INTRODUCTION

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Asthma is a chronic inflammatory disease of the airways of unknown origin which is increasing in prevalence. A profound inflammation is a characteristic feature of fatal asthma. However, recent studies have clearly shown that allergen-mediated inflammation exists even in patients with mild disease (1). Reactive oxygen species (ROS) are likely to participate in asthma. There is a favorable biochemical environment for free radical mediated reactions in the asthmatic airways: (1) the major inflammatory cells involved in asthmatic inflammation such as macrophages and eosinophils produce ROS when activated with different stimuli, and, moreover, cells from asthmatics possess an increased capability to generate free radicals compared with normal cells (2); (2) the oxidant "burst" in the cells can be stimulated by some asthma mediators (5); and (3) antioxidant mechanisms are disturbed in asthmatics (8). Although the commonly generated ROS superoxide radical (O₂) and hydrogen peroxide (H ₂O₂) per se can oxidize biological substrates, the damaging effect is greater when they react with one another or with other reactive species. For example, the reaction of nitric oxide (^.NO) with superoxide (O₂) gives peroxynitrite (ONOO)/peroxynitrous acid (ONOOH), a powerful indiscriminate oxidant and nitrating agent. Recently, it has been shown that peroxynitrite is produced in the asthmatic airway (9). The finding is not surprising because increased amounts of both ^.NO and O₂ are generated in asthma (10). In addition to cells, there are several environmental sources of oxidants, e.g., ozone, a major pollutant, is a potent nonradical oxidant known to generate free radicals in the airways in vivo (11). Nevertheless, because of substantial difficulties in the quantitative measurement of oxidant stress in vivo much of the evidence for the activity of ROS in asthmatic inflammation is indirect or circumstantial. Thus, measurement of increases in ^.NO and H₂O₂ in exhaled gas has been taken as an indication of ROS production, but evidence showing that this results in oxidative consequences in the airway is lacking.

F₂-Isoprostanes (F₂-IsoPs) are recently discovered stable prostaglandin-like compounds that are primarily synthesized by free radical catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase (COX) enzyme. Four F₂-IsoP regioisomers can be formed and each of them can constitute eight racemic diastereomers giving a total of 64 different compounds (12, 13). Unlike prostaglandins, isoprostanes remain in the cell membrane phospholipids until hydrolyzed by specific phospholipases (14). E- and D-ring IsoPs, isothromboxanes, and isoleukotrienes have also been reported. Recently, measurement of F₂-IsoPs has emerged as a reliable tool to assess oxidant status in vitro and in vivo in animals and humans. F₂-IsoPs are elevated in a number of human vascular and inflammatory disorders which have been thought to be associated with an oxidant stress such as coronary reperfusion, atherosclerosis, hypercholesterolemia, hepatorenal syndrome, liver cirrhosis, diabetes mellitus, scleroderma, and in smokers (13). In the human lung, increased formation of F₂-IsoPs has been demonstrated in bronchoalveolar lavage fluid (BALF) after exposure to ozone (15), in patients with interstitial pulmonary fibrosis (16), and in chronic obstructive pulmonary disease (COPD) (17).

In the present study, we demonstrate that inhaled allergen challenge causes the release of F₂-IsoPs into the urine of atopic asthmatics. In addition, F₂-IsoPs are produced in BALF 24 h after segmental allergen challenge, and the increase can be abolished by pretreatment of the volunteers with inhaled corticosteroids. Thus we provide direct evidence that oxidant injury occurs in the setting of allergic inflammation.

	METHODS

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Subjects

Eleven patients with mild atopic asthma, 21 to 44 yr of age, underwent inhaled allergen challenge. Bronchoscopy with bronchoalveolar lavage (BAL) was performed in nine mild allergic asthmatics 24 to 46 yr of age. None of the subjects was using corticosteroids, disodium cromoglycate, or antileukotriene agents. The patients were asked to discontinue theophylline and antihistamines for 48 h, and inhaled short-acting -agonist for at least 12 h before allergen challenges. All volunteers were skin-test positive to at least one allergen.

Study Design

A screening allergen inhalation challenge to determine the dose of allergen causing a fall in the FEV₁ of 20% or greater (the threshold dose), and the measurement of spirometry were carried out as previously described (18). Antigens used included grass, dust mite, and cat (Bayer, Spokane, WA). At least 2 wk after the screening challenge, subjects underwent provocation with the threshold dose of allergen. Pulmonary function was measured at baseline, then every 15 min for the first hour after allergen inhalation, and then hourly for 8 h after inhalation challenge. Urine for analysis of F₂-IsoPs was collected before challenge and then every 2 h for the 8 h of the study. On a separate occasion, inhaled allergen challenge and the collection of urine samples were reproduced in six volunteers after pretreatment with oral aspirin (three doses of 900 mg the day before and 900 mg on the morning of the study) or indomethacin (50 mg in three daily doses for 2 d and 50 mg on the morning of the study and at noon). In another four volunteers urine samples were collected at baseline and in 2-h intervals for 8 h after a challenge with inhaled methacholine causing a decrease in FEV₁ of 20% or greater.

The bronchoscopy study was conducted according to the protocol described elsewhere (19). Nine subjects were randomly assigned to receive either inhaled beclomethasone (6 puffs of 42 µg each 1 h before antigen instillation, and then the same dose 12 and 24 h later) or identical placebo in a double-blind, crossover fashion. After topical lidocaine anesthesia, a fiberoptic bronchoscope was inserted into the airways, and a control BAL was performed in either a lingula or right middle lobe using 50-ml aliquots of warmed normal saline. In the opposite segment, allergen to which the volunteer was skin prick test positive was instilled (5 ml of a solution at 10 allergy units [AU] or 1:10,000). Drug was continued and 24 h later the antigen-challenged segment was similarly lavaged. After a washout period of 3 wk or greater, patients were crossed over to the other arm of the study. All patients tolerated the study without incident. The fluid was filtered through loose gauze, centrifuged, and stored at 70° C until the analysis was done.

Methacholine Challenge

Volunteers had measurements of FEV₁ in duplicate followed by inhalation of doubling concentrations of methacholine every 5 min starting at a concentration 0.075 mg/ml to a maximum of 20.0 mg/ml via DeVilbiss 646 nebulizer coupled to a Rosenthal-French dosimeter (Laboratory of Applied Immunology, Baltimore, MD). Repeat pulmonary function measurements were made 3 min after each dose until the FEV₁ had decreased by 20% from baseline values. All protocols were approved by the Vanderbilt University Committee for the Protection of Human Subjects. All volunteers signed written consent forms before proceeding with the study.

Analytical Methods

F₂-IsoPs in urine and BALF were measured by stable isotope dilution assay that used gas chromatography/negative-ion chemical ionization mass spectrometry (GC-NICI-MS) (20). In brief, the specimens were acidified to pH 3 with 1 M HCl and deuterated internal standard ([²H₄]8-epi-prostaglandin F₂ alpha) was added. Then the samples were extracted on C18 Sep-Pak columns (Waters Chromatography Division, Millipore, Milford, MA) and converted to pentafluorobenzyl (PFB) esters by treatment with a mixture of 10% pentafluorobenzyl bromide and 10% N,N-diisopropylethylamine in acetonitrile (Aldrich Chemical Division, Milwaukee, WI). After evaporation of the reagents under N₂, the residue was subjected to thin layer chromatography (Whatman, Inc., Clifton, NJ). The plates were scraped according to standard and eluted from the silica with ethyl acetate. The samples were dried under N₂ and converted to trimethylsilyl ether derivatives by adding N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) in pyridine (Aldrich Chemical Division). The analysis of F₂-IsoPs was performed using a Nermag R10-10C (Fairfield, NJ) or Hewlett-Packard 5982A mass spectrometer (Palo Alto, CA) and a 15-m DB1701 fused silica capillary column (J&W Scientific, Folsom, CA). Ions were monitored at a mass-to-charge ratio (m/z) of 569 for endogenous F₂-IsoPs and at m/z 573 for the [²H₄]8-epi-PGF₂

PGD-M (9, 11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid), the major urinary metabolite of prostaglandin D₂- PGD₂, was quantified by GC-NICI-MS as previously described (21). Briefly, to 1 ml of urine [¹⁸O₄] PGD-M internal standard was added. Then the sample was acidified to pH 3 with HCl and left to stand at room temperature for 30 min to allow quantitative cyclization of the lower side chain to a hemiketal lactone. After extraction with a C18 Sep-Pak, methylation of the upper carboxyl, and methoxymation of the keto group at C-15, borate buffer (pH 9.1) was added and neutral lipids were extracted with ethyl acetate. The aqueous layer was then acidified to pH 3 with HCl, and PGD-M was extracted with methylene chloride. The lower carboxyl was then converted to a pentafluorobenzyl ester, and the partially derivatized PGD-M was purified on thin layer chromatography. After conversion to a trimethylsilyl ether derivative, quantification of PGD-M was accomplished by selected ion monitoring: mass-to-charge ratio was 514 for endogenous PGD-M and 522 for the internal standard.

Statistical Evaluation

Kolmogorow-Smirnov testing showed normal distribution of the data. Consequently, urinary eicosanoids were analyzed using repeated measures analysis of variance (ANOVA) and Student-Newman-Keuls multiple comparisons test. Analysis of F₂-IsoPs in BALF was performed using the Student's t test. Significance was accepted when the p value was less than 0.05.

	RESULTS

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In all volunteers inhalation of the allergen provoked a 20% or greater decrease in FEV₁ from the baseline. The excretion of F₂-IsoPs was significantly increased at 2 h after allergen inhalation and remained elevated in all urine collections for the 8-h time period of the study (Figure 1). Six patients developed a late response, which was defined as a decrease in FEV₁ of 20% or more from the baseline at 4 to 8 h after allergen inhalation. The concentrations of F₂-IsoPs in these patients were not increased compared with subjects with only an early response.

View larger version (16K): [in this window] [in a new window]   Figure 1.   Urinary concentrations of F2-IsoPs in ng/mg creatinine versus time in 11 atopic asthmatics at baseline and after allergen inhalation challenge. Mean amount of F2-IsoPs for each time period is shown as ± SEM. The differences among columns were significant by ANOVA test.

To demonstrate that the F₂-IsoPs appearing in the urine after allergen challenge were not the COX products of arachidonic acid, oral aspirin or indomethacin was administered in six volunteers before allergen inhalation challenge. COX inhibition was documented by the measurement of the urinary levels of PGD-M, a metabolite of PGD₂, the major COX product of mast cells. As shown in Figure 2, aspirin or indomethacin was effective in blocking the allergen-stimulated increase in PGD-M. In contrast, the levels of F₂-IsoPs were not suppressed, confirming a non-COX origin of the measured compounds (Figure 3). To ensure that the production of F₂-IsoPs was specific to the inhaled allergen, four volunteers underwent a challenge with inhaled methacholine causing a 20% or greater decrease in FEV₁. No alterations in urinary concentration of F₂-IsoPs were generated by methacholine (Figure 4).

View larger version (12K): [in this window] [in a new window]   Figure 2.   Urinary concentrations of PGD-M in ng/mg creatinine in six atopic asthmatics at baseline and after allergen provocation after placebo (open bars) or a COX inhibitor (hatched bars). Mean amount of PGD-M for each time period is shown as ± SEM. Aspirin or indomethacin significantly blocked the allergen-stimulated increase in PGD-M (ANOVA).

View larger version (28K): [in this window] [in a new window]   Figure 3.   Urinary F2-IsoPs in six atopic asthmatics at baseline and after allergen inhalation challenge after a COX inhibitor. Height of the bars indicates mean ± SEM. The differences among columns were significant by ANOVA test.

View larger version (10K): [in this window] [in a new window]   Figure 4.   Urinary F2-IsoPs in four atopic asthmatics at baseline and after methacholine inhalation challenge. Height of the bars indicates mean ± SEM. The release of F2-IsoPs was unchanged by methacholine inhalation causing 20% or greater decrease in FEV1.

Subsequently, F₂-IsoPs were measured in BALF from nine patients with mild atopic asthma at baseline, and from a separate lung segment 24 h after allergen instillation. There was a significant release of F₂-IsoPs into the BALF precipitated by allergen challenge. The response was inhibited by pretreatment of the volunteers with inhaled corticosteroids (Figure 5).

View larger version (16K): [in this window] [in a new window]   Figure 5.   F2-IsoPs in pg/ml BAL fluid from nine atopic asthmatics at baseline and after allergen instillation on placebo (Pl) and 24 h after pretreatment with inhaled corticosteroids (CS). Height of the bars indicates mean ± SEM. Allergen challenge provoked a significant increase in F2-IsoPs on placebo (open bars) (p = 0.007). The increase was suppressed by pretreatment with inhaled beclomethasone (hatched bars) (p = 0.13).

	DISCUSSION

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Our study demonstrates that F₂-IsoPs, the free radical-catalyzed peroxidation lipid products of arachidonic acid, are generated during allergen-induced reaction in atopic asthmatics. Increased concentrations of isoprostanes, determined by the sensitive and specific GC-NICI-MS method, were found in the urine after inhaled allergen challenge and in BALF in response to segmental deposition of the antigen. The formation of F₂-IsoPs appears to be a specific response to allergen because the nonspecific bronchoconstrictor, methacholine, did not cause an increase in F₂-IsoPs. The measured compounds were non-COX products because they were not abrogated by pretreatment of the subjects with either aspirin or indomethacin. The inhibition of the COX enzyme in the subjects proved to be adequate because the synthesis of PGD₂ assessed by the measurement of its major urinary metabolite, PGD-M, was efficiently blocked by either aspirin or indomethacin. COX-dependent formation of F₂-IsoPs has been reported (22), but COX enzymatic activity does not appear to be the source of the urinary F₂-IsoPs generated as a consequence of allergic airway stimulation. It is important to emphasize that the family of F₂-IsoPs contains 64 different compounds which have been identified both in vitro and in vivo (13) and that our methodology does not characterize the whole profile of F₂-ring isoprostanes produced during allergen-evoked inflammation in asthmatics; thus, the total amount of isoprostanes generated is unknown but must be greater than the amount we measured.

The rise of the concentrations of F₂-IsoPs in BALF 24 h after segmental allergen challenge shows that isoprostanes are generated directly in the airways of asthmatic subjects in response to allergen. On the other hand, we did not find consistent release of F₂-IsoPs into BALF 4 min after allergen instillation (data not shown). The lack of F₂-IsoPs early in an allergic reaction could result from a different time course of ROS formation as opposed to mediators such as tryptase and the leukotrienes. It is also known that there is a significant delay from the time of initial lipid peroxidation on the cell membrane to the appearance of free isoprostanes (14). This presumably results from the time necessary for phospholipase activation and subsequent hydrolysis of isoprostanes.

The quantitative assessment of oxidant stress in pathophysiological processes, particularly in vivo, has been associated with major difficulties due to the deficiency of reliable methods. The analysis of lipid peroxidation in human body fluids and tissues based on diene-conjugate and thiobarbituric acid (TBA) assays has been frequently used for that purpose; however, both methods are characterized by a low sensitivity and specificity and, furthermore, can produce confusing artifacts (23). Other traditional approaches, such as an analysis of expired breath condensate hydrogen peroxide and exhaled pentane levels or a measurement of substrate oxidizability or spin trapping of free radical adducts ex vivo suffer from similar limitations. Isoprostanes are stable free radical-catalyzed products of arachidonic acid. There has been a growing number of studies indicating that a specific quantification of F₂-IsoPs in biological samples is a sensitive and a reliable noninvasive method allowing assessment of ROS-caused lipid peroxidation and oxidant stress in vivo (13). Therefore, the elevated concentrations of F₂-IsoPs in the urine and BAL samples from asthmatic subjects after allergen challenge shown in our study can be interpreted as new evidence for an increased oxidant stress in allergen-induced asthmatic reaction. Our finding coincides with the results of earlier studies demonstrating the augmented activity of ROS in asthma (3, 4, 11, 24). The inhibition of allergen-provoked formation of F₂-IsoPs in BALF by inhaled beclomethasone suggests that corticosteroids, which are known to inhibit the late allergic inflammation, may act in part by restraining oxidant stress. This effect of corticosteroids may have been caused by a reduction in the number and activation of cells producing free radicals. Indeed, in a similar experimental model of the late-phase reaction to local allergen challenge, inhaled corticosteroids diminished the influx of eosinophills and reduced the production of inflammatory mediators such as leukotriene B₄ (27).

It is unknown if F₂-IsoPs play a role as a pathophysiological factor in allergen-provoked asthma. Isoprostanes are biologically active compounds. Some of the known effects of F₂-IsoPs could be relevant to the pathophysiology of the lung. For example, one of the F₂-IsoPs, 8-epi-PGF₂, constricts animal and human airways in vitro (28) and causes airflow obstruction and airway plasma exudation in guinea pig in vivo (29). 8-epi-PGF₂ is also a potent vasoconstrictor of the pulmonary artery in rabbits and rats (13, 30). Therefore, although no analogous data exist in humans, F₂-IsoPs could be viewed not only as markers but also as possible mediators of oxidant stress injury in vivo. The mechanism of the action of F₂-IsoPs is unclear. The involvement of the thromboxane receptor as well as a unique isoprostane receptor has been proposed, though the latter hypothesis has not been validated by any experimental data (13). While most of the studies on the biological functions of F₂-IsoPs have been performed with 8-epi-PGF₂, it is likely that other compounds are also bioactive (31). Finally, one can not exclude the possibility that other biologically active isoprostanes (for example, isoprostanes of the E series and isoleukotrienes [13] and even distinct classes of lipid peroxidation products) could also be formed in response to allergen in asthmatics. This notion leads to another general question: could ROS-generated oxidized lipids play a pathophysiological role in allergic inflammation? Recent experimental studies suggest that modest oxidation of membrane lipids may stimulate the expression of selected genes and alter several cellular responses (32). From that perspective, investigation of ROS-mediated lipid peroxidation might be an exciting and important avenue for future studies on the pathogenesis of allergic asthma in humans.

In summary, this study demonstrates that inhaled allergen challenge causes the release of F₂-IsoPs into the urine of atopic asthmatics. F₂-IsoPs are also increased in BALF late after segmental allergen challenge, and this increase can be reduced by pretreatment with inhaled corticosteroids. The pathophysiological role of lipid peroxidation in the pathogenesis of asthmatic inflammation provoked by allergen challenge is currently unknown.