Vitamin D, a secosteroid hormone synthesized in the skin or derived from nutritional sources, serves a variety of functions that include immunomodulation, bone homoeostasis and wound healing (Holick, 1994; van Etten and Mathieu, 2005; Lips, 2006). Deficiency in vitamin D has been linked to autoimmune diseases, carcinogenesis, and importantly, different inflammatory diseases (Mathieu and Badenhoop, 2005; Munger et al., 2006; Mullin and Dobs, 2007; Schwartz and Skinner, 2007). Therapeutic approaches using active vitamin D in chronic inflammatory skin diseases, such as psoriasis, show that treatment of normal human dermal fibroblasts and keratinocytes inhibits chemokine production (Fukuoka et al., 1998a). Others found that vitamin D receptor (VDR)-deficient mice are more susceptible to inflammatory bowel disease (Cantorna et al., 2004). The role of vitamin D, however, in preventing airway inflammatory diseases such as asthma remains unclear (Black and Scragg, 2005; Wjst, 2006; Devereux, 2007).

Asthma, a chronic disease with increasing incidence in the United States, manifests as airway hyper-responsiveness and inflammation. Asthma prevalence continues to increase worldwide and industrialized nations furthest from the equator have highest prevalence (Busse and Lemanske, 2001; Litonjua and Weiss, 2007; Moorman et al., 2007; Rudd and Moorman, 2007). Investigators postulate that the increase in asthma incidence in industrialized populations may relate to the longer time spent indoors, decreasing, therefore, the solar UV-B radiation that penetrates the skin and converts 7-dehydrocholesterol to active vitamin D (Holick, 2007; Weiss and Litonjua, 2007). Although analysis of the data from the third National Health and Nutrition Examination survey demonstrates a strong relationship among serum concentrations of vitamin D, forced expiratory vital capacity and forced expiratory volume in 1 second (Black and Scragg, 2005), the serum concentration of vitamin D in patients suffering from asthma compared with normal controls has not yet been investigated. Other studies, however, suggested that vitamin D deficiency may increase the incidence of asthma. Two prospective cohort studies showed an inverse association between intake of vitamin D and childhood wheezing (Camargo et al., 2007; Devereux et al., 2007). A study on a founder population in Quebec and another using a childhood asthma management network suggest associations among VDR polymorphisms and asthma (Poon et al., 2004; Raby et al., 2004). Accordingly, Topilski et al. (2004) observed a reduction in interleukin (IL)-4 levels and eosinophil counts in the bronchoalveolar lavage in subjects with asthma after vitamin D administration. However, the effect of vitamin D on the immunomodulatory role of airway structural cells in asthma remains unknown.

Although bronchomotor tone is primarily regulated by airway smooth muscle (ASM) and profoundly contributes to the asthmatic diathesis, compelling evidence now suggests that ASM cells also modulate airway inflammation and remodelling in asthma by secreting a variety of inflammatory proteins, such as cytokines and chemokines, and by expressing cell adhesion and Toll-like receptor molecules (see Tliba et al., 2008a). Some studies have investigated the effect of vitamin D on ASM functions. Accordingly, vitamin D alters expression of a variety of genes as determined by microarray (Bosse et al., 2007). Another showed that vitamin D treatment inhibited ASM growth impeding ASM cell cycle traversal (Song et al., 2007). The effect of vitamin D, however, in modulating ASM synthetic function has not been investigated. In this study, we examined the anti-inflammatory effects of vitamin D in comparison with glucocorticoids (GCs) on cytokine-induced chemokine production in human ASM cells.

We explored the anti-inflammatory effect of vitamin D in terms of chemokine secretion from human ASM cells. Vitamin D effectively inhibited chemokine secretion in ASM cells treated with TNFα alone. In steroid-resistant conditions, however, vitamin D differentially modulated chemokine gene expression. Surprisingly, vitamin D significantly reduced the expression of a steroid-resistant gene, FKN. To our knowledge, this is the first report showing that vitamin D may inhibit the expression of steroid-resistant genes in any cell type.

Effects of vitamin D are in part mediated via the stimulation of VDRs (Carlberg and Seuter, 2007). Previous studies in bronchial ASM suggest that vitamin D analogues profoundly increase the expression of CYP24A1 (241-fold) and of VDRs (1.6-fold) levels (Bosse et al., 2007). We also showed that vitamin D increases both the expression of VDRs (1.7-fold) and CYP24A1 in tracheal ASM. These findings demonstrate the ability of ASM, whether bronchial or tracheal, to respond to vitamin D. Most of the effects of vitamin D are to modulate cell function by activating VDRs and by transcriptionally regulating the expression of vitamin D-responsive genes (see van Etten and Mathieu, 2005). Beside this genomic mechanism of action, vitamin D may also act through rapid non-genomic effects (see Marcinkowska, 2001). In agreement with previous studies in immune cells (Yu et al., 1995; Xing et al., 2002), our time course analysis revealed that vitamin D exerted its inhibitory effects on chemokine production through genomic mechanisms that required a minimum of 18 h of exposure (Figure 4). These findings corroborate with those of others who have shown that extended exposure of vitamin D also modulates the expression of ASM contractile genes (Bosse et al., 2007) and proliferation (Song et al., 2007). Our study extends the knowledge of the effects of vitamin D in modulating the biosynthetic functions of ASM cells.

There is evidence that ASM may serve as a source of chemokines in allergen-induced airway inflammation that ultimately recruits and retains inflammatory cells that could amplify pro-inflammatory markers (see Tliba et al., 2008a). Consequently, controlling the expression of chemokines by ASM cells may provide unique therapeutic targets to decrease cell migration/infiltration, and ultimately reverse either airway remodelling or airway inflammation. RANTES is a chemokine that attracts monocytes, eosinophils and T cells during inflammation and immune response (Schall et al., 1990). Interestingly, immunohistochemical and in situ hybridization studies revealed that RANTES is expressed in smooth muscle bundles of bronchial biopsies in subjects with asthma (Fahy et al., 1997). We and others showed that RANTES expression is increased in vitro following TNFα treatment (see Tliba et al., 2008a). In a variety of cell lines, vitamin D differentially modulates chemokine expression (Fukuoka et al., 1998a, 1998b; Xing et al., 2002; Griffin et al., 2004; Equils et al., 2006). In murine dendritic cells, vitamin D significantly inhibits RANTES expression, whereas it also enhances MIP-1α MIP-1β and MCP-1 expression (Xing et al., 2002; Griffin et al., 2004). In human endothelial cells, vitamin D abolishes lipopolysaccharide-induced RANTES secretion and modestly attenuates IL-8 secretion (Equils et al., 2006). In human dermal fibroblasts and epidermal keratinocytes, vitamin D inhibits both RANTES and IL-8 secretion (Fukuoka et al., 1998a, 1998b). In agreement with the latter studies, we showed that vitamin D inhibited TNFα-induced RANTES release in ASM cells in a concentration-dependent manner. Importantly, the inhibition of RANTES expression by vitamin D observed in the current study was of particular interest as neutralizing RANTES expression in murine models of allergen-induced airway hyper-responsiveness dramatically reduces airway mucus secretion as well as leukocyte migration (Chvatchko et al., 2003).

IP-10 (CXCL10) is a potent chemokine for activated T cells, NK cells and mast cells (Luster et al., 1985). Immunohistochemical study showed that IP-10 was preferentially expressed by ASM obtained from subjects with asthma (Brightling et al., 2005). IP-10 neutralization also inhibits mast cell migration towards asthmatic ASM cells (Brightling et al., 2005). We show that IP-10 expression was also increased following ASM treatment with TNFα (Figure 2). The modulatory effect of vitamin D on IP-10 expression in immune cells, however, remains controversial. For instance, vitamin D enhanced IP-10 production in peripheral blood monocytes and Langerhans cells (Vidal et al., 2002; Fujita et al., 2007), and these findings are quite contrary to the observations in other cell types including ASM cells. A number of studies performed either in murine bone marrow-derived macrophages or in pancreatic islets cells have consistently shown decreased IP-10 expression after vitamin D treatment (Gysemans et al., 2005; Helming et al., 2005). Concordantly, we showed that treatment of ASM cells with vitamin D significantly reduced TNFα-induced IP-10 secretion. These conflicting results may be explained by different experimental design using diverse exposure periods to vitamin D (ranging from 18 h to 6 days) and a variety of cell types whose sensitivity to inflammatory cytokines and/or vitamin D responses may vary. The effect of vitamin D on IP-10 expression may also depend on the differentiation status of the cells as suggested by Helming et al. (2005) in myeloid precursors/monocytes and mature macrophages. Further studies will be necessary to address the differential cell-specific effects of vitamin D on IP-10 gene expression.

Inhaled corticosteroids are the most effective agents available for symptomatic control and improvement of pulmonary function in asthma (Busse and Lemanske, 2001). Although most patients with asthma achieve control with inhaled steroids, there remains about 15% of patients who are steroid-insensitive (Adcock and Lane, 2003). Despite high doses of steroids, the therapeutic approach remains ineffective at restoring lung function (Wenzel, 2005). We and others recently showed that treatment of ASM cells with the combination of TNFα and IFNγ impairs the ability of steroids to inhibit the expression of different pro-asthmatic genes (Sukkar et al., 2004, 2006; Tliba et al., 2006, 2008b) by mechanisms, in part, involving the upregulation of the β isoform of the GC receptor and the reduction of GC receptor binding to GC-responsive elements of target DNA (Tliba et al., 2006). In contrast to the broad inhibitory effect of vitamin D on chemokine expression in steroid-sensitive cells (TNFα-treated ASM cells), we found that vitamin D differentially regulated chemokine expression in steroid-resistant cells (TNFα/IFNγ-treated ASM cells). Although calcitriol had little effect on IP-10 secretion and only slightly reduced RANTES secretion, calcitriol inhibited FKN expression (Figure 3f). To our knowledge, our study is the first to report that vitamin D inhibits FKN expression. Of note, no FKN release was observed when ASM cells were treated with TNFα alone. FKN is a CX₃C chemokine that mediates mast cell recruitment to the ASM in asthmatics (El-Shazly et al., 2006). This chemokine is a structurally unique protein in which a chemokine-like domain is located atop a mucin stalk connected to a transmembrane domain. A soluble form of FKN is generated by proteolytic cleavage of the full-length molecule at the membrane proximal site (Tsou et al., 2001). Although the membrane-bound form of FKN acts as an adhesion molecule (Fujimoto et al., 2001), the soluble form of FKN has chemotactic functions (Haskell et al., 2000). In the present study, only the soluble form of FKN was investigated. Interestingly, elevated levels of soluble FKN have been demonstrated in asthmatics before and after allergen challenge (Rimaniol et al., 2003). Importantly, the secretion of FKN from ASM is resistant to steroid treatment, and steroid actually increases FKN production (Figure 3e and Sukkar et al., 2004). The inhibition of FKN mRNA transcription (data not shown) by vitamin D suggests that vitamin D was downregulating both soluble and membrane-bound forms of FKN and was not simply altering the balance of membrane-bound FKN to secreted FKN. Thus, the ability of vitamin D to inhibit the transcriptional induction of FKN (Figure 3f and data not shown), a steroid-resistant chemokine in ASM cells, is of specific interest and may have therapeutic benefit in steroid-resistant states. Xystrakis et al. (2006) have already shown that administration of vitamin D to steroid-resistant asthmatic patients enhanced subsequent responsiveness of T CD4⁺ regulatory cells to dexamethasone. Taken together, vitamin D modulates the expression of steroid-resistant genes in airway structural cells and restores steroid responsiveness in immune cells (Xystrakis et al., 2006), suggesting that vitamin D may hold promise in the treatment of patients with severe asthma.

Because of the efficiency of GCs in asthma, the development of alternative anti-inflammatory agents has been challenging (Barnes and Hansel, 2004). The possibility of combining GCs with other agents may improve the therapeutic index of either drug used alone. In steroid-resistant cells and in combination with steroids, vitamin D showed interesting additive effects in inhibiting RANTES secretion (Figure 5). Similar findings were also reported in dendritic cells where vitamin D and GC additively inhibited RANTES mRNA expression (Xing et al., 2002). A vitamin D analogue, calcipotriol, is used in conjunction with steroids to treat psoriasis, a common chronic inflammatory skin disease characterized by hyper-proliferation and defective differentiation of epidermal keratinocytes (Menter and Griffiths, 2007; Nickoloff et al., 2007). Evidence also suggests that vitamin D administered with high doses of GCs could be useful as prophylaxis against GC-induced osteoporosis (Compston, 2007; Papierska and Rabijewski, 2007). Taking all these findings together, we propose that vitamin D may be useful for asthma control as a potential therapeutic adjunct with steroids.

In conclusion, we demonstrate that vitamin D exerts anti-inflammatory activity in ASM relevant to the pathogenesis of chronic inflammatory airway disease, such as asthma and chronic obstructive pulmonary disease. These findings support a role of vitamin D as an anti-inflammatory agent. Our findings open a new area of investigation, namely, to determine the molecular mechanisms by which vitamin D modulates the transcriptional regulation of pro-inflammatory genes in airway structural cells.

We thank Mary McNichol for assistance in the preparation of the paper. This study was supported by National Institutes of Health Grant 1 K99 HL089409-01 (to OT), RO1-HL081824 (to RAP), 1P30 ES013508 (to RAP), American Lung Association RG-49342-N (to OT), Parker B Francis Family Foundation (to OT) and by GlaxoSmithKline. OT is a Parker B Francis Fellow in Pulmonary Research.

Conflict of interest

The authors state no conflict of interest.