Titanium dioxide particle – induced goblet cell hyperplasia : association with mast cells and IL-13

Excessive mucus secretion is one of the major clinical manifestations of chronic airway diseases such as asthma, chronic bronchitis, and cystic fibrosis [1]. The excessive mucus is attributed to goblet cell hyperplasia (GCH) and submucosal gland hypertrophy, which are hallmarks of airway remodeling in chronic airway diseases [2, 3]. Air pollution aggravates respiratory symptoms in patients with chronic airway diseases. Chronic obstructive pulmonary disease (COPD) patients living in communities exposed to high levels of air pollution have faster rates of decline in lung function than patients living in areas with low pollution [4]. The level of environmental particles is also positively correlated with exacerbation of asthma [5].

Airborne particulate matter less than 10 μm in aerodynamic diameter (PM10) is a complex mixture of organic and inorganic compounds containing sulfates and various metals such as aluminum, calcium, copper, iron, lead, magnesium, titanium, and zinc [6]. Clinically, PM10 particles are thought to provoke airway inflammation with the release of mediators that are capable of exacerbating lung disease in susceptible individuals [5, 7]. This assumption is based on experimental evidence of airway inflammation following direct instillation or inhalation of PM10 particles in animal models [8]. Furthermore, inhaled particles directly stimulate macrophages and epithelial cells to produce inflammatory cytokines such as TNF-α, GM-CSF and IL-8 [9, 10], which induce neutrophil- and eosinophil-mediated airway inflammation, and eventually lead to GCH. Recently, particle exposure favors the antigen – sensitized lung toward Th2 environment with over secretion of IL-13, IL-4 [11] and IL-5 [12]. Beside the inflammatory cell mediated – GCH, IL-13 directly induces GCH and Muc5AC gene expression through the signaling of IL-4Rα and IL-13Rα [13, 14]. Therefore, we hypothesized that particles induce GCH via over-production of IL-13 by recruited inflammatory cells.

Titanium dioxide (TiO₂) particles, one component of PM10, are found in dusty workplaces such as industries involved in the crushing and grinding of the mineral ore rutile [15]. It was reported that 50% of TiO₂-exposed workers had respiratory symptoms accompanied by reduction in pulmonary function [16]. Because acute and chronic exposure to TiO₂ particles induces inflammatory responses in the airways and alveolar spaces of rats [17, 18], TiO₂ – instilled rat may be a good model to study the particle induced – airway injury. In this study, we evaluated the role of neutrophilic and eosinophilic inflammation by pretreatment with cyclophosphamide inducing neutropenia [19] and the association of IL-13 by pretreatment with dexamethasone suppressing IL-13 gene expression [20].

Although air pollution contains heavy metallic environmental particles that increases morbidity and mortality of the patients with chronic airway diseases [4, 5], the underlying mechanisms of mucus hyperproduction causing airway obstruction has not been revealed in detail. In this study, we demonstrated that a single instillation of TiO₂ is able to induce GCH within 24 h. The TiO₂-induced GCH is associated with a dramatic increase in Muc5ac gene and protein expression in the present study (Figure 1 &2). Up regulation of Muc5ac gene in TiO₂ – induced GCH is thought to be a common pathway in the process of GCH because MUC5AC has been demonstrated to be a major MUC gene during the process of GCH observed in the other non-particulates experimental model of airway diseases [22‐25] and the asthmatics [26]. GCH is known as associated with airway inflammation and can be experimentally induced by various inflammatory agents such as LPS [22], neutrophil elastase [27], cathepsin B [23], IL-4 [25], IL-9 [28], and IL-13 [29, 30].

The exact mechanism of GCH, however, may differ in the experimental models. Neutrophils or eosinophils have been implicated in the induction of GCH in some animal models [30, 31]. Neutrophils and eosinophils depleted rats using CPA or specific antibodies inhibit granulocyte in agarose plug-induced and IL-13-induced GCH model [29, 31]. The epidermal growth factor receptor cascades are showed to be involved in underlying mechanism of the neutrophils – induced GCH [29, 31]. However, in the present study we showed that depletion of these inflammatory cells by pretreatment with CPA similar dose used in the previously study [29, 31] did not prevent TiO₂-induced GCH (Figure 4). Because cyclophosphamide effectively suppressed the number of neutrophils and eosinophils in peripheral blood (data not shown) and airways in the present study although not complete (Figure 4), our data indicates that these inflammatory cells may be not responsible for the TiO₂-induced GCH. The dissociation of GCH from airway eosinophilia has been well documented in murine asthma models, in which anti-IL-5 (TRFK-5) [32], or IL-5 deficiency [33] reduced airway eosinophilia without affecting the induction of GCH. Therefore, depending on the experimental models investigated, the induction of GCH may not require neutrophils and eosinophils. Furthermore, IL-13 is known to induce GCH without any help of other inflammatory cells [24] and has been clearly shown to play a single, common pathway by which GCH is induced by CD4+ cells and IL-9 [34]. This process needs IL-4 receptor alpha, but not IL-4 or IL-5 [33, 34]. These data suggested a possibility that IL-13 is also involved in the particle – induced GCH.

In the present study, the levels of IL-13 in BAL fluids increased after TiO₂ instillation concomitantly with the development of GCH and the increase of IL-13 was completely abolished by pretreatment with DEX (0.25 mg/Kg), but not by that with CPA (Figure 4). These results suggest that the elevation of IL-13 may be associated with particles such as TiO₂-induced GCH without any assistance of neutrophils or eosinophils. The in vivo effect of dexamethasone has been also demonstrated in allergic asthma model [35]. Dexamethasone (4 mg / kg) effectively abolishes allergic airway inflammation in mice by suppression of IL-13 m-RNA and protein expression [35]. The exact biochemical mechanism of GCH induction by IL-13 is not fully understood. One possible explanation is that IL-13 converts the bronchial epithelium from an absorptive to a secretory phenotype through loss of an amiloride-sensitive current and an increase in calcium-sensitive apical anion conductance [36]. The increase in apical anion conductance in the airway epithelium is most likely due to the ability of IL-13 to induce expression of hCLCA1/mCLCA3, which encodes a calcium-activated chloride channel. This channel is necessary and sufficient for the development of GCH and mucus hypersecretion in some experiments [37].

Besides Th2 cells, IL-13 is produced by mast cells, eosinophils [38, 39], and macrophages [40]. Since IL-13 was not decreased in rats of which eosinophils depleted by pretreatment of CPA (Figure 4), we can exclude eosinophils as the source of IL-13. Interestingly, serial thin section slides revealed that the IL-13 positive cells are mast cells, as shown by staining with toluidine blue. Also, we found the significant correlation between the IL-13 (+) rate of mast in tissue, concentration of IL-13 in BAL fluid and Muc5ac positive cells (Figure 5 and table 1). Based on these data, mast cells may be the cellular source for IL-13 present in the airways of TiO₂-treated rats. It is well known that mast cells produce IL-13 when stimulated with antigen [39] and that the synthesis can be suppressed by dexamethsone [20]. Our finding showed that TiO₂ instillation increased the numbers of IL-13 expressing mast cells and Muc5ac (+) goblet cells, both of which were decreased by dexamethsone pretreatment is a novel finding to our knowledge. It is not known whether TiO₂ – induced IL-13 overproduction is specific to TiO₂ or generally related to other particulates. However, base on the findings of particles such as diesel exhaust particles or carbon black particle – induced the deviation to Th2 environment in antigen sensitized lung [11, 12], TiO₂ – induced GCH via over production of IL-13 may be a general finding attributed to the particulate matters, but it remains unproven.

We demonstrated that a single intratracheal instillation of TiO₂ particles induces GCH and Muc5ac gene expression within 24 h in rats, and that this process may be associated with elevated amount of IL-13 derived from mast cells. The present study may provide experimental evidences to support that patients with chronic airway disease may aggravate their symptoms and airway functions in the heavily polluted environment of particulate matters.