Introduction

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Predisposition to primary allergen sensitization may be determined in part by hereditary factors, and may be associated with a single gene on chromosome 11q (1). However, there is substantial evidence that environmental factors are also important in the development of atopy.

Exposure to high levels of aeroallergens in early infancy is important for the development of primary sensitization to allergen. Month-of-birth studies have shown that allergy to birch pollen could be almost 30% lower among individuals born other than in the months of February to April (2).

Although sensitization to an allergen can occur at any age, Sporik and colleagues (3) demonstrated the importance of early exposure to house dust mite (HDM) allergen in primary sensitization. These investigators prospectively followed 67 children from birth until the age of 11 yr, and found that exposure to Dermatophagoides pteronyssinus (Der p1; a major allergen of HDM) at levels greater than 10 µg/g of household dust, measured in infancy, was associated with a 4.8-fold relative risk (RR) of developing asthma by the age of 11 yr, and that exposure to high levels of Der p1 at the age of 1 yr was inversely related to the age of onset of asthma in HDM-sensitive children. Price and colleagues also found a strong association between sensitivity (positive skin prick tests and immunoglobulin [Ig]E antibodies) to HDM and the presence of Der p1 in home air (4). The Dust Mite Task Force of the International Association of Allergy and Clinical Immunology proposed that 2-10 µg of Der p1 or Dermatophagoides farinae 1 (Der f1) per gram of dust poses a risk for sensitization and development of asthma, and that levels exceeding 10 µg Der p1 or Der f1 per gram of dust pose a major risk for the development of acute asthma in HDM-allergic patients (5).

Antenatal maternal smoking has been shown to increase the cord blood IgE titer of the resulting offspring (6). Exposure to cigarette smoke (CS) has also been linked with the development of atopy. Increased levels of IgE were found in serum of children aged 3-36 mo of parents who smoked, as compared with those of parents who did not smoke (7). There is also evidence that skin-test reactivity to common aeroallergens increases significantly both in frequency and intensity in children, especially boys, of smoking parents, whereas it remains unchanged in children of nonsmoking parents (8). Many studies have also shown that children of smoking parents, and particularly of smoking mothers, have a higher than normal frequency of respiratory illnesses (9). Other investigators have shown a highly significant increase in wheezing, coughing, and respiratory infections in children with smoking mothers, and that this effect is directly proportional to the number of cigarettes smoked by the mother (12). Furthermore, cigarette smoking is also known to predispose to the development of occupational asthma (13, 14), although the mechanisms underlying this have still not been elucidated.

CS is one of the most damaging indoor pollutants and induces oxidative changes in both the epithelial cell membrane and the cytoskeleton. Li and colleagues have reported that both whole and vapor smoke condensates induced a reversible, concentration-dependent increase in the epithelial permeability of a monolayer of a line of human type II alveolar epithelial cells, and that this was associated with a profound decrease in intracellular glutathione (GSH) (15). These authors have also shown that CS condensate significantly increases rat lung epithelial permeability to [¹²⁵I]bovine serum albumin ([¹²⁵I]BSA) at 6 h after instillation, in association with a significant recruitment of neutrophils into the air spaces. Others, such as Nery and colleagues, have shown that air-space epithelial permeability is increased in smokers as compared with nonsmokers (16). Taken together, these results of epidemiologic and experimental studies link CS both with bronchial epithelial damage and with the development of allergen sensitization.

We have hypothesized that such allergen sensitization results from a CS-induced increase in airway permeability to allergen. In this article we report our findings on the effect of exposure to CS or air in the absence or presence of 300 ng/ml Der p for varying periods, on the permeability of HBEC cultures, as assessed by changes in: (1) electrical resistance (ER); and (2) passage of ¹⁴C-labeled BSA (¹⁴C-BSA) and Der p allergens across the HBEC cultures. Damage to HBEC cultures was assessed by the release of [⁵¹Cr]sodium chromate from prelabeled cultures, and by release of lactate dehydrogenase (LDH). The effects of specific protease inhibitors and the antioxidant GSH on CS- and Der p-induced changes were also investigated.

	Materials and Methods

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All chemicals and reagents were of tissue culture grade, and unless stated otherwise were obtained from the Sigma Chemical Co. (Poole, UK).

Culture of HBEC

Bronchial tissue was obtained from 12 male and three female patients who underwent lobectomy for lung cancer at The London Chest Hospital. All patients were smokers, not allergic to common allergens, and had a mean age of 67.4 ± 2.6 yr (range: 61-78 yr). Following resection, only tissue macroscopically free of tumor was processed further for culture.

Bronchial epithelial cells were cultured according to the explant cell culture technique developed in our laboratory (17). Briefly, the epithelium was dissected away from the underlying lamina propria, and after further dissection, into smaller sections of approximately 0.5 mm³, the epithelium was washed three times with fresh sterile medium 199. Single sections of the epithelium were explanted into 9-mm-diameter Falcon cell culture inserts (Becton Dickinson Ltd., Oxford, UK), and to each insert were added 100-µl aliquots of culture medium supplemented with 2.5% fetal calf serum and a variety of growth factors, including human transferrin (2.5 µg/ml), epidermal growth factor (20 ng/ml), bovine pancreatic insulin (2.5 µg/ml), hydrocortisone (0.36 µg/ml), L-glutamine (0.02 mg/ml), and 1.5% (vol/ vol) antibiotic and antimycotic solution composed of penicillin, streptomycin, and amphotericin B.

The inserts were placed in 24-well culture plates (Becton Dickinson) containing 400 µl of the complete culture medium, as described earlier, in each well and were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air. The medium in the insert and the well was replaced after 3 d with culture medium containing 2.5% NUSERUM IV Culture Supplement (Universal Biologicals Ltd., London, UK) and then again at 48-h intervals. The cultures were observed for epithelial cell outgrowth until the cells had grown to confluency. The explants were removed and the cultures were incubated further until the area left barren by the explant was overgrown with epithelial cells.

The identity of the epithelial cells was confirmed in all cultures by light microscopy, and in randomly selected cultures by electron microscopy and immunocytochemical staining for cytokeratin, using monoclonal antibody preparation CAM 5.2 (Becton Ltd., Oxford, UK). Contaminating cell types were analyzed by staining for T and B lymphocytes, using monoclonal antibodies CD3 and CD37, respectively (Serotec Ltd., Oxford, UK), and for fibroblasts, mast cells, neutrophils and macrophages using monoclonal antibodies 5B5, AA1, NP57, and Ber-MAC3, respectively (Dako Ltd., Bucks, UK).

Preparation of Der p Solution

Aquagen freeze-dried allergen extract (ALK, Horsholm, Denmark), which is known to contain several subgroups of Der p and may therefore be more relevant than a preparation containing one allergen subgroup only, was reconstituted in the specified diluent according to the manufacturer's instructions. This resulted in a stock solution at a concentration of 100,000 Standard Quality U/ml Der p (equivalent to 9,000 ng/ml Der p), which was further diluted with medium 199 containing serum-free supplement (SF-1 medium) (Hyclone Europe Ltd., Northumberland, UK) to provide a working solution at a concentration of 300 ng/ml Der p. This concentration was chosen since it is approximately one-tenth the concentration deemed to pose a risk of sensitization in vivo (7).

Measurement of Protease Activity of Der p Solution

The Aquagen allergen extract is supplied as an immunologically and biologically standardized extract in which the constituent protease activities are not defined. To assess whether the Der p extract contained proteolytically active proteins, we assessed its ability to cleave: (1) BOC-Gln- Ala-Arg-AMC, a substrate for Der p and trypsin like serine proteases (18, 19); and (2) ABZ-Val-Ala-Nle-Ser- (NO₂)Tyr-Asp-NH₂, a substrate for Der p1 (20, 21).

The 300 ng/ml solution of Der p was incubated with an excess (25 µM) of either of the two fluorogenic substrates for 30 min in the absence or presence of 5 µM E64 (trans- epoxysuccinyl-L-leucylamido [4-guanidino]-butane), an irreversible inhibitor of cysteine and serine proteases (22), or in the absence or presence of the serine protease specific inhibitors ₁-antitrypsin (₁-AT) (0.2 µM) (Calbiochem, Nottingham, UK) or soya bean trypsin inhibitor (SBTI) (0.5 µM). Assays were performed in 96-well Teflon plates (Radleys, Essex, UK), and at the end of the incubation period the fluorescence intensity in the reaction mixture was measured in a Floroskan Ascent plate reader (Labsystems, Basingstoke, UK). The endpoint results were expressed as the change in fluorescence intensity (F) after subtraction of time-zero fluorescence readings from the 30-min fluorescence values.

Exposure of HBEC Cultures to CS

HBEC cultures were exposed to CS in an exposure system that we have developed in our laboratory (Figure 1). A continuous, 850-ml/min flow of CS was generated by burning commercially available Superkings cigarettes (12 mg tar, 1.1 mg nicotine, BAT, Nottingham, England, UK) and leading the smoke stream into an airtight polycarbonate exposure chamber with a capacity of 5.5 liters (Billups Rothenberg, Del Mar, CA) and containing the HBEC cultures established on inserts. The continuous flow of CS was achieved by the use of an Airchek 50 Sampling Pump (SKC Ltd., Dorset, UK) capable of generating a flow of 5-3,000 ml/min. The pump was connected to the top of the exposure chamber and the lit cigarette was connected to the bottom outlet of the exposure chamber. The negative pressure created by the sampling pump kept the cigarettes burning with a constant speed. On average, it took 5 min for a cigarette to burn. To achieve even distribution of the CS within the exposure chamber, a small solar energy-powered ventilator was placed and operated inside the chamber, just below the HBEC cultures. The solar panel itself was placed outside the exposure system and was illuminated by a 100 W light bulb positioned 5 cm from the sensor. At intervals of 2.5 s, the exposure chamber was tilted gently on a Luckham 4RT rocking table (Luckham Ltd., Burgess Hill, UK), to an angle of 10° from the horizontal in each quarter of the horizontal plane, thereby providing adequate mixing of the culture media and covering the surface of the HBEC cultures during each tilt. To ensure that the temperature was kept stable at 37°C during exposure, the exposure system was placed in a 60-liter- capacity SI.60 incubator (Stuart Scientific, Redhill, UK) made of acrylic material, thereby providing total visibility.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Schematic diagram of system for exposure of cultures to CS.

HBEC cultures were exposed to CS for 20 min, 1 h, 3 h, and 6 h. Appropriate controls were also prepared by exposing HBEC cultures to air for the same period and then treating them further in the same way as the test cultures.

Effect of Exposure to CS on HBEC Permeability

Changes in permeability of HBEC cultures were investigated by measuring the passage of ¹⁴C-BSA (Amersham International plc, Amersham, UK) across HBEC cultures established in cell culture inserts. Measurements of changes in electrical resistance of HBEC cultures were also made in each culture at each time point studied.

Measurement of passage of ¹⁴C-BSA across HBEC cultures. Before being investigated, HBEC were grown to confluence on cell culture inserts as described earlier, and the explants were removed. The epithelial cells were allowed to grow over the area of the culture insert membrane that was left barren on removal of the explant, and when completely confluent, were used further. Immediately before exposure to CS or air, the cultures were washed gently with fresh culture medium and then incubated for 30 min in the presence of 0.025 µCi ¹⁴C-BSA, which was added into the insert. At the end of this incubation the medium in each insert well was collected and analyzed for total radioactivity through liquid scintillation counting in a Beckman LS6500 scintillation counter (Beckman-RIIC Ltd., High Wycombe, UK). When the total radioactivity passing across the culture was found to be less than 0.5% of the total ¹⁴C-BSA added to the inserts at the beginning of the experiment, the culture was deemed to be fully confluent and the actual experiment was started. HBEC cultures were then exposed to CS or air for 20 min, 1 h, 3 h, and 6 h, in either the absence or presence of 300 ng/ml Der p as described earlier. Immediately after exposure, the medium was collected from each insert well, and after addition of fresh medium, the cultures were incubated further at 37°C in an atmosphere of 5% CO₂ in air. The culture medium from each insert well was collected at 1 h, 3 h, 6 h, and 24 h after exposure, and 25-µl aliquots of each sample were analyzed for radioactivity. After correction for the total amount of radioactivity passing across the epithelial cultures at each time point, results were expressed as a percentage of the total added to the culture at the beginning of the experiment.

Measurement of electrical resistance of HBEC cultures. ER of HBEC cultures was measured before beginning the experiment and then immediately, and at 1 h, 3 h, 6 h, and 24 h after exposure, using an EVOM micro volt-ohm meter incorporating a fixed pair of electrodes (4 mm wide and 1 mm thick) (World Precision Instruments, Owslebury, UK). Data obtained from these experiments were analyzed after calculation of the percentage changes from baseline in each culture at each time point during the 24-h incubation period.

Effect of Exposure to CS on the Passage of Der p across HBEC Cultures

Quantitative determination of Der p in the aliquots obtained from each insert well at various time points from cultures that were incubated in the presence of Der p and exposed to either CS or air was done with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ALK Indoor Allergen Analysis Kit; ALK, Horsholm, Denmark).

Effect of Varying Concentrations of Der p on the Permeability of HBEC Cultures

Sets of confluent HBEC cultures treated with ¹⁴C-BSA were incubated in the absence or presence of 300, 1,000, or 3,000 ng/ml Der p for 24 h, and the passage of ¹⁴C-BSA across the cultures was measured as described earlier.

In a separate experiment, ¹⁴C-BSA-treated HBEC cultures were incubated with Der p that had been preactivated by incubation for 30 min with 1 mM cysteine, and were evaluated for changes in permeability as described previously.

Effect of Antiproteases on CS- and Der p-Induced Changes in the Permeability of HBEC Cultures

Sets of confluent HBEC cultures treated with ¹⁴C-BSA and 300 ng/ml Der p in the absence or presence of: (1) 0.2 µM ₁-AT; (2) 0.5 µM SBTI; (3) 5.0 µM E64; or (4) a combination of the three protease inhibitors were exposed for 20 min to either CS or air, and were then further incubated for 24 h at 37°C in an atmosphere of 5% CO₂ in air. At the end of this incubation period, changes in the passage of ¹⁴C-BSA were measured as described earlier.

Effect of GSH on the Interaction between CS and Der p

Sets of confluent HBEC cultures treated with ¹⁴C-BSA were exposed for 20 min to CS or air in the presence or absence of 300 ng/ml Der p ± 500 µM GSH. After this exposure the cultures were incubated further for up to 24 h, and changes in the passage of ¹⁴C-BSA across the HBEC cultures were measured as detailed earlier. A concentration of 500 µM GSH was chosen to parallel concentrations of 450-500 µM GSH found in epithelial lining fluid in vivo.

Effect of Exposure to CS on HBEC Membrane Damage

⁵¹Cr release studies. HBEC membrane damage was investigated by assessing the release of ⁵¹Cr from cells radiolabeled with [⁵¹Cr]sodium chromate (Amersham International). Prior to exposure to CS, explants were removed from 2- to 3-wk-old, confluent HBEC cultures established on inserts, and the cells were incubated overnight in the presence of 1.0 µCi [⁵¹Cr]sodium chromate. After this incubation the culture medium was decanted from both the inserts and the wells, and the cells were washed gently with medium 199 to remove any radiolabel not incorporated into the cells. Fresh medium 199 was then introduced into both the inserts and the insert wells, and the cultures were exposed to CS for 20 min in the absence or presence of 300 ng/ml Der p introduced into the inserts as described earlier. At the end of the CS exposure, all the medium in the wells, and 50-µl aliquots of the medium in the inserts, were removed. Fresh medium 199 was added to the wells and the cultures were further incubated for up to 24 h at 37°C in an atmosphere of 5% CO₂ in air. During the incubation, the culture medium from the wells and inserts was collected at 1 h, 3 h, 6 h, and 24 h, as described earlier. After the final sampling of the medium from both the wells and the inserts, at 24 h, 500 µl of 1 M NaOH was added to the inserts and the cells were lysed prior to analysis for total radioactivity. Aliquots of 50 µl of all samples collected were mixed with 4.0 ml of Ready-Solv CP scintillation cocktail (Beckman-RIIC) and were assessed for ⁵¹Cr radioactivity with a Beckman LS6500 scintillation counter (Beckman-RIIC). All radioactivity released into the medium at any time point investigated was expressed as a percentage of the total radioactivity in the culture, according to the following formula, and the results were calculated as cumulative release of ⁵¹Cr radioactivity at each time point.

(1)

LDH release studies. Confluent HBEC cultures were equilibrated overnight in phenol red-free medium 199. Following a gentle wash in this medium, fresh medium was added to each culture. Sets of cultures were exposed for 20 min to CS or air in the absence or presence of 300 ng/ml Der p. At the end of exposure, all the medium in the wells, and 50-µl aliquots of the medium in the inserts, were removed. Fresh medium was added to the wells and the cultures were incubated further for up to 24 h at 37°C in an atmosphere of 5% CO₂ in air. During the incubation, the culture medium from the wells and inserts was collected at 1 h, 3 h, 6 h, and 24 h, as described earlier. After the final sampling of the medium, 500 µl of 1% Triton-X was introduced into each insert to lyse the cells, and the cell supernatant was collected. The LDH activity in all medium and cell supernatant samples was determined with an LDH Cytotoxicity Kit (Boehringer Mannheim, Lewes, UK), and the percentage of cell damage was determined according to the formula:

(2)

Statistical Analysis

Data were tested for normality with a normal probability plot and the Shapiro-Wilk test. In cases in which data followed a normal distribution, within-group comparisons were made through two-way analysis of variance (ANOVA) and paired t tests, and between-group comparisons were made with two-sample t tests. For non-normally distributed data, nonparametric ANOVA and the Mann- Whitney U test were used, and results were expressed as median and interquartile range (IQR). All statistical tests were done with a standard computer package (Minitab Data Analysis Software Release 6.1.1, 1987; Minitab Inc., State College, PA), and all values of P < 0.05 were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

The diluent for Der p was used as a control for all experiments. No changes in permeability or release of ⁵¹Cr occurred in HBEC cultures incubated in the presence of Der p diluent.

Assessment of Protease Activity in the Der p Allergen Preparation

Incubation of 300 ng/ml Der p with either of the two substrates, Boc-Gln-Ala-Arg-AMC (Figure 2A) or ABZ- Val-Ala-Nle-Ser-(NO₂)Tyr-Asp-NH₂ (Figure 2B), significantly increased the fluorescence in the reaction mixtures as compared with control incubations without Der p. Concomitant incubation in the presence of the specific inhibitors E64, ₁-AT, and SBTI significantly attenuated the Der p-induced fluorescence when Boc-Gln-Ala-Arg- AMC was used as the substrate (Figure 2A). Concomitant incubation in the presence of ₁-AT and SBTI, but not E64, also significantly attenuated the Der p-induced fluorescence when ABZ-Val-Ala-Nle-Ser-(NO₂)Tyr-Asp- NH₂ was used as the substrate (Figure 2B).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Protease activity in 300 ng/ml Der p as measured by the cleavage of Boc-Gln-Ala-Arg-AMC (A) or ABZ-Val-Ala- Nle-Ser-(NO2)Tyr-Asp-NH2 (B) in the presence or absence of saturating amounts of E64, 1-AT, or SBTI. See the MATERIALS AND METHODS section for inhibitor concentrations. F is the fluorescence value after 30 min minus the fluorescence value at time zero, where background (/) indicates the basal change in fluorescence attributable to the medium and substrate alone.

Assessment of Dose-Response Effect of Der p on the Passage of ¹⁴C-BSA across HBEC Cultures

Incubation of HBEC cultures with 300-3,000 ng/ml Der p significantly increased the passage of ¹⁴C-BSA across HBEC cultures in a dose-dependent manner, and did so maximally after 24 h, as compared with the passage of ¹⁴C-BSA in the absence of Der p (Figure 3). Incubation with Der p activated with 1 mM cysteine further enhanced the passage of ¹⁴C-BSA across HBEC cultures at all concentrations of Der p studied (Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Effect of various concentrations of nonactivated (+0 mM cysteine) Der p (open bars) and activated (+1 mM cysteine) Der p (closed bars) on the passage of 14C-BSA across HBEC cultures after 24 h of incubation (#P < 0.05, ##P < 0.001 versus 300 ng/ ml nonactivated Der p; *P < 0.001 versus 300 ng/ml activated Der p; n = 10 at each concentration).

Effect of Exposure to CS in the Absence or Presence of Der p on HBEC Culture Permeability

Passage of ¹⁴C-BSA. Figure 4 shows the effect of exposure for 20 min to 6 h to air/CS, in the absence or presence of 300 ng/ml Der p, on the passage of ¹⁴C-BSA across HBEC cultures over an incubation period of 24 h.

View larger version (8K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 4.   The effect of 20 min (a and b), 1 h (c), 3 h (d), and 6 h (e) exposure to CS in the absence or presence of 300 ng/ml Der p on the passage of 14C-BSA across HBEC cultures over a period of 24 h (+P < 0.001 versus air, *P < 0.005, **P < 0.001 versus air + Der p; and #P< 0.005, ##P < 0.001 versus CS. For each time point n = 12 for 20-min exposure studies and n = 6 for the 1 h, 3 h, and 6 h studies). (b) Effect of 500 µM GSH on passage of 14C-BSA across HBEC cultures exposed to air or CS for 20 min and incubated in the absence or presence of 300 ng/ml Der p for up to 24 h (n = 8 at each time point).

Changes in ER of HBEC cultures. Figure 5 shows the effect of cell growth on ER of cell culture inserts over a period of 28 d. Bronchial epithelial cell growth progressively increased the ER of cell culture inserts from a median value of 215  (range: 208-221 ) for inserts without cells to a value of 580  [range: 437-640 ) for inserts with confluent cells after culture for 28 d.

View larger version (10K): [in this window] [in a new window]   Figure 5.   Changes in ER of cultures over a period of 4 wk (*P < 0.05, **P < 0.001 versus baseline, n = 20 at each time point).

View larger version (26K): [in this window] [in a new window]   Figure 6.   The effect of 20 min (a), 1 h (b), 3 h (c), and 6 h (d) exposure to CS in the absence or presence of 300 ng/ml Der p on the ER of HBEC cultures over a period of 24 h (+P < 0.05, ++P < 0.001 versus air; *P < 0.05 versus air + Der p. For each time point n = 12 for 20-min exposure studies and n = 9 for the 1 h, 3 h, and 6 h studies).

Effect of Exposure to CS on the Passage of Der p across HBEC Cultures

Figure 7 shows the effect of exposure for 20 min to 6 h to air/CS on the passage of Der p across HBEC cultures over an incubation period of 24 h. When HBEC cultures were incubated in the presence of 300 ng/ml Der p and exposed for 20 min to air, there was a significant time-dependent increase in the passage of Der p across the cultures (Figure 7a).

View larger version (11K): [in this window] [in a new window]   Figure 7.   Effect of 20 min (a), and 1 h, 3 h, and 6 h (b) exposure to CS on the passage of Der p across HBEC cultures over a period of 24 h (+P < 0.001 versus 1 h exposure to air, and #P < 0.001 versus 1 h exposure to CS; n = 12 at each time point).

Exposure to CS for 20 min led to a significant and more pronounced time-dependent increase in the passage of Der p across HBEC cultures than did exposure for 20 min to air. Analysis of the passage of Der p across HBEC cultures exposed either to air or to CS showed that the difference became statistically significant at 3 h after exposure and remained so for up to the full 24-h period studied (P < 0.05).

Exposure to CS for longer periods of 1 h, 3 h, and 6 h led to an even greater increase in the passage of Der p across HBEC cultures than was observed with exposure to air under similar conditions (Figure 7b).

Effect of Antiproteases on Der p-Induced Changes in the Passage of ¹⁴C-BSA across HBEC Cultures Exposed to Air or CS

Addition of: (1) 0.2 µM ₁-AT; (2) 0.5 µM SBTI; (3) 5 µM E64; and (4) a combination of these inhibitors abrogated the Der p-induced increase in the passage of ¹⁴C-BSA across HBEC cultures (Figure 8). The inhibition was most pronounced when the combination of the three inhibitors was used, and was not discerning of either air or CS exposure (Figure 8).

View larger version (21K): [in this window] [in a new window]   Figure 8.   Effect of protease inhibitors on 300 ng/ml Der p-induced increase in the passage of 14C-BSA across HBEC cultures exposed for 20 min to air (open bars) or CS (closed bars), after 24 h of incubation (*P < 0.01 and **P < 0.001 versus air + Der p; #P < 0.01 and ##P < 0.001 versus CS + Der p).

Effect of Exposure to CS in the Absence or Presence of 300 ng/ml Der p on HBEC Membrane Damage

Release of ⁵¹Cr. Investigations of the effect of 20 min exposure to CS on the release of ⁵¹Cr from HBEC cultures in the absence of Der p showed that CS led to a significantly increased release of ⁵¹Cr from these cells from 1 h onward after exposure, and for up to the full 24-h period studied, as compared with exposure for 20 min to air (Figure 9a).

View larger version (10K): [in this window] [in a new window]   Figure 9.   Effect of 20 min exposure to CS in the absence or presence of 300 ng/ml Der p on the release of 51Cr (a) and LDH (b) from HBEC cultures over a period of 24 h (+P < 0.05, ++P < 0.01, +++P < 0.001 versus air; *P < 0.001 versus air + Der p; #P < 0.05 versus CS; n = 7 at each time point for the Cr release studies; n = 6 for the LDH release studies).

Release of LDH. As was the case with ⁵¹Cr release studies, investigations of the effect of 20 min exposure to CS on the release of LDH from HBEC cultures in the absence of 300 ng/ml Der p showed that CS led to a significant increase in the release of LDH from these cultures as compared with exposure to air (Figure 9b). Contrary to ⁵¹Cr release, however, the release of LDH was much lower and occurred considerably later, from 6 h rather than 1 h onward after exposure.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study we showed that exposure of HBEC cultures for 20 min to CS as compared with exposure to air did not significantly alter the permeability of these cultures, as indicated by a lack of effect on either the ER of the cultures or the passage of ¹⁴C-BSA across the cultures. Exposure to CS for longer periods of 1 h, 3 h, and 6 h, however, significantly increased the permeability of these cultures, as indicated by a decrease in ER and an increase in the passage of ¹⁴C-BSA across the cultures. Additionally, exposure to CS as compared with exposure to air led to significant epithelial cell damage, as indicated by significant release of ⁵¹Cr and LDH from these cells. This effect was augmented further when the cultures were incubated with Der p.

Similarly, we also showed that incubation of HBEC cultures with Der p led to a significant, time-dependent increase in the permeability of these cultures, and that these effects were either enhanced significantly by exposure to CS or abrogated by incubation with specific protease inhibitors and GSH. Also, the passage of Der p itself progressively increased with time during incubation, and this was significantly augmented by exposure to CS. To our knowledge, this is the first report on the interaction between CS and Der p with respect to epithelial permeability and damage.

We have previously shown that HBECs cultured to confluence in vitro retain morphologic and biochemical characteristics similar to those found in vivo (23, 24), which therefore renders this model useful in the study of epithelial cell physiology. Characterization of the different epithelial phenotypes that constitute the HBEC cultures revealed that of the total, approximately 0.5% are goblet cells, 40-45% are ciliated epithelial cells, 35-40% are glandular epithelial cells, and 20% are basal epithelial cells (24).

Despite kinetic limitations and the lack of such naturally occurring defense mechanisms as the presence of a "protective" epithelial lining fluid and endogenous "reducing" agents, which may limit the oxidant-induced cell damaging effect of CS, primary cultures from human airway epithelial cells offer a suitable in vitro model in which to study the effects of direct exposure to CS and the mechanism(s) underlying these effects in epithelial cells. Our cell culture system is also useful for studying the mechanisms by which enzymes exhibiting proteolytic activity exert their effect on the integrity of epithelial cells.

A well-balanced human epithelial lining fluid would undoubtedly present the most optimal culture medium for use in such studies; however, such standardized preparations are not now available commercially. A further limitation of the present study was that epithelial cells were cultured from surgical tissue from lung cancer patients with a history of smoking, and that such cells may react differently to oxidant stress when compared with epithelial cells from healthy nonsmokers. The findings of such studies are nonetheless valuable, since they give an indication of the putative mechanisms underlying CS-induced airway epithelial damage and allergen-pollutant interactions.

Despite these limitations, the finding in the present study that Der p leads to a dose-dependent increase in the permeability of HBEC cultures is in agreement with the findings of others, who have shown that Der p1 and other allergens from Dermatophagoides pteronyssinus exhibit proteolytic activity (25). Whereas Der p1 has cysteine and serine protease activity, Der p2 exhibits lysozyme, Der p3 trypsin, Der p4 amylase, Der p6 chymotrypsin, Der p8 serine protease, and Der p9 collagenolytic serine protease activity (26, 27). Additionally, Der p1 may be capable of disregulating the immune system by proteolytically cleaving the low-affinity IgE Fc receptor (CD23) from the surface of B cells (28).

Herbert and colleagues (29) have shown that both Der p1 and mite growth medium extract increased bovine bronchial mucosal permeability to serum albumin. Additionally, these investigators demonstrated that Der p1 at a concentration as low as 100 ng/ml was capable of causing cell detachment of cultured canine kidney and tracheal epithelial cells, and that exposure of the luminal side of bovine bronchial tissue to Der p1 resulted in histologically apparent epithelial injury. The injury was characterized by cell detachment without cytolysis, an effect that could be blocked by the cysteine protease inhibitor E64, suggesting that Der p1 exerted its effect on cell attachment proteins.

The allergen preparation used in our experiments contained a mixture of the Der p subgroups (ALK). At least three protease activities can be distinguished within the Der p extract: (1) E64, ₁-AT, and SBTI sensitive; (2) E64 and ₁-AT insensitive but SBTI sensitive; and (3) E64 insensitive but ₁-AT and SBTI sensitive. This range of activities is consistent with the four serine protease activities previously described in HDM extract (26, 27). Indeed, our findings support the view that the E64-sensitive activity revealed by the Boc-Gln-Ala-Arg-AMC substrate is unlikely for a number of reasons to be a cysteine protease-type Der p1. First, no such activity was detected against the Val-Ala-Nle-Ser-based peptide substrate, previously characterized as an optimal Der p1 substrate (20). Second, when tested against the Boc-Gln-Ala-Arg-AMC substrate, E64 and ₁-AT appeared to be inhibiting the same proteolytic activity, suggesting this to be a serine protease activity, since ₁-AT does not inhibit Der p1 (30). Additionally, there is evidence that E64 can act as a serine protease inhibitor (22).

The results of our studies of the cell damaging effects (release of ⁵¹Cr and LDH) of CS and Der p are interesting in that we found a much greater release of ⁵¹Cr than of LDH in each treatment group, suggesting that the ⁵¹Cr incorporated into HBEC cultures may be readily released from these cultures. This is in agreement with others' findings that the spontaneous release of LDH from cells was considerably weaker than the spontaneous release of ⁵¹Cr (31, 32). Using a bovine endothelial cell culture model, Chopra and colleagues (33) found that any form of injury to the cells led to a much greater and earlier loss of ⁵¹Cr than of LDH from these cells. Substantial loss of ⁵¹Cr was observed even in the absence of ultrastructural damage to endothelial cell membranes. Chopra and colleagues suggested (33) that small molecules, such as adenine nucleotides (⁵¹Cr-labeled), can escape, whereas larger molecules, such as LDH, are retained intracellularly. Others have suggested that LDH release assays are more reliable indicators of damage than is the release of ⁵¹Cr, and therefore are preferable means of measuring cellular cytotoxic reactions (34, 35). Despite this difference, however, there is good correlation between LDH and ⁵¹Cr release assays (36).

Our finding that incubation of HBEC cultures with 300 ng/ml Der p did not lead to damage of these cells is in agreement with the findings of others that proteolytic enzymes generally disrupt epithelial cell linkage by noncytotoxic mechanisms. Histologically, such disruptions are characterized by loss of predominantly lateral, and to a lesser extent basolateral, adhesion between cells, and suggest that the enzymes disrupt intercellular junctions (29, 37).

One interesting observation of ours was that although exposure of HBEC cultures to CS for 20 min doubled the release of both ⁵¹Cr and LDH, there were no changes in the permeability of the cell cultures as assessed by ER or passage of ¹⁴C-BSA. A likely explanation for this observed phenomenon is that although a 20-min exposure to CS may not be sufficient to immediately damage the tight junctions of HBECs, it may have a transient and more subtle adverse effect at the apical surface of these cells, leading to the ready eflux of smaller molecules such as ⁵¹Cr. Indeed, our findings of a correlation between cell damage (increase in the release of ⁵¹Cr/LDH) and an increase in permeability (decrease in ER and increase in passage of ¹⁴C-BSA) when HBEC cultures were exposed for a shorter period of 20 min to CS together with Der p support this hypothesis to a certain extent. It is likely that cleavage of the tight intercellular junctions of HBECs by Der p increases the surface area of individual cells for exposure to CS, and that this subsequently increases the susceptibility of HBEC cultures to both cell damage and permeability.

It is difficult to relate concentrations of Der p used under experimental conditions to concentrations of Der p in the airways, particularly because focal concentration of HDM allergen is more likely to be important. We believe that the 300 ng/ml concentration used in our experiments can easily be encountered during natural exposure. For instance, it has been shown that mite feces contain up to 10 mg Der p1/ml. Once in the airways, allergens elute from these particles and can reach high local concentrations (39). During specific bronchial segmental challenge, similar concentrations of Der p (200 ng/ml) can induce local inflammatory responses (40).

Our finding that although 20-min exposure to CS alone does not significantly influence the permeability of HBEC cultures it does enhance the Der p-induced increase in epithelial permeability and facilitate the movement of Der p through HBECs is both novel and important, since it suggests a likely mechanism for sensitization to HDM in individuals who are exposed to CS.

CS, one of the most damaging indoor air pollutants, is a complex mixture of over 2,000 different compounds, including oxidants. It has been estimated that there are 10¹⁴ free radicals in each puff of CS (15). Our previous studies have shown that ozone (O₃), nitrogen dioxide (NO₂), and particulates, which are all components of CS, can decrease ciliary beat frequency, increase cell damage, increase the permeability of HBEC cultures, and increase the release of inflammatory mediators such as interleukin-8 (41). Additionally, passive or active exposure to tobacco smoke can inactivate ₁-antiprotease in the airway mucosa, allowing the proteolytically active Der p to focally injure the ciliated epithelium. Although the lung has a well-developed antioxidant system, exposure to CS represents a considerable oxidant burden on the respiratory epithelium. Our studies of the effect of GSH on the CS-Der p interaction demonstrated that GSH does have a protective effect, through its antioxidant properties.

Human and animal studies of the effect of exposure to CS have yielded interesting results. Li and colleagues studied the effect of 4 h incubation of monolayers of human type II alveolar epithelial cells (A549 cell line) with both whole- and vapor-smoke condensate, and found a recoverable, concentration-dependent increase in epithelial permeability to [¹²⁵I]BSA, associated with a profound decrease in intracellular GSH, and that the addition of exogenous GSH to the cells reduced the smoke-induced increased epithelial permeability (15). Li and colleagues also reported that both whole- and vapor-smoke condensate significantly increased rat lung epithelial permeability to [¹²⁵I]- BSA at 6 h postinstillation, in association with a significant recruitment of neutrophils into the air spaces. Subsequent studies by the same group of the mechanisms underlying CS-induced damage to the epithelium confirmed the role of the antioxidant GSH (45). Taking their studies collectively, MacNee and colleagues proposed that oxidant stress, causing changes in GSH, was the mechanism underlying the increased epithelial permeability resulting from exposure to CS, and that increased epithelial permeability to CS in vitro was also associated with profound changes in the cytoskeleton (46).

The difference between our own experimental system and that of Li and colleagues may explain why 20-min exposure to CS in our experiments did not influence the permeability of HBEC cultures. It is likely that the 20-min exposure time was not enough to damage HBEC cultures, which, consisting of primary epithelial cells, may behave differently than the A549 cell line used by Li and colleagues.

In conclusion, the present study and the others described here have demonstrated that the Der p extract used in these studies contains at least three proteolytic activities, and that these are likely to lead to cell damage and increase the permeability of HBEC cultures. Additionally, these studies have shown that the interaction between Der p and CS enhances damage to and permeability of HBEC cultures. In view of this interaction, it is tempting to speculate that perturbation of the epithelium in vivo after exposure to CS and Der p allows easier access of the allergen itself: (1) to the dendritic projections of Langerhans cells, which terminate intraepithelially and are normally protected from the external environment by the tight junctions of columnar epithelial cells; and (2) into the subepithelial tissue, where immunocompetent cells reside, therefore increasing the possibility of sensitization to allergens.