INTRODUCTION

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Bradykinin, a nine-amino-acid peptide, is a metabolite of the kallikrein-kinin system that can be generated during inflammatory diseases such as asthma (1). The view that bradykinin is an important inflammatory mediator in asthma is supported by studies showing elevated kinin concentrations in plasma and in the bronchoalveolar lavage fluid of asthmatic patients after allergen challenge (2, 3). Furthermore, both intravenous and inhaled bradykinin cause bronchoconstriction in asthmatic but not in normal subjects (4, 5). Bradykinin not only induces a bronchoconstrictor response, but is also known to cause pain, mucous gland secretion, vasodilation, and airway plasma leakage. Moreover, bradykinin can sensitize airway sensory nerves and can cause airway inflammation (6, 7). Most of these actions are mediated by bradykinin B₂-receptors located on smooth-muscle cells, epithelium, and endothelium, and also on sensory nerves (8). Hoe 140 (icatibant; D-Arg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸]- bradykinin [9-11]) is a specific, selective, and long-acting B₂-receptor antagonist. Bradykinin-induced responses in guinea pig airways can be completely inhibited by Hoe 140, suggesting that B₂-receptors are involved (9). In recent years, animal models have been developed to study some of the characteristics of asthma. Virus-associated bronchial hyperresponsiveness is an important model for studying basic mechanisms in the pathogenesis of asthma. Epidemiologic studies have shown a close temporal association between respiratory viral infections and exacerbations of asthma (12, 13). Abundant evidence indicates that viral respiratory infections induce airway hyperresponsiveness (AHR) to bronchoconstrictor agents in asthmatic and healthy persons (14). Likewise, animal studies have shown that a virus infection increases airway responsiveness to nonspecific stimuli (15, 16). Also, levels of kinins are increased in the airways of both virus-infected animals and humans (17, 18).

We developed a model system in guinea pigs in which long-lasting AHR has been documented in vitro and in vivo after infection with parainfluenza-3 (PI-3) virus. The excessive airway contraction found in virally infected animals after histaminergic or cholinergic receptor stimulation was associated with an increased number of cells in the airways and bronchoalveolar lavage fluid for up to 16 d after intratracheal inoculation of PI-3 virus (19, 20).

In the present study we investigated the role of bradykinin in PI-3 virus-induced AHR in the guinea pig. Airway responsiveness to histamine was studied in vivo in guinea pigs 4 d after PI-3 virus infection. The animals were pretreated with the potent, long-acting B₂-receptor antagonist Hoe 140 (kindly provided by Dr. K. Wirth of Hoechst GmbH, Frankfurt am Main, Germany) or with saline (control) for five consecutive days, starting 1 d before virus inoculation. Also, the efficiency of the Hoe 140 treatment was tested through bradykinin-induced changes in blood pressure (BP). The lungs of the test animals were examined histologically after these experiments in order to establish the effect of Hoe 140 on cell influx into tissue. We also performed lung lavages to examine the number of cells in the animals' bronchoalveolar lavage fluid (BALF). In a separate set of experiments, we gave nebulized bradykinin to captopril-treated animals and measured their airway responsiveness.

	METHODS

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Animals

Specific-pathogen-free, male Dunkin Hartley guinea pigs (Harlan Olac, Ltd., Blackthorn, UK), weighing 400 to 500 g, were housed in isolators under controlled conditions. Water and commercial chow were allowed ad libitum. The guinea pigs were free of respiratory airway infections as assessed by histologic examination. The experiments done in this study were approved by the Animal Care Committee of Utrecht University.

Intratracheal Inoculation

Suspensions of PI-3 virus (1 ml; tissue culture infective dose [TCID₅₀] = 10^8.9/ml) were centrifuged at 100,000 × g, and the pellet was resuspended in sterile pyrogen-free saline in one-tenth of its original volume (0.1 ml) in order to minimize possible mechanical effects of the inoculation procedure. Growth medium was subjected to a similar procedure for use as a control solution. The animals were anesthetized with ether and were laid on their backs on a small table. The animals' mandibles were kept open with two elastic rubber rings, and a needle with a bulbous tip was placed just behind the glottis. The rubber rings were removed and the animals were placed in an upright position. Thereafter, the 0.1 ml of resuspended PI-3 virus was gently injected into the trachea as an inoculum. Control guinea pigs were treated in the same way, with 0.1 ml of control solution, and were housed in a separate isolator (19, 20).

Experimental Protocol

Guinea pigs were pretreated with 0.1 µmol/kg (equivalent to 170 µg/ kg) of Hoe 140 (9) or with saline (control), administered subcutaneously twice a day for five successive days (Day 0 through Day 4). On Day 1, the guinea pigs were inoculated with the control solution or with PI-3 virus. On Day 5 the animals received a single dose of Hoe 140 or saline (control) (21), after which airway responses and BP were measured.

Airway Responsiveness In Vivo after Infection

Airway responsiveness was measured as described previously (15). In short, the guinea pigs were anesthetized with an intraperitoneal injection of urethane (1.3 g/kg) 4 d after being inoculated with control solution or PI-3 virus. The animals were allowed to breathe spontaneously. An anesthesia-induced decrease in body temperature was avoided by placing the animals in a heated chamber, which kept their body temperature at 37° C. The animals were prepared for the measurement of lung resistance (RL) as follows: airflow () and tidal volume (VT) were determined by cannulating and connecting the trachea with a flow head (No. 000; Fleisch, Lausanne, Switzerland) to a pneumotachograph. A pressure transducer (MP45-2; Validyne Corp., Northridge, CA) measured the transpulmonary pressure (PL) by determining pressure differences between the tracheal cannula and a cannula filled with saline and inserted in the esophagus. RL was determined on a breath-by-breath basis with a computerized respiratory analyzer; RL was derived by dividing PL by at isovolumetric points (50%).

A small polyethylene catheter (PE-50) was placed in the right jugular vein for intravenous administrations of drugs.

A dose-response curve for histamine (0.2 to 2.0 µg/100 g B.W.) was constructed. RL is presented as the actual value minus the basal value. Injections of histamine were given at 10-min intervals.

Assessment of Efficiency of Hoe 140 Treatment

To verify the effectiveness of the Hoe 140 pretreatment as described in the experimental protocol, a dose-response curve with bradykinin (0.025 to 2.5 µg/100 g B.W.) was constructed 30 min after generation of the histamine dose-response curve. Injections were given at 10 min intervals. Airway resistance was measured as described earlier, and BP was recorded by inserting a catheter (PE-50) in the left carotid artery and connecting it to a pressure transducer. Mean arterial pressure (MAP) and diastolic arterial pressure (DAP) were calculated.

Morphologic Examination

After the airway responsiveness experiments, the animals were killed with an overdose of sodium pentobarbital (Nembutal; 30 mg/100 g B.W., intraperitoneally). The tracheas were trimmed free of connective tissue, and a small incision was made near the pharynx to cannulate the tracheas. The lungs were then fixed in situ by instilling a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde in phosphate buffer (pH = 7.4; modified Karnovsky's fixative), as previously described (15). The tracheas were ligated to prevent the outflow of fixative, and were placed in the same fixative in toto. After remaining in the fixative for at least 24 h, the lungs were routinely processed before embedding in paraffin. Sections (5 µm thick, 8 sections per animal, four animals per group) were prepared and stained with hematoxylin- eosin or periodic acid-Schiff-hematoxylin for histologic evaluation, as previously described (15).

Lung Lavages

Lung lavages were performed 4 d after inoculation with virus (21). The animals received a lethal dose of sodium pentobarbital (Nembutal; 30 mg/100 g B.W., intraperitoneally). The trachea was trimmed free of connective tissue and a small incision was made to insert a cannula into the trachea. The lungs were filled in situ with 10 ml of lavage fluid (968 mg ethylenediamine tetraacetic acid/L saline). Fluid was withdrawn from the lungs after gentle lung massage, and was collected in a plastic tube on ice. The procedure was repeated until 40 ml of lavage fluid was collected from each animal. The cells were sedimented by centrifugation at 400 × g for 10 min at 4° C, and were washed twice with saline. The first BALF to be recovered was separated from the rest of the lavage fluid, and the supernatant was stored at 80° C for the measurement of bradykinin and albumin. The cells of the first recovered BALF were pooled with the cells of the BALF recovered later from each individual animal. The cells were stained with Türk's solution and counted in a Bürker-Türk bright-line counting chamber. After centrifugation, all cell preparations were analyzed morphologically on microscope slides. Air-dried preparations were fixed and stained with a thiazine/eosin staining kit (Diff-Quik). Differential counts were made through oil immersion microscopy. Cell viability was > 95% as assessed by trypan blue exclusion.

Bradykinin was measured with a radioimmunoassay (RIA) according to the manufacturer's instructions, and albumin was detected with a modification of Lowry's method, using folin-phenol as reagent (21).

Airway Responsiveness In Vivo after Bradykinin

For measuring airway responsiveness in vivo after administration of bradykinin aerosols, we treated guinea pigs as described earlier. Guinea pigs were allowed to take eight breaths from a chamber that was saturated with bradykinin (10⁷ M) or saline (22). This procedure was repeated five times, with a time interval of 5 min between each episode. Histamine dose-response curves were prepared 5 min after the last aerosol administration.

In a separate set of experiments, animals were pretreated with the angiotensin-converting enzyme inhibitor captopril to prevent the breakdown of bradykinin. Captopril (0.3 mg/ml) was added to the animals' drinking water for 14 d, as had been described by Fox and colleagues (6). Body weight and water intake were measured every other day. Histamine dose-response curves were recorded for animals that were receiving normal water or water containing captopril, and were treated with a saline or bradykinin aerosol.

Materials

Nembutal (60 mg/ml sodium pentobarbital) was obtained from Sanofi (Libourne, France). Histamine phosphate was obtained from the Onderlinge Pharmaceutische Groothandel (Utrecht, The Netherlands). Bradykinin was purchased from Sigma Chemical Company (St. Louis, MO). Urethane was from Acros (Geel, Belgium). The Diff-Quik staining kit was purchased from Baxter (Dade AG, Dudingen, Switzerland). The bradykinin RIA was from Peninsula Laboratories (St. Helens, UK). Captopril was from Bristol Myers Squibb BV (The Netherlands). Bovine parainfluenza 3 virus (PI-3) was obtained from J. C. de Jong (National Institute of Public Health and Environmental Protection, Woerden, The Netherlands).

Statistical Analysis

Differences between groups in the increase in RL and in BP found after the construction of dose-response curves were tested with analysis of variance (ANOVA), followed by a Newman-Keuls test. Otherwise, the unpaired Student's t test was applied. All values of p < 0.05 were considered to reflect a statistically significant difference. Responses are presented as mean ± SEM. The dose of histamine that produces 50% of maximal bronchoconstriction (ED₅₀) was used as an index for sensitivity to histamine. The ED₅₀ value was determined for each individual animal, and the mean ED₅₀ for each experimental group was calculated and evaluated statistically as described earlier.

	RESULTS

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Assessment of Efficacy of Hoe 140 Treatment

In order to assess the efficacy of Hoe 140 in this animal model, the BP of the guinea pigs was recorded. MAP values of 69 ± 1.7 mm Hg (n = 5) and 71 ± 3.3 mm Hg (n = 5) were found as average basal values in the control and virus groups (pretreated with saline), respectively. Pretreatment with Hoe 140 did not affect the MAP values in these groups. When bradykinin was administered (0.025 to 2.5 µg/100 g B.W.) to the virus and control groups pretreated with saline, a strong dose- dependent decrease in DAP was observed (Figure 1A). At the highest does of bradykinin (2.5 µg/100 g B.W.), a decrease in DAP of 28 ± 0.80 mm Hg for the control group (n = 5) and a decrease of 26 ± 0.75 mm Hg for the virus group (n = 5) was observed. In all animals pretreated with Hoe 140, this decrease in DAP was prevented almost completely; only at the highest dose of bradykinin (2.5 µg/100 g body weight) could a decrease in pressure be observed (Figure 1B). This decrease amounted to 7.0 ± 2.9 mm Hg for the control group (n = 5) and 10 ± 2.2 mm Hg for the virus group (n = 5). Thus, Hoe 140, administered in the manner and dose described earlier is an effective B₂-receptor antagonist in guinea pigs. Viral infection did not show any effect on the BP. Bradykinin (up to 2.5 µg/100 g B.W., intravenously) induced a slight increase in RL of 0.14 ± 0.05 cm H₂O/ml · s¹ (142% of basal resistance) in both the control and virus-treated animals. This increase in RL was completely inhibited by pretreatment with Hoe 140. 

View larger version (12K): [in this window] [in a new window]   Figure 1.   Tracings recording arterial BP. (A) A typical dose-response curve for bradykinin (0.025 to 2.5 µg/100 g B.W., administered intravenously) in a control animal. (B) A typical dose-response curve for bradykinin after treatment with the B2-receptor antagonist Hoe 140 in a control animal. Similar tracings were obtained for guinea pigs treated with PI-3 virus.

Virus-Induced AHR

The basal values of RL for the control and virus groups were similar (0.33 ± 0.05 cm H₂O/ml · s¹ and 0.32 ± 0.04 cm H₂O/ ml · s¹, respectively, n = 7 animals per group). Histamine induced a dose-dependent increase in RL. The histamine dose- response curve was shifted significantly upward in the virus group as compared with the control group, both of which were pretreated with saline (Figure 2A). In the virus group, an increase in RL of up to 248% of the control group values could be observed. A significant difference was observed when comparing ED₅₀ values of the virus and control groups (p < 0.05, Student's t test; 1.36 ± 0.13 µg/100 g B.W. versus 1.97 ± 0.24 µg/100 g B.W. for the virus and control groups, respectively).

View larger version (11K): [in this window] [in a new window]   Figure 2.   (A) Increase in RL with intravenously administered histamine in guinea pigs 4 d after inoculation with control solution (open bars, n = 7) or PI-3 virus (solid bars, n = 7). RL is expressed as the actual value minus the basal value (mean ± SEM) (*p < 0.05, ANOVA, Newman- Keuls test). (B) Increase in RL with intravenously administered histamine in guinea pigs pretreated with the B2-receptor antagonist Hoe 140 (0.1 µmol/kg, administered subcutaneously twice a day for five successive days) at 4 d after inoculation with control solution (open bars, n = 5) or PI-3 virus (solid bars, n = 6).

Basal resistance did not change when the virus and control groups were pretreated with Hoe 140 (0.1 µmol/kg, administered twice a day for five successive days). Hoe 140 pretreatment did not alter the histamine dose-response curve of the control group (Figure 2B). However, treatment with Hoe 140 completely blocked the virus-induced increase in the histamine dose-response curve (Figure 1B), but the ED₅₀ of the virus and control groups did not differ (1.35 ± 0.16 µg/100 g B.W. versus 1.28 ± 0.15 µg/100 g B.W. for the virus and control groups, respectively.

Morphological Examinations

Under light microscopy, airways of animals inoculated with control solution showed a normal morphology of pseudostratified bronchial epithelium and adjacent alveolar tissue, and no signs of inflammation (Figure 3A). In contrast, airways of animals inoculated with PI-3 virus showed stratification of bronchial epithelium, edema formation, inflammatory infiltration, and accumulation of many mononuclear cells in connective tissue of the lamina propria and in the thickened interalveolar septa. Several airways were in constriction (Figure 3B).

View larger version (114K): [in this window] [in a new window]   View larger version (130K): [in this window] [in a new window]   View larger version (132K): [in this window] [in a new window]   Figure 3.   (A) Light microscopic appearance of lung tissue from vehicle-treated control guinea pigs. Normal morphology of pseudostratified bronchial epithelium and adjacent alveolar tissue, with no sign of inflammation (original magnification: ×44). (B) Light microscopic appearance of lung tissue from guinea pigs inoculated with PI-3 virus (pretreatment with saline). The lung tissue shows stratification of bronchial epithelium, edema formation, inflammatory infiltration, and accumulation of many mononuclear cells in the connective tissue of the lamina propria of airways and in the thickened interalveolar septa. Partial airway constriction can be observed (original magnification: ×44). (C ) Lung tissue of virus-infected animals pretreated with Hoe 140 (0.1 µmol/kg, administered subcutaneously twice a day for five successive days) shows a similar pathology to that depicted in (B) (original magnification: ×44).

Examination of airways of virus-infected animals pretreated with Hoe 140 showed similar pathologic changes to those described for the virus group pretreated with saline (Figure 3C).

Total and Differential Cell Count

The total number of bronchoalveolar cells in the control group was 15.1 ± 0.6 × 10⁶ cells. in the virus-infected group this number was higher, at 19.3 ± 0.6 × 10⁶ cells (an increase of 28%; p < 0.02). Hoe 140 treatment did not affect these numbers.

In the BALF of virus-infected animals, 78% of the cells were alveolar macrophages (AM) (control group: 13.2 ± 0.6 × 10⁶ AM, versus virus group: 15.0 ± 0.36 × 10⁶ AM; p < 0.05). After Hoe 140 treatment there was still a 22% increase in the number of AM after viral infection (control group: 11.0 ± 1.4 × 10⁶ AM, versus virus group: 13.5 ± 1.7 × 10⁶ AM). The number of eosinophils was more than doubled after viral infection (p < 0.05), and was not influenced by Hoe 140 (virus group: 4.0 ± 0.8 × 10⁶, versus Hoe 140-pretreated virus group: 3.3 ± 0.7 × 10⁶). The percentage of neutrophils, lymphocytes, and monocytes was less than 1.5% in each experimental group.

Four days after viral infection, the bradykinin level in BALF tended to be increased (control group: 101 ± pg/100 µl, versus virus group: 132 ± 24 pg/100 µl, n = 5). Moreover, the albumin levels in BALF were not different in the two groups (control group: 255.2 ± 13.7 µg/ml, versus virus group: 289.9 ± 20.6 µg/ml, n = 8).

Airway Responsiveness In Vivo after Bradykinin

The inhalation of saline or bradykinin did not change basal airway resistance, nor did it affect histamine-induced bronchoconstriction (Figures 4A and 4B). Treatment of animals with captopril did not influence basal airway resistance, but tended to increase the airway responsiveness to histamine at high concentrations of the latter; however, this did not reach the level of significance (Figure 4A). Interestingly, administration of nebulized bradykinin to captopril-pretreated animals resulted in an increase in airway responsiveness to histamine as compared with that of animals receiving normal drinking water (Figure 4B). At a dose of 2.0 µg histamine/100 g B.W., the increase in airway resistance was enhanced by 370% (p < 0.05). ED₅₀ values in the four experimental groups did not change. Further, water intake and gain in body weight during the 14-d pretreatment period did not differ among the experimental groups (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 4.   (A) Increase in RL with intravenously administered histamine in guinea pigs given nebulized saline and pretreated with normal tap water (open bars, n = 4) or captopril (solid bars, n = 4). (B) Increase in RL with intravenously administered histamine in guinea pigs given nebulized bradykinin and pretreated with normal tap water (open bars, n = 4) or captopril (solid bars, n = 4). RL is expressed as the actual value minus the basal value (mean ± SEM) (*p < 0.05, ANOVA, Newman- Keuls test).

	DISCUSSION

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The present study shows that the potent and long-acting B₂- receptor antagonist Hoe 140 completely prevents PI-3 virus-induced AHR to histamine in guinea pigs. Interestingly, administration of nebulized bradykinin to captopril-treated animals also induced AHR. This suggests that bradykinin could be a major component of reactions leading to AHR. Previous reports that bradykinin levels are increased in lavage fluid from airways of both animals and humans infected with virus support this view (17, 18).

The efficacy of Hoe 140 treatment in the guinea pig model used in our study was established by its effect on the bradykinin-induced decrease in BP. This bradykinin effect (up to 2.5 µg/100 g B.W.) could be inhibited almost completely by Hoe 140 (0.1 µmol/kg, twice a day for five successive days). Thus, Hoe 140, administered in the manner and dose described here, is an effective B₂-receptor antagonist in this system. Viral infection did not show any effect on BP.

Besides examining the effect of Hoe 140 on virus-induced AHR to histamine, we also investigated the effect of Hoe 140 on the morphology of lung tissue. Pathologic changes were found 4 d after PI-3 virus inoculation of guinea pigs; changes in the airway epithelium, edema formation, and inflammatory cell influx were observed, as found previously (15). Pretreatment with Hoe 140 did not prevent these morphologic changes. Neither did treatment with Hoe 140 significantly affect the virus-induced increase in number of AM or eosinophils in BALF. Although no effect of Hoe 140 could be found on cell-influx in virus-induced AHR, inhibitory effects of Hoe 140 on the activation state of inflammatory cells remain possible.

Earlier studies showed that drugs like the H₁-receptor antagonist levocabastine and the anti-allergic drugs oxatomide and nedocromil inhibit both cell influx and PI-3 virus-induced AHR in guinea pigs (21). However, our results with Hoe 140 indicate that this virus-induced AHR is not necessarily related to the number of inflammatory cells. In contrast, a murine model of pleurisy (23), Hoe 140 did inhibit bradykinin-induced cell influx. Also, the B₂-receptor antagonists D-Arg-[Hyp³, Thi^5,8, D-Phe⁷]-bradykinin (NPC 349), D-Arg-[Hyp³, D-Phe⁷]- bradykinin (NPC 567), and D-Arg-[Hyp³,Thi^5,8,D-Tic⁷,Tic⁸]- bradykinin (NPC 16731) inhibited both AHR and eosinophilia in an allergic model in guinea pigs (24). On the other hand, in this allergic model, pretreatment with capsaicin or a platelet-activating factor antagonist inhibited AHR without affecting eosinophilia (24). Taken together, these results suggest that the presence of certain inflammatory cells is not a prerequisite to the development of AHR.

Viral infection causes epithelial damage in guinea pig airways that can lead to exposure of sensory nerve endings and excessive release of sensory neuropeptides such as substance P (25, 26). Also, epithelial damage induced by PI-3 virus can lead to loss of activity of neutral endopeptidase, which, since this enzyme degrades both bradykinin and neuropeptides, can enable these substances to have prolonged effects. Substance P release has been suggested to occur during PI-3 virus infection in guinea-pig airways (27). Moreover, depletion of sensory neuropeptides, including substance P, by capsaicin treatment prevents virus-induced AHR (25). In guinea pig airways, bradykinin can activate sensory nerves directly, releasing sensory neuropeptides such as substance P (6, 28). This action of bradykinin can be blocked by Hoe 140, which makes it conceivable that such a mechanism might be involved in the inhibition of virus-induced AHR.

Another pathway in which Hoe 140 could interfere with AHR can be deduced from other actions of bradykinin observed in the airways. In guinea pig airways, application of bradykinin leads to a mixed response comprising both contractile and relaxant components (29). Apart from neural mechanisms, as indicated earlier, release of a cyclooxygenase product or nitric oxide (NO) has been described (29). The release of cyclooxygenase products does not seem likely in the guinea pig model, since earlier studies showed that the cyclooxygenase inhibitor suprofen did not affect virus-induced AHR in guinea pigs (21). In contrast, the virus-induced AHR was associated with decreased NO production upon histamine receptor stimulation (22). Interestingly, citric acid-induced bronchoconstriction in guinea pigs was suppressed by Hoe 140 and enhanced by the NO synthase (NOS) inhibitor N^G-monomethyl-L-arginine (34). It cannot be excluded that continuous bradykinin-receptor stimulation leads to the inability of NOS to produce NO and hence to suppress airway responsiveness to histamine. This hypothesis is supported by three studies. In one, atopic asthmatic subjects with an experimental rhinovirus infection remained equally sensitive to repeated nebulization of bradykinin. In contrast, patients without the viral infection became less sensitive to this agent (35). Further, the increase in AHR in atopic asthmatic individuals after a viral infection was found to be inversely correlated with the amount of NO exhaled air. This means that less NO in exhaled air results in a greater decrease in FEV₁ upon histamine provocation (36). Moreover, administration of a nebulized NOS inhibitor to patients with mild asthma resulted in a significant increase in airway responsiveness to bradykinin. These experiments suggest that NO released upon histamine or bradykinin stimulation suppresses bronchoconstriction (37).

In summary, we found that the potent and long-acting B₂-receptor antagonist Hoe 140 can completely block PI-3 virus-induced AHR to histamine in guinea pigs, but cannot block bronchoalveolar cell influx. Bradykinin may be the key player in the development of AHR. A diminished synthesis of NO and/or an increased production of sensory neuropeptides may be the common mechanism behind both virus-and bradykinin-induced AHR.