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. 2012;7(8):e43544.
doi: 10.1371/journal.pone.0043544. Epub 2012 Aug 31.

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells

Véronique Provost  1 , Anick Langlois, François Chouinard, Marek Rola-Pleszczynski, Jamila Chakir, Nicolas Flamand, Michel Laviolette
Affiliations

Leukotriene D4 and interleukin-13 cooperate to increase the release of eotaxin-3 by airway epithelial cells

Véronique Provost et al. PLoS One. 2012.

Abstract

Introduction: Airway epithelial cells play a central role in the physiopathology of asthma. They release eotaxins when treated with T(H)2 cytokines such as interleukin (IL)-4 or IL-13, and these chemokines attract eosinophils and potentiate the biosynthesis of cysteinyl leukotrienes (cysLTs), which in turn induce bronchoconstriction and mucus secretion. These effects of cysLTs mainly mediated by CysLT(1) and CysLT(2) receptors on epithelial cell functions remain largely undefined. Because the release of inflammatory cytokines, eotaxins, and cysLTs occur relatively at the same time and location in the lung tissue, we hypothesized that they regulate inflammation cooperatively rather than redundantly. We therefore investigated whether cysLTs and the T(H)2 cytokines would act in concert to augment the release of eotaxins by airway epithelial cells.

Methods: A549 cells or human primary bronchial epithelial cells were incubated with or without IL-4, IL-13, and/or LTD(4). The release of eotaxin-3 and the expression of cysLT receptors were assessed by ELISA, RT-PCR, and flow cytometry, respectively.

Results: IL-4 and IL-13 induced the release of eotaxin-3 by airway epithelial cells. LTD(4) weakly induced the release of eotaxin-3 but clearly potentiated the IL-13-induced eotaxin-3 release. LTD(4) had no effect on IL-4-stimulated cells. Epithelial cells expressed CysLT(1) but not CysLT(2). CysLT(1) expression was increased by IL-13 but not by IL-4 and/or LTD(4). Importantly, the upregulation of CysLT(1) by IL-13 preceded eotaxin-3 release.

Conclusions: These results demonstrate a stepwise cooperation between IL-13 and LTD(4). IL-13 upregulates CysLT(1) expression and consequently the response to cysLTs This results in an increased release of eotaxin-3 by epithelial cells which at its turn increases the recruitment of leukocytes and their biosynthesis of cysLTs. This positive amplification loop involving epithelial cells and leukocytes could be implicated in the recruitment of eosinophils observed in asthmatics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of IL-13 on the release of eotaxins from airway epithelial cells.
Airway epithelial cells were treated with vehicle (open bars), 10 ng/ml IL-13 (closed bars), or 100 nM LTD4 (gray bars) for 24 hours. Incubations were stopped by removing the supernatants and eotaxin-1, eotaxin-2, and eotaxin-3 were quantified as described in Material and Methods. The data represent the mean (± SEM) of four independent experiments.
Figure 2
Figure 2. Kinetic of eotaxin-3 release from airway epithelial cells.
Airway epithelial cells were incubated with vehicle (open bars), 10 ng/ml IL-13 (closed bars), or 100 nM LTD4 (gray bars) for different times. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in Material and Methods. The data represent the mean (± SEM) of four independent experiments. Insert represents a zoom of the 6 and 24 hours data points. * p = 0.002 vs 6 hours, # p = 0.003 vs 24 hours.
Figure 3
Figure 3. Effect of LTD4 on the IL-13-induced release of eotaxin-3 by airway epithelial cells.
A) Airway epithelial cells were incubated with 10 ng/ml IL-13 alone or in combination with 100 nM LTD4 for up to 48 hours. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in Material and Methods. Data are expressed as % of IL-13-treated cells for each incubation period. The data represent the mean (± SEM) of four independent experiments. NS = not significant. B) Airway epithelial cells were incubated with 50 ng LTD4 for up to 48 hours. Incubations were stopped by adding one volume of cold incubation buffer. Samples were harvested then processed for the analysis of LTD4 and LTE4 by reverse-phase HPLC as described in Material and Methods. Data are the mean (± SEM) of three independent experiments, each performed in duplicate.
Figure 4
Figure 4. Expression of CysLT1 and CysLT2 by airway epithelial cells.
Total mRNA was extracted from resting airway epithelial cells (A549), human primary bronchial epithelial cells (HPBEC) and freshly isolated human eosinophils (Eo). The expression of CysLT1 and CysLT2 mRNA was then analyzed by RT-PCR as described in Material and Methods. The data presented are a typical result of three independent experiments for eosinophils and airway epithelial cells, and four independent experiments for HPBEC.
Figure 5
Figure 5. Effect of IL-13, IL-4 and/or LTD4 on CysLT1 expression by airway epithelial cells.
Airway epithelial cells were treated with vehicle, 10 ng/ml IL-4, 10 ng/ml IL-13 and/or 100 nM LTD4 for 6 hours. Incubations were stopped by removing the incubation media and cells were harvested, processed, and analyzed for CysLT1 expression by flow cytometry as indicated in Material and Methods. A. Representative histograms of CysLT1 expression (control isotype, open bar; baseline expression, gray plot; IL-13-treated airway epithelial cells). B. Data represent the mean (± SEM) of three separate experiments. NS = not significant.
Figure 6
Figure 6. Effect of LTD4 on eotaxin-3 release by airway epithelial cells pretreated with IL-13.
After a 6 hour incubation of airway epithelial cells with 10 ng/ml IL-13, cells were further cultured for an additional 18 hours in the presence or absence of 100 nM LTD4. Incubations were stopped by removing the supernatants and eotaxin-3 was quantified as described in Material and Methods. The data represent the mean (± SEM) of three independent experiments.
Figure 7
Figure 7. Expression of eotaxin-3 by human primary bronchial epithelial cells.
Human primary epithelial cells were treated with vehicle, IL-13 (10 ng/ml) alone or in combination with LTD4 (100 nM) for 6 hours. Total RNA was extracted and the expression of eotaxin-3 mRNA was then analyzed by qPCR as described in Material and Methods. Eotaxin-3 expression was corrected for level of 18s rRNA as housekeeping gene. Data are presented as fold change over vehicle treatment. Data represent the mean (± SEM) of cell lines from four different subjects in duplicates.
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References

    1. Bousquet J, Jeffery PK, Busse WW, Johnson M, Vignola AM (2000) Asthma. From bronchoconstriction to airways inflammation and remodeling. Am J Respir Crit Care Med 161: 1720–1745. - PubMed
    1. Komiya A, Nagase H, Yamada H, Sekiya T, Yamaguchi M, et al. (2003) Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells. Cell Immunol 225: 91–100. - PubMed
    1. Ferland C, Guilbert M, Davoine F, Flamand N, Chakir J, et al. (2001) Eotaxin promotes eosinophil transmigration via the activation of the plasminogen-plasmin system. J Leukoc Biol 69: 772–778. - PubMed
    1. Pease JE, Williams TJ (2001) Eotaxin and asthma. Curr Opin Pharmacol 1: 248–253. - PubMed
    1. Banwell ME, Tolley NS, Williams TJ, Mitchell TJ (2002) Regulation of human eotaxin-3/CCL26 expression: modulation by cytokines and glucocorticoids. Cytokine 17: 317–323. - PubMed

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