Transforming growth factor-β stimulates the expression of eotaxin/CC chemokine ligand 11 and its promoter activity through binding site for nuclear factor-κβ in airway smooth muscle cells

Abstract

Background: Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling.

Objective: We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms.

Methods: HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids.

Results: IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4.

Conclusion: These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

Fig. 1

Regulation of CCR3 ligand mRNA expression in human airway smooth muscle (HASM) cells, (a) CC chemokine ligand 11 (CCL11)/eotaxin, (b) CCL24/eotaxin-2, (c) CCL26/eotaxin-3, and (d) CCL13/monocyte chemoattractant protein-4. RNA was extracted from cells stimulated with 10ng/mL IL-4, IL-13, and/or TGF-β for 24 h and subjected to real-time PCR. Levels of mRNA were calculated as fold induction compared with non-stimulated control cells. The data are presented as the mean±SEM of three independent experiments (*P<0.05).

Fig. 2

CC chemokine ligand 11 (CCL11) protein secretion into the culture medium of human airway smooth muscle cells, (a) Time course of CCL11 secretion, and (b) CCL11 secretion at 24h after stimulation. Medium was collected from cells stimulated with 10ng/mL IL-4, IL-13, and/or TGF-β for the indicated times and subjected to ELISA. The data are presented as the mean±SEM of each three independent experiments (*P<0.05).

Fig. 3

CC chemokine ligand 11 (CCL11) protein secretion into the culture medium of human airway smooth muscle cells. Concentration-dependent secretion of CCL11 in response to IL-4 (a) orIL-13 (b) in the presence of TGF-β. Medium was collected from cells stimulated with the indicated concentrations of IL-4 or IL-13 combined with 10 ng/mL TGF-β for 24 h and subjected to ELISA. The data are presented as the mean±SEM of three independent experiments (*P<0.05).

Fig. 4

Luciferase reporter assays of CC chemokine ligand 11 (CCL11) promoter activity in human airway smooth muscle cells. Cells were transfected with CCL11 promoter-luciferase reporter plasmids illustrated in (a) (pEotx.1363, 478, M1 or M2) and control vector pRL-TK. Forty-eight hours later, cells were incubated for 20h with or without 10 ng/mL IL-4 and TGF-β. (b) pEotx.1363, (c) pEotx.478, (d) pEotx.M1, and (e) pEotx.M2. Relative luciferase activity was calculated as fold induction compared with control value. The data are presented as the mean±SEM of a total of three independent experiments (*P< 0.05).

Fig. 5

Effects of drugs on the expression of CC chemokine ligand 11 (CCL11) mRNA (a and c), CCL11 protein (b), or CCL26 mRNA (d) induced by IL-4 or IL-4 plus TGF-β in human airway smooth muscle cells. Cells were stimulated with IL-4 or IL-4 plus TGF-β (10 ng/mL each) with or without pre-incubation with the indicated drugs, (a and b) Effect of the nuclear factor-κB inhibitor Bay 11-7085, and (c and d) effects of the glucocorticoid fluticasone propionate (FP) and long-acting β₂-agonist salmeterol (SAL) (10⁻⁷M each). The data are presented as the mean±SEM of three independent experiments [*P< 0.05 compared with the cells with same stimulation and treated with diluent dimethyl sulphoxide or dimethyl acetamide (DMA) as indicated].