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

Background Inhalation of particles aggravates respiratory symptoms including mucus hypersecretion in patients with chronic airway disease and induces goblet cell hyperplasia (GCH) in experimental animal models. However, the underlying mechanisms remain poorly understood. Methods To understand this, the numbers of goblet cells, Muc5ac (+) expressing epithelial cells and IL-13 expressing mast cells were measured in the trachea of sham or TiO2 particles – treated rats using periodic acid-Schiff, toluidine blue and immunohistochemical staining. RT-PCR for Muc-1, 2 and 5ac gene transcripts was done using RNA extracted from the trachea. Differential cell count and IL-13 levels were measured in bronchoalveolar lavage (BAL) fluid. In pretreatment groups, cyclophosphamide (CPA) or dexamethasone (DEX) was given before instillation of TiO2. TiO2 treatment markedly increased Muc5ac mRNA expression, and Muc5ac (+) or PAS (+) epithelial cells 48 h following treatment. Results The concentration of IL-13 in BAL fluids was higher in TiO2 treated – rats when compared to those in sham rats (p < 0.05). Pretreatment with cyclophosphamide (CPA) decreased the number of neutrophils and eosinophils in BAL fluid of TiO2 treated – rats (p < 0.05), but affected neither the percentage of PAS (+) cells, nor IL-13 levels in the BAL fluids (p > 0.05). In contrast, pretreatment with dexamethasone (DEX) diminished the percentage of PAS (+) cells and the levels of IL-13 (p < 0.05). TiO2 treatment increased the IL-13 (+) mast cells (p < 0.05) in the trachea, which was suppressed by DEX (p < 0.05), but not by CPA pretreatment (p > 0.05). In addition there were significant correlations of IL-13 (+) rate of mast cells in the trachea with IL-13 concentration in BAL fluid (p < 0.01) and with the percentage of Muc5ac (+) cells in the sham and TiO2 treated rats (p < 0.05). Conclusion In conclusion, TiO2 instillation induces GCH and Muc5ac expression, and this process may be associated with increased production of IL-13 by mast cells.


Background
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 (TiO 2 ) 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 TiO 2 -exposed workers had respiratory symptoms accompanied by reduction in pulmonary function [16]. Because acute and chronic exposure to TiO 2 particles induces inflammatory responses in the airways and alveolar spaces of rats [17,18], TiO 2 -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].

Treatment protocols
Particles of TiO 2 (mean diameter = 0.29 µm, DuPont, Wilmington, DE) were suspended in endotoxin-free saline. The endotoxin concentration of the TiO 2 suspension was less than <0.32 EU/ml as measured with a limulus amebocyte lysate kit (QCL-1000; BioWhittaker, Inc., Walkersville, MD). Seven-week-old male Sprague-Dawley rats (Charles River Technology Inc.) received a single intratracheal instillation of homogeneous suspension of TiO 2 particles (4 mg/kg in 200 µl of endotoxin free water). In a pretreatment group, cyclophosphamide (CPA) (100 mg/kg, i.p.) was given 5 days before instillation of TiO 2 and a second injection of CPA (50 mg/kg, i.p.) 1 day before TiO 2 instillation. In the second pretreatment group, dexamethasone (DEX) (0.25 mg/kg, i.p.; Sigma, St. Louis, MO) was administered 24 h before TiO 2 instillation. The Institutional Animal Care and Use Committee of Soonchunhyang University approved the study protocols.

Preparation of lung tissues and morphological analysis
Rats were sacrificed at 4, 24, 48 and 72 hr after TiO 2 instillation by being anesthetized with pentobarbital sodium (65 mg/kg, i.p.) and bronchoalveolar lavage (BAL) was performed by 5 times instillation of 1 ml normal saline and gentle retrieval. Cell numbers were measured using a hemacytometer and differential cell counts were performed on slides prepared by cyto-centrifugation and Diff-Quik staining (Scientific Products, Gibbstowne, NJ). Immediately following BAL, the trachea was snap-frozen for RNA extraction or fixed with 4% paraformaldehyde in PBS and embedded in paraffin. The tissues were subjected to periodic acid-Schiff (PAS) and toluidine blue staining to permit measurement of goblet cells and mast cells, respectively. Morphometric analysis was performed under light microscopy at ×400 magnification. PAS positive epithelial cells and total epithelial cells were counted on the length of 250 µm basement membrane at each of four predetermined sites (12, 3, 6, 9 o

Measurement of IL-13 concentration in BAL fluids
The levels of IL-13 in the BAL fluids were measured with a quantitative sandwich enzyme-linked immunoassay kit (Biosource, Camarillo, CA). The lower limit of detection was approximately 1.5 pg/ml. Values below this limit were considered as zero for statistical analysis. Inter-and intra-assay coefficients of variance were less than 10%.

Statistical analysis
Differences between independent samples were compared using the Spearman test for continuous data. If differences were found significant, the Mann-Whitney U test was applied to compare differences between two samples. Differences were considered significant when the p value was less than 0.05. Results are expressed as means ± standard error of the mean (SEM) unless otherwise stated. The correlations were analyzed between the ratio of Muc5ac (+) expressing epithelial cell and the concentration of IL-13 in BAL fluid and the number of mast cell and the IL-13 positive rate of mast cells by Spearman's non-parametric correlation using SPSS (version 10.0, Chicago, USA)

Expression of Muc gene transcripts in the trachea of TiO 2 or saline -instilled rats
Total RNA was extracted from the trachea 24 h following treatment with saline or TiO 2 and analyzed for Muc1, Muc2, and Muc5ac transcripts by RT-PCR. As shown in Figure 1, Muc1, Muc2 and Muc5ac mRNAs were practically undetectable in sham-treated rats. In contrast, TiO 2 treatment markedly increased Muc5ac mRNA, but only modestly increased Muc2 mRNA. Muc1 mRNA was not seen in TiO 2 -treated rats.

The effect of TiO 2 instillation on Muc5ac-positive and PAS-positive epithelial cells in trachea
Rats were given a single intratracheal instillation of saline or TiO 2 and the percentage of Muc5ac-positive (Muc5ac (+)) and PAS-positive (PAS (+)) epithelial cells were measured. At 24 h after saline instillation, almost no PAS (+) or Muc5ac (+) epithelial cells were found in the trachea (Fig. 2Aa, b). TiO 2 instillation, however, induced PAS (+) or Muc5ac (+) cells in the trachea at 24 h (Fig. 2Ac, d). The percentage of Muc5ac (+) cells was significantly higher at 24 hr (p < 0.05) and further increased (Fig. 2B) in TiO 2 -instilled rats and maintained until 72 h when compared with those of sham rats (p < 0.01). The percentage of PAS (+) cells was very similar to that of Muc5ac (+) cells at 48 h after TiO 2 instillation ( Figure 2B).

Effects of cyclophosphamide and dexamethasone on the number of inflammatory cells and IL-13 levels in BAL fluid of TiO 2 -treated rats
The numbers of eosinophils and neutrophils are markedly increased in the BAL fluids at 48 h after TiO 2 instillation when compared with those in saline-treated rats (p < 0.05, respectively) ( Fig. 3A and 3B). Also, the levels of IL-13 in BAL fluids were significantly higher in TiO 2 -treated rats than those of sham rats at 48 h after treatment (p < 0.05) (Fig. 3D). Pretreatment with CPA prior to TiO 2 instillation significantly decreased the numbers of neutrophils and eosinophils in BAL fluids when compared with those in rats at 48 h after treatment with TiO 2 alone (p < 0.05, Fig.  3A &3B). Pretreatment with CPA, however, did not affect both the ratio of PAS (+) cells in the trachea and the IL-13 levels in BAL fluids of TiO 2 -treated rats (p > 0.05, Fig. 3C  &3D). Pretreatment with DEX prior to TiO 2 instillation significantly decreased the number of eosinophils in BAL fluid (p < 0.05, Fig. 3A), the ratio of PAS (+) cells in the trachea (p < 0.05, Fig 3C) and the levels of IL-13 in BAL fluid (p < 0.05, Fig. 4D) compared with those of rats instilled by TiO 2 alone.

Effects of cyclophosphamide and dexamethasone on the number and IL-13 expression of mast cells in TiO 2 -treated rats
Toluidine blue -stained mast cells were observed in and around the muscle layer of the trachea in saline-treated rats. The shape of the cells was relatively round with a single nucleus and a large cytoplasm containing granules (Fig. 4Ab). In TiO 2 -instilled rats, some mast cells showed an elongated and branching shape of the cytoplasm (Fig.  4Bb). The trachea of the saline-treated group contained no IL-13 (+) cells (Fig. 4Aa) in spite of the presence of mast cells (Fig. 4Ab). TiO 2 -instilled rats increased the number of mast cells when compared with the saline control group (p < 0.05, Figs. 4Bb and 4E). Serial section slides of the trachea showed that IL-13 protein was expressed exclusively on the mast cells in TiO 2 -treated rats (Fig.  4Ba). CPA pretreatment did not affect the TiO 2 -induced increase in the number of toluidine blue (+) mast cells positive for IL-13 (p > 0.05, Fig. 4Ca, 4Cb &4E). However, DEX pretreatment significantly decreased the number of toluidine blue (+) mast cells expressing IL-13 compared to those of TiO 2 -treated rats (p < 0.05, Fig. 4Da, 4Db &4E).

The correlation between the number of IL-13 expressing mast cells, the concentration of IL-13 in BAL and Muc 5ac positive epithelial cells in the airway
The number of mast cells in the trachea was significantly correlated with percentage of Muc5ac (+) epithelial cells and concentration of IL-13 in BAL fluid of TiO 2 -treated (n = 7) and sham (n = 6) rats (p < 0.001 and p < 0.0001, respectively, Table 1). However, the number of eosinophil and neutrophils in BAL fluids were correlated with neither the percentage of Muc5ac (+) epithelial cells nor the concentration of IL-13 in BAL fluid (p > 0.05, Table 1). In addition, there were significant correlations of IL-13 (+) rate of mast cells in the trachea with IL-13 concentration in BAL fluid (r = 0.782, p < 0.01, Fig. 5A) and with the percentage of Muc5ac (+) cells in the sham and TiO 2 treated rats (r = 0.604, p < 0.05, Fig 5B).

Discussion
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 TiO 2 is able to induce GCH within 24 h. The TiO 2 -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 TiO 2 -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][23][24][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 expression of Muc1, Muc2 and Muc5ac mRNA in TiO 2 treated rats Figure 1 The expression of Muc1, Muc2 and Muc5ac mRNA in TiO 2 treated rats. Rats were treated with TiO 2 , as described in Methods. Twenty-four hours after treatment, the levels of the Muc gene transcripts in the trachea were quantified using RT-PCR. GAPDH was used to ensure an equal loading of RNA samples. This figure is representative of 4 experiments.
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 TiO 2induced 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 TiO 2 -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 TiO 2 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 TiO 2 -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 TiO 2 -treated rats. It is well known The cell distribution in BAL fluid of TiO 2 instilled rats with or without pretreatment Figure 3 The cell distribution in BAL fluid of TiO 2 instilled rats with or without pretreatment. Rats were pretreated with CPA (n = 6) or DEX (n = 6) and then treated intratracheally with TiO 2 . Saline (n = 8) or TiO 2 (n = 8) was treated without pretreatment. At 48 h post-treatment, BAL fluids were collected and analyzed for the numbers of eosinophils (A), neutrophils(B), and the levels of IL-13(D). PAS (+) cells (C) were measured in the trachea as described in Methods. * p < 0.05 as compared with saline -treated group, † p < 0.05 as compared with TiO 2 -treated group.
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 TiO 2 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 TiO 2induced IL-13 overproduction is specific to TiO 2 or generally related to other particulates. However, base on the findings of particles such as diesel exhaust particles or car-bon black particle -induced the deviation to Th2 environment in antigen sensitized lung [11,12], TiO 2 -induced GCH via over production of IL-13 may be a general finding attributed to the particulate matters, but it remains unproven.

Conclusion
We demonstrated that a single intratracheal instillation of TiO 2 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.