Mechanisms of asthma and allergic inflammation
TGF-β differentially regulates TH2 cytokine-induced eotaxin and eotaxin-3 release by human airway smooth muscle cells

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Background

Human airway smooth muscle cells (HASMs) are involved in the pathogenesis of asthma. By producing chemokines, HASMs play a role in the inflammatory processes observed in this disease. Eotaxin, eotaxin-2, and eotaxin-3 are important chemoattractants for eosinophils, and these chemokines are expressed during different phases of the allergic reaction. TH2 cytokines and TGF-β can be found in increased levels in patients with asthma, and these cytokines may be involved in the regulation of chemokine expression.

Objective

The aim of this study was to determine the effect of TH2 cytokines and TGF-β on the regulation of expression of eotaxin, eotaxin-2, and eotaxin-3 by HASMs.

Methods

HASMs were incubated for 24 hours with IL-4, IL-13, TGF-β1, or combinations of these cytokines. Protein and mRNA levels of eotaxin and eotaxin-3 were evaluated by sandwich ELISA and reverse transcriptase-PCR.

Results

IL-4 and IL-13 induced mRNA and protein for both eotaxin and eotaxin-3. Eotaxin-2 mRNA and protein were not detected in HASMs. TGF-β alone did not induce expression of the eotaxins. However, in combination with IL-4 or IL-13, TGF-β enhanced eotaxin production and inhibited TH2 cytokine-induced eotaxin-3 production.

Conclusion

TGF-β differentially regulates TH2 cytokine-induced eotaxin and eotaxin-3 release.

Section snippets

HASM culture

HASMs from 2 donors were purchased from Stratagene (La Jolla, Calif). Cells were characterized and stained positive for α-smooth muscle actin. Cells were cultured in Dulbecco modified Eagle medium/nutrient mixture F12 (1:1; Invitrogen, Carlsbad, Calif) containing 25 mmol/L HEPES (Invitrogen), 10% (vol/vol) FCS (Invitrogen) supplemented with 2.5 mmol/L l-glutamine (Cambrex, East Rutherford, NJ), 1% (vol/vol) nonessential amino acids (Invitrogen), 50 U/mL penicillin, and 50 μg/mL streptomycin

Cytokine-induced eotaxin and eotaxin-3 release

To study the effect of TH2 cytokines on the release of eotaxins by HASMs, serum-starved confluent monolayers were incubated with IL-4, IL-13, and IL-9 in serum-free medium. The TH2 cytokines IL-4 and IL-13 induced eotaxin and eotaxin-3 release by HASMs in a dose-dependent manner (Fig 1, A and B). IL-4 induced eotaxin release at low concentrations, whereas IL-13 was less potent in inducing eotaxin release. Eotaxin-3 release, however, was induced at low concentrations of both IL-4 and IL-13.

Discussion

In this study we have shown that HASMs produce eotaxin and eotaxin-3 upon stimulation with the TH2 cytokines IL-4 and IL-13. HASMs did not release eotaxin-2 protein upon stimulation with the TH2 cytokines IL-4 and IL-13. A low concentration (0.5 ng/mL) of TGF-β increased IL-4–induced and IL-13–induced eotaxin release, whereas it decreased the release of eotaxin-3. At the same concentration, TGF-β alone altered neither eotaxin nor eotaxin-3 release.

We have shown for the first time that IL-4 and

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    Funded by The Netherlands Asthma Foundation (grant 00.17) and AstraZeneca (Lund, Sweden).

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