Abstract
Inhalation of prostaglandin E2 (PGE2) had been reported to prevent allergen-induced bronchoconstrictor responses; however, the effects of inhaled PGE2 on allergen-induced airway inflammation or hyperresponsiveness after allergen are unknown. This study examined the effects of inhaled PGE2 on allergen-induced airway responses and inflammation. Eight mild asthmatics with a dual airway response to inhaled allergen were recruited into a double-blind randomized crossover study comparing the effects of inhaled PGE2 (100 μ g) or placebo, on allergen-induced changes in FEV1 measured for 7 h, induced sputum inflammatory cells, obtained at baseline, 7 and 24 h, and methacholine airway responsiveness measured at 24 h after challenge. Inhaled PGE2 attenuated the allergen-induced early fall in FEV1 from 24.4 ± 3.6% after placebo to 10.3 ± 2.5% after PGE2 (p = 0.002), the late fall in FEV1 from 21.2 ± 2.7% after placebo to 12.6 ± 3.6% after PGE2 (p = 0.03), allergen-induced methacholine airway hyperresponsiveness (p = 0.03) and allergen-induced increases in percent sputum eosinophils from 36.3 ± 8.8% after placebo to 21.0 ± 7.3% after PGE2 (p = 0.01), percentage of EG2 + cells (p = 0.02), and percentage of metachromatic cells (p = 0.02). These results indicate that inhaled PGE2 attenuates allergen-induced airway responses, hyperresponsiveness, and inflammation, when given immediately before inhaled allergen.
Airway inflammation is an important characteristic in patients with current symptomatic asthma. This has been demonstrated by increased numbers of inflammatory cells in airway secretions from asthmatics when compared with nonasthmatics (1, 2). Allergen challenge is a valuable laboratory model for the study of the pathogenesis of airway inflammation in asthma. Allergen inhalation results in acute bronchoconstriction in sensitized subjects, and in 50 to 60% of adult subjects, this is followed by a late bronchoconstrictor response (LAR). The LAR is associated with the development of allergen-induced airway hyperresponsiveness (3), and increases in the number of airway inflammatory cells, particularly eosinophils and metachromatic cells (MCC), which can be measured in induced sputum (4).
Prostaglandin E2 (PGE2) is a cyclo-oxygenase product of arachidonate metabolism present in human airways in the airway epithelium (5) and airway smooth muscle (6), which demonstrates bronchoprotective effects in patients with bronchial asthma. PGE2 has been shown to protect against exercise- induced (7), allergen-induced (8, 9) and aspirin-induced bronchoconstriction (6), as well as bronchoconstrictor agents such as methacholine and histamine (10, 11). PGE2 may represent an endogenous protective mechanism in the airways.
PGE2 has been shown to have inhibitory effects on many inflammatory cells in vitro, including antigen-induced mediator release from mast cells (12), and eosinophil degranulation (13), chemotaxis, and cytokine-stimulated survival (14). Inhaled PGE2 has been demonstrated to prevent early and late allergen-induced bronchoconstrictor responses when given immediately before allergen inhalation challenge (8, 9). In this study, we postulated that PGE2 may not only exert a bronchoprotective effect on allergen-induced bronchoconstriction, but also inhibit allergen-induced airway inflammation, which may be assessed by measurements of inflammatory cells in induced sputum. This study, therefore, has examined the anti- inflammatory role of PGE2 inhaled immediately before allergen challenge.
Eight nonsmoking subjects with mild atopic asthma (Table 1) were selected for the study because of a previously documented allergen- induced early and late bronchoconstrictor response of at least at 15% fall in FEV1, and gave signed consent to participate in the study. This sample size was considered to be sufficient, as a previous study using the same methodology has demonstrated that eight or more subjects can demonstrate a 50% change in the LAR with a power of > 90% (15). The study was approved by the ethics committee of the McMaster University Health Sciences Center. Subjects were not exposed to sensitizing allergens and did not have asthma exacerbations or respiratory tract infections for at least 4 wk prior to entering the study. All subjects had stable asthma with a forced expiratory volume in 1 s (FEV1) greater than 80% of predicted normal on all study days before allergen inhalation. Subjects used no medication other than inhaled β2-agonist as required to treat their symptoms. Medication was withheld for at least 8 h before each visit, and subjects were instructed to refrain from rigorous exercise, tea or coffee in the morning before visits to the laboratory.
| Subject Number | Age (yr) | Gender | FEV1(% predicted ) | Methacholine PC20(mg/ml ) | Inhaled Allergen | Allergen Dilution* | Order of Treatment | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 19 | F | 97.3 | 3.14 | HDM | 1:256 | PGE2 Plac | |||||||
| 2 | 24 | M | 78.8 | 1.89 | Grass | 1:1024 | Plac PGE2 | |||||||
| 3 | 19 | M | 93.1 | 3.84 | Grass | 1:1024 | Plac PGE2 | |||||||
| 4 | 21 | M | 90.7 | 2.59 | Ragweed | 1:256 | PGE2 Plac | |||||||
| 5 | 24 | M | 83.6 | 1.66 | HDM | 1:512 | PGE2 Plac | |||||||
| 6 | 21 | F | 95.0 | 1.51 | HDM | 1:256 | PGE2 Plac | |||||||
| 7 | 23 | M | 81.1 | 1.51 | Cat | 1:16 | Plac PGE2 | |||||||
| 8 | 23 | M | 90.7 | 0.40 | HDM | 1:1024 | Plac PGE2 |
The study was carried out with a double-blind, placebo-controlled, randomized, crossover design. Each subject completed two treatment periods with inhalation of either 100 μg PGE2 or placebo. Each treatment period consisted of 3 visits to the laboratory. Baseline measurements of FEV1, the provocative concentration of methacholine causing a 20% fall in FEV1 (PC20), and induced sputum differential and total cell counts were determined 1 d before allergen challenge. Drug or placebo treatment and allergen challenges were carried out the following morning. Spirometry was measured until 7 h after allergen inhalation, and sputum samples were obtained 7 h after allergen inhalation. Sputum could not be induced earlier than 7 h because inhalation of hypertonic saline can cause bronchoconstriction which would interfere with measures of allergen-induced airway responses. Methacholine PC20 and sputum samples were obtained 24 h postallergen. Each treatment period was separated by a washout period of at least 3 wk.
Stock solution of PGE2 was prepared at a concentration of 2 mg/ml by dissolving 10 mg PGE2 (Sigma Chemical Co, St. Louis, MO) in 5.0 ml of 100% ethanol. This solution was stored at −70° C. Immediately before use, 0.50 ml of stock solution (equivalent to 100 μg PGE2) was added to 4.95 ml physiological saline. Subjects inhaled all of this solution by tidal breathing from a Fisoneb ultrasonic nebulizer (Canadian Medical Products, Ltd., Markham, ON, Canada; output 1 ml/min, aerodynamic mass median diameter 5.6 μm). FEV1 was measured before and after inhalation of PGE2 or placebo, and side effects were recorded. The allergen inhalation challenge was started within 5 min, and was completed within 7 min after PGE2 or placebo inhalation. This dose and timing of administration of PGE2 has been previously described to significantly attenuate allergen-induced bronchoconstrictor responses (8).
In normal subjects, inhalation of PGE2 causes initial bronchoconstriction followed by bronchodilation, which can be accompanied by transient cough, retrosternal soreness, and airway secretions (16). To avoid unblinding of investigators, PGE2 and placebo were inhaled in a closed room, with only one investigator present. This unblinded investigator was responsible only for preparation and administration of drug, and recording side effects, if any. Allergen challenges, measures of methacholine hyperresponsiveness and sputum inflammatory cells were carried out by investigators who remained blinded to the treatment.
Methacholine inhalation challenge was performed as described by Cockcroft (17). Subjects inhaled normal saline, then doubling concentrations of methacholine phosphate from a Wright nebulizer for 2 min. FEV1 was measured at 30, 90, 180, and 300 s after each inhalation. Spirometry was measured with a Collins water-sealed spirometer and kymograph. The test was terminated when a fall in FEV1 of 20% of the baseline value occurred, and the PC20 was calculated.
Allergen challenge was performed as described by O'Byrne and coworkers (18). Allergen extracts were stored at −70° C and diluted in phosphate-buffered saline with 1.5% benzyl alcohol for skin tests, and were later diluted in physiologic saline for allergen inhalation on the day of use. The allergen extract was selected using the results from the skin test, and diluted for inhalation at the concentration determined from a formula described by Cockcroft and coworkers (19) using the results from the skin test and the methacholine PC20. During the screening allergen inhalation, doubling concentrations of allergen were inhaled by tidal breathing (nose clipped) for 2 min, with FEV1 measured 10 min after each inhalation; inhalations were stopped when the FEV1 had fallen by at least 15% from baseline. FEV1 was subsequently measured at 20, 30, 45, 60, 90, and 120 min and at hourly intervals up to 7 h after allergen inhalation. The diluent inhalation challenge was performed in the same manner as the allergen inhalation challenge, with subjects inhaling physiological saline (3 inhalations of 2 min) instead of allergen.
Inhalation challenges were performed using a Wright nebulizer operated by oxygen at 50 psi and at a flow rate that gave an output of 0.13 ml/min and aerodynamic mass median diameter of 1.0 to 1.5 μm. FEV1 was measured using a water-sealed spirometer, with triplicate FEV1 measurements at baseline and single FEV1 measurements postallergen inhalation; volumes were recorded at body temperature, atmospheric pressure, saturated with water vapor. Following the PGE2 or placebo treatments, the allergen challenge was performed as described previously, using only the concentration of allergen utilized during the screening allergen challenge resulting in at least at 15% late fall in FEV1. In order to separate the effects of allergen from the effects of drug, the FEV1 following PGE2 or placebo was used as baseline for the allergen challenge. The early bronchoconstrictor response was taken to be the largest fall in FEV1 within 2 h after allergen inhalation, and the late response was taken to be the largest fall in FEV1 between 3 and 7 h after allergen inhalation.
Sputum was induced and processed using the method described by Popov and coworkers (20). Subjects inhaled 3%, 4%, then 5% saline for 7 min each. The induction was stopped when an adequate sample was obtained, or if the FEV1 dropped 20% from baseline. Cell plugs with little or no squamous epithelial cells were selected from the sample using an inverted microscope, separated from saliva, and weighed. Samples were aspirated in 4 times their volume of 0.1% dithiothreitol (Sputolysin; Calbiochem Corp., San Diego, CA) and 4 times their volume of Dulbecco's phosphate-buffered saline (DPBS; Gibco BRL, Life Technologies, Grand Island, NY). The cell suspension was filtered through a 52-μm nylon gauze (BNSH Thompson, Scarborough, ON, Canada) to remove debris, then centrifuged at 1,500 rpm for 10 min. The total cell count was determined using a hemocytometer (Neubauer Chamber; Hausser Scientific, Blue Bell, PA) and expressed as the number of cells per milliliter sputum. Cells were resuspended in DPBS at 0.75–1.0 × 106/ml. Cytospins were prepared on glass slides using 50 μl of cell suspension and a Shandon III Cytocentrifuge (Shandon Southern Instruments, Sewickly, PA), at 300 rpm for 5 min. Differential cell counts were obtained from the mean of two slides with 400 cells counted per slide stained with Diff-Quik (American Scientific Products, McGaw Park, IL). Metachromatic cell (mast cell and basophil) counts were obtained from slides stained with toluidine blue, from the mean of two slides with 5,000 cells observed on each slide. Cytospins were also prepared on aptex coated slides and fixed in periodate-lysine-paraformaldehyde for immunocytochemical staining. Slides were stained for activated eosinophils using a mouse monoclonal anti-human antibody directed against cleaved eosinophil cationic protein (EG2) at 1 μg/ml (Kabi Pharmacia, Uppsala, Sweden). The EG2 antibody was diluted in 1.0% bovine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO) and wash buffer made up of DPBS, 0.01 M HEPES buffer (Boehringer Manheim Canada Ltd., Burlington, ON, Canada), and 0.01% saponin (Sigma Chemical Co.), and slides were incubated with antibody overnight. Labeling of antibodies was detected by the alkaline phosphatase antialkaline phosphatase method (21). Mouse IgG1 (Sigma Chemical Co.) was used as a negative control. The percentage of EG2+ cells was determined from a count of 500 cells under light microscopy.
All summary statistics are expressed as mean and standard error of the mean (SEM) with the exception of methacholine PC20 measurements which are measured by linear interpolation of log dose response curves resulting in logarithmic values for PC20, and expressed as geometric mean and geometric standard error of the mean (GSEM). One-tailed Student's t test for paired observations was used to compare the early and late airway responses to allergen, in order to test for significant protection on these responses. The effects of placebo and PGE2 treatment on baseline FEV1, and on the allergen- induced change in methacholine PC20, and blood and sputum inflammatory cells were analyzed using a two-factor repeated measures analysis of variance (ANOVA) (22). Statistical analyses were performed using computer software (Statistica 4.5, Stat Soft Inc., Tulsa, OK, 1993).
Inhaled PGE2 significantly reduced allergen-induced early asthmatic responses, the maximal fall in FEV1 being 24.4% (SEM 3.6%) after inhaled placebo and 10.3% (SEM 2.5%) after inhaled PGE2 (p = 0.002) (Figure 1), and also significantly reduced the allergen-induced late asthmatic response, the maximal fall in FEV1 being 21.2% (SEM 2.7%) after inhaled placebo and 12.6% (SEM 3.6%) after inhaled PGE2 (p = 0.03) (Figure 1). The methacholine PC20 decreased from 2.6 mg/ml (1.5 GSEM) before, to 1.0 mg/ml (1.4 GSEM) after allergen with placebo treatment (p = 0.02), and this allergen-induced decrease of methacholine PC20 was significantly attenuated with PGE2 treatment (p = 0.03), being 1.6 mg/ml (1.3 GSEM) before and 1.5 mg/ml (1.4 GSEM) after challenge. There was no change in FEV1 measured 5 min after inhaled placebo or PGE2 (p = 0.41), with the FEV1 being 3.8 ± 0.1 L/s before and 3.8 ± 0.2 L/s after placebo, and 3.7 ± 0.1 L/s before and 3.7 ± 0.2 L/s after PGE2.
Fig. 1. Percent change in FEV1 during the early and late airway responses following diluent or allergen challenge with PGE2 (100 μg) or placebo pretreatment. PGE2 inhalation significantly reduced the allergen-induced early (p = 0.002) and the late (p = 0.03) airway responses after allergen challenge.
[More] [Minimize]The total number of cells per milliliter in induced sputum after inhaled allergen was not statistically different from baseline (p = 0.07), nor was there an effect of PGE2 on the total cell count (p = 0.38) (Table 2). Inhaled allergen increased the percentage of eosinophils after placebo treatment, from 7.9 ± 4.7% before, to 26.4 ± 8.3% at 7 h and to 36.3 ± 8.8% at 24 h after inhaled allergen (p = 0.045) (Figure 2), and this effect was significantly attenuated by inhaled PGE2, from 11.1 ± 4.8% before, to 17.6 ± 4.7% at 7 h and 20.9 ± 7.3% at 24 h after allergen (p = 0.01) (Figure 2). Also, inhaled allergen increased the percentage of EG2+ cells after placebo treatment at 7 h and 24 h after allergen (p = 0.03), and this effect was also significantly attenuated by treatment with inhaled PGE2 (p = 0.02) (Figure 2). Sputum percent MCC increased after inhaled allergen, but this effect was not significant (p = 0.18); however inhaled PGE2 significantly reduced the percent MCC when compared with placebo both at 7 h and 24 h after inhaled allergen (p = 0.02) (Figure 2). Inhaled allergen slightly increased the percentage of neutrophils at 7 h after allergen (p = 0.02), and this effect was not altered by treatment with inhaled PGE2 (p = 0.84) (Table 2).
| Baseline | 7 h Postallergen | 24 h Postallergen | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Placebo | PGE2 | Placebo | PGE2 | Placebo | PGE2 | |||||||
| Eosinophilis, % | 7.9 ± 4.7 | 11.1 ± 4.8 | 26.4 ± 8.3† | 17.6 ± 4.7†,‡ | 36.3 ± 8.8† | 20.9 ± 7.3†,‡ | ||||||
| EG2+, % | 6.9 ± 4.3 | 8.7 ± 3.8 | 19.8 ± 8.7† | 10.5 ± 3.4‡ | 27.4 ± 7.1† | 13.1 ± 3.8‡ | ||||||
| Neutrophils, % | 48.0 ± 9.8 | 34.7 ± 4.8 | 62.9 ± 8.1† | 58.0 ± 10.0† | 40.0 ± 8.7† | 38.5 ± 6.6† | ||||||
| MCC, % | 0.08 ± 0.06 | 0.16 ± 0.08 | 0.37 ± 0.15 | 0.29 ± 0.11‡ | 0.66 ± 0.29 | 0.21 ± 0.06‡ | ||||||
| Total cells, 106/ml | 2.03 ± 0.47 | 2.43 ± 0.56 | 4.95 ± 1.78 | 3.58 ± 0.85 | 3.63 ± 0.96 | 4.15 ± 0.68 | ||||||
Fig. 2. Percent sputum eosinophils (top panel ), EG2+ cells (middle panel ), and metachromatic cells (bottom panel ) with placebo (open bars) and PGE2 (solid bars) treatments shown at baseline, and at 7 and 24 h after allergen challenge. PGE2 treatment significantly reduced the allergen-induced increase in eosinophils, EG2+ cells, and metachromatic cells (p < 0.05). EG2+ cells = activated eosinophils. ▾p < 0.05 allergen-induced change from baseline; *p < 0.05 difference from baseline, placebo versus PGE2.
[More] [Minimize]PGE2 was reported, by some subjects, to cause side effects of transient cough and airway secretions. There were no other adverse effects.
This study has demonstrated that pretreatment with inhaled PGE2 immediately before allergen challenge suppresses allergen-induced airway inflammation in sputum, as indicated by a significant attenuation of allergen-induced increases in sputum eosinophils, EG2+ cells, and MCC up to 24 h after challenge. Using similar methods to Pavord and coworkers (8), this study has confirmed that PGE2 inhalation attenuates the allergen-induced LAR, and suggests a likely mechanism for these physiological findings.
Allergen inhalation by atopic asthmatics results in a sputum eosinophilia which develops during the late response, within 7 h after challenge. Inhaled corticosteroids attenuate both the late response and the increases in sputum eosinophils measured during the late response (23), while regular use of inhaled β2-agonists enhances both the late response and the increases in sputum eosinophils measured during the late response (24), suggesting that eosinophils are, at least in part, associated with the development of the late response after inhaled allergen. The results from the present study confirm these previous observations, this time with the nonsteroidal agent, PGE2, suggesting that inhibition of the late response by PGE2 occurs as a result of attenuating airway eosinophilia.
Bronchodilation following inhaled PGE2 (16) may account, in part, for the attenuation of allergen-induced early responses, as a study with similar methodology has demonstrated maximal bronchodilation 25 min after PGE2 inhalation (8). However, this could not account for the attenuation of the late response by PGE2 because there is no bronchodilation observed after 1 h (8). Cough was experienced by some subjects during PGE2 inhalation and could have unblinded them to the treatment. This could affect measurements of spirometry, but would be unlikely to affect measurements of airway inflammation. Attenuation of the early and late responses to allergen was accompanied by reduced allergen-induced airway inflammation, and protection against allergen-induced airway hyperresponsiveness, measured 24 h after inhaled PGE2. Taken together, the results of the present study indicate that inhaled PGE2 has anti-inflammatory effects in asthmatic airways, unlike the effects of inhaled β2-agonists (24). These results also suggest that, as PGE2 can attenuate allergen-induced airway responses long after its direct pharmacologic activity has resolved (7 h and 24 h after inhaled allergen), its action is through inhibiting very early allergen-induced airway events, such as mast cell degranulation and/or mediator release.
The mechanisms by which PGE2 may regulate airway hyperresponsiveness and airway inflammation after allergen challenge are speculative. The release of arachidonic acid from cell membrane phospholipids following challenge to the airways can result in the production of a wide variety of mediators, including prostanoids, thromboxane, and leukotrienes, which may be relevant in the pathogenesis of asthma. Substances such as thromboxane A2 (TxA2) and the cysteinyl leukotrienes C4, (LTC4), LTD4, and LTE4 are potent bronchoconstrictors in asthmatic airways (25), and can be modulated by PGE2. TxA2 is released after allergen challenge (26), and preincubation with PGE2 has been shown to reduce arachidonic-induced release of thromboxane A2 from human bronchial biopsies (27).
There is more substantial evidence to support the role of cysteinyl leukotrienes in the pathogenesis of asthma. The cysteinyl leukotrienes are released after allergen challenge (28) and recently LTD4 inhalation has been shown to cause bronchoconstriction and increased sputum eosinophils in asthmatic subjects (29). Administration of leukotriene antagonists before challenge is effective in attenuating the allergen-induced early and late responses (30). Eosinophils and mast cells, which are present in greater numbers in the asthmatic lung (2), are the sources of the cysteinyl leukotrienes. There are several ways in which PGE2 may modulate the role of the eosinophil. Endogenous PGE2 inhibits platelet activating factor (PAF)-induced LTC4 synthesis by eosinophils (31) and PGE2 has been shown to inhibit eosinophil degranulation (13). Increased intracellular levels of adenosine 3′,5′-cyclic monophosphate (cAMP) by PGE2 have been shown to suppress leukotriene and prostaglandin production by neutrophils and eosinophils, and cAMP inhibition of release of substrate arachidonic acid from phospholipid pools occurs at levels consistent with interaction with the PGE receptor (32). The findings of the current study, that inhaled PGE2 attenuates the LAR, are consistent with the hypothesis that PGE2 may inhibit allergen-induced synthesis of the potent bronchoconstrictor, LTC4. Unfortunately, however, urinary levels of leukotriene were not measured in this study, which would demonstrate whether allergen-induced LTC4 production was indeed inhibited by PGE2.
There are many events leading to the development of allergic inflammation, which PGE2 may modulate. PGE2 has been shown to regulate the production of peripheral blood mononuclear cell-derived cytokines such as interleukin-2 (IL-2), IL-4, and IL-5 by elevating intracellular levels of cAMP (33), and has been shown to induce shift in the functional profile of cytokine message in T lymphocytes (34), possibly by the same mechanism. The adherence of inflammatory cells to endothelium is one of the initial events necessary for migration of these cells through the vascular wall. PGE2 has been shown to inhibit transendothelial migration of human lymphocytes (35), and inhibit tumor necrosis factor-alpha (TNF-α)-induced intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expression, as well as lymphocyte adhesion to human airway smooth muscle (36). This suggests that PGE2 can modulate cell recruitment indirectly through inhibition of TNF-α.
We have demonstrated that inhaled PGE2 has anti-inflammatory effects in partially protecting against allergen-induced inflammation in asthmatic subjects. This partial protection has also been demonstrated in our studies of the effects of 1 wk of regular treatment with an inhaled corticosteroid on allergen-induced airway inflammation (23). Although PGE2 formulation and administration for the treatment of airway inflammation in asthma may present many challenges, the therapeutic implications of regulating the immune response in the lung, by a mediator known to be synthesized by airway cells, remains an attractive option to study as a possible antiasthma therapy.
The authors thank T. Rerecich, J. Otis, E. Summers, and H. Kerstjens for their skilled contributions to the study.
Supported by a grant-in-aid from MRC, Canada.
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Dr. O'Byrne is a Medical Research Council of Canada Senior Scientist.
