Effects of fluticasone propionate on arachidonic acid metabolites in BAL-fluid and methacholine dose-response curves in non-smoking atopic asthmatics

Hyperresponsiveness of the airways to nonspecific stimuli is a characteristic feature of asthma. Airway responsiveness is usually characterized in terms of the position and shape of the dose–response curve to methacholine (MDR). In the study we have investigated the influence of fluticasone propionate (FP), a topically active glucocorticoid, on arachidonic acid (AA) metabolites in broncho-alveolar lavage (BAL) fluid (i.e. TxB2, PGE2, PGD2, 6kPGF1α and LTC4) on the one hand and MDR curves on the other hand. The effect of FP was studied in a randomized, double-blind, placebo-controlled design in 33 stable nonsmoking asthmatics; 16 patients received FP (500 μg b.i.d.) whereas 17 patients were treated with placebo. We found that the forced expiratory volume in 1s (FEV1 % predicted) increased, the log2PC20 methacholine increased and the plateau value (% fall in FEV1) decreased after a 12 week treatment period. No changes in AA-metabolites could be determined after treatment except for PGD2 which decreased nearly significantly (p = 0.058) within the FP treated group, whereas the change of PGD2 differed significantly (p = 0.05) in the FP treated group from placebo. The levels of the other AA metabolites (i.e. TxB2, PGE2, 6kPGF1α and LTC4) remained unchanged after treatment and were not significantly different from the placebo group. Our results support the hypothesis that although FP strongly influences the position, the shape and also the maximum response plateau of the MDR curve, this effect is not mainly achieved by influence on the level of AA metabolites. Other pro-inflammatory factors may be of more importance for the shape of the MDR curve. It is suggested that these pro-inflammatory factors are downregulated by FP.

HYPERRESPONSIVENESS of the airways to nonspecific stimuli is a characteristic feature of asthma.
Airway responsiveness is usually characterized in terms of the position and shape of the doseresponse curve to methacholine (MDR). In the study we have investigated the influence of fluticasone propionate (FP), a topically active glucocorticoid, on arachidonic acid (AA) metaboHtes in broncho-alveolar lavage (BAL) fluid (i.e. TxB2, PGE2, PGD2, 6kPGFI and LTC4) on the one hand and MDR curves on the other hand. The effect of FP was studied in a randomized, double-blind, placebo-controlled design in 33 stable nonsmoking asthmatics; 16 patients received FP (500 g b.i.d.) whereas 17 patients were treated with placebo. We found that the forced expiratory volume in ls (FEV 1 fro predicted) increased, the log2PC20 methacholine increased and the plateau value (% fall in FEVl) decreased after a 12 week treatment period. No changes in AA-metabolites could be determined after treatment except for PGD2 which decreased nearly significantly (p=0.058) within the FP treated group, whereas the change of PGD2 differed significantly (p 0.05) in the FP treated group from placebo. The levels of the other AA metabolites (i.e. TxB2, PGE2, 6kPGFI= and LTC) remained unchanged after treatment and were not significantly different from the placebo group. Our results support the hypothesis that although FP strongly influences the position, the shape and also the maximum response plateau of the MDR curve, this effect is not mainly achieved by influence on the level of AA metabolites. Other pro-inflammatory factors may be of more importance for the shape of the MDR curve. It is suggested that these pro-inflammatory factors are downregulated by FP.

Introduction
Bronchial hyperresponsiveness (BHR), a prominent feature of asthma, can be demonstrated by generating dose-response curves through inhalation of histamine or methacholine. Usually these curves are sigmoid in shape, with a distinct threshold, a linear slope in the midpart and a maximum response. The provocative concentration producing a fall of 20% in the FEV1 (PC20) is called the sensitivity, whereas the slope in the midpart is defined as reactivity. The plateau value of the curve reflects mammal airway narrowing.
Asthmatics not only show a leftward shift of the dose-response curve, but also higher or even unmeasurable plateau values as compared with normal subjects. Activation of inflammatory 224 Mediators of Inflammation Vol 5 1996 cells and release of mediators, such as arachidonic acid (AA) metabolites in bronchoalveolar lavage (BAL) fluid, may be present in asthma and may influence the shape of the methacholine dose-response (MDR) curve by enhancing BHR. [2][3][4][5] Although it is known that anti-inflammatory therapy with inhaled corticosteroids (ICS) shifts the dose-response curve to the right and reduces the maximum response, 7 data concerning the influence of inflammatory mediators such as AA metabolites are scarce. 4 One may expect that inhibition of their release by ICS may lead to a change in the shape of the dose-response curve. Therefore, in asthmatics, we  In the month preceding the run-in period, m1-1 in normal saline). Methacholine and not patients were only allowed to take inhaled shorthistamine was chosen as bronchoconstrictor acting 2-agonists, on an as needed basis. All stimulus during the study, because it produces other medication was stopped. Patients with a less systemic side effects when given in high history suggesting respiratory infection or exacer-doses. Solutions of methacholine were stored bation of asthma in the month prior to the study at 4C and administered at room temperature. were excluded. All subjects gave informed The aerosols were generated by a De Vilbiss 646 written consent to the study, which was nebulizer (output 0.13 ml min-) and inhaled by approved by the local ethics committee, tidal breathing for 2 min. The response to methacholine was measured as change in FEV Study design: The study was of a randomized, expressed as percentage of initial value and double-blind, placebo-controlled design. After a related tO log2 dose. A test was interrupted if the run-in period of 2 weeks there was a 12 week FEV1 fell by more than 60%, or if unpleasant treatment period. During the 2 week run-in side effects or dyspnoea compelled the patient period patients discontinued use of their usual to stop.
inhaled bronchodilator which was replaced with A recently developed and validated sigmoid salbutamol 400 l.tg as dry powder via the Disk-Cumulative Gaussian Distribution (CGD) funchaler four times daily. Up to four additional tion was fitted to the data. 2 Although the sensidoses were allowed as needed. Baseline data tivity (1og2PC20) was obtained by linear were obtained on two visits with an inteeeal of 2 interpolation of two successive log2 concentraweeks. At each baseline visit that consisted of tion values, 8 the plateau value and the reactivity two morning or afternoon sessions, a flow (defined as slope in the 50% point of the CGD volume curve was constructed, bronchodilator function) were obtained as best fit parameters. response was measured and a provocation test Hence, reactivity denotes the percentual change was carried out. At the second baseline visit, from baseline FEV per doubling dose (%/dd) in intradermal skin testing was performed. When the steepest point of the CGD function. Details patients fulfilled the mentioned criteria, bronchoof the fit procedure and validation of the CGD fit alveolar lavage (BAL) was performed 1 week are according to Aerts eta/. 12 after the last baseline visit. Following the BAL, the patients were randomized to treatment with Bronchoalveolar lavage: Fibre-optic bronchoeither inhaled 500 g FP or placebo, both given scopy was performed according to guidelines of twice daily as dry powder via the Diskhaler. the American Thoracic Society. After pre-Patients continued salbutamol 400 g four times medication with inhaled terbutaline and atroa day, but could take up to four additional doses pine i.m., the bronchoscope (Olympus B1 IT as needed for symptomatic relief. 10, Tokyo, Japan) was introduced into the After 6 and 12 weeks of treatment patients lateral segment of the middle lobe under local attended the clinic on which occasion a MDR curve anaesthesia and placed in wedge position. BAL (see below)was performed. After 13 weeks, 1 was performed with four 50-ml aliquots of week after the last dose-response curve was sterile phosphate buffered saline (PBS)warmed obtained, the BAL procedure was repeated, to 37C. The fluid was then immediately aspir-ated by gently suctioning with -40 cm H20 into Results a siliconized specimen trap placed on melting ice and transported to the laboratory for pro-Sixteen patients (12 men) were randomized cessing and analysis, into the FP group and 17 patients (11 men) into The BAL fluid was centrifuged at 400 x g for the placebo group. Baseline values such as FEV1, 5 min at 4C. The supernatants were decanted reversibility and PC20 histamine were not sigand stored. The cell pellets were then washed in nificantly different between the groups on entry PBS supplemented with 0.5% heat-inactivated to the study (Table 1). Thirty-one of the 33 subbovine serum albumin (BSA). For total leukocyte jects completed the study. One patient receiving numbers in BAL fluid, cell suspensions were placebo and one receiving FP were withdrawn counted in a Coulter Counter and viability was after experiencing a pulmonary exacerbation. assessed by cellular exclusion of trypan blue.
Data of these two patients have not been inclu-Cytospin preparations were stained with May-ded in the analysis. Grnwald-Giemsa stain, and the differential Mean values for FEV1 (as % predicted), sensicounts were performed by counting at least 500 tivity (logiPCi0methacholine), reactivity (%/dd) cells, and plateau (% fall in FEVl) before and after Determination of AA metabolites: Immediately after the BAL procedure, 20 ml of supernatant was processed on C18 SepPak cartridges treatment are shown in Table 2. No statistical significan differences were present between the indices before treatment. In the FP group significan changes occurred after 12 weeks with (Millipore, Bedford, USA) as described pre-respect to means of PC20 (an increase of 3.6 viously, 14  We also determined if there were correlations  No statistical significant differences were present between the indices before treatment. Significance of changes during treatment are indicated (*p < 0.05). Also significant differences in changes between the treatment groups are indicated (** p < 0.01).  AA arachidonic acid; TXB2 thromboxane B2; PGE2 prostaglandin E2; PGD2 prostaglandin 02; 6kPGFI= 6 keto-prostaglandin FI=; LTC4 leukotriene C4. The decrease in PGD2 within the fluticasone group was nearly significant (p 0.058). With respect to the difference in changes between both groups only the change in PGD2 differed significantly (p 0.05) between the fluticasone and the placebo group.
between changes in either treatment group and investigated whether these correlations were different between the treatment groups. In neither of the treatment groups, however, relevant and significant correlations were found between the parameters investigated.

Discussion
We showed that after 12 weeks of treatment fluticasone propionate (FP) significantly decreased both sensitivity to methacholine and the maximal airway narrowing response, whereas it substantially decreased PGD2 levels in BAL fluid. The change in PGD2 level after treatment with FP was significantly larger than the change in the placebo group. We were unable, however, to demonstrate a correlation between these changes in sensitivity and plateau level with the change in PGD2 or one of the other arachidonic acid (AA) metabolites.
To date, several studies have described the relation between airway inflammation, the subsequent release of AA metabolites and bronchial responsiveness. It has to be kept in mind, however, that the bronchial responsiveness in those studies has never been measured by the entire methacholine dose-response (MDR) curve. Bronchial responsiveness as determined by the MDR curve is defined as the sensitivity of the airways to a wide variety of nonsensitizing bronchoconstricting stimuli. It has been demonstrated that the curves from asthmatics could be differentiatied from those of normal subjects by their position, slope and maximal response. MDR curves in asthma have a steeper slope and a higher maximal response at high doses of methacholine as compared to normal subjects. A leftward shift of the cueee can be regarded as being the result of any augmentation of airway narrowing stimuli (i.e. preiunctional mechanisms) such as activation of inflammatory cells and release of mediators such as AA metabolites. [15][16][17] An upward movement of the plateau is the result of an increase in the response of the effector organ (i.e. postjunctional mechanisms) such as smooth muscle contraction and swelling of the airway wall. To our knowledge this is the first study that investigated the possible correlation of sensitivity, reactivity and plateau value of the MDR curve on the one hand and AA metabolites in BAL fluid on the other hand. However, as mentioned above, we could not demonstrate such a correlation, nor could we find a significant correlation between the FP-induced decrease in BHR and the levels of AA metabolites in BAL fluid. This suggests that in addition to AA metabolites other factors, such as epithelial damage, numbers of eosinophils and neutrophils in BAL fluid, pla-In support of this hypothesis, Bel et al.
telet activating factor, histamine and major basic demonstrated that inhaled LTD4 not only caused protein, are important as was shown by other a higher maximal response plateau than methaauthors. [18][19][20][21][22] Our findings are in contrast with choline, but also increased the maximal response those of Oosterhoff et al. who demonstrated that to methacholine for at least 3 days. These findthe levels of PGD2 in BAL fluid are inversely corings could be prevented by the administration of related with the PC20 histamine. A possible inhaled steroids. explanation for this observed discrepancy could Tamaoki et al. 34 found that prednisone be the difference in treatment period; they found reduced the synthesis of eicosanoids by stimua correlation between AA metabolite levels and lated macrophage-rich BAL-fluid cells in vitro.
PC20 histamine after long-term treatment (2.5 However, Dworski et al. showed that oral predyears) with ICS. nisone reduced symptoms but had no significant Prostaglandins D2 and Fia thromboxane B2, effect on BAL fluid eicosanoid levels in vivo. 5 In and leukotrienes B4, C4 and D 4 are potent pro-line with this study, we also failed to find a inflammatory mediators with a wide variety of reduction in AA metabolites in BAL fluid. Only biological activities, including smooth muscle PGD2 appeared to be nearly significantly lower contraction, mucus hypersecretion and leukocyte (p 0.058) in FP treated subjects, whereas the activation.  In a previous study we demon-change in PGD2 before and after 12 weeks of strated that the subclinical inflammation in treatment with FP was significantly larger (p smokers was associated with higher levels of 0.05). It has been demonstrated in canine airway PGF2 and TxB2 in BAL fluid as compared to smooth muscle that PGD2 prejunctionally aug-non_smokers.4 It is conceivable that the ments the parasympathetic contractile increased amounts of PGF2cz and TxB2 are due to response. 4 Stimulation of asthmatics with PGD2 activation of alveolar macrophages in the airways significantly increased the reactivity to histamine of smokers. PGF2a and TxB2 induce airway and methacholine. 2 It was suggested that secretion, constrict isolated human airways and enhanced cholinergic tone underlied these findincrease the sensitivity to contractile stimuli. 26 In ings. Beasley et al. demonstrated that PGD2 and a study of Wardlaw et al. levels of leukotrienes its metabolite 9z, l l[-PGF2 caused a marked (LTC4 and LTB4) were higher in BAL fluid of increase of methacholine induced bronchoconsymptomatic asthmatics with bronchial hyperrestriction that could only partially be prevented by sponsiveness as compared to asymptomancs." 27 an anticholinergic. 2 In addition, several studies Although their methodology to measure AA showed that allergen challenge resulted in a metabolites differed from ours, which makes marked increase in prostaglandin levels in BAL comparison of the results rather difficult, the fluid. <v This suggests that PGD2 may augment levels of LTC4 in our study appeared to be equal the histamine or methacholine induced hyperto those of the asymptomatics in their study. The responsiveness. The effects of prostaglandins on fact that these levels were already low at the start reactivity or plateau value is unknown, because of the study may explain why we were unable to none of the above mentioned authors investidemonstrate an effect of treatment, gated the entire MDR curve and a possible rela-Other workers carried out allergen challenge tion of prostaglandins with reactivity or plateau and measured BAL fluid and urinary levels of value. LTE4, the end product of enzymatically converted In our study PGD2 levels in BAL fluid of asth-LTC4 and LTD4. It was found in asthmatics that matics substantially decreased after treatment the basal levels of leukotrienes were not elevated, with FP. Since PGD2 is a product derived from but increased in vivo after allergen chalmast cells and to a lesser extent from alveolar 38 39 lenge. 28-1 Christie et al. showed that children macrophages, it may be concluded from our with atopic asthma who were resident at high results that ICS, particularly, downregulate mast altitude, exhibit a fall in FEV and an increase in cells to produce PGD2 although the influence on airway responsiveness to histamine upon visiting alveolar macrophages is not excluded.
regions at sea level. This was associated with a In conclusion, in our study we demonstrated three-fold increase in urinary LTE4 excretion. 2 that BHR as determined by the MDR curve is Thus it seems that in asymptomatic asthma downregulated by FP. Although FP strongly influpatients or asthmatics with minor symptoms, the ences the position, the shape and also the levels of leukotrienes in BAL fluid are not maximum response plateau of the MDR curve, it increased. Upon stimulation (tobacco smoke, did not influence levels of AA metabolites in BAL allergen challenge or visit to sea level), however, fluid except for PGD2. Our results indicate that leukotriene levels rapidly increase resulting in the effect of FP on BHR is not achieved mainly bronchial hyper-responsiveness, by its influence on the level of the AA metabo-lites. It is suggested that other pro-inflammatory factors are of more importance for the shape of the MDR curve.