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The Journal of Immunology, 2002, 168: 4764-4771.
Copyright © 2002 by The American Association of Immunologists

IFN-{gamma}, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis

Zha O-Quan Shi*, Mark J. Fischer*, George T. De Sanctis{ddagger}, Mark R. Schuyler{dagger} and Yohannes Tesfaigzi1,*

* Lovelace Respiratory Research Institute, and {dagger} Department of Medicine, University of New Mexico School of Medicine, Albuquerque, NM 87108; and {ddagger} Aventis Pharmaceutical, Inc., Bridgewater, NJ 08870.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammatory responses induced by allergen exposure cause mucous cell metaplasia (MCM) by differentiation of existing and proliferating epithelial cells into mucus-storing cells. Airway epithelia have various mechanisms that resolve these changes to form normal airway epithelia. In this report, we first investigated the state of mucous cell metaplasia and the mechanisms by which MCM is reduced despite continued exposures to allergen. After 5 days of allergen exposure, extensive MCM had developed but was reduced when allergen challenge was continued for 15 days. During this exposure period, IL-13 levels decreased and IFN-{gamma} levels increased in the bronchoalveolar lavage fluid. In contrast, IL-13 levels decreased but IFN-{gamma} was not detected at any time point during the resolution of MCM following cessation of allergen exposure. Instillation of IFN-{gamma} but not anti-Fas caused accelerated resolution of MCM and MCM was not resolved in Stat1-deficient mice exposed to allergen for 15 days, confirming that IFN-{gamma} is crucial for reducing MCM during prolonged exposures to allergen. IFN-{gamma} but not anti-Fas induced apoptotic cell death in proliferating normal human bronchial epithelial cells and in human bronchial epithelial cells from subjects with asthma. The apoptotic effect of IFN-{gamma} was caspase dependent and was inhibited by IL-13, indicating that the Th2 milieu in asthmatics may maintain MCM by preventing cell death in metaplastic mucous cells. These studies could be useful in the understanding of deficiencies leading to chronicity in airway changes and designing novel therapies to reverse MCM and airway obstruction in asthmatics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The normal tracheobronchial epithelium contains ciliated, basal, and secretory cells, which are maintained at fixed ratios by homeostatic mechanisms. The proportion of these cell types is perturbed following various inflammatory responses or mechanical injury to the epithelium due to proliferation (1). Various studies have shown that nonciliated columnar cells are the main cell type that is recruited to the cell cycle in larger numbers (2). Following cessation of allergen exposure, airway epithelia return to the original proportion of cell types (3). Therefore, various mechanisms must exist that regulate the numbers of mucous cells to be adjusted to the original state following mucous differentiation of proliferating epithelial cells.

IFN-{gamma} plays an important role in airway inflammation and immune reaction. It down-regulates IgE secretion in B cells and is a negative growth factor for Th2 lymphocytes and, thus, counteracts Th2-mediated allergic reactions (4). Delivery of IFN-{gamma} to the airways prevents airway hyperreactivity during secondary exposure to allergen (5), reduces Th2 cytokine secretion, and inhibits Th2-induced airway eosinophilia (6, 7). Repeated exposure of rodents to allergen for extended periods causes airway inflammation to decrease from initially high levels, and this tolerance to inhaled allergen is dependent on TCR-{gamma}{delta}+ cells that produce high levels of IFN-{gamma} (8).

In asthma, Th2 cells and their cytokines mediate the appearance of mucus-storing cells in the bronchiolar regions, which are normally devoid of these cells, also termed mucous cell metaplasia (MCM)2 (9, 10). Although IFN-{gamma} blocks the production of mucus in airway epithelia (11), it is not known whether IFN-{gamma} acts directly on epithelial cells or reduces allergen-induced mucous cells by antagonizing cells producing Th2 cytokines. IFN-{gamma} induces apoptosis in a variety of cell types, including colon adenocarcinoma cells (12), A549 lung epithelial cells (13), primary human keratinocytes (14), HeLa cells (15), breast tumor cells (16), and fibroblasts (17). Therefore, in the present study, we investigated whether IFN-{gamma} may resolve allergen-induced mucous cell metaplasia by directly affecting epithelial cells in mice. Results show that during repeated exposures to allergen, IFN-{gamma} plays a pivotal role in resolving MCM through the Stat1 pathway and instillation of IFN-{gamma} accelerates reduction of allergen-induced MCM. Furthermore, we show that IFN-{gamma} induces apoptotic cell death in normal human bronchial epithelial cells (HBEs) and in HBEs from subjects with asthma by activation of caspases. Together, these studies demonstrate that IFN-{gamma} plays a critical role in reducing allergen-induced mucous cells and may provide a means to arrest and possibly reverse pathological sequelae that contribute to the persistence of MCM and ultimately the formation of life-threatening mucous plugs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male pathogen-free wild-type (+/+) C57BL/6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Stat1-/- C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). Mice, 6–8 wk of age, were housed in isolated cages under specific pathogen-free conditions. Unless otherwise specified, mice entered the experimental protocol at 7–9 wk of age. Mice were either sensitized by i.p. injection with 1 µg of OVA/100 µg of Al(OH)3 (grade III; Sigma-Aldrich, St. Louis, MO) in 0.5 ml of water or received 0.5 ml of water/100 µg of Al(OH)3 along with a booster injection on day 7. On day 14, mice were exposed 5 h/day to OVA aerosol at a concentration of 2.3 mg/m3 for 5, 10, or 15 consecutive days. The exposed mice were sacrificed either immediately or 16 h after the end of the last exposure. Immunized mice were exposed to allergen for 5 days and instilled with a 50-µl volume of IFN-{gamma}, IL-13 (R&D Systems, Minneapolis, MN), or anti-mouse Fas mAb, Jo2 (BD PharMingen, San Diego, CA), in saline or saline only and sacrificed 24 h later.

Histopathological evaluation

Mice were sacrificed by i.p. injection of 10 mg of sodium pentobarbital (Abbott Laboratories, Chicago, IL) and exsanguination via the renal artery. The thoracic contents were exposed, and the lungs were perfused by cardiac puncture with 0.9% saline (w/v) (McGaw, Irvine, CA). The trachea was cannulated with a 23-gauge blunt needle tipped with surgical tubing, and the lungs were lavaged three times with 0.5 ml of ice-cold PBS to collect the bronchoalveolar lavage fluid (BALF). The right lungs were removed, snap frozen in liquid nitrogen, and stored at -80°C for protein analysis. The left lungs were inflated with 10% zinc Formalin (Stephens Scientific, Riverdale, NJ) at a constant pressure of 25 cm for 3 h and processed, as described previously (18), to prepare five or six slices of about 0.3-cm thickness for embedding in paraffin and sectioning at 5-µm thickness for various staining procedures.

Morphometry of mucous cell numbers and stored intraepithelial mucosubstances

Tissue sections were stained with Alcian blue (AB) and periodic-acid Schiff (PAS) as described previously (19). The length of the basal lamina underlying the surface epithelium was calculated from the contour length of the digitized image using a Zeiss microscope equipped with the morphometry software system from Intelligent Imaging (Denver, CO). We counted the number of AB/PAS-positive cells lining the surface epithelium and normalized the number to 1-mm length of basal lamina. We also estimated the volume of stored AB/PAS-stained mucosubstances per unit of surface area of epithelial basal lamina as described (20). In all cases, both methods of quantification for MCM showed similar results, indicating that increases were due to MCM and not only enlargement of existing mucous cells.

TUNEL assay

We analyzed lung tissues from each group for cells with internucleosomal DNA fragmentation using the TUNEL assay, as described elsewhere (21). Briefly, terminal deoxynucleotidyltransferase was used to incorporate biotin-16-dUTP into the ends of DNA fragments. TUNEL signals were visualized in cell cultures using FITC conjugated to avidin and fluorescent microscopy (Zeiss). For lung tissues from each group for cells with internucleosomal DNA fragmentation TUNEL signals were visualized using the Vectastain avidin-biotin complex kit and the peroxidase substrate diaminobenzidine (Vector Laboratories, Burlingame, CA) as described by the manufacturer.

Cytokine detection

Lungs of mice were lavaged three times with 0.5 ml of PBS via the tracheal tube, and BALF was stored at -80°C until use. IL-13 and IFN-{gamma} were measured using the Quantikine M Murine kits (R&D Systems, Minneapolis, MN). Thirty-five microliters of BALF was added to each anticytokine-coated plate and incubated at 4°C overnight. The cytokine concentrations in the BALF were determined by standard ELISA techniques as described in the manufacturer’s manual. Detection limits were 3.9 pg/ml for IL-13 and 4.7 pg/ml for IFN-{gamma}.

Study subjects

Subjects were recruited using informed consent for a protocol approved by the University of New Mexico and the Lovelace Respiratory Research Institute Human Studies Review Board. Subjects had a mean age of 29.2 years with a SE of 4.1 years, were nonsmokers, not exposed to chronic medications, allergy immunotherapy, or corticosteroid therapy for at least 6 months, and had no experience of symptomatic asthma or upper respiratory tract infection within 4 wk prior to enrollment into the study. Asthmatic subjects had either a >15% increase in FEV1 in response to 200 µg of albuterol or >20% decrease of FEV1 after inhalation of <25 mg/ml methacholine. Asthmatic subjects met clinical diagnostic criteria for asthma.

Primary culture of HBEs

Endobronchial brushings were obtained from three different right lower lobe basilar segments with a cytology brush from all subjects using a fiberoptic bronchoscope. The brushings were collected in ice-cold serum-free L15 medium and washed in DMEM. The viability of epithelial cells was determined by trypan blue exclusion.

HBEs from a 28-year-old nonsmoker were purchased (catalog no. CC-2540, lot no. 8F1805; Clonetics, San Diego, CA) and expanded in vented T75 tissue culture flasks by growing them to 75–80% confluence in bronchial epithelial basal medium (Clonetics) and DMEM (Life Technologies, Rockville, MD) containing 25 ng/ml human recombinant epidermal growth factor, 65 ng/ml bovine pituitary extract, 5 x 10-8 M all trans-retinoic acid, 1.5 mg/ml BSA, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamicin, and 50 µg/ml amphotericin B (Clonetics). Cells were maintained at 37°C in an atmosphere of 5% CO2 and air. Cells were dissociated with trypsin/EDTA and frozen as passage 2 according to the manufacturer’s description.

For generating confluent cultures, HBEs were cultured in an air-liquid interface system on Transwell clear culture inserts (24.5 mm, 0.45-µm pore size; Costar, Cambridge, MA) that were thin coated with rat-tail collagen, type I (Collaborative Research, Bedford, MA). Cells were cultured under submerged conditions for the first 5–7 days in culture medium and when cultures reached 90% confluence, the air-liquid interface was created by removing the apical medium and exposing cells only to medium from their basal surface. At this time, the plates were switched to an incubator with 3% CO2. Medium in the lower compartment of the Transwell system was changed daily, containing the respective cytokine treatment.

For low confluent cultures, ~5000 HBEs were seeded on the rat-tail collagen-coated Transwell system to determine morphology, on 96-well dishes for MTT assays, or on four-well Lab-Tek tissue culture chamber slides (Nunc, Naperville, IL) for staining procedures. In all cases, the same medium was used for culturing, and cells were treated with the respective cytokines immediately after being placed in culture. For cross-linking and activating the Fas receptor, the anti-FasR mAb DX2 (BD PharMingen) was used.

Statistical analyses

Grouped results from at least four different mice were expressed as mean ± SEM, and differences between groups were assessed for significance by Student’s t test when data were available in only two groups. When data were available in more than two groups, ANOVA was used to perform pairwise comparisons. A p value of <0.05 was considered to indicate statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduction of MCM during prolonged exposure to allergen and concurrent increase in IFN-{gamma}

The respiratory tract of mice normally exhibits very few mucous secretory cells, but systemic sensitization to OVA followed by repeated exposure to aerosols generated from OVA solutions induces allergic inflammation and MCM (22). However, pulmonary inflammation decreases from these initially high levels when exposure continues for extended periods (23). The number of mucous cells per millimeter of basal lamina peaked during the first 5 days of OVA exposure, remained elevated for 10 days, and decreased significantly after 15 days of repeated daily allergen exposure (Fig. 1Go, A–C). AB-positive and -negative epithelial cells with nuclei positive for the TUNEL reaction were observed in mice exposed for 15 days (Fig. 1Go, D and E). We determined the levels of Th2 and Th1 cytokines in the BALF after allergen challenge for 5 and 10 days. Although IL-13 levels were high at 5 days, they were below detection levels at 10 days of allergen challenge (Fig. 1GoF). In contrast, IFN-{gamma} levels were low at 5 days but were increased at 10 days of allergen exposure (Fig. 1GoF).



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  		FIGURE 1. A), Continuous 15-day exposure to allergen causes MCM to decrease from higher levels at 5 and 10 days. Mice were immunized with water or OVA and challenged with OVA aerosols for 5, 10, and 15 days. The number of AB/PAS-positive cells per millimeter of basal lamina of the airways in the left lung lobe of each mouse was determined. Bars, Group means ± SD from the mean (n = 4–7 mice/group). *, Significantly different from day 5. Representative photomicrographs of airways at 5 days (B) and 15 days (C) of allergen exposure. Mucous cells with TUNEL-positive nuclei were absent in mice exposed to allergen for 5 days (D) but present in mice exposed for 15 days (E). IL-13 and IFN-{gamma} levels in BALF of C57BL/6J mice immunized and challenged with OVA for 5, 7, and 10 days. IL-13 levels decreased and IFN-{gamma} levels increased during repeated exposure to allergen (F).

 
IFN-{gamma} levels do not increase during resolution of MCM after cessation of allergen exposure

Decremental decreases in airway hyperreactivity, inflammatory cell counts in the airway lumen, and MCM also occurs following cessation of allergen exposure (3). Cytokine levels were determined during resolution of MCM following cessation of allergen exposure for comparison to the resolution during prolonged exposure to allergen. MCM was generated in sensitized mice by exposure to allergen for 5 days. MCM decreased by more than half during a recovery period of 10 days and remained at that level at 14 and 17 days after challenge (Fig. 2GoA). IL-13 levels in the BALF were elevated after 5 days of allergen exposure, but decreased to background levels when mice had recovered for 5 days; however, unlike the resolution in the context of prolonged allergen exposure, IFN-{gamma} was not detectable during this entire recovery period (Fig. 2GoB).



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  		FIGURE 2. A, Resolution of MCM during recovery from a 5-day (d) allergen exposure. Mice were immunized with water (W d0) or OVA (Od0) and challenged with OVA aerosols for 5 days. The number of AB/PAS-positive cells per millimeter of basal lamina of the airways in the left lung lobe of each mouse was determined after allergen exposure at 5, 10, 14, and 17 days in the absence of allergen. Bars, Group means ± SEM (n = 4–7 mice/group). *, Significantly different from water-immunized controls. B, IL-13 and IFN-{gamma} levels in BALF of sensitized mice challenged with OVA for 5 days and allowed to recover for 5, 10, 14, and 17 days in the absence of allergen. IL-13 levels decreased to background levels after 5 days, and IFN-{gamma} was barely detectable at any time point. Bars, Group means ± SD from the mean (n = 4 mice/group). *, Significantly different from other days.

 
Instillation of mice with IFN-{gamma} causes a decrease of MCM by causing cell death

The observation that IFN-{gamma} levels were elevated during prolonged exposure to allergen led to the hypothesis that IFN-{gamma} may be crucial to reduce MCM. To test this hypothesis, we exposed mice initially to allergen for 5 days and then intranasally instilled them with vehicle, 100 ng of IL-13 or 50 or 100 ng of IFN-{gamma} in a volume of 50 µl of saline on the sixth day. One day later, we quantified the numbers of mucous cells and found no significant changes in mice instilled with 100 ng of IL-13 or 50 ng of IFN-{gamma} compared to the saline-instilled controls (Fig. 3GoA). However, MCM levels decreased significantly in mice instilled with 100 ng of IFN-{gamma} (Fig. 3GoA). Three days after instillation, mice instilled with 50 ng of IFN-{gamma} also showed significantly decreased MCM levels (data not shown), similar to the numbers observed after prolonged exposure to allergen. Furthermore, similar to mice exposed to allergen for 15 days, we detected TUNEL positivity in epithelial cells of mice instilled with IFN-{gamma} but not in mice instilled with IL-13 (data not shown).



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  		FIGURE 3. A, MCM in sensitized mice exposed to allergen for 5 days, intranasally instilled with IFN-{gamma} the following day, and sacrificed 1 day later. Mice instilled with saline, IL-13, or 50 ng of IFN-{gamma} in saline showed no decrease in MCM, while the mice instilled with 100 ng of IFN-{gamma} showed significantly decreased MCM. Bars, Group means ± SEM (n = 4–7 mice/group). *, Significantly different from saline-instilled controls. B, Stat1-deficient mice do not resolve MCM after prolonged exposure to allergen. Mice were immunized with OVA on days 1 and 7 and exposed to OVA aerosols for 15 days. The number of AB/PAS-positive cells per millimeter of basal lamina of the airways in the left lung lobe of each mouse was determined. Bars, Group means ± SEM (n = 5 mice/group). *, Significantly different from wild-type (WT) controls. KO, Knockout.

 
Stat1 is essential for the reduction of MCM during prolonged exposure to allergen

Stat1, which plays an obligate and dedicated role in mediating IFN-{gamma}-dependent responses, is a downstream signaling protein of IFN-{gamma} (24). To determine whether IFN-{gamma} is responsible for the reduction of MCM, we compared the extent of MCM in Stat1+/+ and Stat1-/- mice after 15 days of exposure to allergen. MCM was drastically decreased in Stat1+/+ while in Stat1-/- mice, MCM remained at levels observed in Stat1+/+ mice at 5 days of exposure (Fig. 3GoB), indicating that IFN-{gamma} mediates the reduction of MCM. Furthermore, we detected TUNEL-positive mucous cells in Stat1+/+ mice but not in Stat1-/- mice (data not shown).

IFN-{gamma} causes apoptotic cell death in HBEs

To further test whether IFN-{gamma} directly acts on epithelial cells, we examined the effect of IFN-{gamma} on HBEs. AB/PAS staining showed that HBEs, under our culture conditions, produce mucosubstances at low and high confluence (data not shown). IFN-{gamma}-induced cell death was highly dependent on the state of confluence. Viability of HBEs was reduced to 40% by day 4 and to 10% by day 8 when cells were treated with 50 ng/ml IFN-{gamma} immediately after placing cells in culture or at <40% confluence (Fig. 4GoA). However, viability was reduced to only 75% of untreated cells, when HBEs were treated at 80–100% confluence even after 7 days of treatment and when IFN-{gamma} concentration was increased to 100 ng/ml (Fig. 4GoB). To exclude any undefined cytokine effects, cells were treated with IL-9 and IL-13 as control. IL-9 and IL-13 caused cells to grow as compared to untreated cells regardless of whether the cells were treated at 30 or 80% confluence (data not shown).



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  		FIGURE 4. IFN-{gamma}-induced cell death is maximized when HBEs are treated at low confluence. HBEs were treated with 50 ng/ml IFN-{gamma} either immediately or at <40% confluence (A) or with up to 100 ng/ml IFN-{gamma} at 100% confluence (B). Viability was assessed using the MTT assay as percentage of untreated controls. After 7 days of treatment, viability was reduced to 10% or only to 75% when cells were treated with IFN-{gamma} at low or high confluence, respectively. Bars, Group means ± SEM (n = 6 repeats of experiment).

 
To further verify whether the cell death occurs by apoptotic pathways, IFN-{gamma}-treated cells were analyzed for chromatin condensation and DNA fragmentation. After 4 days of IFN-{gamma} treatment, similar percentages of cells showed condensed nuclei and were positive by the TUNEL assay (Fig. 5Go). Typical nuclear fragmentation was observed throughout the culture. Furthermore, IFN-{gamma}-induced apoptosis was at least partially inhibited by zVAD-fmk, which inhibits the activity of caspases 1, 3, 6, 7, 8, and 9 (Fig. 5GoG).



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  		FIGURE 5. IFN-{gamma}-induced cell death in HBEs has features of apoptotosis. Phase-contrast micrographs (A and D), Hoechst staining of nuclei (B and E), and TUNEL positivity detected by fluorescence of FITC (C and F) of HBEs that were treated on Lab-Tek slides with nothing as control (A–C) or IFN-{gamma} (D–F). G, IFN-{gamma}-induced apoptosis is reduced by zVAD-fmk, an inhibitor of caspases 1, 3, 7, 8, and 9. Confluent cultures of HBEs maintained in the presence or absence of zVAD-fmk and IFN-{gamma}, and viability was assessed after Hoechst staining and counting the percentage of condensed nuclei. Bars, Group means ± SEM (n = 6 repeats of experiment). *, Significantly different from IFN-{gamma}-treated cells.

 
In keratinocytes, IFN-{gamma} induced apoptosis by up-regulating expression of Fas ligand and sensitizing cells to Fas-induced cell death (25). Therefore, we investigated whether anti-Fas Ab would synergistically increase IFN-{gamma}-induced cell death. Anti-Fas did not induce cell death in HBEs at low confluence (Fig. 6GoA), and only 15% of HBEs showed condensed nuclei when treated at high confluence for 3 days (Fig. 6GoB). IFN-{gamma}-induced apoptosis was greater than that observed for Fas, both at low and high confluence, suggesting that HBEs are more susceptible to IFN-{gamma}- than Fas-induced apoptosis (Fig. 6Go). Interestingly, in low confluent cultures, addition of 500 ng/ml anti-Fas had an inhibitory effect on IFN-{gamma}-induced apoptosis. Although the same concentration of anti-Fas combined with IFN-{gamma} induced cell death in an additive manner, increasing the concentrations of anti-Fas to 1000 and 2000 ng/ml reduced the percentage of IFN-{gamma}-induced apoptosis to 6% (data not shown). Substituting the anti-Fas with an irrelevant Ab, mouse IgG, as control had no effect on IFN-{gamma}-induced apoptosis.



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  		FIGURE 6. Fas is not an effective inducer of apoptosis in HBEs. HBE cultures were treated with 500 ng/ml Fas, 50 ng/ml IFN-{gamma}, or with both Fas and IFN-{gamma} at <40% confluence (A) or at 100% confluence (B). Viability was assessed using the MTT assay as percentage of controls (C). (n = 6 repeats of experiment).

 
IL-13 inhibits IFN-{gamma}-induced apoptosis in HBEs

The absence of TUNEL-positive cells in IL-13-instilled mice suggested that IL-13 may have inhibitory function on IFN-{gamma}-induced apoptosis. To test this hypothesis, IFN-{gamma}-treated cells were exposed to 5 or 50 ng/ml IL-13. Although 5 ng/ml was sufficient in low confluent cultures (Fig. 7GoA), 50 ng/ml IL-13 was necessary to reduce IFN-{gamma}-induced cell death in HBEs in confluent cultures (Fig. 7GoB).



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  		FIGURE 7. IL-13 inhibits IFN-{gamma}-induced apoptosis in HBEs. At low confluence (A), 5 ng/ml IL-13 reduces IFN-{gamma}-induced apoptosis. In confluent cultures (B), 50 ng/ml of IL-13 is necessary to reduce the effect of IFN-{gamma}. C, Control.

 
IFN-{gamma} induces cell death in HBEs from asthmatics

Sampath et. al (26) had reported that Stat1 is constitutively activated in bronchial epithelial cells in asthmatics compared with normal control subjects. Therefore, we examined whether there is any difference in susceptibility toward IFN-{gamma}-induced cell death in HBEs obtained by bronchial brushings from six asthmatics. Cells were exposed to IL-9, IL-13, or IFN-{gamma} in Transwell cultures at low confluence. Although both IL-9 and IL-13 induced cell growth in these cultures, all cultures maintained in the presence of 50 ng/ml IFN-{gamma} displayed condensed nuclei and died.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies of tracheobronchial injury and repair have demonstrated that mucous-secreting cells proliferate and play a significant role in the normal development and repair process of the tracheobronchial epithelium in both rodents (27, 28) and monkeys (29). MCM is a result of existing and proliferating epithelial cells differentiating into mucous cells in areas that are devoid of mucous cells (30, 31). The present study shows that IFN-{gamma} may be a crucial component of the regulatory system to eliminate excess mucous cells during resolution of metaplasias.

A panel of cytokines secreted by Th2 cells cause MCM in human and mouse airway epithelial cells (9, 10, 32, 33). Our results indicate that 60–80 mucous cells/mm basal lamina represent maximum attainable MCM after allergen exposure of mice. Instillation of allergen-exposed mice with additional IL-13 did not further increase the number of mucous cells, suggesting that these numbers represent the maximum mucous cell density that the airway epithelium can accommodate.

Exposure of mice (23) and rats (34, 35) to allergen for extended periods leads to a significant decrease in inflammation from their previous peak levels. Although tolerance, a state of nonresponsiveness to an Ag, can result by several mechanisms (36), extended exposures of rodents to inhaled Ags stimulates "tolerance" via immune deviation (8). During repeated allergen challenge for 10 days, IL-13 levels decreased and IFN-{gamma} levels increased in BALF, confirming that this process is a result of immune deviation. The presence of IFN-{gamma} during prolonged exposure to allergen and the development of immune deviation suggested that this cytokine induced the reduction of MCM. This hypothesis was verified by the fact that instillation of 100 ng of IFN-{gamma} accelerated the decrease of MCM in a lung environment dominated by a Th2 milieu.

The decrease of MCM could also be a result from increased mucin secretion that may surpass the rate of mucin synthesis. However, the presence of TUNEL-positive mucous cells demonstrates that MCM was decreased by death of these cells. Many of the TUNEL-positive cells contained mucosubstances. Some do not show mucous contents and may represent cells that have secreted all their contents in the process of undergoing apoptosis. This hypothesis is supported by the observation that, in general, the mucous contents in TUNEL-positive cells is less than in nonpositive cells, suggesting that these cells have secreted part of their contents. Because we cannot identify the nonmucous cells, our current studies do not exclude the possibility that other types of epithelial cells underwent apoptosis in response to IFN-{gamma}. However, the presence of TUNEL-positive mucous cells shows that mucous cells are the predominant cell type that dies after instillation with IFN-{gamma}.

Our study was the first to show that MCM levels decrease by about 50% following increase of IFN-{gamma}. The observed partial reduction of MCM suggests that other mechanisms maintained the remaining mucous cells, or they may belong to the cells that eventually constitute the recovered epithelium after secreting their mucous contents. TUNEL positivity and activation of caspases in epithelial cells was always associated with the decrease of MCM in the presence of IFN-{gamma}, suggesting that the reduction of MCM results partly by inducing programmed cell death. The fact that allergen-induced MCM is reduced when Th1 cells are adoptively transferred along with Th2 cells (11) supports the involvement of IFN-{gamma} in reducing MCM. This dose-dependent inhibitory effect of Th1 cells is abolished when Th1 and Th2 cells are transferred into IFN-{gamma} receptor -/- mice, indicating that IFN-{gamma} receptor signaling is crucial for the inhibitory role of Th1 cells (11, 37). Several reports (38, 39, 40) have shown that HBEs in the presence of retinoids, as we have in our culture conditions in this study, differentiate into mucous-producing cells. We have also observed AB/PAS-positive material in these HBE cells in culture. Our data with HBEs demonstrate that IFN-{gamma} directly affects mucous cells to undergo apoptosis and that proliferation is critical for this process. Decreases in apoptosis when cells reach confluence have been observed for various cell types (41, 42), although the mechanism for this phenomenon remain unclear.

Although Fas receptor (43) and Fas ligand (44) are expressed in HBE cells, there are no reports documenting Fas-induced apoptosis in HBEs. In our experiments, Fas was ineffective in causing apoptosis in low confluent cultures, but induced apoptosis in approximately 15% of confluent HBEs. Similarly, only 6% apoptosis was observed in lung epithelial A549 cells that were treated with anti-Fas (13). IFN-{gamma} increases intracellular as well as surface-bound Fas ligand expression in keratinocytes (14, 25) and thereby enhances the susceptibility of these cells to Fas-induced apoptosis. However, as tested by Western blot analysis, expression of Fas receptor was not enhanced in HBEs by IFN-{gamma} (our unpublished observation) and the combined effect of Fas and IFN-{gamma} was only additive. In addition, anti-Fas at concentrations that show cell death in keratinocytes (14) had an inhibitory effect on IFN-{gamma}-induced apoptosis in HBEs. These results demonstrate that IFN-{gamma} directly induces apoptosis without the involvement of the Fas pathway and that Fas does not induce apoptosis in HBEs. Furthermore, instillation of anti-Fas, Jo2, did not reduce allergen-induced MCM in mice (our unpublished observation). Expression of Fas and Fas ligand appears to be primarily involved in controlling bronchial inflammation (44), whereas IFN-{gamma} may be the primary factor reducing MCM. IFN-{gamma} may not affect resting cells, but plays a role to control and eliminate excess cells following proliferation in response to allergen.

The absence of TUNEL-positive cells after IL-13 instillation indicates that high levels of IL-13 may inhibit the effect of IFN-{gamma} and prevent the reduction of MCM. The presence of Th2 cytokines in the lungs of mice exposed to allergen may be why instillation of 100 ng but not 50 ng of IFN-{gamma} reduced MCM significantly, while 50 ng/ml IFN-{gamma} was sufficient to induce apoptosis in proliferating HBEs. The finding that the presence of IL-13 reduces IFN-{gamma}-induced apoptosis in HBEs suggests that the establishment of a predominantly Th2 milieu in asthmatic lungs may maintain MCM by inhibiting apoptotic signaling. Serum levels of IFN-{gamma} can increase during severe asthma (45), and IFN-{gamma} is sometimes found in BALF from mild asthmatics (46). Why IFN-{gamma} in these asthma patients does not drive the resolution of airway hyperreactivity and MCM may be explained by IFN-{gamma} levels not being high enough to reverse the inhibitory effects of Th2 cytokines.

Although low levels of IFN-{gamma} were detected in airway tissue of asthmatics subjects, Sampath et al. (26) found that epithelial Stat1 is activated, and that IFN regulatory factor 1 and Stat1 were increased in expression in airway epithelial cells of asthmatics compared with normal control subjects or chronic bronchitis subjects. These findings led the authors to conclude that the Stat1 pathway is abnormal in airway epithelia of asthmatics. Although we have not examined the state of Stat1 activation in our samples, the study subjects used were similar to those described by Sampath et al. (26). All brushings from asthmatics in our study showed growth in the presence of IL-13 and IL-9, but died in the presence of IFN-{gamma}, indicating that the regulation of mucous cell numbers in asthmatics may be controlled by the ratio of IL-13 to IFN-{gamma} rather than the HBEs from asthmatics having an intrinsic irresponsiveness to IFN-{gamma}-induced cell death.

MCM was not reduced in Stat1-deficient mice, suggesting that Stat1 is crucial for IFN-{gamma}-induced apoptosis. Stat1 has been linked to IFN-{gamma}-induced apoptosis in various cell lines such as NIH3T3 and Me180 cells (17). Furthermore, Stat1 activation promotes cell death in cardiomyocytes (47) by affecting the promoter activities of the anti-apoptotic proteins, Bcl-2 and Bcl-xL genes and reducing their expression (48). The involvement of Stat1 and IFN regulatory factor 1 in the reduction of Bcl-xL in bronchial epithelial cells is currently under investigation.

That Stat1-/- mice could not resolve MCM after prolonged allergen exposure supports the hypothesis that within a given subpopulation of asthmatics, a deficiency in the Janus kinase/Stat-signaling pathway could render IFN-{gamma} incapable of inducing cell death in epithelial cells. Further support for this hypothesis is that polymorphisms in two genes directly related to this pathway, IFN-{gamma} and IFN regulatory factor 1, confer genetic susceptibility to atopic asthma in Japanese children (49). These findings indicate that deficiencies in the Janus kinase/Stat pathway may contribute sustained increased levels of MCM in humans with asthma.

The presence of TUNEL-positive cells during the resolution of MCM after extended exposures to allergen and its absence when MCM is decreased during recovery after allergen exposure suggest that the mechanisms of resolution are different. In the latter case, MCM was resolved because of a reduced inflammatory response that decreased biosynthesis of mucus and storage in airway epithelial cells. How excess cells are eliminated in this system is unknown. Spontaneous resolution of inflammatory response in the airway lumen and MCM after allergen exposure has been reported for BALB/c mice (3). In those studies, mice were challenged by intratracheal instillation of OVA, and the score for mucous cell numbers declined 4-fold until day 30 and reached control levels after 50 days of recovery (3). The shorter resolution period in our study may stem from differences in the strains analyzed or in the allergen challenge protocol. Intratracheal instillation of allergen delivers larger amounts to the lung than can be delivered by inhalation (3). In spite of the differences in the length of time needed for the resolution, the rapid initial declines in mucous cell numbers are strikingly similar in both studies.

In summary, IFN-{gamma} is crucial in eliminating metaplastic mucous cells by inducing apoptosis. Apoptosis induced by this cytokine but not by the Fas ligand appears to play a major role in reducing MCM and restoring the normal proportion of cell types in airway epithelia following purturbation due to allergic inflammatory responses.


   Acknowledgments
 
We thank Yoneko Knighton (Lovelace Respiratory Research) for preparing tissue samples and for excellent technical assistance by Leigh Schutzberger. Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Yohannes Tesfaigzi, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive, SE, Albuquerque, NM 87108. E-mail address: ytesfaig{at}lrri.org Back

2 Abbreviations used in this paper: MCM, mucous cell metaplasia; HBE, human bronchial epithelial cell; BALF, bronchoalveolar lavage fluid; AB, Alcian blue; PAS, periodic-acid Schiff. Back

Received for publication November 2, 2001. Accepted for publication February 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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