 U/ml). ICAM-1 was expressed constitutively. IL-17 alone did not enhance ICAM-1 expression on NCI-H 292 cells. However, IL-17 synergistically enhanced ICAM-1 expression induced by IFN-gamma. These results suggest that IL-17 has an effect on ICAM-1 expression of bronchial epithelial cells in airway inflammation.     

Th2-type cytokines modulate IL-6 release by human bronchial epithelial cells

The multifunctional cytokine IL-6, which can be locally produced by human bronchial epithelial cells (HBECs), has been found to play a role in IL-4 dependent IgE synthesis. Since the allergic reaction in bronchial asthma is associated with the upregulation of IL-4 and Th2 type of immune response, the purpose of our study was to assess whether IL-4 and related cytokines IL-10 and IL-13 regulate IL-6 release by HBEC s. HBECs were obtained by bronchial brushing, cultured in LHC-9/RPMI 1640. At the third passage the cells were stimulated with cytokines (0.1-20 ng/ml) diluted in unsupplemented media for 24 h. The supernatants were tested for IL-6 content by sandwich ELISA. Unstimulated HBECs produced detectable amounts of IL-6 (368+/-25 pg/ml). Exposure to IL-10 (368+/-22 pg/ml) and IL-13 (395+/-6 pg/ml) resulted in little changes. IL-4 caused a slight but significant increase in IL-6 release (530+/-45 pg/ml), P<0.05, TNFalpha (1657+/-85 pg/ml) and IFNgamma (1953+/-37 pg/ml) showed strong induction of IL-6 release in HBECs (P<0.005 and P<0.001, respectively). Both IL-4 and IL-13 significantly inhibited TNF induced IL-6 release (P<0.01 for both) while augmenting the effect of IFNgamma (P<0.005 and P<0.01, respectively.). IL-10 was without a significant effect. We conclude that Th2-type cytokines IL-4 and IL-13 affect the release of IL-6 by HBECs in response to TNFalpha (inhibition) and IFgamma (augmentation). IL-10 had no effect on the regulation of IL-6 release. Modulation of IL-6 levels by Th2-type cytokines may play a role in allergic reactions through the IL-6 promoting effect on IL-4 mediated IgE production.     

IL-27 regulates IL-4-induced chemokine production in human bronchial epithelial cells

IL-4 coordinates the Th2-type immune response in inflammatory diseases such as asthma. IL-27 can inhibit the development of both Th2 and Th1 cells. However, IL-27 can also drive naïve T cells to differentiate toward the Th1 phenotype. In this study, we investigated the effects of IL-27 on the activation of IL-4-induced human bronchial epithelial cells (BEAS-2B). Compared to controls, both IL-4 and IL-27 (25–100 ng/mL) increased the concentrations of CCL2 and IL-8 in a dose-dependent manner. However, compared to cells stimulated individually with IL-4 or IL-27, treatment with a combination of both cytokines reduced CCL2 and IL-8 concentrations in a dose- and time-dependent manner. IL-4 increased the activation of p38 MAPK, ERK1/2, STAT6 and NF-κB, while IL-27 increased the activation of p38 MAPK and ERK1/2 but not STAT6 and NF-κB. Compared to IL-4-stimulated cells, cells treated with both IL-27 and IL-4 displayed decreased activation of STAT6 and NF-κB but not ERK1/2 and p38 MAPK. Taken together, these results suggest that IL-27 plays a pro-inflammatory role when administered alone but downregulates bronchial epithelial cell activation when combined with IL-4. Therefore, IL-27 may be an interesting target for the treatment of Th2 inflammatory diseases.     

The IL-17F signaling pathway is involved in the induction of IFN-gamma-inducible protein 10 in bronchial epithelial cells

BACKGROUND: IL-17F is involved in airway inflammation, but its biologic activity and signaling pathway remain incompletely defined. Interferon-gamma-inducible protein 10 (IP-10) is widely expressed and plays a role in airway inflammatory diseases. OBJECTIVE: We sought to investigate the functional linkage between IL-17F and IP-10 expression in bronchial epithelial cells. METHODS: Bronchial epithelial cells were cultured in the presence or absence of IL-17F, and/or a T(H)1 cytokine, T(H)2 cytokines, proinflammatory cytokines, various kinase inhibitors, or a Raf1 dominant-negative mutant to analyze the expression of IP-10. Moreover, the involvement of p90 ribosomal S6 kinase (p90RSK) and cyclic AMP response element-binding protein (CREB) in IL-17F-induced IP-10 expression were investigated. RESULTS: IL-17F induces the gene and protein expression of IP-10. The addition of IFN-gamma, IL-1beta, and TNF-alpha augmented IL-17F-induced IP-10 expression. The mitogen-activated protein kinase kinase (MEK) inhibitors PD98059, U0126, and Raf1 kinase inhibitor I significantly inhibited its production. In contrast, a p38 inhibitor, a JNK inhibitor, protein kinase C inhibitors, and a phosphatidylinositol 3-kinase inhibitor, showed no inhibitory effect. Furthermore, overexpression of a Raf1 dominant-negative mutant inhibited its expression. Of interest, IL-17F phosphorylated p90RSK and CREB, and transfection of the cells with a short interfering RNA for p90RSK or CREB inhibited its expression, suggesting p90RSK and CREB as novel signaling molecules of IL-17F. CONCLUSION: IL-17F is a potent inducer of IP-10 in bronchial epithelial cells through the activation of the Raf1-MEK1/2-extracellular signal-regulated kinase 1/2-p90RSK-CREB pathway, supporting its regulatory role in airway inflammation. CLINICAL IMPLICATIONS: The IL-17F-IP-10 axis might be a novel and critical therapeutic target for airway inflammatory diseases.     

Agaphelin modulates the activation of human bronchial epithelial cells induced by lipopolysaccharide and IL-4

Sand fly saliva presents molecules with potential to development of compounds for treatment of inflammatory diseases. Agaphelin, isolated from the saliva of the mosquito Anopheles gambiae, demonstrates anti-inflammatory properties such as neutrophils chemotaxis inhibition. Here, we extend these results and evaluated the role of agaphelin (0.1−100 nM) in an in vitro model consisting in the activation of human bronchial epithelial cells (BEAS-2B) by IL-4 (50 ng/mL) or lipopolysaccharide (LPS; 10 ng/mL). Agaphelin is non-cytotoxic for BEAS-2B cells. Notably, agaphelin markedly reduces CCL2 and IL-8 production induced by IL-4 or LPS, without altering the IL-10 production. The TLR4 expression and STAT1 phosphorylation induced by LPS were inhibited by agaphlin. In addition, agaphelin decreased the phosphorylation of STAT6 induce by IL-4, whose effect was independent of IL-4-binding activity. Taken together, these findings identify agaphelin as a potential anti-inflammatory therapeutic agent for airway inflammations.     

Regulation of epithelial immunity by IL-17 family cytokines

Cutaneous and mucosal epithelial cells function as both a physical barrier and as immune sentinels against environmental challenges, such as microbial pathogens, allergens and stress. The crosstalk between epithelial cells and leukocytes is essential for orchestrating proper immune responses during host defense. Interleukin (IL)-17 family cytokines are important players in regulating innate epithelial immune responses. Although IL-17A and IL-17F promote antibacterial and antifungal responses, IL-17E is essential for defense against parasitic infections. Emerging data indicate that another member of this family, IL-17C, specifically regulates epithelial immunity. IL-17C production serves as an immediate defense mechanism by epithelial cells, utilizing an autocrine mechanism to promote antibacterial responses at barrier surfaces.     

Inhibition of NFkappaB activation and IL-8 expression in human bronchial epithelial cells by acrolein

Lipid oxidation and environmental pollutants are major sources of alpha,beta-unsaturated aldehydes such as acrolein and 4-hydroxynonenal. Acrolein (2-propenal), a major product of organic combustion such as tobacco smoke, represents the most reactive alpha,beta-unsaturated aldehyde, with high reactivity toward nucleophilic targets such as sulfhydryl groups. To investigate how acrolein affects respiratory tract cell activation, we exposed either primary (NHBE) or immortalized human bronchial epithelial cells (HBE1) to 0-25 microM acrolein, and determined effects on basal and tumor necrosis factor-alpha (TNFalpha)-induced production of the chemokine interleukin (IL)-8. Cell exposure to acrolein dose-dependently suppressed IL-8 mRNA levels in HBE1 cells (26, 40, and 79% at 5, 10, and 25 microM acrolein concentrations, respectively) and resulted in corresponding decreases in IL-8 production. Studies of nuclear factor-kappaB (NFkappaB) activation, an essential event in IL-8 production, showed decreased TNFalpha-induced NFkappaB activation by acrolein, illustrated by inhibition of nuclear translocation of NFkappaB and reduced IkappaBalpha degradation. Immunochemical analysis of IkappaB kinase (IKK), a redox-sensitive regulator of NFkappaB activation, indicated direct modification of the IKK beta-subunit by acrolein, suggesting that acrolein may act directly on IKK. In summary, our results demonstrate that acrolein can suppress inflammatory processes in the airways by inhibiting epithelial IL-8 production through direct or indirect inhibitory effects on NFkappaB activation.     

Intestinal neuroendocrine cells and goblet cells are mediators of IL-17A-amplified epithelial IL-17C production in human inflammatory bowel disease

Interleukin (IL)-17C is a novel member of the IL-17 cytokine family. Its function in human inflammatory bowel disease (IBD) remains elusive as its role in colonic inflammation is entirely derived from murine models. We aimed to analyze the role of IL-17C in human IBD, focusing on T helper type 17 (Th17) cell- and intestinal epithelial cell (IEC)-dependent mechanisms. IL-17C mRNA (P=0.005), serum levels (P=0.008), and colonic staining intensity (P=0.004) is increased in active IBD. Serum IL-17C levels are modified by IL23R genotypes and IL-17C mRNA correlates (r>0.5, P<0.001) with IL-17A, tumor necrosis factor (TNF)-α, C-C motif chemokine ligand 20 (CCL20) and IL-23 mRNA in the inflamed colon of IBD patients. In the inflamed colon, IL-17C is produced by enteroendocrine and goblet cells, with contrary polar cytosolic localization of IL-17C within the cellular axis. In these two cell types, IL-17A strongly amplifies TNF-α-induced IL-17C production. On the molecular level, IL-17C production in IECs is dependent on TNF-α-activated nuclear factor-κB, extracellular signal–regulated kinase-1/2 and p38, and IL-17A-activated Akt, monocyte chemotactic protein–induced protein 1, and C/EBPδ. IL-17C upregulates the Th17 chemoattractant CCL20 in IECs. In summary, our findings support the involvement of IL-17A-amplified IL-17C production by enteroendocrine and goblet cells in the pathogenesis of active IBD, revealing an interaction between the neuroendocrine system and the Th17 pathway in human IBD.     

PLTP 在香烟诱导HBECs 合成IL-8 中的作用

目的:研究磷脂转运蛋白(PLTP)对香烟诱导人支气管上皮细胞(HBECs)合成白介素8(IL-8)的影响。方法:Wistar 大鼠连续3 d 被动吸烟,收集支气管肺泡灌洗液(BALF),苏木精-伊红(HE)染色,免疫组织化学法(IHC)检测蛋白表达;体外培养HBECs,不同浓度烟草提取物(CSE)刺激;PLTP siRNA 干扰后,进行CSE 刺激。MTT 法检测细胞增殖,同时检测IL-8 含量及PLTP 表达。结果:烟熏组BALF 白细胞计数以及分类计数较对照组均明显升高(P<0.01),IHC 显示烟熏组PLTP 和IL-8 蛋白表达高于对照组;高浓度CSE(2%、4%)抑制HBECs 增殖(P<0.001);不同浓度的CSE 作用24 h 后,IL-8mRNA 及蛋白表达量增加,而PLTP 蛋白表达量下降,并且IL-8 蛋白分泌呈现时间依赖性;PLTP siRNA 处理后,IL-8 表达量明显升高(P<0.001)。结论:PLTP 基因缺失促进香烟诱导HBECs 合成IL-8。     

Grain dust induces IL-8 production from bronchial epithelial cells: the effect of dexamethasone on IL-8 production.

BACKGROUND: Recent publications have suggested an active participation of neutrophils to induce bronchoconstriction after inhalation of grain dust (GD). OBJECTIVE: To further understand the role of neutrophils in the pathogenesis of GD-induced asthma, this investigation was designed to determine whether human bronchial epithelial cells could produce IL-8 production and to observe the effect of dexamethasone on IL-8 production. MATERIALS AND METHODS: We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four concentrations (1 to 200 microg/mL) of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant, which was derived from the culture of PBMC from a GD-induced asthmatic subject under the exposure to 10 microg/mL of GD, and compared with those cultured without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. To evaluate the effect of dexamethasone on IL-8 production, four concentrations (5 to 5000 ng/mL) of dexamethasone were pre-incubated for 24 hours and the same experiments were repeated. RESULTS: There was significant production of IL-8 from bronchial epithelial cells with additions of GD in a dose-dependent manner (P < .05), which was significantly augmented with additions of PBMC supernatant (P < .05) at each concentration. Compared with the untreated sample, pretreatment of dexamethasone could induced a remarkable inhibitions (15% to 55%) of IL-8 production from bronchial epithelial cells in a dose-dependent manner. CONCLUSION: These results suggest that IL-8 production from bronchial epithelial cells may contribute to neutrophil recruitment occurring in GD-induced airway inflammation. The downregulation of IL-8 production by dexamethasone from bronchial epithelial cells may contribute to the efficacy of this compound in reducing cellular infiltration and ultimately to its anti-inflammatory property.     

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.     

Grain dust induces IL-8 production from bronchial epithelial cells: effect on neutrophil recruitment.

BACKGROUND: There have been several investigations suggesting an involvement of activated neutrophils in the development of grain dust (GD)-induced occupational asthma. Interleukin-8 in the sputa from GD-induced asthmatic patients increased significantly after the exposure to GD. OBJECTIVE: To confirm IL-8 production from bronchial epithelial cells when exposed to GD, and to evaluate the role of IL-8 on neutrophil recruitment. MATERIALS AND METHOD: We cultured Beas-2B, a bronchial epithelial cell line. To observe GD-induced responses, four different concentrations ranging from 1 to 200 microg/mL of GD were incubated for 24 hours and compared with those without incubation of GD. To evaluate the effect of pro-inflammatory cytokines on IL-8 production and neutrophil chemotaxis, epithelial cells were incubated with peripheral blood mononuclear cell (PBMC) culture supernatant derived from subjects with GD-induced asthma exposed to 10 microg/mL of GD, and then compared with those without addition of PBMC supernatant. The level of released IL-8 in the supernatant was measured by enzyme-linked immunosorbent assay. Neutrophil chemotactic activity of the culture supernatant was determined by modified Boyden chamber method. RESULTS: Interleukin-8 production and neutrophil chemotactic activity from bronchial epithelial cells significantly increased with additions of GD in a dose-dependent manner (P < .05, respectively), and were significantly augmented with additions of PBMC supernatant (P < .05, respectively) at each concentration. Close correlation was noted between neutrophil chemotactic activity and IL-8 level (r = 0.87, P < .05). Compared with the untreated sample, pre-treatment of anti-IL-8 antibody induced a significant suppression (up to 67.2%) of neutrophil chemotactic activity in a dose-dependent manner. CONCLUSION: These results suggest that IL-8 produced from bronchial epithelial cells may be a major cytokine, which induces neutrophil migration into the airways when exposed to GD.     

CD40 expression by human bronchial epithelial cells

CD40 is a 50-kDa protein expressed on B cells, dendritic cells, monocytes and epithelial cells, but the distribution of CD40 expression in humans is not completely known. It binds to a ligand (CD40L) which is expressed essentially on activated T cells. The interaction between CD40 and CD40L plays important roles in immune responses. CD40 expression was investigated on bronchial tissues and human bronchial cell lines using immunohistochemistry, immunofluorescence staining and analysis with a cytometer, respectively. Constitutive CD40 expression, but not that of CD40L, was slightly detectable on normal human bronchial epithelial cells (HBEC) in situ and on an adult lung adenocarcinoma (SKLU1) cell line, while another cell line, a bronchial transformed SV40 cell line (WI26VA4), was negative for CD40. Among the various cytokines tested, only interferon (IFN)-gamma was found to induce CD40 expression on WI26VA4. Tumour necrosis factor (TNF)-alpha was the best cytokine able to up-regulate CD40 in SKLU1 cells. A combination of IFN-gamma and TNF-alpha was slightly more effective than the cytokine alone at up-regulating CD40 expression on both cell lines. We further investigated the functional consequences of CD40 ligation on both cell lines. These bronchial cells expressed CD40, HLADR and CD54 under basal conditions or when stimulated by cytokines. Stimulation through CD40 did not affect cell-surface-antigen expression on either cell line. The production of cytokines such as interleukin (IL)-6 and granulocyte macrophage-colony stimulating factor (GM-CSF) by HBEC has been described. SKLU1 and WI26VA4 cells released IL-6 and GM-CSF spontaneously. Whatever the case, CD40 engagement did not modulate spontaneous or TNF-alpha-induced production of these two cytokines. These data indicate for the first time that normal HBEC express CD40 in situ. Further investigations are required in order to determine the role of CD40 on normal HBEC.     

Anti-inflammatory Effect of Resveratrol and Polydatin by In Vitro IL-17 Modulation

Interleukin-17 (IL-17) is a proinflammatory cytokine produced, although not exclusively, by T helper 17 recently identified as a distinct T helper lineage mediating tissue inflammation. IL-17 is known to be involved in a number of chronic disorders although the mechanisms regulating its production in inflammatory disease are still unclear. The beneficial properties of the polyphenolic compound resveratrol including its anti-inflammatory, antioxidant, and antitumor effects, its role in the aging process and in the prevention of heart and neurodegenerative diseases are well-known. In addition, derivatives of resveratrol, including glucosylated molecules as polydatin have been linked to similar beneficial effects. We have investigated the effects of resveratrol and polydatin on the in vitro production of IL-17 in a model of inflammation in vitro. The results obtained by activated human peripheral blood mononuclear cells, stimulated with anti-CD3/anti-CD28 monoclonal antibodies and treated with these polyphenolic compounds at different concentrations show that both decrease IL-17 production in a concentration-dependent manner. This study confirms the anti-inflammatory activity of resveratrol and its derivatives and suggests a potential clinical relevance in the therapy of inflammatory diseases.     

Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha.

We have tested the hypothesis that the bronchial epithelium has the capacity to generate and release cytokines that could contribute to inflammatory events associated with inflammatory lung diseases. Messenger RNA (mRNA) for interleukin-6 (IL-6), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was identified in human bronchial epithelial cell primary cultures, characterized on the basis of staining for cytokeratin, using both in situ hybridization and Northern blotting. Using in situ hybridization we have shown that the majority of the cells expressed mRNA for IL-6 and IL-8, whereas fewer than 20% of cells expressed message for GM-CSF. The numbers of cells expressing message were increased by culture with tumour necrosis factor-alpha (TNF-alpha) (20 ng/ml, 24 hr). These observations were substantiated by Northern blotting, which showed that both TNF-alpha and IL-1 beta were able to induce a dose-dependent increase in IL-8-specific mRNA. Immunoreactive IL-6 and GM-CSF were detected and quantified in the culture supernatants by ELISA, and IL-8 by radioimmunoassay. The levels of immunoreactivity were increased by incubation of epithelial cells with either IL-1 beta or TNF-alpha for 24 hr. A transformed tracheal epithelial cell line (9HTEo-) expressed mRNA for IL-6, IL-8 and GM-CSF but, whereas levels of immunoreactive IL-6 in culture supernatants were comparable with those in primary cell cultures, levels of IL-8 were low and GM-CSF trivial. These observations indicate that the bronchial epithelium has the potential to be a major source of IL-8 and a number of other cytokines, and that production can be amplified substantially by IL-1 beta and TNF-alpha. The bronchial epithelium is ideally situated to modulate inflammatory and immunological events in and around the airways, and these observations suggest that it could contribute to promote and sustain inflammatory and immunological processes in inflammatory lung diseases such asthma.     

Measurement of IL-13-induced iNOS-derived gas phase nitric oxide in human bronchial epithelial cells

Exhaled nitric oxide (NO) is altered in numerous diseases including asthma, and is thought broadly to be a noninvasive marker of inflammation. However, the precise source of exhaled NO has yet to be identified, and the interpretation is further hampered by significant inter-subject variation. Using fully differentiated normal human bronchial epithelial (NHBE) cells, we sought to determine (1) the rate of NO release (flux, pl.s(-1.)cm(-2)) into the gas; (2) the effect of IL-13, a prominent mediator of allergic inflammation, on NO release; and (3) inter-subject/donor variability in NO release. NHBE cells from three different donors were cultured at an air-liquid interface and stimulated with different concentrations of IL-13 (0, 1, and 10 ng/ml) for 48 h. Gas phase NO concentrations in the headspace over the cells were measured using a chemiluminescence analyzer. The basal NO flux from the three donors (0.05 +/- 0.03) is similar in magnitude to that estimated from exhaled NO concentrations, and was significantly increased by IL-13 in a donor-specific fashion. The increase in NO release was strongly correlated with inducible nitric oxide synthase (iNOS) gene and protein expression. There was a trend toward enhanced production of nitrate relative to nitrite as an end product of NO metabolism in IL-13-stimulated cells. NO release from airway epithelial cells can be directly measured. The rate of release in response to IL-13 is strongly dependent on the individual donor, but is primarily due to the expression of iNOS.