Mechanisms of asthma and allergic inflammation
Cysteinyl leukotrienes synergize with growth factors to induce proliferation of human bronchial fibroblasts

https://doi.org/10.1016/j.jaci.2006.08.028Get rights and content

Background

Cysteinyl leukotrienes (cys-LTs) are potent asthma-related mediators that function through their G protein–coupled receptors, cys-LT receptor type 1 (CysLT1R) and cys-LT receptor type 2 (CysLT2R).

Objective

Because many G protein–coupled receptors transactivate the epidermal growth factor receptor (EGFR) through metalloprotease-mediated ligand shedding, we investigated the effects of cys-LTs on signal transduction and proliferation of bronchial fibroblasts.

Methods

Human bronchial fibroblasts were grown from biopsy specimens of healthy subjects. Mitogenesis was assessed on the basis of tritiated methylthymidine incorporation.

Results

Leukotriene (LT) D4 alone did not increase mitogenesis but dose-dependently increased thymidine incorporation and cell proliferation in the presence of epidermal growth factor (EGF). The enhancement was not prevented by CysLT1R antagonists (MK-571 and montelukast) or by a dual antagonist (BAY u9773), which is consistent with the lack of detectable mRNA for CysLT1R and CysLT2R in bronchial fibroblasts. LTD4 did not cause EGFR transphosphorylation nor was the synergism blocked by the metalloprotease inhibitor GM6001. The EGFR-selective kinase inhibitor AG1478 suppressed the synergy between LTD4 and EGF but had no effect on synergistic interactions of LTD4 with other receptor tyrosine kinase growth factors. The effect of LTD4 involved a pertussis toxin–sensitive and protein kinase C–mediated intracellular pathway, leading to sustained growth factor–dependent phosphorylation of extracellular signal–regulated kinase 1/2 and protein kinase B (PKB/Akt).

Conclusion

Cys-LTs do not transactivate EGFR but have a broader capability to synergize with receptor tyrosine kinase pathways.

Clinical implications

This study implies a critical role of cys-LTs in airway fibrosis in asthma and other chronic airway diseases, which might not be blocked by therapy with current LT receptor antagonists.

Section snippets

Cell cultures

Primary human bronchial fibroblasts were grown from bronchial biopsy specimens obtained from healthy subjects, as previously reported.16 The detailed methods are described in the Methods section in the Online Repository at www.jacionline.org.

Reagents

LTD4, LTC4, and MK-571 were from Cayman Chemical (Ann Arbor, Mich). Montelukast sodium was a kind gift from Dr Jilly Evans (Merck, West Point, Pa). Other reagents are described in the Methods section in the Online Repository at www.jacionline.org.

Mitogenesis analyses

Bronchial

LTD4 synergizes with EGF to induce mitogenesis

To assess whether cys-LTs have mitogenic activity, we analyzed the incorporation of tritiated thymidine into primary cultures of bronchial fibroblasts. LTC4 or LTD4 alone had little mitogenic effect. However, when coincubated with EGF, both LTC4 and LTD4 dose-dependently augmented EGF-induced mitogenesis. Thus 0.5 μmol/L of LTD4 or LTC4 increased mitogenesis by 408% ± 93% (P = .002) or 421% ± 97% (P = .002), respectively, compared with EGF (1 ng/mL) alone. The dose-response curves show very

Discussion

Airway remodeling is an important component of chronic asthma involving proliferation and activation of airway fibroblasts.20 Previous studies have shown that LTC4 and LTD4 stimulate mitogenesis of human skin fibroblasts in vitro.21 Furthermore, targeted deletion of the CysLT1R gene unexpectedly augmented bleomycin-induced lung fibrosis and alveolar septal thickening,22 whereas deletion of CysLT2R prevented these effects.23 These data suggest that CysLT1R and CysLT2R have distinct functions

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    Supported by Asthma UK.

    Disclosure of potential conflict of interest: H. Yoshisue and D. E. Davies have received grant support from Asthma UK. A. P. Sampson has consultant arrangements with Merck Sharp & Dohme; has received grant support from Merck & Co, Ono Pharma UK Ltd, and AstraZeneca; and has received honoraria from Merck & Co. The rest of the authors have declared that they have no conflict of interest.

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