Increased expression of transforming growth factor-beta1 in small airway epithelium from tobacco smokers and patients with chronic obstructive pulmonary disease (COPD).

Tobacco smoke is believed to cause small airway disease and then chronic obstructive pulmonary disease (COPD), but the molecular mechanisms by which small airway obstruction occurs remain unknown. To study the gene expression levels of transforming growth factor (TGF)-beta1, a potent fibrogenic factor, in small airway epithelium from smokers and patients with COPD, we harvested highly pure samples of epithelial cells from small airways under direct vision by using an ultrathin bronchofiberscope BF-2.7T (outer diameter 2.7 mm with a biopsy channel of 0.8 mm in diameter). The expression levels of TGF-beta1 were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of TGF-beta1 corrected by beta-actin transcripts were significantly higher in the smoking group and patients with COPD than those in nonsmokers (p < 0.01). Furthermore, among smokers and patients with COPD, TGF-beta1 mRNA levels correlated positively with the extent of smoking history (pack-years) and the degree of small airway obstruction as assessed by measurements of flow-volume curves. Immunocytochemistry of the cells demonstrated more intense stainings for TGF-beta1 in samples from smokers and patients with COPD than from nonsmokers. Spontaneously released immunoreactive TGF-beta1 levels from cultured epithelial cells were more elevated in subjects with a history of smoking and patients with COPD than in nonsmokers. Our study showed a close link between smoking and expression of TGF-beta1 in small airways. Our results also suggested that small airway epithelial cells might be involved in obstructive changes found in smokers and patients with COPD.

Benzene-extracted components are important for the major activity of diesel exhaust particles: effect on interleukin-8 gene expression in human bronchial epithelial cells.

Epidemiologic and experimental studies suggest that diesel exhaust particles (DEPs) may be related to increasing respiratory mortality and morbidity. We have shown that DEPs augmented the production of inflammatory cytokines by human airway epithelial cells in vitro. To better understand the mechanisms of their proinflammatory activities, we studied the effects of several components extracted from DEPs on interleukin (IL)-8 expression in human bronchial epithelial cell line BEAS-2B and normal human airway epithelial cells obtained from very peripheral airways by an ultrathin bronchoscope. We used several agents active on signal transduction pathways in cytokine expression, such as the protein kinase C inhibitor staurosporin, antioxidant agents including N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Benzene-extracted components showed effects mimicking DEPs on IL-8 gene expression, release of several cytokines (IL-8; granulocyte macrophage colony-stimulating factor; and regulated on activation, normal T cells expressed and secreted) and nuclear factor (NF)-kappa B activation. We also found that NAC, PDTC, and SB203580 suppressed the activities of DEPs and their benzene extracts, suggesting the roles of oxidants-mediated NF-kappa B activation and p38MAPK pathways. Finally, benzo[a]pyrene, one of the important compounds included in the benzene component, replicated the activities shown by DEPs.

Diesel exhaust particles activate human bronchial epithelial cells to express inflammatory mediators in the airways: a review.

OBJECTIVE: Epidemiological as well as experimental studies suggest that particulate air pollutants, including diesel exhaust particles (DEP), may play a role in the recent increase of respiratory morbidity and mortality. We studied the effect of DEP on the production of inflammatory cytokines and mediators including IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) by human airway epithelial cells in vitro. METHODOLOGY: Suspended DEP were added to cultured normal human bronchial epithelial cells or transformed BEAS-2B cells. The release of cytokines and mediators was evaluated by enzyme-linked immunosorbent assay. The transcriptional levels of IL-8 mRNA was studied by northern blot analysis and run-on transcription assay. Activation of transcription factors was assessed by electrophoretic mobility shift assay. RESULTS: Non-toxic doses of suspended DEP showed a significant stimulatory effect on IL-8 and GM-CSF production by airway epithelial cells. Diesel exhaust particles increased the steady-state levels of IL-8 mRNA, which was suggested to be largely due to increased transcriptional rates. Electrophoretic mobility shift assay demonstrated that DEP induced increased binding to the specific motif of nuclear factor (NF)-kappaB, but not of transcription factor AP-1. Both N-acetylcysteine and pyrrolidine dithiocarbamate attenuated the action of DEP on IL-8 mRNA expression, suggesting that oxidant-mediated pathway might be involved in its processes. Transient transfection of airway epithelial cells with wild and NF-kappaB binding motifs indicated that the activation of NF-kappaB was essential for IL-8 gene upregulation by reporter gene assay. CONCLUSIONS: These results suggested that DEP activate NF-kappaB, which might be an important pathway for the expression of inflammatory cytokines in vitro.

Diesel exhaust particles up-regulate expression of intercellular adhesion molecule-1 (ICAM-1) in human bronchial epithelial cells.

Epidemiological and experimental studies suggest that diesel exhaust particles (DEP) may play an active role in the increased respiratory mortality and morbidity. We have shown that DEP augmented the production of inflammatory cytokines by human airway epithelial cells in vitro. ICAM-1 has been shown to play an important role in the local accumulation of inflammatory cells. We studied the effect of DEP on ICAM-1 gene expression and surface expression in human bronchial epithelial cell line BEAS-2B. DEP (5-50 microg/ml) showed a stimulatory effect on ICAM-1 mRNA levels as evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Flow cytometric analysis demonstrated an increased ICAM-1 expression on the epithelial cell surfaces. The soluble form of ICAM-1 molecules was also increased by the stimulation of DEP. In vitro neutrophil attachment onto DEP-stimulated epithelial cells was augmented, which was partially blocked by anti-ICAM-1 neutralizing antibody. Finally, these events were significantly inhibited by pretreatment with anti-oxidants pyrrolidine dithiocarbamate and N-acetyl cysteine, and p38 mitogen activated protein kinase (MAPK) inhibitor SB203580. These findings suggested that DEP induced up-regulation of ICAM-1 gene, and this process might be largely dependent on oxidant-mediated NF-kappaB activation and p38-MAPK pathways.

Increased expression of inflammatory mediators in small-airway epithelium from tobacco smokers.

To study the inflammatory responses of small-airway epithelium in smokers, we harvested enough living epithelial cells (1.97 x 10(6) +/- 0.74 x 10(6)) with a new ultrathin fiberscope from the very peripheral airways of 22 current smokers and 17 subjects who never smoked after informed consent was obtained. The cells were keratin positive and composed mainly of nonciliated cells. The expression levels of inflammatory markers [interleukin (IL)-8 and intercellular adhesion molecule (ICAM)-1] were evaluated with RT-PCR. The magnitude of the mRNA levels corrected by beta-actin transcripts of IL-8 and ICAM-1 was significantly higher in the smokers than in the nonsmokers (P < 0.001). Furthermore, among current smokers, IL-8 mRNA levels correlated positively with the extent of smoking history [in pack. years (packs/day x no. of years of smoking); r = 0.754, P < 0.001]. Spontaneously released IL-8 and soluble ICAM-1 levels (n = 12) from cultured epithelial cells were elevated in subjects with a smoking history than in those without it (IL-8, 1,580 +/- 29.6 vs. 354 +/- 39.4 pg. 10(6) cells(-1). 24 h(-1); P < 0.001; soluble ICAM-1, 356.0 +/- 45.9 vs. 112.9 +/- 12.9 pg. 10(6) cells(-1). 24 h(-1); P < 0.01 by Student's t-test ). In contrast, the epithelial cells from the main bronchi did not show such differences between smokers and nonsmokers. Our study highlighted a close link between smoking and the expression of inflammatory mediators such as IL-8 and ICAM-1 in small airways. Our results also suggested that this new ultrathin bronchofiberscope promised a good approach for the evaluation of cellular changes in the small airways.

Erythromycin suppresses nuclear factor-kappaB and activator protein-1 activation in human bronchial epithelial cells.

Erythromycin (EM), and related 14-member macrolide antibiotics, has attracted attention for its effectiveness in airway diseases including diffuse panbronchiolitis and sinobronchial syndrome. However, its molecular mechanisms remain unknown. We evaluated the effects of EM on activation of several transcription factors, including nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) in human bronchial epithelial cell line BET-1A, which are known to regulate the expression of many proinflammatory cytokines and chemokines such as interleukin-8 (IL-8). BET-1A cells were cultured with hormonally defined Ham's F12 medium, and were stimulated by phorbol myristate acetate (PMA). EM suppressed mRNA expression as well as the release of IL-8 at therapeutic and noncytotoxic concentrations (% inhibition of IL-8 protein release: 42.2 +/- 5.5%, at 10(-6) M). Furthermore, electrophoretic mobility shift assays revealed that EM inhibited the activations of NF-kappaB and AP-1 induced by PMA in BET-1A cells. These data indicate that EM has inhibitory effects not only on the mRNA expression and release of IL-8, but also on the activation of transcription factors NF-kappaB and AP-1. Our findings support the concept that the recruitment of neutrophils in airway diseases may be regulated by NF-kappaB and AP-1.

Diesel exhaust particles induce NF-kappa B activation in human bronchial epithelial cells in vitro: importance in cytokine transcription.

Fine particles derived from diesel engines (diesel exhaust particles, DEP) have attracted attention, since their density in industrial countries seems related to the increased prevalence of pulmonary diseases. Previous studies have suggested that DEP have a potential to directly activate airway epithelial cells to produce and release inflammatory cytokines and mediators, and thus facilitate inflammatory responses in the lung. To elucidate the molecular mechanisms of their action, we studied here IL-8 gene expression, one of the important cytokines in inflammatory responses, by Northern blot analysis and run-on transcription assay. Suspended DEP (1-50 microgram/ml) increased the steady state levels of IL-8 mRNA, which was suggested to be largely due to increased transcriptional rates. Electrophoretic mobility shift assay demonstrated that DEP induced increased binding to the specific motif of NF-kappa B, but not of transcription factor AP-1. The luciferase reporter gene assay using wild-type and mutated NF-kappa B-binding sequences showed that DEP-induced NF-kappa B activation was involved in IL-8 transcription. Finally, both N-acetylcysteine and pyrrolidine dithiocarbamate attenuated the action of DEP on IL-8 mRNA expression, suggesting that oxidant-mediated pathway might be involved in its processes. These results suggested that DEP activate NF-kappa B, which might be an important mechanism of its potential to increase the expression of inflammatory cytokines in vitro.

Erythromycin and clarithromycin attenuate cytokine-induced endothelin-1 expression in human bronchial epithelial cells.

Erythromycin and its fourteen-member macrolide analogues have attracted attention for their efficacy in bronchial asthma. However, their mechanisms of action remain unclear. We evaluated the effects of the macrolide antibiotics on endothelin-1 (ET-1) expression in normal and transformed human bronchial epithelial cells, one of the sources of this potent bronchoconstrictor important in the pathogenesis of asthma. Human bronchial epithelial cells were obtained from the resected bronchi, and the effect of several antimicrobial and antiasthmatic drugs on the production and messenger ribonucleic acid (mRNA) levels of ET-1 was evaluated. Bronchoepithelial cells were also isolated from the mucosa of asthmatic patients under fibreoptic bronchoscopy, and the modulating effects of the drugs were studied. Erythromycin and clarithromycin uniquely suppressed mRNA levels as well as the release of ET- at therapeutic and non-cytotoxic concentrations (percentage inhibition of ET-1 protein release: 26.4+/-5.22% and 31.2+/-7.45%, respectively, at 10(-6) M). Furthermore, erythromycin and clarithromycin inhibited ET-1 expression in bronchoepithelial cells from patients with chronic, stable asthma. A glucocorticosteroid, dexamethasone, also inhibited ET-1 expression. In contrast, theophylline, salbutamol and FK506 had no effect on ET-1 production. Our findings demonstrated that these fourteen-member macrolide antibiotics had an inhibitory effect on endothelin-1 expression in human bronchial epithelial cells. Moreover, this new mode of action may have some relevance to their clinical efficacy in bronchial asthma.

Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro.

BACKGROUND: Epidemiologic and experimental studies suggest that air pollution such as diesel exhaust particles (DEPs), one of the important air pollutants, may play a role in the increasing prevalence of allergic airway diseases. OBJECTIVE: We studied the effect of suspended particulate matter (SPM) and its main component, DEPs, on the production of IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human airway epithelial cells in vitro. METHODS: SPM obtained from high-volume air samplers and DEPs were added to cultured human nasal polyp-derived upper airway, normal bronchial, and transformed bronchial epithelial cells. Production of GM-CSF and IL-8 by airway epithelial cells was evaluated. RESULTS: Nontoxic doses of DEPs showed a significant stimulatory effect on IL-8 and GM-CSF production by these three kinds of epithelial cells in a dose- and time-dependent fashion. SPM had a stimulatory effect on GM-CSF, but not IL-8, production. These effects were abrogated by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that the process required a de novo protein synthesis. On the double-chamber plates, airway epithelial cells responded to DEPs only when they were stimulated from the apical sides, which can be a model for in vivo environments. Neither charcoal nor graphite showed such stimulatory effects, indicating that the activity of DEPs did not derive from their particulate nature. Benzo(a)pyrene, one of the main aromatic hydrocarbons contained in DEPs, showed a stimulatory effect on the release of the cytokines, and this organic substance might have a causative effect on of the potency of DEPs. CONCLUSION: We conclude that SPM and DEPs, its main component, might be important air pollutants in the activation of airway epithelial cells for the release of cytokines relevant to allergic airway inflammation.

Roxithromycin inhibits cytokine production by and neutrophil attachment to human bronchial epithelial cells in vitro.

We evaluated the effect of roxithromycin on cytokine production and neutrophil attachment to human airway epithelial cells. Roxithromycin suppressed production of interleukin 8 (IL-8), IL-6, and granulocyte-macrophage colony-stimulating factor. It inhibited neutrophil adhesion to epithelial cells. Roxithromycin modulates local recruitment and activation of inflammatory cells, which may have relevance to its efficacy in airway diseases.

The effect of diesel exhaust particles on cell function and release of inflammatory mediators from human bronchial epithelial cells in vitro.

Animal studies have reported that diesel exhaust particles (DEP), which constitute an important fraction of particulate air pollution, lead to inflammation and/or damage of the airways. To investigate the mechanisms underlying DEP-induced airway disease in humans, we have cultured human bronchial epithelial cells (HBEC) from surgically obtained bronchial explants and investigated the effects of purified DEP on the permeability and ciliary beat frequency (CBF) of HBEC, and on the release of inflammatory mediators from these cells. Exposure to 10-100 microg/ml DEP and a filtered solution of 50 microg/ml DEP significantly increased the electrical resistance of the cultures, reaching a maximum of 200% over baseline after 6 h incubation with 100 microg/ml DEP. In contrast, movement of 14C-labeled bovine serum albumin across cell cultures was not significantly altered by incubation of HBEC with DEP. Exposure to 50 microg/ml DEP, filtered DEP solution, and 100 migrog/ml DEP significantly attenuated the CBF of these cells by 51%, 33%, and 73%, respectively, from baseline after 24 h incubation. Similarly, 50 microg/ml DEP, filtered DEP solution, and 100 microg/ml DEP significantly increased the release of interleukin-8 from 12.9 pg/microg cellular protein to 41.6, 114.9, and 44.3 pg/microg cellular protein, respectively, after 24 h incubation. The release of granulocyte-macrophage colony stimulating factor (GM-CSF) and soluble intercellular adhesion molecule-1 (sICAM-1) was also significantly increased after exposure for 24 h to 50 microg/ml DEP (GM-CSF from 0.033 pg/microg cellular protein to 0.056 pg/mug cellular protein and sICAM-1 from 7.2 pg/microg cellular protein to 12.5 pg/microg cellular protein). These results suggest that exposure of HBEC to DEP may lead to adverse functional changes and release of proinflammatory mediators from these cells, and that these effects may influence the development of airway disease.

Erythromycin modulates IL-8 expression in normal and inflamed human bronchial epithelial cells.

Erythromycin (EM) and its 14-member macrolide analogues have attracted attention for its effectiveness in a variety of airway diseases, including diffuse panbronchiolitis (DPB), sinobronchial syndrome, and chronic sinusitis. However, its mechanisms of action remain unelucidated. We evaluated the effects of several antibiotics on IL-8 expression by normal and transformed human bronchial epithelial cells, an important source of this potent chemokine involved in cell recruitment into the airways. EM and clarithromycin (CAM) uniquely suppressed mRNA levels as well as the release of IL-8 at the therapeutic and noncytotoxic concentrations (% inhibition of IL-8 protein release: 25.0 +/- 5.67% and 37.5 +/- 8.99%, respectively, at 10(-6) M). The other antimicrobes, including a 16-member macrolide josamycin, showed no effect. Bronchial epithelial cells from very peripheral airways as well as from main bronchi were obtained from patients with chronic airway inflammatory diseases, and EM and CAM inhibited IL-8 release from these cells. Among five patients who underwent bronchoscopy before and after macrolide treatment, four showed decreased levels of IL-8 expression in airway epithelium as assessed by reverse transcription and polymerase chain reaction. Our findings showed these 14-member macrolides had inhibitory effect on IL-8 expression in human bronchial epithelial cells, and this new mode of action may have relevance to their clinical effectiveness in airway diseases.

Eosinophil adhesion to human bronchial epithelial cells: regulation by cytokines.

BACKGROUND: Eosinophil infiltration into the airways and interaction with bronchial epithelial cells are important in the pathogenesis of asthma. The purpose of the present study was to elucidate the regulatory mechanisms of eosinophil adhesion to human bronchial epithelial cells. METHODS: We cultured a human bronchial epithelial cell line, BEAS-2B, on a collagen-coated glass slide. Highly purified human eosinophils were added to each well to allow attachment to epithelium for 30 min. The number of attached eosinophils was counted. RESULTS: Eosinophil adhesion to epithelial cells was increased when the BEAS-2B cells were pretreated with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Although IFN-gamma upregulated ICAM-1 expression as shown by flow cytometry, specific neutralizing antibody to ICAM-1 failed to block eosinophil adhesion. Dexamethasone significantly suppressed eosinophil adhesion to bronchial epithelial cells. CONCLUSION: Eosinophil adhesion to bronchial epithelium was dynamically regulated by cytokines, and this process might be a target for therapeutic intervention in the treatment of asthma.

Eosinophil degranulation in the presence of bronchial epithelial cells. Effect of cytokines and role of adhesion.

It has been suggested that eosinophils (Eos) are responsible for damage to bronchial epithelial cells by releasing toxic eosinophil granule proteins in bronchial asthma. We examined eosinophil cationic protein (ECP) release from human Eos cultured in the presence of human bronchial epithelial cell line BEAS-2B (Ep). ECP release was potentiated only when both Eos and Ep were activated by IL-5 and TNF, respectively, while it was not potentiated when either Eos or Ep were activated. ECP release from Eos activated by IL-5 was also enhanced when Ep was stimulated by IFN-gamma. Paraformaldehyde fixation of Ep had no effect on ECP enhancement, excluding the possibility that soluble factors from Ep contribute to ECP potentiation. Coculture of Eos and Ep with cytokine treatment resulted in the enhancement of eosinophil adhesion and ECP release, and eosinophil adhesion preceded ECP release in the kinetic study. The enhancement of ECP release was partially inhibited by anti-CD18 mAb, which caused partial and comparable inhibition on the potentiation of eosinophil adhesion. These results suggest that the activation of Ep may profoundly affect the ability of cocultured Eos to release ECP and that CD18-dependent adhesion of Eos to Ep may be considered as one of the mechanisms of ECP enhancement.

Interleukin 6-receptor expression on human bronchial epithelial cells: regulation by IL-1 and IL-6.

Airway epithelial cells have a potential to participate in regulation of local homeostasis by releasing active compounds including cytokines and growth factors. Several factors such as transforming growth factor-beta and endothelin have been shown to regulate airway epithelial cell functions through an autocrine mechanism. We studied the expression of the specific receptor for a multifunctional cytokine interleukin 6 (IL-6), which is expressed and released by airway epithelial cells. Specific binding assay demonstrated a single set of binding sites on human primary and transformed bronchial epithelial cells. Human interleukin-1alpha (IL-1alpha) increased maximal binding sites to IL-6. Northern blot analysis demonstrated that airway epithelial cells constitutively expressed mRNA for IL-6 receptor (IL-6R), and IL-1alpha and IL-6 itself upregulated IL-6R gene expression. Moreover, exogenously added human recombinant IL-6 had a stimulatory effect on IL-8 release from human bronchial epithelial cells. These results indicated that human bronchial epithelial cells expressed IL-6R, and IL-6 might be involved in the regulation of the epithelial functions via an autocrine as well as a paracrine mechanism.

Histamine activates bronchial epithelial cells to release inflammatory cytokines in vitro.

Airway epithelial cells have a potential to produce cytokines which are relevant to airway inflammation. To elucidate the mechanisms of their regulation, we focused on the effects of three chemical mediators [histamine, platelet-activating factor (PAF) and endothelin-1] important in the pathogenesis of bronchial asthma. Histamine, but not PAF or endothelin-1, showed a dose-dependent stimulatory effect on the release of interleukin-6, interleukin-8 and granulocyte-macrophage colony-stimulating factor by normal and transformed human bronchial epithelial cells when studied 6 h after the treatment. The process required protein synthesis as evaluated by the effect of cycloheximide, and was mainly via H1 receptor. We concluded that histamine might be involved in the activation of airway epithelial cells to release inflammatory cytokines in allergic responses.

Erythromycin suppresses interleukin 6 expression by human bronchial epithelial cells: a potential mechanism of its anti-inflammatory action.

We evaluated the effects of several antibiotics on IL-6 expression by human bronchial epithelial cells, potent sources of this proinflammatory cytokine important in airway inflammation. Among those tested, erythromycin (EM) and clarithromycin (CAM) uniquely suppressed mRNA levels as well as the release of IL-6 at the therapeutic and non-cytotoxic concentration (10(-6)M). Our findings suggested that these macrolide antibiotics had suppressive effect on cytokine expression in human cells, and this new mode of action may have relevance to their clinical effectiveness in airway inflammatory diseases.

Induction of eosinophil cytokine generation by chemoattractants.

Recent studies have shown that eosinophils are capable of generating and releasing cytokines, illustrating a novel biologic aspect of eosinophils in regulating allergic inflammation by either autocrine or paracrine mechanisms. The effect of chemotactic agonists on eosinophil cytokine generation was examined by determination of interleukin-8 (IL-8) as a main parameter. Both complement C5a and N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated eosinophils to release IL-8, but platelet activating factor (PAF) did not exert any significant effects. The generation of IL-8 by chemoattractants was absolutely dependent on the presence of cytochalasin B. Pertussis toxin completely attenuated C5a- and FMLP-induced IL-8 production, indicating the involvement of pertussis toxin-sensitive G proteins in the signal transduction process. In situ hybridization showed that both C5a and FMLP promoted eosinophil IL-8 production via transcriptional gene activation. Furthermore, C5a and FMLP, but not PAF, induced significant secretion of granulocyte-macrophage colony-stimulating factor from eosinophils. These results indicate that C5a and FMLP stimulate eosinophils to elaborate cytokines, which could be an important mechanism in the regulation of allergic inflammation.

[Cytokine production by human airway epithelial cells and its modulation].

There is increasing evidence that airway epithelial cells play an active role in allergic inflammation, including bronchial asthma. We showed that human airway epithelial cells in culture release GM-CSF, G-CSF, M-CSF, IL-6, and IL-8, using a serum-free culture system. These cytokines are known to modulate the bioactivities of inflammatory cells that accumulate at the site of inflammation. Among them, GM-CSF, IL-8, or both may be important because they influence the bioactivities of eosinophils, which are characteristic of allergic inflammation. Here we report on the effects of air pollutants such as suspended particulate matter and diesel exhaust particulates on release of cytokines from airway epithelial cells. All air pollutants we tested stimulated epithelial cells to release GM-CSF. These results suggest that one cause of the recent increase in the prevalence of allergic disorders is direct stimulation of airway epithelial cells by air-pollutants. Furthermore, anti-inflammatory agents such as steroids and anti-allergic drugs were found to suppress the release of GM-CSF from airway epithelial cells in vitro.