Ketotifen

Background: It is well-known that the expression of T helper (Th) type 2 cytokines such as interleukin (IL)-4 and IL-5, and their receptors, is up-regulated within the airways of allergic asthmatic patients. Furthermore, higher numbers of cells producing GATA-3, c-MAF, and signal transducer and activator of transcription factor (STAT)-6, which are transcription factors (TFs) that are implicated in the regulation and signaling of the Th2 cytokines, have been observed in bronchial biopsy specimens from asthmatic patients but not in those of healthy control subjects.

Methods: We examined whether these mediators also can be detected in induced sputum. Immunoreactivity for IL-4R[alpha], IL-5R[alpha], GATA-3, c-MAF, and STAT-6 was investigated in samples of induced sputum from asthmatic patients (n = 8) and healthy control subjects (n = 8).

Results: Our results showed that the numbers of cells expressing IL-4 receptor et (Ret) and IL-5R[alpha] were higher in samples from asthmatic patients compared to those of control subjects (p < 0.01). More cells exhibiting GATA-3, c-MAF, and STAT-6 immunoreactivity also were found in asthmatic patients vs those in control subjects (p < 0.005). Furthermore, the expression of STAT-6 and IL-4R[alpha], GATA-3 and IL-5R[alpha], and c-MAF with both IL-4R[alpha] and IL-5R[alpha] was correlated (p < 0.05).

Conclusions: This study demonstrated that induced sputum provides sufficient sensitivity for examining the pathways of cytokine signaling, cytokine receptor signaling, and intracellular signaling. Furthermore, these data show correlations between the expression of Th2 cytokine receptors and associated TFs in the human lung, which has not been documented previously.

Key words: asthma; induced sputum; transcription factors

Abbreviations: APAAP = alkaline phosphatase anti-alkalinephosphatase; ICC = immunocytochemistry; IL = interleukin; MBP = major basic protein; R[alpha] = receptor or; STAT = signal transducer and activator of transcription factor; TBS = trisbuffered saline: TF = transcription factor

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Asthma (1) is characterized by reversible airway obstruction, bronchial hyperresponsivness, production of IgE, increased numbers of eosinophils and CD4 T cells, as well as elevated levels of T helper (Th) type 2 cytokines, especially interleukin (IL)-4 and (2) IL-5, (1,2) within samples of bronchial mucosa and BAL fluid. IL-4 elicits its actions by binding specifically its cell surface receptor IL-4 receptor (Ra), which is also part of the IL-13 receptor complex. The interaction of the IL-4R[alpha] with the [gamma]c chain of the IL-2 receptor results in increased binding affinity. (3) Similarly, IL-5 has its own specific cell surface receptor, IL-5Rot, while the [beta]-subunit of the receptor complex is also shared with IL-3 and granulocyte-macrophage colony-stimulating factor receptors. (4) Like their ligands, the number of cells expressing IL-4R[alpha] and IL5R[alpha] is increased in the bronchial and nasal mucosal biopsy specimens of subjects with allergen-induced rhinitis and atopic asthma, respectively. (5,6)

The production of IL-4 and IL-5 is mediated by the transcription factors (TFs) GATA-3 and c-MAF. These factors are found at low levels in naive T cells, and their numbers are increased during Th2 phenotype activation. (7,8) GATA-3 is selectively expressed in Th2-type cytokines (but not Th1-type cytokines) producing T cells, (9) and blocking GATA-3 in Th2-type cells inhibited the expression of both IL-4 and IL-5 production. The effects of c-MAF appear to be more selective, since in vitro studies have demonstrated that this factor can activate the promoter for IL-4, but not for IL-5 or IL-13. (10,11) Signal transducer and activator of transcription (STAT)-6 is an integral component of IL-4-induced allergic inflammation. (12) In mice, the disruption of the STAT-6 gene resulted in the failure of IL-4 signaling (13) and in a loss of Th2 responses such as isotype switching to IgE. (14)

Increased numbers of cells expressing GATA-3 messenger RNA have been observed within bronchial biopsy specimens of atopic asthmatic patients, which is a situation that has been associated with IL-5 expression and airway hyperresponsiveness. (15) Similarly, a higher number of GATA-3, c-MAF, and STAT-6 immunoreactive cells have been observed within the airways of asthmatic patients compared to those of control subjects. (16) However, despite in vitro and animal studies demonstrating the relationship between Th2 cytokines and related TFs, (17-21) these associations with the corresponding receptors have not been well-documented in the human lung.

Sputum induction by hypertonic saline solution represents a safe and noninvasive tool with which to investigate the pathology of airway inflammation in asthma patients. As such, it is now a widely used technique and involves relatively little inconvenience to the patient. Here, we examined whether samples of induced sputum provide an accurate reflection of the inflammatory milieu observed within bronchial tissue, in regard to the expression of IL-4R[alpha], IL-5R[alpha], GATA-3, c-MAF, and STAT-6 in samples of induced sputum obtained from asthmatic and non-asthmatic control subjects. We also determined the phenotype of these cells and the relationship between the receptors and TFs.

MATERIALS AND METHODS

Subjects

Eight patients who fulfilled the American Thoracic Society criteria for asthma, had been classified as having mild asthma, and had been recruited from the Asthma Clinic at the Montreal Chest Institute, McGill University (Montreal, QC, Canada), and eight nonasthmatic, nonatopic, nonsmoker control subjects were included in this study. Asthmatic subjects had shown airway reversibility to inhaled [[beta].sub.2]-agonists or increased airway responsiveness to methacholine (ie, FE[V.sub.1], < 8 mg/mL). Asthmatic medications included only inhaled short-acting selective [[beta].sub.2]-agonists. Atopy was based on skin-test reactivity to a series of common allergens. The clinical characteristics of the subjects are given in Table 1. Asthmatic patients were nonsmokers or ex-smokers who had stopped smoking for at least 12 months and had smoked < 5 pack-years in their lifetime. Exclusion criteria included the use of inhaled or oral corticosteroids, nonsteroidal anti-inflammatory medications (ie, cromolyn and ketotifen), theophylline, long-acting B2 agonists, leukotriene antagonists, or antihistamines within the 3 months prior to entering the study, the occurrence of respiratory tract infection within the 6 weeks prior to entering the study, or immunotherapy within the previous 12 months.

Study Design

The Ethics Review Board of the Montreal Chest Institute, McGill University, approved the study, and all subjects gave written informed consent. The patients and control subjects were screened by a questionnaire, spirometry, and a methacholine inhalation test (if FE[V.sub.1] was > 70%). Allergy skin-prick tests were performed, and atopy was defined as the appearance of a skin wheal of > 3 mm in response to > 1 of 13 common allergens. Blood was drawn for the performance of a CBC count, differential cell count, and total serum IgE measurement. Sputum was induced in the laboratory at least 48 h after methacholine bronchoprovocation. The difference in FEV1 on the 2 test days did not exceed 10%.

Sputum Induction

Sputum induction was performed using a modification of the method of Pin and coworkers. (22) Subjects were administered salbutamol (200 [micro]g) to inhibit possible bronchoconstriction during sputum induction followed by the inhalation of a 4.5% hypertonic saline solution that was generated by an ultrasonic nebulizer (DeVilbiss Ultra-Neb 99; Sunrise Medical; Somerset, PA) for 15 min. The procedure was interrupted every 2 min to measure peak expiratory flow. Subjects were asked to rinse the mouth and blow the nose to minimize contamination with saliva and postnasal drip, and also were instructed to cough sputum into a sterile container. If a fall in peak expiratory flow of > 10% occurred, the procedure was terminated.

Sputum Examination

Sputum sample volumes were recorded. Sputum that was macroscopically free of salivary contamination was selected to minimize squamous cell contamination and was processed within 2 h by a modification of the technique described by Pizzichini and coworkers. (23) Briefly, sputum was selected from the expectorate, and a weighed aliquot was dispersed with four volumes of freshly prepared 0.1% dithiothreitol followed by four volumes of phosphate-buffered saline solution, after which the dispersed sample was filtered through a 48-[micro]m nylon mesh. The sample was centrifuged at 300g for 10 min. The cell pellet was resuspended in phosphate-buffered saline solution, and total cell counts of leukocytes and cell viability were determined through the use of trypan blue exclusion. For immunocytochemistry (ICC), cells were adhered to microscope slides using a cytocentrifuge at 650 revolutions per minute for 6 min (Shandon Somerest; Cheshire, UK) and then were fixed in acetone/ methanol (60:40) for 7 rain, air dried, and stored at -20[degrees]C until further use. A sample was considered adequate if, on differential cell counting, the slides contained < 20% squamous epithelial cells.

Antibodies

Monoclonal antibodies that were raised against specific cellular markers for eosinophils (ie, major basic protein [MBP]) [gift from R. Moqbel, MD; University of Alberta; Edmonton, AB, Canada], T lymphocytes (ie, CD3 cells; Becton Dickinson; Mississauga, ON, Canada), epithelial cells (ie, anti-cytokeratin CAM-5.2 [a murine monoclonal antibody]; Becton Dickinson), macrophages (ie, CD68 cells; Dako Diagnostics Canada Inc; Mississauga, ON, Canada), and neutrophils (ie, elastase; Dako Diagnostics Canada Inc) were used. Cytokine receptor monoclonal antibodies against IL-4R[alpha] (Immunex; Seattle, WA), and IL-5R[alpha] (a gift from J. Taveruier, MD; Roche Research; Brussels, Belgium) were employed, while GATA-3, STAT-6, and c-MAF polyclonal antibodies (rabbit antihuman antibodies; Santa Cruz Biotech; Santa Cruz, CA) were used.

ICC

ICC was performed using a modified alkaline phosphatase anti-alkaline phosphatase (APAAP; Dako Diagnostics Canada Inc) method, as previously reported. (24) Briefly, slides were incubated with the specific antibodies overnight at 4[degrees]C. After washing in tris-buffered saline (TBS) solution, the secondary rabbit antimouse antibodies (1:60 dilution; Becton Dickinson) were applied, and the slides were washed in TBS and incubated with APAAP mouse antibodies (1:60 dilution; Becton Dickinson). The reaction was visualized by dissolving the Fast Red TR (Sigma Chemicals Co) chromogen in an alkaline-phosphatase substrate. This enzymatic reaction results in the precipitation of red particles onto cells bound to the antiphosphatase antibody, and thereby identifies cells bound to the primary antibody. All the slides were counterstained with hematoxylin and were mounted before examination. Negative control experiments were performed in a similar manner but in the absence of the primary antibody. The primary antibody was replaced by nonspecific mouse Ig or by a TBS solution.

Double Immunostaining

To identify the phenotype of GATA-3, c-MAF, and STAT-6 immunoreactive-positive cells, colocalization studies were performed on preparations from induced sputum samples. Briefly, endogenous peroxidase activity was blocked using 1% [H.sub.2][O.sub.2] in TBS for 30 min. (25) The primary antibody was applied to the cytospin consisting of GATA-3, STAT-6, or c-MAF rabbit antihuman polyclonal antibodies, which were used to detect TF immunoreactivity, and the appropriate monoclonal antibodies to determine cellular phenotype. After incubation with the appropriate secondary antibodies (ie, biotinylated swine anti-rabbit antibody; Dako Diagnostics Canada Inc), a tertiary layer of streptavidin peroxidase and murine APAAP conjugate then was applied. Cytospins were developed sequentially in Fast Red (APAAP substrate) and diaminobenzidine (peroxidase substrate). GATA-3, c-MAF, and STAT-6 immunoreactive cells stained brown, and cells immunoreactive for CD3, MBP, and cytokeratin stained red. Double-immunoreactive cells stained reddish-brown. To colocalize c-MAF, STAT-6, and GATA-3 with IL-4R[alpha] and IL-5R[alpha], we also used the double-ICC technique by applying an appropriate monoclonal antibody to detect IL-4R[alpha] and IL-5R[alpha], and polyclonal antibody to detect c-MAF, STAT-6, and GATA-3. The negative control subjects included omission of the primary antibodies or the use of irrelevant isotype and species-matched primary antibodies.

Data Analysis

The numbers of positive cells were counted per 1,000 total cells, excluding squamous cells with the help of a phase contrast lens. Positive cells were reported as a mean percentage [+ or -] SD. To avoid observer bias, slides were coded prior to analysis and were read in a blinded fashion. At least two cytospins were counted for each immunocytochemical marker, and the mean value of these slides was reported. Significant differences were detected by analysis of variance and Mann-Whitney U test. Correlation coefficients were determined using the Spearman correlation test. Significance was accepted at the 95% level of confidence.

RESULTS

Study subjects and patients were well-matched for age, sex, and clinical characteristics (Table 1). Cellular phenotypes in induced sputum were compared between asthmatic patients and control subjects (Table 2). The total volume of sputum in asthmatic patients was 3.4 mL (range, 1.7 to 4.9 mL) compared to 1.9 mL (range, 1.1 to 3.3) in control subjects (p < 0.05). The total cell count in asthmatic subjects was 4.4 x [10.sup.6] cells/mL (range, 2.4 to 10.5 x [10.sup.6] cells/mL) compared to 2.3 x [10.sup.6] cells/mL (range, 1.4 to 4.7 x [10.sup.6] cells/mL) in control subjects. Cell viability was > 85% in all samples. The percentage of MBP-positive cells in asthmatic patients (5.1%; range, 1.8 to 9.6%) was significantly greater than that in nonasthmatic subjects (0.6%; range, 0.1 to 1.2%; p < 0.05). Similarly, the percentage of epithelial cells was significantly higher in asthmatic patients (7.1%; range, 4.0 to 9.0%) compared to nonasthmatic subjects (1.4%; range, 0.9 to 1.8%; p < 0.01). There was no difference between asthmatic patients and healthy control subjects in the percentage of T cells (CD3+), macrophages (CD68+), or neutrophils (elastase +).

Th2 Cytokine Receptors and TFs in Induced Sputum

Figure 1 illustrates the constitutive expression of IL-4R[alpha] and IL-5R[alpha] (3.1% [+ or -] 0.9% and 3.2% [+ or -] 1.1%, respectively), since immunoreactivity was observed in induced sputum samples obtained from healthy control subjects. However, the percentages of cells expressing these receptors were significantly higher in samples from asthmatic patients (12.2% [+ or -] 1.5% vs 10.2% [+ or -] 1.1%, respectively; p [less than or equal to] 0.01). Immunoreactivity for GATA-3, c-MAF, and STAT-6 also was present in all subjects (Fig 2). In healthy control subjects, STAT-6 (mean, 4.5% [+ or -] 0.8%), c-MAF (mean, 3.5% [+ or -] 0.9%), and GATA-3 (mean, 1.7% [+ or -] 0.6%) were well-represented, although in sputum from asthmatic patients the proportion of cells expressing all three of these TFs was significantly higher (14.4% [+ or -] 2.2%, 12.5% [+ or -] 1.9%, and 9.4% [+ or -] 1.5%, respectively; p < 0.005) [Fig 3].

[FIGURES 1-3 OMITTED]

Phenotype of Cells Expressing GATA-3, STAT-6, and c-MAF

Double immunostaining was performed on samples of induced sputum from asthmatic patients to determine the phenotypes of cells expressing GATA-3, c-MAF, and STAT-6 (Table 3). The majority of cells expressing GATA-3 immunoreactivity were CD3+ T cells and eosinophils. T cells also accounted for a moderate percentage of c-MAF and STAT-6 immunoreactive cells, a small percentage of which was attributed to eosinophils. In addition to the inflammatory cell component, all three TFs were expressed by a minor percentage of cytokeratin-expressing epithelial cells.

Colocalization of Th2-Associated TFs and Th2 Cytokine Receptors

Double immunostaining also was employed to determine the percentage of cells coexpressing GATA-3, c-MAF, or STAT-6 and IL-4R[alpha] or IL-5R[alpha] immunoreactivity (Table 4). The majority of cells expressing GATA-3 also expressed the IL-5R[alpha] and, to a lesser extent, the IL-4R[alpha]. On the other hand, most of the STAT-6-positive cells colocalized with IL-4R[alpha], and few cells colocalized with IL-5R[alpha]. c-MAF immunoreactivity appeared to colocalize equally with both the IL-4R[alpha] and IL-5R[alpha].

Correlation of [alpha]IL-4R and [alpha]IL-5R With GATA-3, c-MAF, and STAT-6

Because there was increased expression of both the Th2-associated TFs and cytokine receptors in the induced sputum of asthmatic subjects, we examined whether the expressions of these mediators were correlated. There were significant correlations between the percentage of IL-4R[alpha] and STAT-6 immunoreactive cells ([r.sup.2] = 0.71; p < 0.01) and between IL-5R[alpha] and GATA-3 immunoreactive cells ([r.sup.2] = 0.64; p < 0.02) in induced sputum samples of asthmatic subjects. The percentages of cells expressing IL-4R[alpha] and IL-5R[alpha] also were correlated ([r.sup.2] = 0.61, p < 0.09 and [r.sup.2] -0.67, p < 0.02, respectively) with c-MAF immunoreactivity in induced sputum samples of asthmatic subjects.

DISCUSSION

Previously, we reported (16) an increase in GATA-3, c-MAF, and STAT-6 expression within the bronchial mucosa of asthmatic patients at levels that were significantly higher than those in healthy control subjects. The present study was designed to determine whether the expression of these mediators of allergic inflammation also could be detected using the noninvasive sampling technique of induced sputum. We demonstrate here the constitutive expression of IL-4R[alpha], IL-5R[alpha], GATA-3, c-MAF, and STAT-6 in sputum samples obtained from healthy subjects as well as significantly higher numbers of cells expressing these receptors and TFs within sputum samples from asthmatic patients. These findings provide clear evidence that, in addition to the expression of the soluble mediators that have been reported previously, (26,27) cell surface receptors and intracellular TFs also can be detected within samples of induced sputum. Furthermore, this work illustrates the close relationship that exists between the expression of Th2 cytokine receptors and the related TFs.

GATA-3 is necessary and sufficient for the expression of IL-5, (7) and the inhibition of its expression in Th2 cells has been associated with decreased levels of IL-4, IL-5, and IL-13. (9) Although we did not examine IL-5 expression here, we did observe that 74% of cells expressing IL-5R[alpha] also express GATA-3 and that there was a strong correlation between GATA-3 and IL-5R[alpha] expression. This could suggest that, in addition to regulating IL-5 expression, GATA-3 also may participate in the transcriptional regulation of the IL-5 receptor. Indeed, there are a number of putative GATA-3 binding sites within the promoter of the IL-5R gene. (28) The lack of correlation between IL-4R[alpha] and GATA-3 indicates that, at the very least, these factors do not possess similar patterns of expression. In light of our previous observation that IL-4R[alpha] is found in a vast number of different cell types within the lung, (5) it was not particularly surprising that there was no correlation between IL-4R[alpha] immunoreactivity and the more tightly regulated expression of GATA-3. On the other hand, both IL-4R[alpha] and IL-5R[alpha] expression were significantly correlated with c-MAF, which reportedly is a critical factor for IL-4 production, but not IL-5 production. (10) One may speculate that, like B cells, the [alpha]IL-4R may be up-regulated by its own ligand (IL-4) on T cells, (29) leading to Th2 development and the subsequent eosinophil expression of the IL-5R. This is supported by our colocalization data indicating that the majority of c-MAF immunoreactive cells were T cells (43%) and eosinophils (12%). However, while the majority of GATA-3 immunoreactivity was attributed to T cells, eosinophils, and epithelial cells (80%), more than half the c-MAF immunoreactive cells (56%) could not be accounted for by these cell types. It has been reported that c-MAF is also expressed by mast cells, (30) and as such it is possible that these cells may contribute some of the c-MAF immunoreactivity that we observed.

STAT-6 immunoreactivity also was increased within samples of induced sputum from asthmatic patients compared to healthy control subjects. We observed that STAT-6 was significantly correlated with the IL-4R[alpha], in accord with in vivo and in vitro findings that STAT-6 mediates signaling of the IL-4 receptor. (22,23) Although our colocalization data demonstrate that STAT-6 expression was attributed in large part to T cells (44%), eosinophils (10%), and epithelial cells (5%), these cell types appear to account for only 50 to 60% of STAT-6 expression. Since STAT-6 is expressed constitutively in the cytoplasm of most cells, (31,32) including macrophages, (33) it is likely that these cell types also contribute to the observed STAT-6 immunoreactivity. Both IL-4 and IL-13 use the IL-4R[alpha] as a receptor component, (34) and the coexpression of human IL-13R[alpha] with IL-4R[alpha] results in substantial binding activity for IL-13. (35) STAT-6 also mediates IL-13 signaling, (36) and, while this cytokine does not appear to act on T cells, it has been reported (37) to activate human B lymphocytes and monocytes. As such, it is important to note that not all the STAT-6 that we observed here may be specifically related to IL-4 signaling. While the 5% of STAT-6 immunoreactivity attributed to epithelial cells may seem to be small, there is much evidence that this cell type plays an integral role in the allergic response. We and others have shown that airway epithelial cells within bronchial biopsy and induced sputum samples express RANTES (regulated on activation normal T cell expressed and secreted), MCP-4, eotaxin, IL-16, and IL-8. (27,38-40)

It is now known that TFs such as GATA-3 and STAT-6 operate by binding to sequence-specific elements within a promoter, which results in the activity of that particular gene. Subsequently, the study of the regulation of Th2 responses has shifted focus slightly, and now the question of importance addresses the issue of chromatin structure. (41) Which factor, or factors, is the master switch that is responsible for remodeling the chromatin so that the binding sites become open and available for TF binding? Independent investigations (42,43) have proposed GATA-3 and STAT-6 as candidate determinants. The data in this study are relevant to understanding how GATA-3, STAT-6, and c-MAF work together during the Th2 response in vivo.

In conclusion, this study has demonstrated that appreciable differences can be seen in the expression of the TFs STAT-6, GATA-3, and e-MAF in samples of induced sputum from asthmatic patients compared to those of control subjects and has also demonstrated that their expression is correlated with IL-4R[alpha] and IL-5R[alpha] expression. These findings indicate that the noninvasive technique of induced sputum can be useful as a means to determine the efficacy of certain asthma therapies against specific TFs or cytokines receptors as well as inhibiting the synthesis of these genes by corticosteroid treatment.

ACKNOWLEDGMENT: We thank Ms. Elsa Schotman and Ms. Cathy Fugere for technical assistance.

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* From the Meakins-Christie Laboratories and Montreal Chest Research Institute (Drs. Taha, Hamid, and Olivenstein), McGill University, Montreal, QC, Canada; and Respiratory Sciences Center (Dr. Cameron), University of Arizona, Tempe, AZ. Supported by: The J.T. Costello Memorial Research Fund, the Medical Research Council of Canada, and Inspiraplex.

Manuscript received January 17, 2002; revision accepted November 11, 2002.

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Correspondence to: Ron Olivenstein, MD, Montreal Chest Institute, McGill University, 3650 St. Urbain St, Montreal, QC, Canada H2X 2P4; Tel: (514) 849-5201; e-mail: rolive3@po-box. mcgill.ca

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