INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Nitric oxide (NO) plays a key role in the physiologic regulation of airway function and has been implicated in the pathophysiology of inflammatory airway diseases, including bronchial asthma (1).

Nitric oxide is synthesized from the semi-essential amino acid L-arginine by the enzyme NO synthase, and three isoforms of this enzyme have been identified (2). Two constitutive NO synthase isoforms (collectively called cNOS) are expressed in inhibitory nonadrenergic noncholinergic neurons (neuronal NOS or nNOS), endothelial cells (endothelial NOS or eNOS), and epithelial cells (nNOS and eNOS) of the airways (4). These isoforms are activated by depolarization- or agonist-induced intracellular Ca²⁺ changes, and small (picomolar) amounts of NO are generated. This activation is short-lived, and the NO produced serves as a diffusible signaling molecule mediating various processes, including relaxation of airway and vascular smooth muscle (2). These effects are mainly mediated by activation of soluble guanylyl cyclase by binding of NO to its prosthetic heme group (8).

An inducible isoform of NO synthase (iNOS) is induced by pro-inflammatory cytokines in a variety of cells, including macrophages and epithelial cells (1). iNOS is distinguished from the cNOS isoforms by its prolonged Ca²⁺-independent production of relatively large (nanomolar) amounts of NO, while its expression, unlike that of cNOS, is inhibited by glucocorticosteroids (9). In these high concentrations, iNOS- derived NO not only activates soluble guanylyl cyclase, but may additionally have cytostatic and cytotoxic effects, which may be important in the host defense against invading micro-organisms and malignant cells (10, 11). High concentrations of iNOS-derived NO are also produced in asthmatic airway inflammation. Thus, NO is detectable in the exhaled air from humans and various experimental animals (12), and its concentration is increased in exhaled air of patients with chronic asthma (13). The increase in exhaled NO can be normalized after the administration of oral glucocorticosteroids (14) or inhalation of the selective iNOS inhibitor aminoguanidine (15), indicating that high exhaled NO concentrations in these patients may reflect inflammation-induced enhanced expression of iNOS. Indeed, immunohistologic studies have indicated increased expression of iNOS in the airway epithelium and some inflammatory cells in airway biopsies from patients with asthma (16). In addition, both in allergic patients with asthma (17) and in sensitized rats (18) and guinea pigs (19), it has been demonstrated that allergen provocation results in enhanced endogenous NO production during the late asthmatic response, which is due to the induction of iNOS during this response (18).

The role of iNOS-induced NO in allergen-induced airway hyperreactivity (AHR) has thus far not been solved. Since NO is a bronchodilator, increased levels of NO could have beneficial effects on the airway reactivity to bronchoconstrictive stimuli. However, high levels of NO could also have detrimental effects by causing edema due to increased bronchial blood flow and plasma exudation in the airways (21). In addition, high concentrations of NO as well as peroxynitrite, which can be generated from NO and oxygen radicals during the asthmatic inflammation (3), may cause epithelial damage and hence AHR (22, 23). Furthermore, the allergic inflammatory response may be exacerbated by a selective suppressive effect of NO on the T helper cells, type 1 (Th1), and this might promote the proliferation of T helper cells, type 2 (Th2), which are specifically involved in asthmatic airway inflammation (24).

Using a guinea pig model of acute allergic asthma, characterized by early (EAR) and late (LAR) asthmatic reactions, airway inflammation and AHR after both reactions (25), we have recently established both in vivo (26) and ex vivo (27) that a deficiency of (presumably cNOS-derived) NO may contribute to the allergen-induced AHR to histamine and methacholine after the EAR. In contrast, by examining the direct effect of inhalation of the nonselective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) on the histamine-induced airway obstruction after the LAR, it was observed that renewed production or effectiveness of NO is involved in the partial reversal of AHR after the LAR by its functional antagonism of the histamine-induced response (26).

Using inhalation of the selective iNOS inhibitor aminoguanidine instead of L-NAME, we have now investigated whether the partial reversal of allergen-induced AHR is due to the bronchodilatory action of iNOS-derived NO. In addition, the possible detrimental effects of iNOS activation on airway inflammation and the airway reactivity to histamine were investigated by administration of the NOS inhibitor shortly before the onset of the LAR. A recent study by Yates and coworkers (15) in patients with asthma indicated that inhalation of aminoguanidine may be very useful as a selective inhibitor of iNOS in the airways.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Specified pathogen-free guinea pigs of either sex (Charles River, SAVO, Kiszlegg, Germany) were used in this study. All animals, weighing approximately 250 g, were sensitized to ovalbumin at 4 wk of age as described previously (28). To obtain a shift to immunoglobulin E class antibodies, an allergen solution containing 100 µg ovalbumin (OA) and 100 mg Al(OH)₃ per ml saline was used. The mixture of allergen solution and Al(OH)₃ was gently rotated for 60 min to obtain an alu-gel, and 0.5 ml was injected intraperitoneally, while another 0.5 ml was divided over seven intracutaneous injection sites in the proximity of lymph nodes in the paws, lumbar regions, and neck (29). Animals were operated on 3 wk after sensitization and used experimentally 4 to 8 wk after sensitization. The animals were housed in individual cages in climate-controlled animal quarters and were given water and food ad libitum. All protocols described were approved by the University of Groningen Animal Health Committee, which is responsible for the care and proper use of experimental animals.

Measurement of Airway Function

Airway function was assessed by measurement of pleural pressure (Ppl), as described previously (28). In short, a small, fluid-filled latex balloon, connected to a saline-filled cannula, was surgically implanted inside the thoracic cavity. The free end of the cannula was driven subcutaneously to the neck of the animal, where it was exposed and attached permanently. Via an external fluid-filled cannula, the pleural balloon was connected to a pressure transducer (Ohmeda DTX, SpectraMed, Bilthoven, the Netherlands). Ppl (in cm H₂O) was continuously measured using an on-line computer system. Ppl data were sampled for 10 s every minute. Breath-by-breath variation was normally less than 10%; incidental large variations caused by sudden movements or deep sights were rejected from the calculation. We have previously shown that changes in Ppl are linearly correlated to changes in airway resistance and hence can be used as a sensitive index for allergic and nonallergic bronchoconstriction (28).

During the experimental protocol (1-5 wk after surgery), baseline Ppl measurements remained stable and no signs of inflammation were observed at the sites of surgery. Airway function can be monitored repeatedly and continuously for periods of at least 24 h, while the animals are unaware of the measurements being taken.

Provocation Procedures

Ovalbumin and histamine provocations were performed by inhalation of aerosolized solutions. These provocations were carried out in a specially designed perspex cage of 9 L, in which the guinea pigs could move freely. A DeVilbiss nebulizer (type 646, DeVilbiss, Somerset, PA) driven by an airflow of 8 L/min, provided the aerosol with an output of 0.33 ml/min.

The animals were habituated to the experimental conditions and the provocation procedures on two sequential days at least 1 wk after surgery, when preoperative weight was restored. On the first day, the animals were placed in the provocation cage unconnected to the pressure transducer. After an adaptation period of at least 30 min, three consecutive provocations with saline were performed, each provocation lasting 3 min and separated by a 7-min interval. The next day, this procedure was repeated with the animals connected to the measurement system.

On the experimental days following the habituation procedure, allergen and histamine provocations were performed as indicated below. All provocations were preceded by an adaptation period of at least 30 min, followed by two consecutive control provocations with saline as described above. A baseline Ppl value was calculated by averaging the Ppl values from the last 20 min of the adaptation period.

In order to assess the airway reactivity for histamine, provocations with the agonist were performed starting with a 25-µg/ml solution in saline, followed by increasing dosage steps of 25 µg/ml. Histamine provocations lasted 3 min and were separated by 7-min intervals. Animals were challenged until Ppl was increased by more than 100% above baseline for at least three consecutive minutes. The concentration histamine causing a 100% increase of Ppl (PC₁₀₀) was derived by linear intrapolation of the concentration-Ppl curve and was used as an index for airway reactivity toward histamine. Ppl returned to baseline within 15 min after the last histamine provocation.

Allergen provocations were performed by inhalation of increasing concentrations of 1.0, 3.0, and 5.0 mg/ml OA in saline for 3 min, separated by 7-min intervals. Allergen inhalations were discontinued when an increase in Ppl of more than 100% was observed. Using these conditions, none of the animals developed anaphylactic shock after allergen provocation.

Provocation Protocols

In a first group of animals, the concentration and time dependence of aminoguanidine-induced effects on basal histamine reactivity were established. Thirty minutes after the assessment of the basal histamine PC₁₀₀, aminoguanidine was inhaled for 3 min using various nebulizer concentrations (0.1-5 mM); saline inhalation was used as a control. After a resting period of 10 min, the next histamine PC₁₀₀ measurement was started, such that histamine-induced bronchoconstriction occurred approximately 30 min after aminoguanidine inhalation. In separate experiments, using 2.5 mM aminoguanidine, subsequent histamine provocations were performed at 30 min and at 3, 6, 12, and 24 h after inhalation of the NOS inhibitor. Aminoguanidine was dissolved in acidified saline of which the pH was readjusted to normal saline pH.

In a second group of animals, baseline histamine PC₁₀₀ was assessed 24 h before OA provocation. After 30 min, aminoguanidine (0.1 or 2.5 mM, 3 min) was inhaled and a subsequent histamine PC₁₀₀ measurement was assessed 30 min later to determine the effect of aminoguanidine on baseline histamine reactivity, as indicated above. The next day, at 5 h and 23 h after OA provocation, histamine PC₁₀₀ was measured again to determine the allergen-induced AHR after the EAR and the LAR, respectively. To investigate the direct regulatory role of endogenous NO in the observed histamine reactivity at these time points, aminoguanidine was inhaled at 5.5 h and 23.5 h after OA provocation, and subsequent histamine PC₁₀₀ values were reassessed 30 min afterwards at 6 h and 24 h after OA provocation, respectively.

In addition, to investigate the possible effects of 0.1 and 2.5 mM aminoguanidine inhalations, given at 5.5 h after OA provocation, on AHR to histamine after the LAR (indirect effect of aminoguanidine), the same animals inhaled saline instead of aminoguanidine at 5.5 h after allergen provocation, at a different occasion. Both protocols were randomly alternated and performed with a 1-wk interval.

For the quantitative assessment of the EAR (between 0 h and 5 h after allergen provocation) and the LAR (between 8 h and 23 h after allergen provocation) in the abovementioned experiments, airway function was continuously measured during the whole procedure. Between the measurements of PC₁₀₀ values at 5 h and 23 h, the animals were placed in their home cage (0.16 m²), in which water and food were freely accessible and where they could move freely. During this transfer the animals remained connected to the measurement system.

Bronchoalveolar Lavage

At 25 h after OA provocation or saline inhalation (control), a bronchoalveolar lavage (BAL) was performed in all groups of animals, as described previously (25). In short, the animals were anesthetized with 20 mg/kg Brietal, 35 mg/kg Ketalar, and 6 mg/kg Rompun, administered intraperitoneally. The trachea was exposed and cannulated, and the lungs were lavaged gently using 5 ml of sterile saline at 37° C, followed by three subsequent aliquots of 8 ml of saline. The recovered lavage samples were cooled on ice and centrifuged at 200 × g for 10 min at 4° C. The pellets were combined and resuspended to a final volume of 1.0 ml in phosphate-buffered saline and total cell numbers were counted using a Coulter counter. For cytologic examination, cytospin preparations were stained with May-Grünwald and Giemsa. A cell differentiation was performed by counting at least 400 cells in duplicate.

Data Analysis

The magnitudes of the allergen-induced EAR and LAR were expressed as the area under the Ppl time-response curve (AUC) between 0 h and 5 h after allergen provocation for the EAR, and between 8 h and 23 h after provocation for the LAR. Ppl was expressed as percentage change from baseline, and AUC was calculated by trapezoid integration over discrete (5-min) time periods. Based on saline control provocations, threshold values of AUC (mean + 2 × SD; 99% confidence interval) were defined as 1,185% × 5 min for a positive EAR and 2,790% × 5 min for a positive LAR, respectively (25). Using these criteria, animals were characterized as single early responder or dual responder (i.e., animals expressing an early as well as late asthmatic response). Inherent to the research question, only dual responders (27 of 35 animals; 77%) were included in this study.

The effect of single aminoguanidine inhalation on airway reactivity to histamine in time has been statistically analyzed by a one-way analysis of variance (ANOVA) with repeated measure, whereas allergen- and aminoguanidine-induced changes in airway reactivity to histamine, and changes in allergen-induced asthmatic reactions have been analyzed by a one-way ANOVA. When significance was observed (p < 0.05), a complementary analysis was undertaken (Student Newman-Keuls test) to identify differences between groups. Changes in allergen-induced cell infiltration have been statistically analyzed by a Kruskal-Wallis ANOVA. When a significance was found (p < 0.05), a Mann-Whitney U test was performed to determine the significance between different groups. All data are presented as mean ± SEM.

Materials

Histamine hydrochloride, ovalbumin (grade III), aminoguanidine, May-Grünwald stain, and Giemsa stain were obtained from Sigma Chemical Co. (St. Louis, MO). Al(OH)₃ was obtained from J. T. Baker Chemical Co. (Phillipsburg, NJ). Brietal (methohexital sodium) was purchased from Eli Lilly (Nieuwegein, the Netherlands), Ketalar (ketamine hydrochloride) from Parke-Davis (Hoofddorp, the Netherlands), Rompun (2-(2,6-xylidino)-5,6-dihydro-4H-1,3-thiazine-hydrochloride, methylparaben) from Bayer (Leverkusen, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of Aminoguanidine on Basal Airway Reactivity to Histamine

Aerosol inhalation of aminoguanidine increased the airway reactivity to histamine in a dose-dependent manner (Figure 1). A maximal 2.03 ± 0.24-fold increase (p < 0.001) in histamine reactivity was observed at a dose of 2.5 mM aminoguanidine, the effect being very similar to that of the nonselective NOS inhibitor L-NAME (2.02 ± 0.25-fold increase [p < 0.01], not shown) and predominantly representing inhibition of cNOS in these animals (19). A significant increase in histamine reactivity was found for up to 6 h after inhalation of this aminoguanidine concentration (Figure 2). At a concentration of 1 mM aminoguanidine, a submaximal 1.64 ± 0.30-fold increase (p < 0.05) in airway reactivity was observed, while inhalation of 0.1 mM aminoguanidine did not affect the basal airway reactivity to histamine at all (Figure 1).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Effect of inhalation of various concentrations of aminoguanidine (solid bars) and saline (open bar) on basal airway reactivity to histamine. The airway reactivity to histamine is expressed as a ratio of PC100 values observed before and 30 min after treatment, respectively. Data represent mean values ± SEM of three to six animals. Statistical analysis by one-way ANOVA, followed by a Student Newman-Keuls test: *p < 0.05; p < 0.01; p < 0.001.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Time dependence of the effect of a single inhalation of aminoguanidine (2.5 mM, 3 min) on the basal airway reactivity (AR) to histamine. Inset shows the semi-logarithmic plot of the AR increase with time; based on first-order kinetics the effect half-life amounted to 2.0 h. Airway reactivity to histamine is expressed as a ratio of PC100 values observed before (control, open bar) and at various time points after inhalation of aminoguanidine (solid bars). Data represent mean values ± SEM of six animals. Statistical analysis by one-way repeated measures ANOVA followed by a Student Newman-Keuls test: *p < 0.05; p < 0.01; p < 0.001.

Based on the 10- to 100-fold selectivity of aminoguanidine for the inducible NOS isoenzyme (30), 0.1 mM was chosen as an iNOS-selective concentration of the inhibitor, whereas 2.5 mM aminoguanidine was used for total NOS (cNOS and iNOS) inhibition. Inhalation of aminoguanidine aerosol did not affect baseline Ppl at all concentrations used. In addition, administration of saline instead of aminoguanidine did not affect subsequent histamine PC₁₀₀ values (results not shown).

Effect of Aminoguanidine on Allergen-induced Airway Hyperreactivity to Histamine

At 24 h before OA challenge, 0.1 mM aminoguanidine did not affect the airway reactivity to histamine (Figure 3). After the EAR, at 5 h after OA exposure, a significant 2.74 ± 0.48-fold increase (p < 0.01) in the airway reactivity to histamine had developed (Figure 3, Table 1). Subsequent inhalation of 0.1 mM aminoguanidine at 5.5 h after OA provocation had no effect on this AHR (Figure 3, Table 2). A reduced but still significant AHR to histamine (1.44 ± 0.16-fold, p < 0.05) was observed after the LAR, at 23 h after OA exposure (Figure 3, Table 1). Subsequent inhalation of 0.1 mM aminoguanidine at 23.5 h now caused a significant 1.59 ± 0.09-fold increase (p < 0.01) in AHR to histamine as monitored at 24 h (Figure 3, Table 2). Inhalation of 0.1 mM aminoguanidine at 5.5 h after OA provocation did not affect the allergen-induced AHR observed at 23 h after provocation (Table 1), nor did it affect the acute enhancing effect of the iNOS inhibitor given at 23.5 h, as indicated by the similar values for these parameters obtained when the same animals inhaled saline after the EAR instead of aminoguanidine (Table 2).

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effects of 0.1 mM aminoguanidine inhalations on the histamine PC100 value at 24 h before ovalbumin (OA) challenge, and after the allergen-induced early (EAR) and late (LAR) asthmatic reactions. Two subsequent PC100 measurements were performed 30 min before (open bars) and 30 min after (solid bars) inhalation of aminoguanidine. Data represent mean values ± SEM of eight animals. Statistical analysis by one-way ANOVA followed by a Student Newman-Keuls test: *p < 0.05; p < 0.01.

Inhalation of 2.5 mM aminoguanidine caused a significant 2.03 ± 0.24-fold increase (p < 0.001) in airway reactivity to histamine at 24 h before allergen provocation (Figure 4). After the EAR, at 5 h following allergen inhalation, a significant 4.13 ± 0.83-fold increase (p < 0.001) in the airway reactivity to histamine had developed (Figure 4, Table 1). Subsequent inhalation of 2.5 mM aminoguanidine at 5.5 h did not significantly affect this enhanced reactivity to histamine (Figure 4, Table 2). By contrast, the allergen-induced AHR after the LAR (1.41 ± 0.11-fold increase [p < 0.01] in the airway reactivity to histamine) (Figure 4, Table 1) was significantly enhanced after subsequent inhalation of 2.5 mM aminoguanidine at 23.5 h after allergen provocation (Figure 4, Table 2), to a similar extent as with 0.1 mM aminoguanidine (Figure 3, Table 2). Inhalation of 2.5 mM aminoguanidine after the EAR, before the LAR, at 5.5 h after allergen provocation, significantly reduced the allergen-induced AHR after the LAR (at 23 h) as compared with an inhalation of saline instead of aminoguanidine by the same animals (Table 1). Inhalation of 2.5 mM aminoguanidine after the EAR at 5.5 h did not affect the acute (bronchodilating) effect of aminoguanidine (given at 23.5 h) on the AHR after the LAR monitored at 24 h, as indicated by a similar value for this parameter when saline was inhaled after the EAR instead of aminoguanidine (1.82 ± 0.16-fold and 1.57 ± 0.17-fold, respectively; NS) (Table 2).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Effects of 2.5 mM aminoguanidine inhalations on the histamine PC100 value at 24 h before ovalbumin (OA) challenge, and after the allergen-induced early (EAR) and late (LAR) asthmatic reactions. Two subsequent PC100 measurements were performed 30 min before (open bars) and 30 min after (solid bars) inhalation of aminoguanidine. Data represent mean values ± SEM of eight animals. Statistical analysis by one-way ANOVA followed by a Student Newman-Keuls test: *p < 0.01; p < 0.001.

In a control group of animals, inhalations of saline at 5.5 h and 23.5 h instead of aminoguanidine did not affect the airway reactivity to histamine observed at 5 h and 23 h after allergen challenge (Figure 5, Table 2).

View larger version (16K): [in this window] [in a new window]   Figure 5.   Effects of saline inhalations on the histamine PC100 value at 24 h before ovalbumin (OA) challenge, and after the allergen-induced early (EAR) and late (LAR) asthmatic reactions. Two subsequent PC100 measurements were performed 30 min before (open bars) and 30 min after (solid bars) saline inhalation. Data represent mean values ± SEM of eight animals. Statistical analysis by one-way ANOVA followed by a Student Newman-Keuls test: *p < 0.01; p < 0.001.

Effect of Aminoguanidine on the Allergen-induced Late Asthmatic Response

Table 3 shows the EAR and LAR of the different groups of dual responding animals described previously. EARs were comparable between these groups (Table 3). Administration of aminoguanidine in either concentration (0.1 mM or 2.5 mM) at 5.5 h after allergen provocation did not affect the severity of the subsequent LAR (Table 3).

Effect of Aminoguanidine on Allergen-induced Airway Inflammation

Total and differential BAL cell counts from saline- and allergen-challenged guinea pigs are presented in Figure 6. In all groups the recovery of the lavage fluid was high, with an overall average of 81.4 ± 0.3% (n = 32). In the control group of animals that received saline both after the EAR and LAR, allergen provocation increased the number of cells in the BAL, which was accompanied by an increased number of eosinophils (p < 0.001), neutrophils (p < 0.01), and epithelial cells (p < 0.05). The low dose of aminoguanidine (0.1 mM, 3 min) administered at 5.5 h and 23.5 h after allergen provocation did not affect the allergen-induced increase in inflammatory or epithelial cells as compared with the control group receiving saline at both time points. However, with the high dose of aminoguanidine (2.5 mM, 3 min), administered at both 5.5 h and 23.5 h after allergen provocation, the numbers of lymphocytes (p < 0.05), neutrophils (p < 0.01), and epithelial cells (p < 0.05) in the BAL were reduced significantly (Figure 6). In contrast, no significant effect was seen at all in the group of animals receiving saline at 5.5 h and high aminoguanidine (2.5 mM, 3 min) at 23.5 h after allergen provocation, excluding an acute effect of aminoguanidine inhalation on the presence of inflammatory and epithelial cells in the airway lumen.

View larger version (24K): [in this window] [in a new window]   Figure 6.   Total and differential cell counts in the bronchoalveolar lavage (BAL) fluid of ovalbumin-sensitized guinea pigs 25 h after saline (open bars), or ovalbumin (OA) inhalation. The OA-challenged animals had received either saline (shaded bars), 0.1 mM aminoguanidine (hatched bars), or 2.5 mM aminoguanidine (solid bars) both at 5.5 h and 23.5 h after OA provocation, or saline at 5.5 h and 2.5 mM aminoguanidine at 23.5 h after OA provocation (cross-hatched bars). Data represent mean values ± SEM of five to seven animals. Statistical analysis by Kruskal-Wallis one-way ANOVA, followed by a Mann-Whitney U test: *p < 0.05; p < 0.01; p < 0.001.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Using a chronically instrumented, freely moving guinea pig model of acute allergic asthma, we showed for the first time that iNOS-derived NO has both beneficial and detrimental effects on allergen-induced AHR to histamine that is observed after the LAR. Thus, iNOS-derived NO was found to be involved in the functional antagonism of the histamine-induced bronchoconstriction after the allergen-induced LAR, while it also contributed to the development of AHR to histamine after the LAR, possibly by causing enhanced infiltration of inflammatory cells in the airways and damage of the airway. In addition, the latter observation indicates a differential role for NO in the development of AHR after the EAR and LAR, respectively, since the present investigation, as well as previous studies from our laboratory (26, 27), indicated that a deficiency of NO is involved in the AHR after the EAR.

Thus, in an ex vivo study, we recently showed that luminal perfusion of intact guinea pig tracheal tube preparations with the nonselective NOS inhibitor L-NAME caused an increased airway constriction to both intraluminally and extraluminally applied histamine and methacholine, indicating that endogenous (cNOS-derived) NO counteracts the contractile agonist-induced response, as has also been observed by others (34). Very remarkably, similar increases in histamine and methacholine responses were found in tracheal preparations of OA-sensitized guinea pigs 6 h after allergen challenge, i.e., after the EAR, while L-NAME was without effect on these responses, indicating that a reduced production or effectiveness of NO in the airways may be important in the development of allergen-induced AHR at this time point (27). This was confirmed by the in vivo observation that inhalation of L-NAME by OA-sensitized guinea pigs caused increased bronchial responsiveness to inhaled histamine before inhalation of allergen, whereas the effect of L-NAME had completely vanished at 6 h after allergen challenge, when the animals are hyperreactive (26). Similarly, the present study indicated that inhalation of both an iNOS-selective (0.1 mM) and nonselective (2.5 mM) concentration of aminoguanidine had no effect on the AHR to histamine after the EAR.

The allergen-induced AHR to histamine after the LAR was shown to be less pronounced than after the EAR. In a previous study (26), it was demonstrated that the partial reversal of the AHR after the LAR could be completely overcome by inhalation of L-NAME at this time point, indicating that renewed NO synthesis or effectiveness is involved in this reversal by its bronchodilating effect. The present study indicates that the NO involved in the reversal of hyperreactivity is derived from iNOS, since this reversal was equally inhibited by inhalation of an iNOS-selective (0.1 mM) concentration of aminoguanidine and a nonselective concentration (2.5 mM) of this NOS inhibitor. Several studies have indicated that iNOS may be induced during the allergen-induced LAR (17). Most of the expression is localized to the epithelium (20), and presumably involves a number of pro-inflammatory cytokines that are released upon allergen challenge, including interferon gamma (IFN-), interleukin-1, and tumor necrosis factor alpha (16, 35). Very interestingly, IFN- has also been shown to cause inhibition of cNOS expression in various cells (36, 37), indicating that a cytokine-induced switch from cNOS to iNOS may be involved in the observed changes in NOS expression after allergen challenge. Finally, in support of our observation that iNOS-derived NO may have a beneficial effect on allergen-induced AHR, it was recently demonstrated that LPS- induced airway hyporeactivity to carbachol in rats was mediated by iNOS-derived NO (38).

The deleterious effect of iNOS-derived NO on AHR to histamine after the LAR was indicated by the observation that inhalation of 2.5 mM aminoguanidine at 5.5 h after allergen provocation, i.e., after the EAR but before the LAR, reduced the AHR after the LAR. This may reflect a protective effect of the NOS inhibitor on airway inflammation during the LAR, which is presumably involved in the development of AHR after this reaction (39). BAL studies indicated that this protection might involve a reduced number of neutrophils, lymphocytes, and ciliated epithelial cells in the airways, indicating that airway inflammation and epithelial damage induced by iNOS-derived NO may contribute to the AHR after the LAR. This was supported by the observation that inhalation of the lower dose of aminoguanidine (0.1 mM) at 5.5 h after allergen challenge had neither an effect on the BAL cell counts nor on the AHR after the LAR. Although 0.1 mM aminoguanidine was shown to inhibit iNOS activity during at least 30 min, the duration of action of this dose may have been too short to have a significant effect on the inflammatory response during the LAR. By preparing a semi-logarithmic plot of the time course of the 2.5 mM aminoguanidine effect on the basal histamine reactivity (Figure 2, inset), which is mediated by inhibition of cNOS (19), a half-life of this effect of 2.0 h was calculated. Assuming a 26-fold selectivity (40) and a similar half-life of the inhibitor for iNOS, the duration of action on iNOS (15% inhibition remaining) by the higher concentration of 2.5 mM aminoguanidine could be calculated to be 16.9 h (i.e., up to 22.4 h after allergen provocation), which could explain its effectiveness on inflammation during the LAR. In addition, based on these calculations, a direct residing effect of 2.5 mM aminoguanidine, given at 5.5 h after OA provocation, on the AHR measured at 23 h after OA provocation may be considered negligible. This is also indicated by the observation that no difference was found in the acute effects of aminoguanidine on the AHR toward histamine after the LAR (aminoguanidine administered at 23.5 h after OA provocation), when 2.5 mM aminoguanidine or saline were inhaled before the LAR at 5.5 h after OA provocation (Table 2).

Previous studies have indicated that NO may be involved in the chemotaxis of neutrophils (41, 42), which could explain the reduced number of these cells in the BAL after aminoguanidine inhalation. Since neutrophils have the capacity to generate enhanced levels of superoxide anions (O₂) in patients with asthma, which correlates with the airway reactivity to methacholine in these patients (43), enhanced production of peroxynitrite in the vicinity of epithelial cells that express iNOS could be one mechanism of the observed NO-induced epithelial cell damage and subsequent hyperreactivity. Thus, it was recently demonstrated that exogenous peroxynitrite may cause epithelial damage and hyperreactivity of guinea pig airways (23). Moreover, peroxynitrite was demonstrated to increase the release of major basic protein from eosinophils in tracheal preparations (23), which may similarly cause epithelial damage in the airways (44).

In contrast, the number of eosinophils in the BAL was not affected by aminoguanidine inhalation before the LAR. Although the cellular composition of BAL fluid was not determined after the EAR in the present study, in a previous study using the same animal model it has been shown that the major part of eosinophil infiltration is already occurring during the early asthmatic response (25), and may hence not be affected significantly by inhalation of aminoguanidine after the EAR.

Very remarkably, inhalation of 2.5 mM aminoguanidine before the LAR significantly reduced the number of lymphocytes in the BAL obtained after the LAR, although OA provocation did not significantly affect this cell number, as has also been observed in previous studies both in guinea pigs (45) and man (48). A similar reduction of lymphocytes in the BAL and of AHR was also observed in OA-challenged guinea pigs pretreated with a sub-bronchodilating concentration of rolipram, a selective type 4 phosphodiesterase inhibitor (46), or mepyramine, a histamine H ₁-receptor antagonist (47). Since lymphocytes may be involved in the activation of eosinophils (49), reduced lymphocyte number could contribute to the reduced AHR after the LAR.

In conclusion, the present study has shown that iNOS- derived NO may have both beneficial and deleterious effects on allergen-induced AHR to histamine after the LAR, by functional antagonism of histamine-induced bronchoconstriction on the one hand, and by promoting airway inflammation and epithelial damage on the other hand. The net outcome of this dual action in chronic asthma remains to be established.