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. 2013 Jun;139(2):245-55.
doi: 10.1111/imm.12077.

Cigarette smoke increases BLT2 receptor functions in bronchial epithelial cells: in vitro and ex vivo evidence

Elisabetta Pace  1 , Maria Ferraro, Serena Di Vincenzo, Andreina Bruno, Antonino Giarratano, Valeria Scafidi, Luana Lipari, Denise Valentina Di Benedetto, Serafina Sciarrino, Mark Gjomarkaj
Affiliations

Cigarette smoke increases BLT2 receptor functions in bronchial epithelial cells: in vitro and ex vivo evidence

Elisabetta Pace et al. Immunology. 2013 Jun.

Abstract

Leukotriene B(4) (LTB(4)) is a neutrophil chemotactic molecule with important involvement in the inflammatory responses of chronic obstructive pulmonary disease (COPD). Airway epithelium is emerging as a regulator of innate immune responses to a variety of insults including cigarette smoke, the major risk factor for COPD. In this study we have explored whether cigarette smoke extracts (CSE) or soluble mediators present in distal lung fluid samples (mini-bronchoalveolar lavages) from smokers alter the expression of the LTB(4) receptor 2 (BLT2) and peroxisome proliferator-activated receptor-α (PPAR-α) in bronchial epithelial cells. We also evaluated the effects of CSE on the expression of intercellular adhesion molecule 1 (ICAM-1) and on the binding of signal transducer and activator of transcription 1 (STAT-1) to ICAM-1 promoter as well as the adhesiveness of neutrophils to bronchial epithelial cells. CSE and mini-bronchoalveolar lavages from smokers increased BLT2 and ICAM-1 expression as well as the adhesiveness of neutrophils to bronchial epithelial cells and decreased PPAR-α expression. CSE induced the activation of STAT-1 and its binding to ICAM-1 promoter. These findings suggest that, in bronchial epithelial cells, CSE promote a prevalent induction of pro-inflammatory BLT2 receptors and activate mechanisms leading to increased neutrophil adhesion, a mechanism that contributes to airway neutrophilia and to tissue damage.

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Figures

Figure 1
Figure 1
Mini-bronchoalveolar lavages (mini-BALs) from smokers increase BLT2 expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured with or without mini-BALs from smokers (n = 5) and from non-smokers (n = 3) for 18 hr and were used to assess BLT2 expression by flow cytometry (see Materials and methods for details). (a) Representative examples of flow cytometric analysis. 1 = negative control; 2 = BLT2 baseline expression; 3 = BLT2 expression after a mini-BAL from smoker exposure. (b) The results are expressed as percentage of positive cells and as GeoMean fluorescence intensity ± SD. Two replicates were performed from each mini-BAL sample.*P < 0·05
Figure 2
Figure 2
Mini-bronchoalveolar lavages (mini-BALs) from smokers decrease peroxisome proliferator-activated receptor-α (PPAR-α) expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured with/without mini-BALs from smokers (n = 5) and from non-smokers (n = 3) for 18 hr and were used for assessing PPAR-α expression by flow cytometry (see Materials and methods for details). (a) Representative histogram plots. 1 = negative control; 2 = baseline expression of PPAR-α; 3 = a mini-BAL from smoker. (b) The results are expressed as percentage of positive cells and as GeoMean fluorescence intensity ± SD.Two replicates were performed from each mini-BAL sample. *P < 0·05
Figure 3
Figure 3
Expression of BLT2 and peroxisome proliferator-activated receptor-α (PPAR-α) molecules in bronchial epithelial cells from surgical samples of smokers. Immunohistochemistry for BLT2 (a) and PPAR-α (b) in central airways from surgical samples of smokers (n = 5) and of non-smokers (n = 3) was performed. Representative BLT2 and PPAR-α immunostaining at 400 × magnification was shown.
Figure 4
Figure 4
Mini-bronchoalveolar lavages (mini-BALs) from smokers increase the adhesiveness between bronchial epithelial cells and neutrophils. Bronchial epithelial cells (16HBE) were cultured with/without mini-BALs from smokers (n = 5) for 18 hr and were used for assessing neutrophil adhesion to bronchial epithelial cells (see Materials and methods for details). Two replicates were performed from each mini-BAL sample. The results are expressed as % of the fluorescence ratio of bound cells on total cells ± SD. *P < 0·05
Figure 5
Figure 5
Mini-bronchoalveolar lavages (mini-BALs) from smokers increase intercellular adhesion molecule 1 (ICAM-1) expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured with/without mini-BALs from smokers (n = 5) and in the presence of an inhibitor for BLT2 (LY255283; 1 hr pre-treatment) or an inhibitor for peroxisome proliferator-activated receptor-α (PPAR-α) (MK886; 1 hr pre-treatment) for 18 hr and were used for assessing ICAM-1 expression by flow-cytometry (see Materials and methods for details). The results are expressed as GeoMean fluorescence intensity ± SD. Two replicates were performed from each mini-BAL sample. *P < 0·05
Figure 6
Figure 6
Effect of cigarette smoke extract (CSE) in the apoptosis of bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE (10%, 20%) and for 18 hr and then were used for assessing cell necrosis and cell apoptosis by annexin V/propidium iodide (PI) method by flow cytometry (see Materials and methods for details). Representative dot plots showing the percentage of annexin V, PI and annexin V/PI positive 16HBE at baseline and following the exposure of CSE (10%, 20%) are shown.
Figure 7
Figure 7
Cigarette smoke extracts (CSE) increase BLT2 expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE 10% for 18 hr and were used for assessing BLT2 expression by flow cytometry (see Materials and methods for details). (a) Representative histogram plots. 1 = negative control; 2 = baseline expression of BLT2; 3 = CSE 10%. (b) The results are expressed as GeoMean fluorescence intensity ± SD. *P < 0·05
Figure 8
Figure 8
Cigarette smoke extracts (CSE) decrease peroxisome proliferator-activated receptor-α (PPAR-α) expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE 10% for 18 hr (n = 6) and were used for assessing PPAR-α expression by flow cytometry (see Materials and methods for details). (a) Representative histogram plots. 1 = negative control; 2 = baseline expression of PPAR-α; 3 = CSE 10%. (b) The results are expressed as GeoMean fluorescence intensity ± SD. *P < 0·05
Figure 9
Figure 9
Cigarette smoke extracts (CSE) increase neutrophil adhesion to bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE 10% for 18 hr (n = 6) and were used for assessing neutrophil adhesion to bronchial epithelial cells. The results are expressed as % of the fluorescence ratio of bound cells on total cells. *P < 0·05
Figure 10
Figure 10
Cigarette smoke extracts (CSE) increase intercellular adhesion molecule 1 (ICAM-1) expression in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE 10%, of an inhibitor for BLT2 (LY255283; 1 hr pre-treatment) or of an inhibitor for peroxisome proliferator-activated receptor-α (PPAR-α) (MK886; 1 hr pre-treatment) (n = 6) and then were used for assessing ICAM-1 expression by flow cytometry. In some experiments (n = 2), RNA interference for BLT2 or PPAR-α was also performed. The results are expressed as GeoMean fluorescence intensity ± SD.*P < 0·05
Figure 11
Figure 11
Cigarette smoke extracts (CSE) increase signal transducer and activator of transcription 1 (STAT-1) activation and its interaction with the promoter of intercellular adhesion molecule 1 (ICAM-1) in bronchial epithelial cells. Bronchial epithelial cells (16HBE) were cultured in the presence and in the absence of CSE 10% for 18 hr (n = 3) and were used for assessing STAT-1 or pSTAT-1 by Western blot analysis and for chromatin immunoprecipitation assay (ChIP). (a) Representative Western blots of STAT-1, pSTAT-1 and β-actin expression. Lane 1 = baseline; lane 2 = CSE 10%. (b) Signals corresponding to pSTAT-1 or to STAT-1 were semi-quantified by densitometric scanning, normalized for β-actin and results were expressed as pSTAT-1/STAT-1 ratio.*P < 0·05. (c) ChIP assay using anti-STAT1 antibody and PCR using primers spanning the promoter region 3537 of intercellular adhesion molecule 1 (ICAM-1) gene were performed (see Materials and methods for details) (n = 3). Lane 1 = DNA marker; lane 2 = negative control of PCR; lane 3 = baseline; lane 4 = CSE 10%.
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References

    1. Proud D, Leigh R. Epithelial cells and airway diseases. Immunol Rev. 2011;242:186–204. - PubMed
    1. Crooks SW, Bayley DL, Hill SL, Stockley RA. Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4. Eur Respir J. 2000;15:274–80. - PubMed
    1. O'Donnell R, Breen D, Wilson S, Djukanovic R. Inflammatory cells in the airways in COPD. Thorax. 2006;61:448–54. - PMC - PubMed
    1. Hill AT, Bayley D, Stockley RA. The interrelationship of sputum inflammatory markers in patients with chronic bronchitis. Am J Respir Crit Care Med. 1999;160:893–8. - PubMed
    1. Gompertz S, O'Brien C, Bayley DL, Hill SL, Stockley RA. Changes in bronchial inflammation during acute exacerbations of chronic bronchitis. Eur Respir J. 2001;17:1112–9. - PubMed

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