Vitamin D and glucocorticoids differentially modulate chemokine expression in human airway smooth muscle cells

Abstract

Background and purpose: Chemokines play a critical role in the pathogenesis of asthma and facilitate the recruitment of inflammatory cells in the airways. Evidence now suggests that airway smooth muscle (ASM) may serve as a source of chemokines in inflamed airways. Although vitamin D has potent anti-inflammatory properties in vitro in some cell types, its effects on ASM cells remain unclear. Here, we investigated whether 1alpha, 25-dihydroxy vitamin D3 (calcitriol) modulated chemokine production in ASM.

Experimental approach: Human ASM cell cultures were derived from tracheal samples taken during surgery. ASM cells were treated with tumour necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma) for 24 h in the presence of calcitriol and/or the glucocorticoid fluticasone added 2 h before. RANTES (regulated upon activation, normal T-cell expressed and secreted), interferon-inducible protein 10 (IP-10) and fractalkine (FKN) levels in cell supernatants were measured by ELISA.

Key results: In TNFalpha-treated cells, calcitriol inhibited RANTES and IP-10 secretion in a concentration-dependent manner. FKN levels were negligible. In TNFalpha/IFNgamma-treated cells, whereas fluticasone or calcitriol alone partially inhibited RANTES secretion (by 38 and 20%, respectively), the combination of both drugs additively inhibited RANTES secretion (by 60%). No effect was observed on IP-10 secretion. Whereas fluticasone enhanced FKN secretion (by 50%), calcitriol significantly decreased FKN levels (by 50%). Interestingly, calcitriol blocked the stimulatory effect of fluticasone on FKN secretion, which was inhibited by 60% with the combination of calcitriol and fluticasone.

Conclusions and implications: These findings suggest that vitamin D uniquely modulates human ASM expression of chemokines and may exert some beneficial effects in the treatment of steroid-resistant patients with asthma.

Figure 1

Effect of calcitriol on vitamin D receptor (VDR) and 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) mRNA expression in human airway smooth muscle (ASM) cells. Cells were treated for 24 h with 100 nM of calcitriol. Total mRNA (2 μg) was then subjected to reverse transcription-PCR using VDR (top gel), CYP24A1 (middle gel) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (lower gel) primers. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Data are representative of three different cell lines.

Figure 2

Calcitriol inhibits tumour necrosis factor alpha (TNFα)-induced secretion of chemokines in airway smooth muscle (ASM) cells. Cells were stimulated for 24 h with TNFα (10 ng mL⁻¹) in the presence or absence of calcitriol (0.001–100 nM) (a, c) or fluticasone (Fp) (0.01–100 nM) (b, d) added 2 h before. Secretion of RANTES (a, b) and IP-10 (c, d) was analysed as described under Materials and methods. Values shown are mean±s.e.mean of three separate experiments. ^*P<0.05 compared with cells treated with TNFα alone; ^**P<0.01 compared with cells treated with TNFα alone; ^***P<0.001 compared with cells treated with TNFα alone.

Figure 3

Calcitriol differentially regulates chemokine secretion in airway smooth muscle (ASM) cells treated with tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) combination. Cells were stimulated for 24 h with TNFα (10 ng mL⁻¹) and IFNγ (500 IU mL⁻¹) in the presence or absence of fluticasone (Fp) (0.001–100 nM) (a, c, e) or calcitriol (0.001–100 nM) (b, d, f) added 2 h before. Secretion of RANTES (a, b), IP-10 (c, d) and fractalkine (FKN) (e, f) was analysed as described under Materials and methods. Values shown are mean±s.e.mean of three separate experiments. ^*P<0.05 compared with cells treated with TNFα and IFNγ; ^**P<0.01 compared with cells treated with TNFα and IFNγ.

Figure 4

Time course analysis of the inhibitory effects of calcitriol on chemokine secretion in tumour necrosis factor alpha (TNFα)/interferon gamma (IFNγ)-treated airway smooth muscle (ASM) cells. Cells were stimulated for 24 h with TNFα (10 ng mL⁻¹) and IFNγ (500 IU mL⁻¹), and 100 nM of calcitriol was added either 1 h before, simultaneously, or 6, 12 and 18 h after cytokine treatment. Secretion of RANTES (a) and fractalkine (FKN) (b) was analysed as described under Materials and methods. Values shown are mean±s.e.mean of three separate experiments. ^*P<0.05 compared with cells treated with TNFα/IFNγ; ^**P<0.01 compared with cells treated with TNFα and IFNγ.

Figure 5

Effects of the combination of fluticasone with calcitriol on chemokine secretion in tumour necrosis factor alpha (TNFα)/interferon gamma (IFNγ)-treated airway smooth muscle (ASM) cells. Cells were pretreated with calcitriol (100 nM), fluticasone (Fp) (100 nM) or a combination of both agents for 2 h before incubation with TNFα (10 ng mL⁻¹) and IFNγ (500 IU mL⁻¹) for an additional 24 h. Secretion of RANTES (a), IP-10 (b) and fractalkine (FKN) (c) was analysed as described under Materials and methods. Values shown are mean±s.e.mean of three separate experiments. ^*P<0.05 compared with cells treated with TNFα and IFNγ; ^**P<0.01 compared with cells treated with TNFα and IFNγ.