Cigarette Smoke Suppresses the Surface Expression of c-kit and FcεRI on Mast Cells

Chronic obstructive pulmonary disease (COPD) is a multicomponent disease characterized by emphysema and/or chronic bronchitis. COPD is mostly associated with cigarette smoking. Cigarette smoke contains over 4,700 chemical compounds, including free radicals and LPS (a Toll-Like Receptor 4 agonist) at concentrations which may contribute to the pathogenesis of diseases like COPD. We have previously shown that short-term exposure to cigarette smoke medium (CSM) can stimulate several inflammatory cells via TLR4 and that CSM reduces the degranulation of bone-marrow-derived mast cells (BMMCs). In the current study, the effect of CSM on mast cells maturation and function was investigated. Coculturing of BMMC with CSM during the development of bone marrow progenitor cells suppressed the granularity and the surface expression of c-kit and FcεRI receptors. Stimulation with IgE/antigen resulted in decreased degranulation and release of Th1 and Th2 cytokines. The effects of CSM exposure could not be mimicked by the addition of LPS to the culture medium. In conclusion, this study shows that CSM may affect mast cell development and subsequent response to allergic activation in a TLR4-independent manner.


Introduction
The incidence of chronic respiratory diseases like chronic obstructive pulmonary disease (COPD) and asthma is increasing dramatically and currently affect the lives of approximately 300 and 200 million people, respectively, worldwide [1,2]. COPD is characterized by a complex interaction between inflammatory and structural cells, all of which have the capacity to release multiple inflammatory mediators [3]. Cigarette smoke (CS) is the major player in the pathogenesis of COPD [3]. Exposure to CS activates an inflammatory cascade in the airways resulting in the production of a number of potent cytokines and chemokines with accompanying damage to the lung epithelium, increased permeability, and recruitment of macrophages and neutrophils [4]. CS contains high levels of reactive oxygen species [5] and LPS [6,7]. LPS is a strong Toll-Like Receptor (TLR) 4 agonist [8]. TLRs are an evolutionarily conserved family of cell surface molecules which participate in innate immune response [9,10]. The effects of smoking on inflammatory cell maturation and differentiation have not been well described. Upon encountering pathogens and/or proinflammatory mediators, cells undergo a transformation process termed "maturation, " which, for example, enhances dendritic cell (DC) Ag-presenting capacity or ability to release inflammatory cytokines. The role of TLRs in maturation and development of DCs [11] and B cells [12] has been extensively described. In this regard, it has been reported that CS exposure leads to (a) decreased sputum mature DCs in healthy smokers and patients with COPD [13], (b) impaired DC maturation and Tcell proliferation in thoracic lymph nodes of mice [14] and (c) suppressed generation of IL-12 and IL-23 from DCs mediated through ERK-dependent pathways [15].
Parental smoking during childhood and personal cigarette smoking in teenage and early adult life are associated with a lower risk of allergic sensitization in those with a family history of atopy. The underlying mechanisms for this association remain to be determined, but the findings are consistent with the hypothesis that the immune-suppressant effects of CS protect against atopy [16]. So far few studies have reported on the role of mast cells in human smokers and in animal models of emphysema. Mast cells normally reside close to epithelia, blood vessels, nerves, smooth muscle cells, and mucus-producing glands [17]. Mast cells play a crucial role in allergic reactions [18]. Interestingly, emerging evidence also suggests a role of mast cells in the pathogenesis of emphysema [19,20]. Kalenderian et al. [19,20] found that the levels of mast cell mediators, such as histamine and tryptase, are considerably elevated in BALF from smokers. The importance of mast cells is further supported by the fact that mast cell tryptase activity is correlated with the severity of COPD [21], and in COPD patients an accumulation of mast cells in the airways has been observed [22]. Mast cells located here could be exposed to inhaled environmental challenges, and mast cell activation results in the coordinated release of proinflammatory mediators into the surrounding tissue; activation of this cell type may result in pathology associated with chronic inflammatory stimuli [23,24]. Mast cells play a crucial role in acute and allergic inflammation and have Fc RI on their surface [24]. Cross-linking of surface IgE molecules results in exocytosis of preformed mediators such as amines and proteases, as well as the release of newly generated mediators including leukotrienes, prostaglandins, and a variety of cytokines such as Th1 (TNF-, IL-6) or Th2 cytokines (IL-13, IL-4, IL-5) [24].
Previously, we showed that CSM (without IgE/Ag activation) does not trigger degranulation of bone marrowderived mast cells (BMMCs) but does induce the release of chemokines [2]. In addition, CSM exposure suppresses IgE-mediated mast cell degranulation and cytokine release but had no effect on leukotriene release. This suggests that exposure to CSM may lead to a reduced allergic activation of mast cells without affecting their response to other stimuli [25]. In contrast, CS exposure in vivo enhanced OVA-specific IgE levels, Penh values, and recruitment of inflammatory cells including mast cells in OVA-exposed allergic mice [26].
In the current study, we investigated the effect of CSM exposure on the mast cell development from bone marrow progenitor cells.

Production of Cigarette Smoke Medium (CSM).
Cigarette smoke-conditioned medium (CSM) was produced as described previously [25]. CSM was generated by burning reference cigarettes 2R4F (University of Kentucky, Lexington, KY), using the TE-10z smoking machine (Teague Enterprises, Davis, CA, USA), which is programmed to smoke cigarettes according to the Federal Trade Commission protocol (35 mL puff volume drawn for 2 s, once per minute). Briefly, this machine was used to direct main-and side-stream smoke from one cigarette through a 5 mL culture medium (RPMI without phenol red). Hereafter, absorbance was measured spectrophotometrically, and the media were standardized to the absorbance at 320 nm. The pH of the resultant extract was titrated to pH 7.4 and diluted with medium. This concentration (optical density [OD] = 4.0) was serially diluted with untreated media to 0.75%, 1.5%, and 3% OD and used in the indicated experiments. In preliminary experiments, a CSM concentration of 1.5% was found optimal in the culture experiments.

Mouse Bone Marrow-Derived Mast Cell (BMMC) Cultures and CSM Treatment.
BMMCs were generated from bone marrow of male BALB/cBy mice as described previously [27]. Cells were cultured in RPMI medium supplemented with mitogen-stimulated spleen cell conditioned medium (see below) [28].
Cells were used for the experiments after 3 weeks when a mast cell purity >95% was achieved. Bone marrow cells were cocultured (1 × 10 6 /mL) with either 1.5% CSM or LPS (100 ng/mL) during the third week of culture.

Toluidine Blue
Staining. The granularity of the mast cells was determined by toluidine blue staining [29]. In brief, the cells were cytospun, fixed with Carnoy's fluid, and then stained by either 2 minutes with acid toluidine blue (pH = 2.7). Cells were examined by light microscopy.

Mast Cell Degranulation
Assay. The degranulation assay was performed as described before [28]. Briefly, approximately 2-3 × 10 6 cells from each group were resuspended in culture medium (enriched medium) and incubated with 1 g/mL anti-DNP-IgE for 2 hr. After that, cells were washed and resuspended at a density of 0.6 × 10 6 cells/mL. Cells were aliquoted in 96 well plates (

Statistical Analysis.
Experimental results are expressed as mean ± S.E.M. Results were tested statistically by an unpaired two-tailed Student's -test or one-way ANOVA, followed by a Newman-Keuls test for comparing all pairs of groups. Analyses were performed by using GraphPad Prism (version 5.04). Results were considered statistically significant when < 0.05.

CSM Reduced the Granularity of Mast Cells during
Culturing. Bone marrow cells were cultured with CSM or LPS during the third week of mast cell development. Cell granularity was analyzed by staining with toluidine blue (Figure 1(a)). Coculturing cells with CSM (1.5%) decreased the granularity of mast cells (Figure 1(b)). LPS (1 g/mL) did not affect the granularity of cultured mast cells (Figure 1(c)).

Long-Term Exposure to CSM Modulates IgE/Ag-Mediated Degranulation and Cytokine Production by Mast Cells.
Stimulation with IgE/Ag caused a dose-dependent degranulation of mast cells in the control group (Figure 3). Coculturing mast cells with CSM (1.5%) reduced IgE-mediated degranulation ( Figure 3). In contrast, LPS, a TLR4 agonist, did not affect IgE/antigen-induced BMMC degranulation (Figure 3).

Discussion
In the current studies, we further investigated the effects of CSM on the development and function of primary cultured bone-marrow-derived mast cells. We show that BMMC exposed to CSM during development from progenitor cells inhibited mast cell development as determined by toluidine staining and expression on c-kit and Fc RI. Furthermore, the release of both Th1 and Th2 cytokines in response to Fc RI activation was reduced. Interestingly, the TLR4 agonist LPS did not affect these parameters and even slightly increased IL-4 production.
Mast cells are important in allergic airway diseases, but they have remained poorly studied in nonallergic inflammatory airway diseases like COPD. Mast cells are of particular interest due to their ability to promote airway remodeling and mucus hypersecretion. Clinical data show increased levels of mast cell tryptase and degranulated mast cells in the lavage and bronchial tissue of smokers [30][31][32][33]. Moreover, CS exposure facilitates allergic sensitization in mice [34].
We have previously reported that short-term exposure of mature mast cells to CSM attenuated their response to allergic stimuli [28]. Kim et al. also showed an inhibitory effect of CSM on mast cell activation which suggests that CS may contribute to the reduced allergic symptoms observed in smokers [26].
Mast cells are functionally dynamic effector cells in innate and adaptive immunity [24]. Two mast cell surface receptors c-Kit and Fc RI mediate activation via innate and adaptive immune mechanisms, respectively [17,23,24]. c-Kit is expressed on both mature mast cells and on the earliest mast cell progenitors [35,36]. c-Kit is expressed both as a soluble form and on the cell membranes [36]. Although c-Kit represents a major growth and differentiation factor for both murine and human mast cells [35,37] it also promotes c-Kit-dependent mast cell mediator release [38] as well as the release of mast cells mediators via IgEdependent mechanisms [39]. IgE-dependent allergic diseases are initiated by multivalent binding of allergens to IgE that is bound to Fc RI on mast cells [40]. Fc RI plays a critical role in allergic reactions. It is the major surface receptor through which mast cells direct immunologically specific secretory effects such as the release of preformed cytoplasmic granuleassociated mediators and the generation and release of lipid mediators and cytokines [41]. Thus, the suppression of c-Kit and Fc RI on the mast cells by CSM could account for decreased responsiveness of mast cells to IgE/Ag activation.
Our study suggests that LPS may not be involved in the mechanism by which CSM affects mast cell maturation and activation.
Mast cells express functional TLRs [42] which may account for the protection conferred by mast cells against bacterial and parasitic infections [43]. Activated mast cells release an array of potent inflammatory mediators by rapid discharge of preformed mediators in granules, the generation of inflammatory lipids from arachidonic acid, and the production of numerous Th2-type cytokines and chemokines [44]. All these responses are evoked by allergens via Fc RI, while stimulation of mast cells via TLR2 and TLR4 receptors results primarily in generation of cytokines such as IL-4, IL-5, IL-6, IL-10, IL-13, and TNF- [43]. Saluja et al. recently showed that prolonged exposure of mast cells to LPS amplifies Fc RI-mediated degranulation, lipid mediator generation, and cytokine production [45]. This is in contrast to the effects found in this study, where LPS treatment did not increase IgEmediated mast cell activation. The discrepancy may be due to the different mouse strains used in both studies and the time of treatment during mast cell development.
In conclusion, our study suggests that CSM, independent of TLR4 signaling, suppresses the maturation and function of mast cells. This suppressive effect of cigarette smoke on mast cells may account for the reduced allergic response seen in animal models of cigarette-smoke-induced emphysema [46].