Allergy
ORIGINAL ARTICLE
AIRWAY DISEASES
Induced sputum eicosanoids during aspirin bronchial challenge of asthmatic patients with aspirin hypersensitivity jcik, K. Wo jcik, A. Gielicz, R. Januszek, A. Cholewa, P. Strez k & M. Sanak L. Mastalerz, N. Celejewska-Wo Department of Medicine, School of Medicine, Jagiellonian University, Cracow, Poland
jcik N, Wo jcik K, Gielicz A, Januszek R, Cholewa A, Strez k P, Sanak M. Induced sputum eicosanoids during aspirin To cite this article: Mastalerz L, Celejewska-Wo bronchial challenge of asthmatic patients with aspirin hypersensitivity. Allergy 2014; DOI: 10.1111/all.12512.
Keywords aspirin-exacerbated respiratory disease; eicosanoids; induced sputum; leukotrienes; prostaglandin E2. Correspondence Prof. Marek Sanak, MD, PhD, Department of Medicine, School of Medicine, Jagielloska 8, 31 – 066 nian University, ul. Skawin w, Poland. Krako Tel.: +48 12 430 51 69 Fax: +48 12 430 52 03 E-mail: [email protected] Accepted for publication 11 August 2014 DOI:10.1111/all.12512 Edited by: Michael Wechsler
Abstract Background: Altered metabolism of eicosanoids is a characteristic finding in aspirin-exacerbated respiratory disease (AERD). Bronchial challenge with lysylaspirin can be used as a confirmatory diagnostic test for this clinical condition. Induced sputum allows to measure mediators of asthmatic inflammation in bronchial secretions. Objectives: To investigate the influence of inhaled lysyl-aspirin on sputum supernatant concentration of eicosanoids during the bronchial challenge test. Subjects with asthma hypersensitive to nonsteroidal anti-inflammatory drugs were compared with aspirin-tolerant asthmatic controls. Methods: Induced sputum was collected before and following bronchial challenge with lysyl-aspirin. Sputum differential cell count and sputum supernatant concentrations of selected lipoxygenases products: 5-,12-,15-hydroxyeicosatetraenoic acid, cysteinyl leukotrienes, leukotriene B4, 11-dehydro-thromboxane B2, and prostaglandins E2, D2, and F2a and their metabolites, were measured using validated methods of chromatography–mass spectrometry. Results: Aspirin precipitated bronchoconstriction in all AERD subjects, but in none of the aspirin-tolerant asthmatics. Phenotypes of asthma based on the sputum cytology did not differ between the groups. Baseline sputum eosinophilia correlated with a higher leukotriene D4 (LTD4) and leukotriene E4 (LTE4) concentrations. LTC4, PGE2, and 11-dehydro-TXB2 did not differ between the groups, but levels of LTD4, LTE4, and PGD2 were significantly higher in AERD group. Following the challenge, LTD4 and LTE4 increased, while PGE2 and LTB4 decreased in AERD subjects only. Conclusions: During the bronchial challenge, decrease in PGE2 and its metabolite is accompanied by a surge in bronchoconstrictory cysteinyl leukotrienes produced at the expense of LTB4 in AERD subjects. Bronchial PGE2 inhibition in AERD seems specific and sensitive to a low dose of aspirin.
Abbreviations 5-,12-,15-HETE, 5-,12-,15-hydroxyeicosatetraenoic acids; 5-LO, arachidonic acid 5-lipoxygenase; AA, arachidonic acid; AERD, aspirin-exacerbated respiratory disease; ATA, L-ASA-tolerant asthma; COX, cyclooxygenase; cys-LTs, cysteinyl leukotrienes; LASA, lysyl-acetylsalicylic acid; LTB4, leukotriene B4; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4; NSAIDs, nonsteroidal anti-inflammatory drugs; PGD2 metabolites, 9a,11bPGF2a – 13,14-dihydro,15-keto-PGD2; PGD2, prostaglandin D2; PGE2 metabolite, 13,14-dihydro,15-keto-PGE2; PGE2, prostaglandin E2.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inhalation of hypertonic sodium chloride solution facilitates expectoration of sputum. It is used as a simple noninvasive diagnostic procedure for sampling of bronchial secretions, useful in monitoring of lower airway inflammation. Induced sputum (IS) has been validated for studies on inflammatory cells and mediators in the assessment of both asthma and other pulmonary diseases (1). Eicosanoids, including 5-lipoxygenation products leukotrienes (LTs) and cyclooxygenase metabolites prostaglandins (PGs), are highly bioactive oxylipins involved in the asthmatic
Sputum eicosanoids in aspirin bronchial challenge
inflammation (2). Aspirin-exacerbated respiratory disease (AERD) is a distinct asthma phenotype characterized by persistent course of the disease, chronic rhinosinusitis with nasal polyposis, and hypersensitivity to aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) (3). There is a consensus on nonallergic mechanism responsible for asthmatic attacks in AERD patients, involving abnormal metabolism of arachidonic acid (AA) by cyclooxygenase (COX) and lipoxygenase (LO) pathways (3–5). Inhalation of exogenous PGE2 prevents bronchoconstriction provoked by aspirin and inhibits urinary excretion of cys-LTs (6–8). These effects seem not related to myorelaxant activity of PGE2 on bronchial smooth muscle, and PGE2 is fast-inactivated to 13,14-dihydro,15-keto-PGE2. It is rather release of mediators from inflammatory cells, the most likely mast cells and eosinophils, which is inhibited by PGE2 (9, 10). Epithelial cells from surgically removed nasal polyps of patients with AERD produce less PGE2 than the same cells from aspirin-tolerant controls (11), and peripheral blood cells from patients with AERD release less PGE2 at baseline than healthy controls (12). PGE2 production by bronchial fibroblasts in AERD is lower, as compared to aspirin-tolerant (ATA) controls (13). Moreover, lower expression of COX-2 in nasal polyp tissue from aspirin hypersensitive patients, accompanied by decreased production of PGE2, contributes to imbalance between anti- and pro-inflammatory eicosanoids (14, 15). Expression of inhibitory EP2 receptor for PGE2 is decreased on inflammatory cells infiltrating the nasal mucosa of patients with AERD (16), and a particular promoter variant of the gene encoding EP2 receptor is more frequent among AERD subjects (17). However, after oral administration of aspirin to patients with AERD, no decrease in the systemic production of PGE2 could be demonstrated using measurements of stable PGE2 metabolites in urine (18). Data on local PGE2 response to aspirin challenge are scarce and contradictory (19–21). 5-LO is the key enzyme in leukotriene synthesis transforming AA into leukotriene A4 (LTA4). This intermediate can generate bioactive eicosanoids by hydrolysis into leukotriene B4 (LTB4) or by conjugation with glutathione into leukotriene C4 (LTC4). Next steps of extracellular metabolism produce leukotriene D4 (LTD4) and leukotriene E4 (LTE4). LTB4 is a potent chemoatractant for neutrophils, stimulating their adhesion to epithelia and production of reactive oxygen species. Cysteinyl leukotrienes (cys-LTs), LTC4, LTD4, and LTE4, are mediators of allergic inflammation potently contracting smooth muscles, causing vascular leak and mucus production (22). In numerous studies, the key role of cys-LTs overproduction in AERD was well documented on the systemic level, and increased urinary excretion of LTE4 is a hallmark of this disease (18, 23–26). Following ingestion of NSAIDs, cys-LTs released from eosinophils and mast cells cause a violent bronchoconstriction and sometimes extrapulmonary signs of facial edema and skin erythema (9, 25, 27). A pharmacological mechanism of hypersensitivity has been proposed, in which COX-1 inhibition by aspirin and other NSAIDs decreases biosynthesis of PGE2. This may lead to
Mastalerz et al.
enhanced or disinhibited synthesis of cys-LTs by inflammatory cells (28). Specific synthases convert cyclic endoperoxides of AA not only to PGE2 but also to other prostaglandins: D2 (PGD2), F2a (PGF2a), and prostanoids: thromboxane A2 (TXA2) and prostacyclin. Both PGD2 and its metabolite 9a,11b-PGF2a, and PGF2a are potent bronchoconstrictors (22), while more abundant metabolite 13,14-dihydro,15keto-PGD2 is not active. Elevated levels of PGD2 or its metabolite were reported in plasma (29), urine (9, 25) and in exhaled breath condensate (30) (EBC) from patients with AERD. However, no change in PGE2 or PGD2 could be detected in EBC after oral or bronchial aspirin provocation (31, 32). Inhibitory effect of aspirin on PGE2 biosynthesis in AERD subjects was shown in bronchoalveolar lavage fluid (20, 33) after segmental provocation and in nasal washings after topical administration of aspirin (21). In contrast with PGE2, level of PGD2 metabolite 9a,11bPGF2a increased following aspirin challenge in both urine and plasma (29). The aim of this study was to investigate effects of aspirin bronchial challenge on prostanoids and leukotrienes in the induced sputum supernatant (ISS) from AERD subjects. We hypothesized that the eicosanoid profile in IS is specific for aspirin hypersensitivity and could reveal mechanisms underlying bronchoconstriction on the bronchial level. Eicosanoid measurements were taken using a high sensitivity and specific mass spectrometry analysis.
Methods Subjects The study groups consisted of 27 patients with asthma, whose aspirin hypersensitivity was diagnosed by oral provocation test and 16 asthmatics who tolerated aspirin well. The asthma diagnosis and control was ascertained according to GINA 2012 update. Definition of severe asthma complied with ATS/ERS 2013 recommendations. The clinical characteristics of the study participants are presented in Table 1. Aspirin-tolerant asthmatic (ATA) controls occasionally used NSAIDs without any adverse reactions. All the study subjects had stable asthma, and their baseline FEV1 was >70% the predicted value on the challenge day. None had experienced any exacerbation or a respiratory tract infection within the 6-week period preceding the study. The study participants were instructed to withhold medications decreasing bronchial responsiveness prior to the aspirin challenge. Short-acting b2-agonists were not used at least 8 h before the challenge, and long-acting b2-agonists and theophylline were withdrawn for 24 h. Short-acting antihistamines and cromones were discontinued 5 days before the challenge. Inhaled steroids were allowed at dose not exceeding 2000 lg fluticasone equivalent per day. None was treated with systemic corticosteroids or leukotriene-modifying drugs. Baseline and postchallenge sputum induction was carried out in all study participants. Informed consent was obtained from all
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Sputum eicosanoids in aspirin bronchial challenge
Table 1 Clinical characteristics of the study participants
Age (SD) ACT median (min–max) Duration of CRS, median (min–max) Inhaled steroids yes/no Dose of inhaled steroids (lg/day) fluticason eq., median (min–max) FEV1 baseline (%predicted) Allergic rhinitis yes/no Chronic rhinosinusitis yes/no Total serum IgE (IU/ml), median [interquartile range] Peripheral blood eosinophilia count (mm 3), median [interquartile range] IS cellular phenotype Eosinophilic Neutrophilic Mixed Paucicellular
all AERD (n = 27)
AERD with extrabronchial symptoms (n = 11)
ATA (n = 16)
40.6 9.8 22 (16–25) 7 (1–27) 27/0 1000 (100–2000)
38.0 10.6 22 (16–25) 5 (1–26) 11/0 500 (100–1000)
48.9 17.3 25 (19–25)* 11 (2–30) 11/5*** 400 (0–1110)
92.6 14.8 10/17 27/0 104 [49.2–212] 397 [260.5–555]
91.7 14.5 4/7 11/0 176 [50.5–364.8] 356 [154.5–441.5]
100.1 16.2** 6/10 11/5*** 141 [65.2–282] 390 [210.3–749.5]
13 6 1 7
4 4 0 3
5 4 2 5
ACT, asthma control test; CRS, chronic rhinosinusitis; IS, induced sputum. *AERD vs ATA P < 0.05. **AERD vs ATA P < 0.01. ***AERD vs ATA P < 0.005.
subjects, and the study was approved by the Jagiellonian University Ethics Committee (KBET/7/B/2010).
participants were monitored for FEV1 and extrabronchial symptoms every 30 min for the next 6 h after the challenge.
Study design The study was conducted in two consecutive days. On the first day, baseline IS samples were collected. On the second day, a standard bronchial single-blind placebo-controlled challenge test with lysyl-aspirin (Kardegic, Sanofi Aventis, Prague) was carried out in all study participants (34). The test began with the inhalation of seven breaths of placebo (saline), followed by FEV1 measurements 10 and 20 min later. The postsaline FEV1 obtained at 20 min was used as the prechallenge baseline value. By increasing concentrations and incrementing numbers of breaths, doses of lysyl-aspirin (0.18, 0.36, 0.90, 2.34, 7.20, 16.2, 39.60, 115.2 mg) were inhaled every 30 min, up to the cumulative dose of 181.98 mg. At intervals of 10, 20, and 30 min after each dose, FEV1 was measured. The challenge procedure was interrupted if a bronchospastic reaction occurred (FEV1 decrease by 20% or more), extrabronchial signs of hypersensitivity became evident, or the maximum cumulative dose of aspirin was reached. The cumulative dose of aspirin causing a 20% fall in FEV1 was reported as the provocative dose (PD20). Collection of IS was started after the last dose of aspirin. If needed, the rescue inhalation of (Salbutamol, 400 lg) was short-acting b2-mimetic administered once before (25). In patients with positive bronchial aspirin challenge, induced sputum sample was collected 30 min after symptoms appeared, while in ATA controls, 30 min after the last dose (cumulative dose of 181.98 mg), lysyl-aspirin was administered. The study
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lung function Parameters of pulmonary function were measured using a flow-integrating computerized pneumotachograph (Pneumoscreen, Jaeger, Germany). Induced sputum collection Collection of IS was performed according to ERS recommendations (35). Sputum induction was performed by inhalation of hypertonic saline solution at concentrations increasing from 3% to 5%, using ultrasonic nebulizer (Ultraneb 2000; DeVilibiss, Somerset, PA, USA). Sputum was expectorated onto ice-cooled Petri dish and immediately transferred to the laboratory. After manual separation from saliva, mucus plugs were processed to obtain cytospin slides for differential cell count and supernatant for measurements of eicosanoids (36). Briefly, mucus plugs were incubated with Sputolysin reagent (EMD Millipore Merck, Darmstad, Germany) at 1 : 4 weight/volume dilution for 1 h at 37°C. The solubilized material was diluted 1 : 1 vol/ vol in the phosphate-buffered saline (PBS), filtered through the filter (48 lm mesh), and separated by centrifugation at 700 g for 10 min. Cell counts were ascertained using MayGr€ unwald-stained cytospin preparations from the pellet resuspended in PBS, while ISS was aliquoted and stored in 70°C until analysis. ISS corresponded to the mucous plug diluted in 1 : 10 weight to volume ratio.
Sputum eicosanoids in aspirin bronchial challenge
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Biochemical assays
Results
Total protein concentration in ISS was measured using Vitros 350 (Ortho Clinical Diagnostics, Rochester, NY, USA) biochemical analyzer and expressed in milligrams per milliliter. The concentration of prostanoids in ISS was measured by gas chromatography/mass spectrometry (GC-MS) for prostaglandins or by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS2) for leukotrienes, HETEs, and prostaglandin metabolites. Organic-phase extraction was performed on samples acidified to pH 3.5 with acetic acid and spiked with chemically identical deuterated internal standards (500 pg each; Cayman Chemical Co., Ann Arbor, MI, USA). Concentrations of eicosanoids were calculated using a stable isotope dilution method from the area under the peak. Eicosanoid concentrations were expressed in picograms per milliliter of ISS. Detailed analytical method was presented elsewhere (37). Limits of quantification for eicosanoid measurements are presented in Table 2, together with results. Statistical analysis Summary statistics were expressed as arithmetic mean and standard deviation or quartiles depending on the data distribution. Spearman’s rank correlation was used. Between-groups comparisons were carried out using nonparametric Mann–Whitney rank-sum test, and Wilcoxon test was used for within-group analyses. Calculations were done using IBM SPSS Statistics 21 (IBM, Armonk, NY, USA). Type I statistical error P-value ≤ 0.05 was assumed significant.
Clinical characteristics and response to the bronchial challenge with aspirin The study groups differed in the frequency of chronic rhinosinusitis and therapy with inhaled corticosteroids (ICS), both were more common in AERD subjects (Table 1). None of the study participants reacted to the saline inhalation. In ATA controls, the full dose of inhaled L-ASA (181.98 mg) was tolerated well. In AERD group, 20 individuals (74.1%) reacted with bronchoconstriction (average FEV1 decrease by 25.9%; PD20 = 17 mg [5–97]), and in seven subjects, the challenge resulted in extrapulmonary symptoms. In addition, two AERD subjects, who completed the challenge with a positive result, developed extrabronchial symptoms of mucosal and facial skin edema after inhalation of the last tolerated dose of aspirin. In seven AERD subjects, the challenge caused intense edema of facial skin and mucosa: six of them received the full dose of lysyl-aspirin without FEV1 decrease by 20%, and in one, the challenge was terminated after the cumulative dose 39.6 mg of lysyl-aspirin (FEV1% decrease by 18.2 mg%). These AERD subgroups averaged FEV1 decrease by 10.1%. A significant negative correlation between lysyl-aspirin PD20 and postchallenge FEV1% decrease was noted in AERD group (q = 0.740; P < 0.001). All the bronchial symptoms were relieved by short-acting b2-agonists. Induced sputum cell counts and total protein Differential cell count in IS had a similar distribution in the study groups. Eosinophilic (>3% eosinophils), neutrophilic
Table 2 Concentration of eicosanoids in induced sputum supernatant Eicosanoid, (LLOQ) [pg/ml ISS] LTB4 (2.96) LTC4 (2.42) LTD4 (2.85) LTE4 (3.22) PGE2 (1.62) 13,14-dihydro, 15-keto-PGE2 (7.3) PGD2 (2.09) 13,14-dihydro, 15-keto-PGD2 (6.8) 9a,11b-PGF2a (1.2) 5-HETE (0.82) 12-HETE (0.63) 15-HETE (2.18) PGF2a (1.52) 11-dehydro-TXB2 (1.84)
AERD (n = 27)
ATA (n = 16)
Baseline
Postchallenge
P
308.9 40.1 28.1 59.9 69.5 34.5
164.2 66.2 45.7 94.1 52.6 31.7
P = 0.04 0.01 0.03 0.031 0.049 0.11
[181.9–1234.4] [5.4–63.0] [13.0–60.0] [28.9–91.8]* [48.8–101.0] [22.8–62.2]
33.9 [24.5–53.3]* 21.6 [15.9–31.5] 3.8 521.4 1313 907.1 8.0 19.2
[1.8–7.8] [268.2–1906.9] [728.1–2483] [358.6–1739] [3.8–16.8] [15.7–24.1]
[114.8–749.8] [3.3–100.1] [26.3–124.4]** [38.4–374.3]** [45.1–78.1] [25.0–38.9]
40.9 [19.0–74.1]* 21.2 [17.8–25.7] 2.7 381.8 799.1 634.0 5.9 18.2
[1.7–8.1] [168.4–536.6] [496.5–1231.2] [290.1–1313] [3.1–12.3] [14.3–22.7]*
Baseline
NS NS
389.8 26.5 17.7 17.5 54.8 41.4
[114.8–749.8] [3.1–104.8] [10.6–100.6] [5.4–78.5] [45.0–116.9] [28.2–66.0]
24.6 [11.9–35.4] 19.7 [17.4–24.6]
NS 0.08 0.01 NS 0.067 NS
2.3 530.0 1569.1 837.1 9.3 18.3
[1.4–7.1] [307.8–974.9] [536.7–8713] [305.0–1234] [6.7–13.1] [12.8–22.1]
Postchallenge
P
318.7 30.8 10.3 16.0 42.8 26.9
NS NS NS NS NS 0.011
[75.3–574.7] [2.4–53.4] [5.1–49.3] [8.2–36.3] [34.8–101.1] [22.7–45.5]
23.5 [12.3–42.6] 20.9 [15.7–27.0] 3.7 [1.3–6.3] 466.5 [105.2–767.1] 719.0 [462.5–1529] 492.8 [133.0–858.8] 6.8 [3.1–12.3] 14.8 [13.6–17.0]
NS NS NS NS 0.04 NS NS 0.013
LLOQ: the lowest limit of quantification (signal to noise ratio > 10). *AERD vs ATA P < 0.05. **AERD vs ATA P < 0.01.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Sputum eicosanoids in aspirin bronchial challenge
(>50% neutrophils), or paucigranulocytic (noneosinophilic, nonneutrophilic) asthma was present with a similar frequency. A difference in IS eosinophil count was not significant (median AERD 3.8% vs ATA 2.0%; P = 0.24). Neutrophil count was lower in AERD group, but this was not significant (median AERD 28.4% vs ATA 44.1%; P = 0.13). Following the bronchial challenge, IS eosinophilia decreased (P = 0.049) in AERD subjects from 3.77% [0.77–12.83] to 2.13% [0.39–8.05]. Baseline and postchallenge IS eosinophilia correlated positively (AERD, q = 0.697, P < 0.001; ATA q = 0.535, P = 0.033). A correlation was also present for baseline and postchallenge ISS total protein (AERD: R = 0.795; P < 0.001 vs ATA: R = 0.666; P = 0.005). Recalculation of eicosanoids on the total protein concentrations
had no effect on any comparisons. Statistical analysis was conducted using the raw data. Leukotrienes in ISS Baseline ISS concentrations of LTC4 and LTD4 did not differ between AERD and ATA group, whereas median LTE4 was 3.4 times higher in AERD subjects (P = 0.04) than in controls (Table 2, Fig. 1). Following the challenge, each cys-LT increased significantly (LTC4, P = 0.008; LTD4, P = 0.03; LTE4, P = 0.03) only in AERD (Table 2). Baseline ISS concentration of LTB4 did not differ between AERD subjects and ATA controls. Postchallenge LTB4 decreased only in AERD group (P = 0.044).
LTB 4 10 000
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Figure 1 Concentration of leukotrienes in sputum supernatant before and after bronchial aspirin challenge. AERD—aspirinexacerbated respiratory disease, dashed lines and open circles—
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pre
Post L-ASA tolerant
extrapulmonary reaction during the challenge. ATA—aspirin-tolerant asthma. Side bars represent median.
Sputum eicosanoids in aspirin bronchial challenge
Inhaled steroid dose had no effect on cys-LT concentrations at baseline or after the challenge. Prostanoids in ISS Baseline PGE2 did not differ between AERD and ATA groups. Postchallenge ISS PGE2 decreased only in AERD subjects (P = 0.049). An inactive metabolite of PGE2 13,14dihydro,15-keto-PGE2 decreased (P = 0.005) in all subjects after the challenge, and this decrease was significant in ATA but not in AERD group (Table 2, Fig. 2). Baseline PGD2 was significantly higher in AERD subjects (P = 0.021) than in ATA, but no differences were present in 9a,11b-PGF2a or 13,14-dihydro,15-keto-PGD2 (PGD2 metabolites). Postchallenge ISS concentrations of PGD2 or its metabolites did not change in either study group. However, a higher PGD2 concentration in AERD group was still present (P = 0.05) in the postchallenge ISS. Baseline ISS PGF2a did not differ between AERD and ATA groups and did not change after the challenge. Baseline concentration of a TXA2 metabolite 11-dehydro-TXB2 did not differ between the study groups. Postchallenge ISS 11-dehydro-TXB2 remained unchanged in AERD subjects, but decreased in ATA (P = 0.013), resulting in a lower postchallenge 11-dehydro-TXB2 (P = 0.031) in this group when compared to AERD. HETEs in ISS Baseline 5-, 12-, and 15-lipoxygenation products of AA–HETEs did not differ between the study groups. Postchallenge 5-HETE and 15-HETE did not change, whereas 12-HETE ISS concentration decreased significantly in both the study groups (AERD, P = 0.01; ATA, P = 0.044). Correlation of cell counts and eicosanoid concentrations A highly significant positive correlation was found between peripheral blood eosinophilia and induced sputum eosinophilia at baseline (all subjects q = 0.471, P = 0.002; AERD q = 0.460, P = 0.018). Peripheral blood eosinophilia correlated positively with ISS concentrations of LTE4 and LTD4 in all subjects (q = 0.457, P = 0.002 and q = 0.421, P = 0.005) and in ATA (q = 0.671, P = 0.004 and q = 0.706, P = 0.002), but not in AERD. Eosinophilic asthma phenotype was characterized by a higher ISS LTD4 (median 59.2 pg/ml vs 16.5; P = 0.01) and LTE4 (median 63.9 pg/ml vs 23.5; P = 0.01). Baseline IS eosinophilia was a good predictor of LTE4 and LTD4 in all subjects (q = 0.744, P < 0.001 and q = 0.463, P = 0.002) and in ATA (q = 0.676, P = 0.004 and q = 0.624, P = 0.01). However, in AERD, a moderate significant positive correlation was only between baseline IS eosinophilia and LTD4 (q = 0.393, P = 0.047). Postchallenge concentrations of LTE4 and LTD4 correlated positively with IS eosinophilia exclusively in ATA controls (q = 0.685 and q = 0.647, P < 0.01). A negative correlation between baseline IS neutrophilia and LTB4 was observed in all subjects (q = 0.415,
Mastalerz et al.
P = 0.006). This was limited only to AERD in a group analysis (q = 0.550, P = 0.004). As percentage counts were analyzed, in which neutrophils and macrophages are the most common cells, concentration of LTB4 in ISS was positively correlated with IS macrophage count in all study participants (q = 0.449, P = 0.003) and in AERD subjects (q = 0.514, P = 0.007). Postchallenge correlations between IS neutrophil count and LTB4 became positive for all study participants (q = 0.321, P = 0.038) and in AERD subjects (q = 0.513, P = 0.007). No correlation was present between peripheral blood eosinophilia or any IS differential cell count and ISS PGD2. AERD subgroup with and without 20% decrease in FEV1 following the aspirin challenge No difference was present in basal or postchallenge concentrations of cys-LTs between AERD subjects who reacted with bronchoconstriction or extrabronchial symptoms. Increased postchallenge ISS cys-LTs (total LTC4, LTD4, and LTE4) characterized both subgroups, although in asthmatics with extrabronchial symptoms, changes from baseline values were not significant (Fig. 1). The subgroup with bronchoconstriction had lower baseline LTB4 than AERD asthmatics with extrapulmonary symptoms (193.5 pg/ml [125.2–309.9] vs 1451 [350.9–1618], P = 0.001). In the same subgroup, postchallenge decrease in PGE2 was lessened than in those with extrapulmonary symptoms (Fig. 2). Moreover, both baseline and postchallenge PGD2 in ISS were higher than in the subgroup with extrapulmonary symptoms (50.5 pg/ml [2.4– 116.7] vs 22.6 [21.4–52.0], P = 0.005; and 70.14 pg/ml [35.0– 100.4] vs 21.18 [12.44–69.96], P = 0.026). Discussion We investigated concentrations of selected eicosanoids in bronchial secretions of asthmatics stratified by their hypersensitivity to aspirin. Sampling of IS was performed before the challenge and repeated the next day, immediately after the aspirin bronchial provocation, to study changes induced by inhibition of cyclooxygenases. A highly sensitive and specific high-performance liquid or gas chromatography/ mass spectrometry method of quantification of eicosanoids was used, allowing compensation for analytical errors (30, 37). By measurement of ISS, we confirmed that aspirin hypersensitive asthmatics had overproduction of LTE4 in the bronchial compartment. They had also elevated concentration of PGD2, which seemed to distinguish asthmatics more prone to respond with bronchoconstriction during the challenge. An overproduction of cys-LTs following the challenge was accompanied by a decline in PGE2 concentration; however, the magnitude of these changes did not correlate. These results are in agreement with the studies on intrabronchial (20, 33) or intranasal administration of the drug (21, 38), also causing a decrease in PGE2 and an increase in cys-LTs. We suggest that pharmacological activity of aspirin inhibiting PGE2 in AERD is altered. Despite 4.6-fold lower dose of © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Sputum eicosanoids in aspirin bronchial challenge
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Figure 2 Concentration of prostaglandins E2 and D2 and its fast inactivation metabolites in sputum supernatant before and after bronchial aspirin challenge. AERD—aspirn-exacerbated respiratory
disease, dashed lines and open circles—extrapulmonary reaction during the challenge. ATA—aspirin-tolerant asthma. Side bars represent median.
inhaled aspirin, a magnitude of PGE2 decrease in AERD group was similar to ATA (AERD by 24.3%, ATA by 21.9%). At the same time, 13,14-dihydro,15-keto-PGE2,
PGE2 fast inactivation metabolite, also decreased in AERD group (AERD by 62.0%, ATA by 44.2%). Thus, both PGE2 and 13,14-dihydro,15-keto-PGE2 biosynthesis diminished due
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Sputum eicosanoids in aspirin bronchial challenge
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to cyclooxygenases inhibition by aspirin regardless of a sharp difference in the dose of the drug (Fig. 2). The time elapsed between the last inhalation of aspirin and sampling of IS was comparable in both groups of the study. PGE2 metabolite requires two subsequent intracellular enzymatic reactions, catalyzed by 15-hydroxy prostaglandin dehydrogenases and 15-oxoprostaglandin D13-reductases abundant in the lung (39). In conclusion, bronchial production of PGE2 in AERD subjects is more sensitive to inhibition and both the parent and the metabolized prostaglandin decrease at lower aspirin dose. But we failed to detect any postchallenge decrease in 11-dehydro-TXB2 in AERD group. This effect of aspirin was previously described after segmental bronchial challenge (33) or nasal aspirin provocation (38). Such a decrease occurred also in our study, but only in ATA. Possible explanation of this discrepancy is much lower dose of aspirin inhaled by AERD subjects. However, the correlation between the provocative dose of aspirin and bronchoconstriction was negative, which is inconsistent with pharmacological dose response of COX pathway. A high sensitivity of cyclooxygenases to the inhibition supports the critical role of PGE2 biosynthesis in AERD. Aspirin-triggered bronchoconstriction in AERD is accompanied by a decrease in PGE2 and enhanced biosynthesis of cys-LTs in bronchi, as described previously (19, 20, 23–25, 33). In our data, lack of changes in PGD2, PGF2a, or 11-dehydro-TXB2 is consistent with alternative
cyclooxygenase activities. COX-1 isoenzyme is irreversibly inhibited by aspirin. But PGD2 concentration did not change after the cumulative aspirin dose even in ATA. Perhaps, PGD2 in bronchi is produced by COX-2 rather than COX-1 and is not suppressed by inhalation of aspirin, in contrast to PGE2 in AERD subjects or 11-dehydro-TXB2 in ATA controls. This conclusion would be in disagreement with systemic PGD2 production, decreasing after administration of coxibs, a class of NSAIDs tolerated in AERD (40). Aspirin hypersensitive asthmatics in this study had typical features of chronic rhinosinusitis. Regardless of ICS treatment in all, they had suboptimal control of asthma and lower FEV1%. However, eosinophilic inflammation of airways, as ascertained by IS cell count, only tended to be more common in AERD subjects, probably due to ICS controller therapy. Nevertheless, baseline ISS PGD2 and LTE4 were elevated. We did not observe PGD2 changes following the challenge. Previous studies documented increased baseline levels of PGD2 in AERD patients, as compared with ATA, in EBC during the stable disease (29, 30). In a recent study, Fajt et al. (41). Showed that higher PGD2 in bronchoalveolar lavage fluid was associated with worse control of asthma. In our data, blood and sputum eosinophilia correlated positively with PGD2. It is doubtful, however, that higher PGD2 could contribute to bronchoconstriction during the challenge as its concentration did not change.
Total 5-LO products 10 000
pg/ml
1000
100
10
1
cys-LTs index
100
10 1
0.1
0.01
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Figure 3 Total 5-lipoxygenase products (5-HETE+LTB4+LTC4+ LTD4+LTE4) in sputum supernatant and total cysteinyl leukotrienes (C4, D4, and E4) as a fraction of leukotriene B4 reflecting relative activity of leukotriene C4 synthase. Pre- and postchallenge ratio in
Pre
Post L-ASA tolerant
aspirin-exacerbated respiratory disease (AERD) and aspirin-tolerant asthma (ATA) subjects. Dashed lines and open circles—AERD extrapulmonary reaction during the challenge. Side bars represent median.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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A novel finding on AERD is decrease in LTB4 concentration of ISS after the challenge. Asthmatics responding with bronchoconstriction only manifested a change in leukotrienes metabolism from LTB4 to cys-LTs in response to very low dose (median 17 mg) of aspirin inhaled. We suggest that a shift in metabolism of a 5-hydroperoxidetetraenoic acid (5-HETE parent compound) is responsible for this observation. 5-HETE can be either conjugated with glutathione forming LTC4 or hydrolyzed to LTA4 and LTB4. As LTC4 synthase is expressed in blood platelets (42) and contribution of leukocyte-adherent platelets has been shown mandatory for aspirin sensitivity in a PGE-synthase-deficient mouse model of the disease (43), this alteration in leukotrienes may reflect enhanced transcellular biosynthesis of cys-LTs triggered by L-ASA. Interestingly, a total of 5-LO products (5-HETE+LTB4+LTC4+LTD4+LTE4) did not differ between prechallenge and postchallenge ISS in either study group (AERD: 1060.5 [739.1–3476.9] vs 1066.7 [765.9–3482]; L-ASA tolerant: 1022.4 [854.9–2296.5] vs 1029.5 [865.1–2301.6]). Consequently, we question the role of 5-LO activation in the bronchoconstriction triggered by L-ASA or generally in AERD. Some other 5-LO products might have not been included in this comparison; however, Wilcoxon’s paired data comparison showed in most subjects a decrease in total ISS 5-LO products following the challenge (L-ASA tolerant P = 0.004; AEDR P < 0.001) (Fig. 3). Baseline concentration of total cysteinyl leukotrienes (LTC4+LTD4+LTE4) was 29% [9–114] of LTB4 in AERD group and 33% [12–80] in ATA. After the challenge, this difference became significant (129% [38–377] vs 37% [17–74] in ATA; P = 0.025). A decrease in LTB4 was recently described in IS of asthmatics after allergen challenge (44). No change in 5-HETE concentration of ISS in the current study supports our observation on lack of activation in 5-lipoxygenase pathway during L-ASA precipitated bronchoconstriction.
In conclusion, we confirmed that cys-LTs are overproduced at the bronchial level in AERD and their increased concentration is reflected in ISS (45). Noninvasively obtained IS eicosanoids do not discriminate well between AERD and ATA due to an overlap in their concentrations with eosinophilic phenotype of asthma. Following the challenge, AERD phenotype is clearly delineated by changes in ISS eicosanoids and specific inhibition of PGE2 biosynthesis, which shifts 5-lipoxygenase pathway from LTB4 to cys-LTs. Acknowledgments We thank Iwona Lipiarz, Magorzata Głuszek, and Anita Borkowska-Mosur for technical assistance and dr Aleksander Kania for IS cytospin analyses. Funding This work was supported by the grant N N402592440 from the Polish Ministry of Science and by a grant from Switzerland through the Swiss Contribution to the enlarged European Union (PSPB-072/2010). Author contributions LM conceived the study and drafted the manuscript. NC-W, KW, and RJ performed challenge tests and contributed to writing. AG did eicosanoid measurements. AC and PS enrolled patients. MS did statistical analysis and wrote final version of the manuscript. Conflicts of interest The authors declare that they have no conflicts of interest.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
ORIGINAL ARTICLE
AIRWAY DISEASES
Induced sputum eicosanoids during aspirin bronchial challenge of asthmatic patients with aspirin hypersensitivity jcik, K. Wo jcik, A. Gielicz, R. Januszek, A. Cholewa, P. Strez k & M. Sanak L. Mastalerz, N. Celejewska-Wo Department of Medicine, School of Medicine, Jagiellonian University, Cracow, Poland
jcik N, Wo jcik K, Gielicz A, Januszek R, Cholewa A, Strez k P, Sanak M. Induced sputum eicosanoids during aspirin To cite this article: Mastalerz L, Celejewska-Wo bronchial challenge of asthmatic patients with aspirin hypersensitivity. Allergy 2014; DOI: 10.1111/all.12512.
Keywords aspirin-exacerbated respiratory disease; eicosanoids; induced sputum; leukotrienes; prostaglandin E2. Correspondence Prof. Marek Sanak, MD, PhD, Department of Medicine, School of Medicine, Jagielloska 8, 31 – 066 nian University, ul. Skawin w, Poland. Krako Tel.: +48 12 430 51 69 Fax: +48 12 430 52 03 E-mail: [email protected] Accepted for publication 11 August 2014 DOI:10.1111/all.12512 Edited by: Michael Wechsler
Abstract Background: Altered metabolism of eicosanoids is a characteristic finding in aspirin-exacerbated respiratory disease (AERD). Bronchial challenge with lysylaspirin can be used as a confirmatory diagnostic test for this clinical condition. Induced sputum allows to measure mediators of asthmatic inflammation in bronchial secretions. Objectives: To investigate the influence of inhaled lysyl-aspirin on sputum supernatant concentration of eicosanoids during the bronchial challenge test. Subjects with asthma hypersensitive to nonsteroidal anti-inflammatory drugs were compared with aspirin-tolerant asthmatic controls. Methods: Induced sputum was collected before and following bronchial challenge with lysyl-aspirin. Sputum differential cell count and sputum supernatant concentrations of selected lipoxygenases products: 5-,12-,15-hydroxyeicosatetraenoic acid, cysteinyl leukotrienes, leukotriene B4, 11-dehydro-thromboxane B2, and prostaglandins E2, D2, and F2a and their metabolites, were measured using validated methods of chromatography–mass spectrometry. Results: Aspirin precipitated bronchoconstriction in all AERD subjects, but in none of the aspirin-tolerant asthmatics. Phenotypes of asthma based on the sputum cytology did not differ between the groups. Baseline sputum eosinophilia correlated with a higher leukotriene D4 (LTD4) and leukotriene E4 (LTE4) concentrations. LTC4, PGE2, and 11-dehydro-TXB2 did not differ between the groups, but levels of LTD4, LTE4, and PGD2 were significantly higher in AERD group. Following the challenge, LTD4 and LTE4 increased, while PGE2 and LTB4 decreased in AERD subjects only. Conclusions: During the bronchial challenge, decrease in PGE2 and its metabolite is accompanied by a surge in bronchoconstrictory cysteinyl leukotrienes produced at the expense of LTB4 in AERD subjects. Bronchial PGE2 inhibition in AERD seems specific and sensitive to a low dose of aspirin.
Abbreviations 5-,12-,15-HETE, 5-,12-,15-hydroxyeicosatetraenoic acids; 5-LO, arachidonic acid 5-lipoxygenase; AA, arachidonic acid; AERD, aspirin-exacerbated respiratory disease; ATA, L-ASA-tolerant asthma; COX, cyclooxygenase; cys-LTs, cysteinyl leukotrienes; LASA, lysyl-acetylsalicylic acid; LTB4, leukotriene B4; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4; NSAIDs, nonsteroidal anti-inflammatory drugs; PGD2 metabolites, 9a,11bPGF2a – 13,14-dihydro,15-keto-PGD2; PGD2, prostaglandin D2; PGE2 metabolite, 13,14-dihydro,15-keto-PGE2; PGE2, prostaglandin E2.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Inhalation of hypertonic sodium chloride solution facilitates expectoration of sputum. It is used as a simple noninvasive diagnostic procedure for sampling of bronchial secretions, useful in monitoring of lower airway inflammation. Induced sputum (IS) has been validated for studies on inflammatory cells and mediators in the assessment of both asthma and other pulmonary diseases (1). Eicosanoids, including 5-lipoxygenation products leukotrienes (LTs) and cyclooxygenase metabolites prostaglandins (PGs), are highly bioactive oxylipins involved in the asthmatic
Sputum eicosanoids in aspirin bronchial challenge
inflammation (2). Aspirin-exacerbated respiratory disease (AERD) is a distinct asthma phenotype characterized by persistent course of the disease, chronic rhinosinusitis with nasal polyposis, and hypersensitivity to aspirin or other nonsteroidal anti-inflammatory drugs (NSAIDs) (3). There is a consensus on nonallergic mechanism responsible for asthmatic attacks in AERD patients, involving abnormal metabolism of arachidonic acid (AA) by cyclooxygenase (COX) and lipoxygenase (LO) pathways (3–5). Inhalation of exogenous PGE2 prevents bronchoconstriction provoked by aspirin and inhibits urinary excretion of cys-LTs (6–8). These effects seem not related to myorelaxant activity of PGE2 on bronchial smooth muscle, and PGE2 is fast-inactivated to 13,14-dihydro,15-keto-PGE2. It is rather release of mediators from inflammatory cells, the most likely mast cells and eosinophils, which is inhibited by PGE2 (9, 10). Epithelial cells from surgically removed nasal polyps of patients with AERD produce less PGE2 than the same cells from aspirin-tolerant controls (11), and peripheral blood cells from patients with AERD release less PGE2 at baseline than healthy controls (12). PGE2 production by bronchial fibroblasts in AERD is lower, as compared to aspirin-tolerant (ATA) controls (13). Moreover, lower expression of COX-2 in nasal polyp tissue from aspirin hypersensitive patients, accompanied by decreased production of PGE2, contributes to imbalance between anti- and pro-inflammatory eicosanoids (14, 15). Expression of inhibitory EP2 receptor for PGE2 is decreased on inflammatory cells infiltrating the nasal mucosa of patients with AERD (16), and a particular promoter variant of the gene encoding EP2 receptor is more frequent among AERD subjects (17). However, after oral administration of aspirin to patients with AERD, no decrease in the systemic production of PGE2 could be demonstrated using measurements of stable PGE2 metabolites in urine (18). Data on local PGE2 response to aspirin challenge are scarce and contradictory (19–21). 5-LO is the key enzyme in leukotriene synthesis transforming AA into leukotriene A4 (LTA4). This intermediate can generate bioactive eicosanoids by hydrolysis into leukotriene B4 (LTB4) or by conjugation with glutathione into leukotriene C4 (LTC4). Next steps of extracellular metabolism produce leukotriene D4 (LTD4) and leukotriene E4 (LTE4). LTB4 is a potent chemoatractant for neutrophils, stimulating their adhesion to epithelia and production of reactive oxygen species. Cysteinyl leukotrienes (cys-LTs), LTC4, LTD4, and LTE4, are mediators of allergic inflammation potently contracting smooth muscles, causing vascular leak and mucus production (22). In numerous studies, the key role of cys-LTs overproduction in AERD was well documented on the systemic level, and increased urinary excretion of LTE4 is a hallmark of this disease (18, 23–26). Following ingestion of NSAIDs, cys-LTs released from eosinophils and mast cells cause a violent bronchoconstriction and sometimes extrapulmonary signs of facial edema and skin erythema (9, 25, 27). A pharmacological mechanism of hypersensitivity has been proposed, in which COX-1 inhibition by aspirin and other NSAIDs decreases biosynthesis of PGE2. This may lead to
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enhanced or disinhibited synthesis of cys-LTs by inflammatory cells (28). Specific synthases convert cyclic endoperoxides of AA not only to PGE2 but also to other prostaglandins: D2 (PGD2), F2a (PGF2a), and prostanoids: thromboxane A2 (TXA2) and prostacyclin. Both PGD2 and its metabolite 9a,11b-PGF2a, and PGF2a are potent bronchoconstrictors (22), while more abundant metabolite 13,14-dihydro,15keto-PGD2 is not active. Elevated levels of PGD2 or its metabolite were reported in plasma (29), urine (9, 25) and in exhaled breath condensate (30) (EBC) from patients with AERD. However, no change in PGE2 or PGD2 could be detected in EBC after oral or bronchial aspirin provocation (31, 32). Inhibitory effect of aspirin on PGE2 biosynthesis in AERD subjects was shown in bronchoalveolar lavage fluid (20, 33) after segmental provocation and in nasal washings after topical administration of aspirin (21). In contrast with PGE2, level of PGD2 metabolite 9a,11bPGF2a increased following aspirin challenge in both urine and plasma (29). The aim of this study was to investigate effects of aspirin bronchial challenge on prostanoids and leukotrienes in the induced sputum supernatant (ISS) from AERD subjects. We hypothesized that the eicosanoid profile in IS is specific for aspirin hypersensitivity and could reveal mechanisms underlying bronchoconstriction on the bronchial level. Eicosanoid measurements were taken using a high sensitivity and specific mass spectrometry analysis.
Methods Subjects The study groups consisted of 27 patients with asthma, whose aspirin hypersensitivity was diagnosed by oral provocation test and 16 asthmatics who tolerated aspirin well. The asthma diagnosis and control was ascertained according to GINA 2012 update. Definition of severe asthma complied with ATS/ERS 2013 recommendations. The clinical characteristics of the study participants are presented in Table 1. Aspirin-tolerant asthmatic (ATA) controls occasionally used NSAIDs without any adverse reactions. All the study subjects had stable asthma, and their baseline FEV1 was >70% the predicted value on the challenge day. None had experienced any exacerbation or a respiratory tract infection within the 6-week period preceding the study. The study participants were instructed to withhold medications decreasing bronchial responsiveness prior to the aspirin challenge. Short-acting b2-agonists were not used at least 8 h before the challenge, and long-acting b2-agonists and theophylline were withdrawn for 24 h. Short-acting antihistamines and cromones were discontinued 5 days before the challenge. Inhaled steroids were allowed at dose not exceeding 2000 lg fluticasone equivalent per day. None was treated with systemic corticosteroids or leukotriene-modifying drugs. Baseline and postchallenge sputum induction was carried out in all study participants. Informed consent was obtained from all
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
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Table 1 Clinical characteristics of the study participants
Age (SD) ACT median (min–max) Duration of CRS, median (min–max) Inhaled steroids yes/no Dose of inhaled steroids (lg/day) fluticason eq., median (min–max) FEV1 baseline (%predicted) Allergic rhinitis yes/no Chronic rhinosinusitis yes/no Total serum IgE (IU/ml), median [interquartile range] Peripheral blood eosinophilia count (mm 3), median [interquartile range] IS cellular phenotype Eosinophilic Neutrophilic Mixed Paucicellular
all AERD (n = 27)
AERD with extrabronchial symptoms (n = 11)
ATA (n = 16)
40.6 9.8 22 (16–25) 7 (1–27) 27/0 1000 (100–2000)
38.0 10.6 22 (16–25) 5 (1–26) 11/0 500 (100–1000)
48.9 17.3 25 (19–25)* 11 (2–30) 11/5*** 400 (0–1110)
92.6 14.8 10/17 27/0 104 [49.2–212] 397 [260.5–555]
91.7 14.5 4/7 11/0 176 [50.5–364.8] 356 [154.5–441.5]
100.1 16.2** 6/10 11/5*** 141 [65.2–282] 390 [210.3–749.5]
13 6 1 7
4 4 0 3
5 4 2 5
ACT, asthma control test; CRS, chronic rhinosinusitis; IS, induced sputum. *AERD vs ATA P < 0.05. **AERD vs ATA P < 0.01. ***AERD vs ATA P < 0.005.
subjects, and the study was approved by the Jagiellonian University Ethics Committee (KBET/7/B/2010).
participants were monitored for FEV1 and extrabronchial symptoms every 30 min for the next 6 h after the challenge.
Study design The study was conducted in two consecutive days. On the first day, baseline IS samples were collected. On the second day, a standard bronchial single-blind placebo-controlled challenge test with lysyl-aspirin (Kardegic, Sanofi Aventis, Prague) was carried out in all study participants (34). The test began with the inhalation of seven breaths of placebo (saline), followed by FEV1 measurements 10 and 20 min later. The postsaline FEV1 obtained at 20 min was used as the prechallenge baseline value. By increasing concentrations and incrementing numbers of breaths, doses of lysyl-aspirin (0.18, 0.36, 0.90, 2.34, 7.20, 16.2, 39.60, 115.2 mg) were inhaled every 30 min, up to the cumulative dose of 181.98 mg. At intervals of 10, 20, and 30 min after each dose, FEV1 was measured. The challenge procedure was interrupted if a bronchospastic reaction occurred (FEV1 decrease by 20% or more), extrabronchial signs of hypersensitivity became evident, or the maximum cumulative dose of aspirin was reached. The cumulative dose of aspirin causing a 20% fall in FEV1 was reported as the provocative dose (PD20). Collection of IS was started after the last dose of aspirin. If needed, the rescue inhalation of (Salbutamol, 400 lg) was short-acting b2-mimetic administered once before (25). In patients with positive bronchial aspirin challenge, induced sputum sample was collected 30 min after symptoms appeared, while in ATA controls, 30 min after the last dose (cumulative dose of 181.98 mg), lysyl-aspirin was administered. The study
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Lung function Parameters of pulmonary function were measured using a flow-integrating computerized pneumotachograph (Pneumoscreen, Jaeger, Germany). Induced sputum collection Collection of IS was performed according to ERS recommendations (35). Sputum induction was performed by inhalation of hypertonic saline solution at concentrations increasing from 3% to 5%, using ultrasonic nebulizer (Ultraneb 2000; DeVilibiss, Somerset, PA, USA). Sputum was expectorated onto ice-cooled Petri dish and immediately transferred to the laboratory. After manual separation from saliva, mucus plugs were processed to obtain cytospin slides for differential cell count and supernatant for measurements of eicosanoids (36). Briefly, mucus plugs were incubated with Sputolysin reagent (EMD Millipore Merck, Darmstad, Germany) at 1 : 4 weight/volume dilution for 1 h at 37°C. The solubilized material was diluted 1 : 1 vol/ vol in the phosphate-buffered saline (PBS), filtered through the filter (48 lm mesh), and separated by centrifugation at 700 g for 10 min. Cell counts were ascertained using MayGr€ unwald-stained cytospin preparations from the pellet resuspended in PBS, while ISS was aliquoted and stored in 70°C until analysis. ISS corresponded to the mucous plug diluted in 1 : 10 weight to volume ratio.
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Biochemical assays
Results
Total protein concentration in ISS was measured using Vitros 350 (Ortho Clinical Diagnostics, Rochester, NY, USA) biochemical analyzer and expressed in milligrams per milliliter. The concentration of prostanoids in ISS was measured by gas chromatography/mass spectrometry (GC-MS) for prostaglandins or by high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS2) for leukotrienes, HETEs, and prostaglandin metabolites. Organic-phase extraction was performed on samples acidified to pH 3.5 with acetic acid and spiked with chemically identical deuterated internal standards (500 pg each; Cayman Chemical Co., Ann Arbor, MI, USA). Concentrations of eicosanoids were calculated using a stable isotope dilution method from the area under the peak. Eicosanoid concentrations were expressed in picograms per milliliter of ISS. Detailed analytical method was presented elsewhere (37). Limits of quantification for eicosanoid measurements are presented in Table 2, together with results. Statistical analysis Summary statistics were expressed as arithmetic mean and standard deviation or quartiles depending on the data distribution. Spearman’s rank correlation was used. Between-groups comparisons were carried out using nonparametric Mann–Whitney rank-sum test, and Wilcoxon test was used for within-group analyses. Calculations were done using IBM SPSS Statistics 21 (IBM, Armonk, NY, USA). Type I statistical error P-value ≤ 0.05 was assumed significant.
Clinical characteristics and response to the bronchial challenge with aspirin The study groups differed in the frequency of chronic rhinosinusitis and therapy with inhaled corticosteroids (ICS), both were more common in AERD subjects (Table 1). None of the study participants reacted to the saline inhalation. In ATA controls, the full dose of inhaled L-ASA (181.98 mg) was tolerated well. In AERD group, 20 individuals (74.1%) reacted with bronchoconstriction (average FEV1 decrease by 25.9%; PD20 = 17 mg [5–97]), and in seven subjects, the challenge resulted in extrapulmonary symptoms. In addition, two AERD subjects, who completed the challenge with a positive result, developed extrabronchial symptoms of mucosal and facial skin edema after inhalation of the last tolerated dose of aspirin. In seven AERD subjects, the challenge caused intense edema of facial skin and mucosa: six of them received the full dose of lysyl-aspirin without FEV1 decrease by 20%, and in one, the challenge was terminated after the cumulative dose 39.6 mg of lysyl-aspirin (FEV1% decrease by 18.2 mg%). These AERD subgroups averaged FEV1 decrease by 10.1%. A significant negative correlation between lysyl-aspirin PD20 and postchallenge FEV1% decrease was noted in AERD group (q = 0.740; P < 0.001). All the bronchial symptoms were relieved by short-acting b2-agonists. Induced sputum cell counts and total protein Differential cell count in IS had a similar distribution in the study groups. Eosinophilic (>3% eosinophils), neutrophilic
Table 2 Concentration of eicosanoids in induced sputum supernatant Eicosanoid, (LLOQ) [pg/ml ISS] LTB4 (2.96) LTC4 (2.42) LTD4 (2.85) LTE4 (3.22) PGE2 (1.62) 13,14-dihydro, 15-keto-PGE2 (7.3) PGD2 (2.09) 13,14-dihydro, 15-keto-PGD2 (6.8) 9a,11b-PGF2a (1.2) 5-HETE (0.82) 12-HETE (0.63) 15-HETE (2.18) PGF2a (1.52) 11-dehydro-TXB2 (1.84)
AERD (n = 27)
ATA (n = 16)
Baseline
Postchallenge
P
308.9 40.1 28.1 59.9 69.5 34.5
164.2 66.2 45.7 94.1 52.6 31.7
P = 0.04 0.01 0.03 0.031 0.049 0.11
[181.9–1234.4] [5.4–63.0] [13.0–60.0] [28.9–91.8]* [48.8–101.0] [22.8–62.2]
33.9 [24.5–53.3]* 21.6 [15.9–31.5] 3.8 521.4 1313 907.1 8.0 19.2
[1.8–7.8] [268.2–1906.9] [728.1–2483] [358.6–1739] [3.8–16.8] [15.7–24.1]
[114.8–749.8] [3.3–100.1] [26.3–124.4]** [38.4–374.3]** [45.1–78.1] [25.0–38.9]
40.9 [19.0–74.1]* 21.2 [17.8–25.7] 2.7 381.8 799.1 634.0 5.9 18.2
[1.7–8.1] [168.4–536.6] [496.5–1231.2] [290.1–1313] [3.1–12.3] [14.3–22.7]*
Baseline
NS NS
389.8 26.5 17.7 17.5 54.8 41.4
[114.8–749.8] [3.1–104.8] [10.6–100.6] [5.4–78.5] [45.0–116.9] [28.2–66.0]
24.6 [11.9–35.4] 19.7 [17.4–24.6]
NS 0.08 0.01 NS 0.067 NS
2.3 530.0 1569.1 837.1 9.3 18.3
[1.4–7.1] [307.8–974.9] [536.7–8713] [305.0–1234] [6.7–13.1] [12.8–22.1]
Postchallenge
P
318.7 30.8 10.3 16.0 42.8 26.9
NS NS NS NS NS 0.011
[75.3–574.7] [2.4–53.4] [5.1–49.3] [8.2–36.3] [34.8–101.1] [22.7–45.5]
23.5 [12.3–42.6] 20.9 [15.7–27.0] 3.7 [1.3–6.3] 466.5 [105.2–767.1] 719.0 [462.5–1529] 492.8 [133.0–858.8] 6.8 [3.1–12.3] 14.8 [13.6–17.0]
NS NS NS NS 0.04 NS NS 0.013
LLOQ: the lowest limit of quantification (signal to noise ratio > 10). *AERD vs ATA P < 0.05. **AERD vs ATA P < 0.01.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mastalerz et al.
Sputum eicosanoids in aspirin bronchial challenge
(>50% neutrophils), or paucigranulocytic (noneosinophilic, nonneutrophilic) asthma was present with a similar frequency. A difference in IS eosinophil count was not significant (median AERD 3.8% vs ATA 2.0%; P = 0.24). Neutrophil count was lower in AERD group, but this was not significant (median AERD 28.4% vs ATA 44.1%; P = 0.13). Following the bronchial challenge, IS eosinophilia decreased (P = 0.049) in AERD subjects from 3.77% [0.77–12.83] to 2.13% [0.39–8.05]. Baseline and postchallenge IS eosinophilia correlated positively (AERD, q = 0.697, P < 0.001; ATA q = 0.535, P = 0.033). A correlation was also present for baseline and postchallenge ISS total protein (AERD: R = 0.795; P < 0.001 vs ATA: R = 0.666; P = 0.005). Recalculation of eicosanoids on the total protein concentrations
had no effect on any comparisons. Statistical analysis was conducted using the raw data. Leukotrienes in ISS Baseline ISS concentrations of LTC4 and LTD4 did not differ between AERD and ATA group, whereas median LTE4 was 3.4 times higher in AERD subjects (P = 0.04) than in controls (Table 2, Fig. 1). Following the challenge, each cys-LT increased significantly (LTC4, P = 0.008; LTD4, P = 0.03; LTE4, P = 0.03) only in AERD (Table 2). Baseline ISS concentration of LTB4 did not differ between AERD subjects and ATA controls. Postchallenge LTB4 decreased only in AERD group (P = 0.044).
LTB 4 10 000
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Figure 1 Concentration of leukotrienes in sputum supernatant before and after bronchial aspirin challenge. AERD—aspirinexacerbated respiratory disease, dashed lines and open circles—
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Pre
Post L-ASA tolerant
extrapulmonary reaction during the challenge. ATA—aspirin-tolerant asthma. Side bars represent median.
Sputum eicosanoids in aspirin bronchial challenge
Inhaled steroid dose had no effect on cys-LT concentrations at baseline or after the challenge. Prostanoids in ISS Baseline PGE2 did not differ between AERD and ATA groups. Postchallenge ISS PGE2 decreased only in AERD subjects (P = 0.049). An inactive metabolite of PGE2 13,14dihydro,15-keto-PGE2 decreased (P = 0.005) in all subjects after the challenge, and this decrease was significant in ATA but not in AERD group (Table 2, Fig. 2). Baseline PGD2 was significantly higher in AERD subjects (P = 0.021) than in ATA, but no differences were present in 9a,11b-PGF2a or 13,14-dihydro,15-keto-PGD2 (PGD2 metabolites). Postchallenge ISS concentrations of PGD2 or its metabolites did not change in either study group. However, a higher PGD2 concentration in AERD group was still present (P = 0.05) in the postchallenge ISS. Baseline ISS PGF2a did not differ between AERD and ATA groups and did not change after the challenge. Baseline concentration of a TXA2 metabolite 11-dehydro-TXB2 did not differ between the study groups. Postchallenge ISS 11-dehydro-TXB2 remained unchanged in AERD subjects, but decreased in ATA (P = 0.013), resulting in a lower postchallenge 11-dehydro-TXB2 (P = 0.031) in this group when compared to AERD. HETEs in ISS Baseline 5-, 12-, and 15-lipoxygenation products of AA–HETEs did not differ between the study groups. Postchallenge 5-HETE and 15-HETE did not change, whereas 12-HETE ISS concentration decreased significantly in both the study groups (AERD, P = 0.01; ATA, P = 0.044). Correlation of cell counts and eicosanoid concentrations A highly significant positive correlation was found between peripheral blood eosinophilia and induced sputum eosinophilia at baseline (all subjects q = 0.471, P = 0.002; AERD q = 0.460, P = 0.018). Peripheral blood eosinophilia correlated positively with ISS concentrations of LTE4 and LTD4 in all subjects (q = 0.457, P = 0.002 and q = 0.421, P = 0.005) and in ATA (q = 0.671, P = 0.004 and q = 0.706, P = 0.002), but not in AERD. Eosinophilic asthma phenotype was characterized by a higher ISS LTD4 (median 59.2 pg/ml vs 16.5; P = 0.01) and LTE4 (median 63.9 pg/ml vs 23.5; P = 0.01). Baseline IS eosinophilia was a good predictor of LTE4 and LTD4 in all subjects (q = 0.744, P < 0.001 and q = 0.463, P = 0.002) and in ATA (q = 0.676, P = 0.004 and q = 0.624, P = 0.01). However, in AERD, a moderate significant positive correlation was only between baseline IS eosinophilia and LTD4 (q = 0.393, P = 0.047). Postchallenge concentrations of LTE4 and LTD4 correlated positively with IS eosinophilia exclusively in ATA controls (q = 0.685 and q = 0.647, P < 0.01). A negative correlation between baseline IS neutrophilia and LTB4 was observed in all subjects (q = 0.415,
Mastalerz et al.
P = 0.006). This was limited only to AERD in a group analysis (q = 0.550, P = 0.004). As percentage counts were analyzed, in which neutrophils and macrophages are the most common cells, concentration of LTB4 in ISS was positively correlated with IS macrophage count in all study participants (q = 0.449, P = 0.003) and in AERD subjects (q = 0.514, P = 0.007). Postchallenge correlations between IS neutrophil count and LTB4 became positive for all study participants (q = 0.321, P = 0.038) and in AERD subjects (q = 0.513, P = 0.007). No correlation was present between peripheral blood eosinophilia or any IS differential cell count and ISS PGD2. AERD subgroup with and without 20% decrease in FEV1 following the aspirin challenge No difference was present in basal or postchallenge concentrations of cys-LTs between AERD subjects who reacted with bronchoconstriction or extrabronchial symptoms. Increased postchallenge ISS cys-LTs (total LTC4, LTD4, and LTE4) characterized both subgroups, although in asthmatics with extrabronchial symptoms, changes from baseline values were not significant (Fig. 1). The subgroup with bronchoconstriction had lower baseline LTB4 than AERD asthmatics with extrapulmonary symptoms (193.5 pg/ml [125.2–309.9] vs 1451 [350.9–1618], P = 0.001). In the same subgroup, postchallenge decrease in PGE2 was lessened than in those with extrapulmonary symptoms (Fig. 2). Moreover, both baseline and postchallenge PGD2 in ISS were higher than in the subgroup with extrapulmonary symptoms (50.5 pg/ml [2.4– 116.7] vs 22.6 [21.4–52.0], P = 0.005; and 70.14 pg/ml [35.0– 100.4] vs 21.18 [12.44–69.96], P = 0.026). Discussion We investigated concentrations of selected eicosanoids in bronchial secretions of asthmatics stratified by their hypersensitivity to aspirin. Sampling of IS was performed before the challenge and repeated the next day, immediately after the aspirin bronchial provocation, to study changes induced by inhibition of cyclooxygenases. A highly sensitive and specific high-performance liquid or gas chromatography/ mass spectrometry method of quantification of eicosanoids was used, allowing compensation for analytical errors (30, 37). By measurement of ISS, we confirmed that aspirin hypersensitive asthmatics had overproduction of LTE4 in the bronchial compartment. They had also elevated concentration of PGD2, which seemed to distinguish asthmatics more prone to respond with bronchoconstriction during the challenge. An overproduction of cys-LTs following the challenge was accompanied by a decline in PGE2 concentration; however, the magnitude of these changes did not correlate. These results are in agreement with the studies on intrabronchial (20, 33) or intranasal administration of the drug (21, 38), also causing a decrease in PGE2 and an increase in cys-LTs. We suggest that pharmacological activity of aspirin inhibiting PGE2 in AERD is altered. Despite 4.6-fold lower dose of © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mastalerz et al.
Sputum eicosanoids in aspirin bronchial challenge
PGE2
1000
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Post AERD
Pre
Post L-ASA tolerant
Figure 2 Concentration of prostaglandins E2 and D2 and its fast inactivation metabolites in sputum supernatant before and after bronchial aspirin challenge. AERD—aspirn-exacerbated respiratory
disease, dashed lines and open circles—extrapulmonary reaction during the challenge. ATA—aspirin-tolerant asthma. Side bars represent median.
inhaled aspirin, a magnitude of PGE2 decrease in AERD group was similar to ATA (AERD by 24.3%, ATA by 21.9%). At the same time, 13,14-dihydro,15-keto-PGE2,
PGE2 fast inactivation metabolite, also decreased in AERD group (AERD by 62.0%, ATA by 44.2%). Thus, both PGE2 and 13,14-dihydro,15-keto-PGE2 biosynthesis diminished due
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Sputum eicosanoids in aspirin bronchial challenge
Mastalerz et al.
to cyclooxygenases inhibition by aspirin regardless of a sharp difference in the dose of the drug (Fig. 2). The time elapsed between the last inhalation of aspirin and sampling of IS was comparable in both groups of the study. PGE2 metabolite requires two subsequent intracellular enzymatic reactions, catalyzed by 15-hydroxy prostaglandin dehydrogenases and 15-oxoprostaglandin D13-reductases abundant in the lung (39). In conclusion, bronchial production of PGE2 in AERD subjects is more sensitive to inhibition and both the parent and the metabolized prostaglandin decrease at lower aspirin dose. But we failed to detect any postchallenge decrease in 11-dehydro-TXB2 in AERD group. This effect of aspirin was previously described after segmental bronchial challenge (33) or nasal aspirin provocation (38). Such a decrease occurred also in our study, but only in ATA. Possible explanation of this discrepancy is much lower dose of aspirin inhaled by AERD subjects. However, the correlation between the provocative dose of aspirin and bronchoconstriction was negative, which is inconsistent with pharmacological dose response of COX pathway. A high sensitivity of cyclooxygenases to the inhibition supports the critical role of PGE2 biosynthesis in AERD. Aspirin-triggered bronchoconstriction in AERD is accompanied by a decrease in PGE2 and enhanced biosynthesis of cys-LTs in bronchi, as described previously (19, 20, 23–25, 33). In our data, lack of changes in PGD2, PGF2a, or 11-dehydro-TXB2 is consistent with alternative
cyclooxygenase activities. COX-1 isoenzyme is irreversibly inhibited by aspirin. But PGD2 concentration did not change after the cumulative aspirin dose even in ATA. Perhaps, PGD2 in bronchi is produced by COX-2 rather than COX-1 and is not suppressed by inhalation of aspirin, in contrast to PGE2 in AERD subjects or 11-dehydro-TXB2 in ATA controls. This conclusion would be in disagreement with systemic PGD2 production, decreasing after administration of coxibs, a class of NSAIDs tolerated in AERD (40). Aspirin hypersensitive asthmatics in this study had typical features of chronic rhinosinusitis. Regardless of ICS treatment in all, they had suboptimal control of asthma and lower FEV1%. However, eosinophilic inflammation of airways, as ascertained by IS cell count, only tended to be more common in AERD subjects, probably due to ICS controller therapy. Nevertheless, baseline ISS PGD2 and LTE4 were elevated. We did not observe PGD2 changes following the challenge. Previous studies documented increased baseline levels of PGD2 in AERD patients, as compared with ATA, in EBC during the stable disease (29, 30). In a recent study, Fajt et al. (41). Showed that higher PGD2 in bronchoalveolar lavage fluid was associated with worse control of asthma. In our data, blood and sputum eosinophilia correlated positively with PGD2. It is doubtful, however, that higher PGD2 could contribute to bronchoconstriction during the challenge as its concentration did not change.
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Figure 3 Total 5-lipoxygenase products (5-HETE+LTB4+LTC4+ LTD4+LTE4) in sputum supernatant and total cysteinyl leukotrienes (C4, D4, and E4) as a fraction of leukotriene B4 reflecting relative activity of leukotriene C4 synthase. Pre- and postchallenge ratio in
Pre
Post L-ASA tolerant
aspirin-exacerbated respiratory disease (AERD) and aspirin-tolerant asthma (ATA) subjects. Dashed lines and open circles—AERD extrapulmonary reaction during the challenge. Side bars represent median.
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
Mastalerz et al.
Sputum eicosanoids in aspirin bronchial challenge
A novel finding on AERD is decrease in LTB4 concentration of ISS after the challenge. Asthmatics responding with bronchoconstriction only manifested a change in leukotrienes metabolism from LTB4 to cys-LTs in response to very low dose (median 17 mg) of aspirin inhaled. We suggest that a shift in metabolism of a 5-hydroperoxidetetraenoic acid (5-HETE parent compound) is responsible for this observation. 5-HETE can be either conjugated with glutathione forming LTC4 or hydrolyzed to LTA4 and LTB4. As LTC4 synthase is expressed in blood platelets (42) and contribution of leukocyte-adherent platelets has been shown mandatory for aspirin sensitivity in a PGE-synthase-deficient mouse model of the disease (43), this alteration in leukotrienes may reflect enhanced transcellular biosynthesis of cys-LTs triggered by L-ASA. Interestingly, a total of 5-LO products (5-HETE+LTB4+LTC4+LTD4+LTE4) did not differ between prechallenge and postchallenge ISS in either study group (AERD: 1060.5 [739.1–3476.9] vs 1066.7 [765.9–3482]; L-ASA tolerant: 1022.4 [854.9–2296.5] vs 1029.5 [865.1–2301.6]). Consequently, we question the role of 5-LO activation in the bronchoconstriction triggered by L-ASA or generally in AERD. Some other 5-LO products might have not been included in this comparison; however, Wilcoxon’s paired data comparison showed in most subjects a decrease in total ISS 5-LO products following the challenge (L-ASA tolerant P = 0.004; AEDR P < 0.001) (Fig. 3). Baseline concentration of total cysteinyl leukotrienes (LTC4+LTD4+LTE4) was 29% [9–114] of LTB4 in AERD group and 33% [12–80] in ATA. After the challenge, this difference became significant (129% [38–377] vs 37% [17–74] in ATA; P = 0.025). A decrease in LTB4 was recently described in IS of asthmatics after allergen challenge (44). No change in 5-HETE concentration of ISS in the current study supports our observation on lack of activation in 5-lipoxygenase pathway during L-ASA precipitated bronchoconstriction.
In conclusion, we confirmed that cys-LTs are overproduced at the bronchial level in AERD and their increased concentration is reflected in ISS (45). Noninvasively obtained IS eicosanoids do not discriminate well between AERD and ATA due to an overlap in their concentrations with eosinophilic phenotype of asthma. Following the challenge, AERD phenotype is clearly delineated by changes in ISS eicosanoids and specific inhibition of PGE2 biosynthesis, which shifts 5-lipoxygenase pathway from LTB4 to cys-LTs. Acknowledgments We thank Iwona Lipiarz, Magorzata Głuszek, and Anita Borkowska-Mosur for technical assistance and dr Aleksander Kania for IS cytospin analyses. Funding This work was supported by the grant N N402592440 from the Polish Ministry of Science and by a grant from Switzerland through the Swiss Contribution to the enlarged European Union (PSPB-072/2010). Author contributions LM conceived the study and drafted the manuscript. NC-W, KW, and RJ performed challenge tests and contributed to writing. AG did eicosanoid measurements. AC and PS enrolled patients. MS did statistical analysis and wrote final version of the manuscript. Conflicts of interest The authors declare that they have no conflicts of interest.
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