INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Inhalation of air pollutants induces airway hyperresponsiveness. This enhancement in bronchial reactivity is a cause of concern, especially in patients whose airways are already compromised by obstructive diseases or sensitized by allergies such as asthmatic patients. A large number of studies suggest that allergic airway diseases are associated with an increased susceptibility to air pollution (1). However, although epidemiological evidence indicates that an increase in exacerbation of allergic diseases such as asthma may be linked to increased air pollution (2, 3), there is little experimental data to support it (4). Clinical studies as well as experimental animal studies have shown that exposure to various air pollutants can increase bronchial responsiveness to allergen stimulation or bronchial reactivity (5). However, this issue remains controversial (4, 10). Therefore, the interaction between air pollution and allergen sensitization requires further investigations with complementary approaches, especially in humans.

We have reported that a variety of gas pollutants such as nitrogen dioxide (NO₂) (11), ozone (O₃) (12), or acrolein (an aliphatic aldehyde) (13) administered ex vivo to the human lung alters the subsequent in vitro responsiveness of bronchial smooth muscle. This in vitro exposure technique has been used to determine the dosimetric relationship between the dose of pollutant and the response in terms of human bronchial smooth muscle contraction (12, 13) or calcium signaling (14).

On the other hand, we have previously developed a model of human isolated airways exhibiting in vitro hyperresponsiveness, i.e., human tissues passively sensitized with asthmatic serum (15). As is the case for airway tissues from spontaneously or actively sensitized animals, immunological passive sensitization produces hyperresponsiveness of human isolated airways and provides the opportunity to study the interaction between allergic factors and smooth muscle behavior.

The aim of the present study was thus to investigate the interaction between passive sensitization and exposure to air pollutants in human isolated airways. We have examined the effect of a preexposure to O₃ or acrolein on the contractile response of human sensitized bronchi to a specific allergenic stimulation, as well as the effect of passive sensitization on the contractile response to nonspecific agonists in human bronchi preexposed to acrolein. On the basis on our previous dosimetric studies (12, 13) we have used two different exposure regimes for these two pollutants corresponding to submaximal and maximal changes in human isolated bronchial reactivity.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Tissue Donors

Human lung was obtained from 25 patients undergoing resection for carcinoma. As in previous studies (13, 18, 20), specimens were selected from patients whose lung function was within a normal range, i.e., whose forced expiratory volume in one second (FEV₁) was above 80% of predicted. Moreover, analysis of medical records of the patients revealed that all were nonatopic (total serum IgE < 100 international units [IU]/ml and negative specific IgE for usual inhaled allergens). Finally, they reported neither a clinical history of allergy or asthma, nor did they receive any antiasthmatic medication.

Tissue Preparation

After resection, the specimens were immediately transferred to the laboratory in an ice-cold oxygenated modified Krebs solution (composition in mM: 118.4 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25.0 NaHCO₃, 11.1 D-glucose, pH 7.4). From a macroscopically tumor-free part of each of the specimens, segments of human bronchi (third to fifth generation; 3 to 5 mm in internal diameter) were carefully dissected from surrounding parenchyma. Segments were then cut into rings measuring 4 to 5 mm in length.

Passive Sensitization of Human Isolated Bronchial Rings

Immunological passive sensitization was carried out as previously described (15). Human bronchial rings were immunologically sensitized by incubation overnight at room temperature in a nondiluted serum pool from atopic asthmatic patients whose concentration of both total and specific IgE to Dermatophagoides pteronyssinus (D. pter.) was above 1,000 IU/ml and 17.5 Phadebas radioallergosorbent test units (PRU)/ml (i.e., +4 RAST titer), respectively. Nonsensitized rings were incubated overnight in a nondiluted serum pool from nonasthmatic, nonatopic subjects whose total IgE concentration was below 10 IU/ml. At the end of each experiment, when baseline tone had reestablished on repeated wash-out of the tissue, 0.05 ml (500 U) of D. pteronyssinus was administered to all of the bronchial rings to verify that it induced a contractile response only in rings that were immunologically sensitized.

In Vitro Exposure to Pollutants

Exposure to acrolein or ozone was performed as previously described in tissues mounted between two stainless steel clips in vertical 20-ml organ baths of a computerized isolated organ bath system (12, 13). Passive sensitization, when required, was always carried out overnight before exposure to pollutants which was performed the following day. Briefly, exposure to acrolein was performed for 10 or 20 min using acrolein in solution in the organ bath at the concentration of 0.3 µM (final concentration in the bath). For exposure to O₃, a Teflon tube was attached to the vertical part of the L-shaped holder of the lower clip. The distal end of the Teflon tube formed a right angle so that it opened into the lumen of the airway rings when attached between the two clips. The proximal end of the Teflon tube was connected to an O₃ generator (ozonator) coupled to an analyzer (photometer; ultraviolet photometric O₃ calibrator 49PS; Thermoenvironmental Instruments, Franklin, MA). The output of the ozonator is 25 parts per billion (ppb) to 1 part per million (ppm) at 6 to 8 L/min with a stability of ± 4 ppb. The concentration of O₃ delivered to the lumen of the airway rings was continuously monitored by means of the photometer, the precision of which is 2 ppb. We have previously shown in human isolated bronchi that exposure to clean air does not alter the subsequent response to agonists compared with that of unexposed tissues remaining immersed in modified Krebs solution (11).

Protocol

Isometric contraction was measured in airway smooth muscle rings that were mounted between two stainless steel clips in vertical 20-ml organ baths of a computerized isolated organ bath system (IOS₁; EMKA Technologies, Paris, France) previously described (11). Baths were filled with modified Krebs solution maintained at 37° C and bubbled with a 95% O₂-5% CO₂ gas mixture. The upper stainless clip was connected to an isometric force transducer (EMKA Technologies, Paris, France). The lower clip was maintained attached to the horizontal part of an L-shaped holder that was placed in the vertical organ bath. Tissues were set at optimal length by equilibration against a passive load of 1.5 g, as previously determined for these types of preparation (17). At the beginning of each experiment, a supramaximal stimulation with acetylcholine chloride (ACh) (10³ M final concentration in the bath) was administered to each of the rings to elicit a reference response that was used to normalize subsequent contractile responses. This reference response to ACh was not different among the various tissue groups (Tables 1 and 2) and was elicited always before exposure to the pollutant. After washing the rings with fresh modified Krebs solution to eliminate the ACh response, two rings were exposed to acrolein or ozone, and two were left unexposed and used as paired temporal control. After exposure to the pollutant, tissues were washed twice with fresh modified Krebs solution, and 10 min after completion of exposure, a cumulative concentration-response curve (CCRC) to the desired agonist, i.e., the antigen D. pter. or the nonspecific agonists carbachol or histamine, was constructed.

Chemicals and Drugs

ACh, carbamylcholine chloride (carbachol), histamine, and acrolein, minimum 90% pure and stabilized with 0.1% hydroquinone, were purchased from Sigma (Saint Quentin Fallavier, France). House dust mite (D. pter.) 1:100 wt/vol, 10,000 protein nitrogen U/ml was obtained from Institut Pasteur (Paris, France). All drug solutions were prepared using distilled water. It was verified that hydroquinone alone at the appropriate concentration had no effect on the responsiveness of human isolated bronchial rings.

Data Analysis and Statistics

For each ring, the contractile response was expressed as a percentage of the maximal reference ACh response in that ring. Because duplicate airway rings were studied in each experimental condition, from the individual CCRC constructed in each ring, a mean CCRC was obtained for the two rings, either control or test, to be representative of that specimen and repeated on five to eight different individuals. Overall mean CCRC were generated in control and test tissues, and paired comparisons between the curves were made. The parameters derived from the CCRC were as follows. F_max, i.e., the contractile force on the CCRC in response to the maximal agonist concentration, was expressed as the mean ± SEM. The concentration of agonist producing 50% of the maximal response (EC₅₀) was expressed as pEC₅₀ (log EC₅₀) ± SEM. pEC₅₀ was determined for each individual tissue by fitting the CCRC with the logistic equation writing F/F_max = 1/[1 + e^{2.3h(C  pEC50)}], (h being the Hill coefficient, a factor related to the slope of the sigmoidal curve, and C the log concentration of the agonist or antigen dilution) using Origin software (Microcal, Northampton, MA). The change in airway smooth muscle responsiveness was defined as F_max, i.e., the difference between F_max in test and control rings expressed as a percentage of F_max in the control ring. Statistical comparison of paired mean CCRC was carried out using first a two-way analysis of variance (ANOVA) along the whole curve to determine whether the curves were different from each other. Then, when the F test was significant, modified Student's paired t tests (two-tailed) using the Bonferroni correction were carried out to determine the concentrations for which the responses were statistically different. pEC₅₀ values were compared using Student's paired t tests. Results were considered significant at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effects of Exposure to Acrolein or Ozone on the Response to the Antigen D. pter. of Human Isolated Bronchi

In this series of experiments, we measured the contractile response of passively sensitized bronchial rings to the antigen D. pter. in a dilution range of antigen solution from 10⁶ to 10², corresponding 0.001 to 10 U/ml D. pter. which induced concentration-dependent contractile responses in these sensitized tissues. In control rings, i.e., rings unexposed to pollutant, F_max was 87.1 ± 4.8% of the reference response to ACh (n = 10). The maximal response was achieved for a 1/1,000 antigen dilution, i.e., 1 U/ml. pEC₅₀ was 4.12 ± 0.26 antigen dilution (log).

Preexposure to 0.3 µM acrolein for either 10 min or 20 min significantly increased the maximal contractile response to D. pter. (Table 1, Figure 1). The maximal change in the contractile response was observed after a 20-min exposure duration, i.e., for an exposure regime to acrolein, assessed as the product of exposure concentration and exposure time, that induces a maximal change in the reactivity to carbachol in human nonsensitized bronchial rings (13) (Figure 2). Exposure to acrolein did not modify the EC₅₀ value.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Effect of exposure to 0.3 µM acrolein on the subsequent CCRC to D. pter. in human passively sensitized isolated bronchial rings. Exposure duration was 10 min (a) and 20 min (b). Open circles: mean values of contractile force (expressed as percentage of reference response to ACh) for seven experiments in acrolein- exposed tissues. Closed circles: mean values for paired experiments in unexposed tissues. Abscissa: antigen dilution from 106 to 102, corresponding 0.01 to 10 U/ml D. pter. Vertical bars are SEM. *p < 0.05.

View larger version (13K): [in this window] [in a new window]   Figure 2.   Change in reactivity of sensitized bronchial rings to antigen stimulation induced by exposure to pollutants. Abscissa: time of exposure to 0.3 µM acrolein (open circles) or 1 ppm ozone (open squares) in min. Ordinate: change in maximal contraction to D. pter. of exposed tissues relative to control (Fmax%). Each symbol represents a mean value from 6 to 8 different specimens. Vertical bars are SEM. *p < 0.05.

Exposure to ozone was performed at 1 ppm for 20 or 40 min, i.e., exposure conditions to ozone that induce submaximal and maximal changes in the reactivity to carbachol in human nonsensitized bronchial rings, respectively (12). As for acrolein, preexposure to ozone increased the amplitude of the contractile response to D. pter. (Table 1, Figure 3), without altering the EC₅₀ value. However, unlike for acrolein, changes in airway responsiveness were similar whatever the duration of exposure (Figure 2).

View larger version (12K): [in this window] [in a new window]   Figure 3.   Effect of exposure to 1 ppm ozone on the subsequent CCRC to D. pter. in human passively sensitized isolated bronchial rings. Exposure duration was 20 min (a, n = 6) and 40 min (b, n = 8). Open squares: mean values of contractile force (expressed as percentage of reference response to ACh) in ozone-exposed tissues. Closed squares: mean values for paired experiments in unexposed tissues. Abscissa: antigen dilution from 106 to 102, corresponding 0.01 to 10 U/ml D. pter. Vertical bars are SEM. *p < 0.05.

Effects of Passive Sensitization on the Response to Carbachol or Histamine in Acrolein-Exposed Human Isolated Bronchi

In a first series of experiments, we assessed the effect of passive sensitization on the contractile response to carbachol of bronchial rings preexposed to 0.3 µM acrolein (Table 2, Figure 4). For a 10-min duration of exposure to acrolein, the amplitude of the maximal carbachol-induced response was 114 ± 11% of the reference ACh response, i.e., was not different from control, nonexposed tissues as previously observed (13). However, the contractile response of sensitized rings was significantly greater than that of nonsensitized ones, the F_max being 33.5 ± 6.2% (n = 5). When tissues were exposed to 0.3 µM acrolein for a 20-min duration, as expected, the maximal response was higher than that observed for a 10-min exposure to acrolein in control tissues. The amplitude of the maximal carbachol-induced response was 197 ± 14% of the reference ACh response, a value close to that we observed previously comparing acrolein-exposed and nonexposed human isolated airways (13). However, there was no difference in contractile response of sensitized rings versus control rings. Whatever the duration of exposure to acrolein, passive sensitization did not induce any change in the potency of carbachol.

View larger version (11K): [in this window] [in a new window]   Figure 4.   Effect of passive sensitization on the subsequent (CCRC) to carbachol in human isolated bronchial rings preexposed to 0.3 µM acrolein. Exposure duration was 10 min (a) and 20 min (b). Open circles: mean values of contractile force (expressed as percentage of reference response to ACh) for five experiments in passively sensitized tissues. Closed circles: mean values for paired experiments in nonsensitized tissues. Vertical bars are SEM. *p < 0.05.

We then assessed the effect of sensitization in rings exposed to 0.3 µM acrolein for 10 min on the contractile response to histamine (Table 2, Figure 5). As for carbachol, sensitized rings exhibited a greater response to histamine than control rings, F_max being 32.5 ± 5.1% (n = 5), without change in the EC₅₀ values.

View larger version (17K): [in this window] [in a new window]   Figure 5.   Effect of passive sensitization on the subsequent CCRC to histamine in human isolated bronchial rings preexposed to 0.3 µM acrolein for 10 min. Open circles: mean values of contractile force (expressed as percentage of reference response to ACh) for five experiments in passively sensitized tissues. Closed circles: mean values for paired experiments in nonsensitized tissues. Vertical bars are SEM. *p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present results indicate that exposure to acrolein and ozone potentiates the contractile response of passively sensitized human bronchus to specific antigen stimulation. These results also show that passive sensitization and exposure to acrolein act synergistically on human bronchial smooth muscle reactivity in response to nonspecific agonists. This synergistic effect depends on the duration and intensity of the pollutant exposure. To the best of our knowledge, this is the first study that provides a proof of principle in vitro for a combined effect of immunological sensitization and exposure to pollutants in human bronchial responsiveness.

Epidemiological studies indicate that an increase in exacerbation of allergic diseases such as asthma may be linked to air pollution (2, 3). Moreover, clinical studies have shown that exposure to various air pollutants can increase bronchial responsiveness to allergen stimulation or bronchial reactivity (7, 9). However, this issue remains controversial (4, 10, 21) and both epidemiological and clinical studies conducted in patients are somewhat limited (1). This is the reason why we designed the present study to investigate the interaction between passive sensitization and exposure to air pollutants in human isolated airways. One should be cautious in extrapolating in vitro data and comparison of our in vitro conditions with those in vivo is difficult because several phenomena, which are not present in vitro such as the breathing pattern, the role of the upper airways, and very importantly, the ongoing inflammatory reaction, may play a role in vivo. Nevertheless, we believe that the present experiments simulate a clinically relevant condition for the following reasons. First, regarding the ex vivo exposure to ozone or acrolein, we have previously compared the range of doses of pollutant producing an effect in vitro with existing data in vivo (12, 13). In human subjects exposed to O₃, the maximal decrease in FEV₁ was observed for a product concentration by time of exposure (C × T) value of approximately 50 ppm × min which is in the same range as that used in the present study (22). In a previous study (12) we observed that in vitro exposure to an O₃ concentration lower than that used in the present study (i.e., 0.5 and 1 ppm, respectively) yielded similar results in terms of bronchial responsiveness provided that the duration of exposure was longer. Moreover, cellular and biochemical changes were observed in the lung of humans exposed to low concentrations of O₃ (0.08 ppm) for a duration of 6.6 h. Again, these changes were more pronounced when the concentration was increased to 0.1 ppm inhaled for the same exposure duration (23). These data correspond to 32 and 40 ppm × min, respectively, a range of doses again similar to that used in the present study. For acrolein, the range of doses used in this study is approximately on the order of one thousandth of that required to produce airway hyperresponsiveness in live animals (i.e., 1 ppm) (24) owing to the absence of a form of protection in the present experimental conditions, i.e., absorption by upper airways and by the mucus layer. Environmental exposure to acrolein may vary from approximately 10 ppb for urban air pollution up to 80 ppm for an average smoker consuming 20 cigarettes per day. Second, regarding immunological sensitization, as is the case for airway tissues from spontaneously or actively sensitized animals, we and others have observed that human tissues passively sensitized with asthmatic serum exhibit in vitro hyperresponsiveness (15, 25).

The in vitro synergistic effect of immunological sensitization and exposure to pollutants was observed in response to two distinct types of agonist. First, exposure to ozone or acrolein increased the response to the specific antigen (i.e., D. pter.) against which were sensitized the patients whose sera were used to passively sensitize the human lung specimens collected in this study. Antigen-induced response in unexposed human bronchial ring was similar in terms of both reactivity and sensitivity to previous findings reported by different investigators (17, 26). There was a difference in the way the two pollutants examined in this study increased the reactivity to D. pter. This increase occurred for doses of pollutants that have been shown to increase in vitro bronchial responsiveness (12, 13). However, the effect of acrolein on antigen-induced contraction was graded unlike that of ozone which remained consistent for two different exposure durations. The reason for this difference is not clear and requires further investigation. Second, passive sensitization increased the response to nonspecific agonists (i.e., carbachol and histamine) in bronchial rings exposed to acrolein. This increase also depended on the dose of acrolein. It occurred only for a duration of acrolein exposure that induced a nonmaximal potentiation of bronchial responsiveness, i.e., 0.3 µM for 10 min (13). When tissues were exposed to this concentration of acrolein but for 20 min, as previously observed (13), the response to the highest concentration of carbachol was maximally increased to approximately 200% of the ACh response and passive sensitization had no further effect.

From the present experiments a definitive interpretation about the cellular and molecular mechanisms of action for this synergistic effect of sensitization and pollutants on human bronchial responsiveness to both specific antigen and nonspecific agonists cannot be proposed because it obviously requires complementary investigations. We assume that this effect is related to an action of each of two conditions, i.e., pollution and sensitization on distinct excitation-contraction coupling pathways in bronchial smooth muscle, i.e., pharmaco- and electromechanical coupling, respectively. This assumption is based on existing information about the mechanism of action of these two conditions in airway smooth muscle. On the one hand, there is evidence that these two pollutants have little effect on the electromechanical coupling of airway smooth muscle, i.e., on the contractile activity which depends on surface membrane potential changes since neither acrolein (13) nor ozone (12) alters the response to KCl. Conversely, these pollutants increase the contractile responses to agonists that, as part of their mechanism of action, produce contraction via activation of pharmacomechanical coupling (i.e., a coupling that is independent of changes in the membrane potential of the smooth muscle cell) because they interact with the release of intracellular calcium ions (12, 14, 31). On the other hand, passive sensitization alters the mechanical response induced by compounds that modulate voltage-dependent mechanisms, i.e., the electromechanical coupling, such as potassium chloride and histamine or ion channel modulators (15, 18) and induces changes in both membrane potential and force generation (32, 33). Whether the synergistic effect on the contractile activity observed in the present study can be ascribed to an effect on distinct excitation-contraction coupling pathways in bronchial smooth muscle requires a direct confirmation.

In conclusion, this study has provided a proof of principle in vitro for a synergistic effect of sensitization and pollutants on human bronchial responsiveness to both specific antigen and nonspecific agonists. Further investigations are required to determine the cellular and molecular mechanisms implicated in this combined effect in order to design appropriate in vivo protocols to address this issue of the association of allergic airway diseases and susceptibility to air pollution.