Abstract
Induced sputum 8-iso-prostaglandin F2α (PGF2α) concentrations may be a useful marker of oxidative stress in airways disease. This study examines oxidative stress (measured by 8-iso-PGF2α) in airway disease according to disease type (asthma and bronchiectasis), disease activity (stable and acute asthma), and disease pattern (intermittent, mild, moderate, and severe persistent asthma). We compared subjects with stable asthma (n = 71) and bronchiectasis (n = 23) with healthy control subjects (n = 29). Another group of patients with asthma (n = 39) were assessed during and after acute exacerbation. Induced sputum 8-iso-PGF2α concentrations were validated and found to be elevated in subjects with stable asthma and bronchiectasis versus control subjects (median [interquartile range] 216 [103–389] and 698 [264–1,613] ng/L vs. 123 [41–290] ng/L, p < 0.001) and increased as clinical asthma pattern worsened (intermittent 115 [42–153], mild persistent 116 [89–229] ng/L, moderate persistent 183 [110–317] ng/L, severe persistent 387 [102–587] ng/L; p = 0.010). Sputum 8-iso-PGF2α concentrations were elevated during acute asthma and decreased with recovery (458 [227–950] ng/L vs. 214 [148–304] ng/L, p = 0.0002). We conclude that 8-iso-PGF2α is involved in the pathophysiology of inflammatory airway diseases, being related to disease type, pattern, and activity. Analysis of 8-iso-PGF2α concentrations in induced sputum provides a useful tool for monitoring oxidative stress and investigating strategies aimed at reducing oxidative stress in airways disease.
Oxidative stress is believed to play an important role in the pathophysiology of respiratory disease (1–6). The most commonly recognized mechanism leading to oxidative stress in respiratory disease is chronic inflammation, which involves recruitment and activation of inflammatory cells. During the respiratory burst, inflammatory cells produce excessive quantities of free radicals, which overwhelm host antioxidant defenses, leading to oxidative stress (7). Oxidative stress can have many detrimental effects on airway function, including airway smooth muscle contraction (8), induction of airway hyperresponsiveness, mucus hypersecretion, epithelial shedding, and vascular exudation (reviewed in Reference 7). Furthermore, reactive oxygen species can induce cytokine and chemokine production through activation of intracellular signaling cascades. For example, the transcription factor NF-κB has been shown to be activated by oxidative stress in bronchial epithelial cells (9).
Oxidative damage to lipids (lipid peroxidation) leads to the production of isoprostanes. Isoprostanes, of which 8-iso-prostaglandin F2α (PGF2α) is the best-characterized isomer, are produced independently of the cyclooxygenase enzyme by the peroxidation of arachidonic acid, catalyzed by free radicals. They are considered to be a reliable index of in vivo oxidative stress because they are structurally stable and are produced in vivo (10, 11). In recent years, many studies have demonstrated that increased concentrations of 8-iso-PGF2α are a common feature of respiratory disease (11). Elevated 8-iso-PGF2α concentrations have been reported in patients with asthma (12–14), chronic obstructive pulmonary disease (15, 16), cystic fibrosis (17–19), interstitial lung disease (20), pulmonary hypertension (21), and acute respiratory distress syndrome (22), and in infants with respiratory failure (23). These reports highlight the involvement of lipid peroxidation in respiratory diseases.
Induced sputum is collected from the intrapulmonary airways, at the site directly adjacent to asthma pathology, and thus may accurately reflect conditions at the site of oxidative damage (24). Other methods used for sampling the airways are limited: the collection of bronchoalveolar fluid is extremely invasive and expired air collection has questionable reproducibility (25). Induced sputum samples contain a range of biomarkers useful for studying the lower respiratory tract (26), and sample collection is noninvasive. Thus, induced sputum may be a useful medium for examining oxidative stress in the airways. However, to date there are no reports of 8-iso-PGF2α in induced sputum. This study investigates oxidative stress in inflammatory airway diseases according to disease type, pattern, and activity using induced sputum 8-iso-PGF2α as a biomarker. We tested the hypothesis that induced sputum 8-iso-PGF2α would be elevated in inflammatory airway diseases (asthma and bronchiectasis) and would increase in asthma as disease pattern and activity worsened. Some of these results have previously been presented in the form of a conference abstract (27).
Adults with stable asthma (n = 71), acute asthma exacerbation (n = 39), bronchiectasis (n = 23), and healthy control subjects (n = 29) were recruited. Subjects with stable asthma were recruited from the John Hunter Hospital Asthma Clinic. Asthma was diagnosed on the basis of current episodic respiratory symptoms, doctor's diagnosis of asthma, and airway hyperresponsiveness to hypertonic saline. The clinical asthma pattern was categorized according to Global Initiative for Asthma guidelines (28). Subjects with acute asthma were recruited and studied on hospital admission and again 4 to 6 weeks after exacerbation, as previously reported (29). Subjects with bronchiectasis confirmed by chest computed tomography (17 previously described with allergic bronchopulmonary aspergillosis, a hypersensitivity reaction to the fungus Aspergillus fumigatus, which causes severe asthma [30]) were recruited from the John Hunter Hospital Respiratory Clinic. Healthy control subjects without asthma were recruited by advertisement. The study was approved by the Hunter area and University of Newcastle human research ethics committees.
Spirometry (Minato Autospiro AS-600; Minato Medical Science, Osaka, Japan) and combined bronchial provocation and sputum induction with hypertonic saline (4.5%) were performed (31) with the standard 11.5-minute induction time. Subjects with acute asthma were pretreated with supplemental β2-agonist before administration of nebulized 0.9% saline (32). Sputum was selected from saliva (26, 31) and dispersed with dithiothreitol, and a total cell count of leukocytes and viability was performed. Cytospins were prepared and stained (May-Grunwald geimsa), and a differential cell count was obtained from 400 nonsquamous cells. The remaining solution was centrifuged (400 × g, 10 minutes, 4°C) and the cell-free supernatant was aspirated and stored at −70°C.
8-iso-PGF2α concentrations were detected in 100% of sputum supernatant samples by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) with standard curves using purified 8-iso-PGF2α in 0.1% dithiothreitol solution. The assay has a 4-ng/L detection limit, uses antiserum that has 100% cross-reactivity with 8-isoprostane and less than 0.3% with prostaglandin analogs, and has been used in plasma (12), bronchoalveolar lavage (20), and expired breath (13). The intraassay (n = 201) and interassay (n = 17) coefficients of variation were 3 and 8%, respectively. The assay was validated according to European Respiratory Society guidelines (33). Spiking experiments in sputum supernatant yielded 100% recovery. The within-patient reproducibility (samples collected 2 days apart) was good. All values were within the limits of agreement according to Bland-Altman, with a mean difference of 13.7 pg/ml and limits of agreement of −12.5 to 39.9 pg/ml. A strong association was observed between sputum supernatant isoprostane measurements using gas chromatography-mass spectroscopy (GC-MS) and enzyme immunoassay (r = 0.912, p < 0.001; Figure 1)
Figure 1. Induced sputum isoprostane concentrations analyzed using gas chromatography–mass spectroscopy (GC-MS) versus enzyme immunoassay (EIA) ranks. R = 0.912, p < 0.001. See online supplement for description of method.
[More] [Minimize]Data are reported as mean ± SE (normal data) and median (interquartile range [nonparametric data]). PD15 was log-transformed and presented as geometric mean (log SD). Group comparisons were performed using the Kruskal-Wallis test, with post hoc testing using the Mann-Whitney U test and the Bonferonni correction (Minitab, State College, PA). Associations were examined using Pearson's and Spearman's rank correlation coefficients for normal and nonparametric data, respectively. 8-iso-PGF2α was log-transformed for multiple regression analysis using disease group, age, sex, inhaled corticosteroid (ICS) use, and %predicted FEV1 as predictors of log108-iso-PGF2α. Significance was accepted if p was less than 0.05.
The clinical characteristics of the subjects with stable asthma, subjects with bronchiectasis, and healthy control subjects are shown in Table 1
Healthy Control Subjects | Stable Asthma | Asthma Exacerbation (On Admission) | Bronchiectasis | |
|---|---|---|---|---|
| N | 29 | 71 | 39 | 23 |
| Age, yr | 43 ± 3 | 46 ± 2 | 44 ± 3 | 55 ± 3 |
| Sex, M/F | 12/17 | 13/58 | 12/27 | 10/13 |
| FEV1, % predicted | 100 ± 2 | 77 ± 2‡ | 63 ± 4‡,‖ | 55 ± 4‡,¶ |
| FVC, % predicted | 103 ± 2 | 92 ± 2§ | 68 ± 4‡,¶ | 77 ± 3‡,¶ |
| FEV1/FVC, % | 81 ± 1 | 68 ± 1‡ | 74 ± 3‖ | 57 ± 3‡,¶,†† |
| PD15, ml*,† | N/A | 0.55 (0.61) | N/A | N/A |
| Inhaled corticosteroid dose, μg/d beclamethasone equivalent | N/A | 1,000 (800–2,000) | 1000 (0–2,000)** | 2000 (750–3,000) |
| Atopy, +/− (% +) | 16/8 (67) | 44/4 (92) | 22/15 (59) | 22/1 (96) |
Induced sputum 8-iso-PGF2α concentrations were elevated in subjects with bronchiectasis and asthma compared with control subjects (Table 2
Healthy Controls | Stable Asthma | Bronchiectasis | p Value | |
|---|---|---|---|---|
| 8-iso-prostaglandin F2α, ng/L | 123 (41–290) | 216 (103–389)* | 698 (264–1613)‡,§ | < 0.001 |
| Total cell count, × 106/ml | 1.44 (0.83–2.50) | 2.25 (1.35–5.22)† | 7.74 (4.59–13.50)‡,§ | < 0.001 |
| Neutrophils, % | 21.9 (8.5–33.4) | 30.1 (13.0–51.8) | 67.0 (37.2–84.3)‡,§ | < 0.001 |
| Eosinophils, % | 0.0 (0.0–0.3) | 1.0 (0.5–2.5)‡ | 0.5 (0.0–4.8)† | < 0.001 |
| Macrophages, % | 71.8 (61.9–82.5) | 59.3 (41.6–78.0)† | 26.1 (14.1–38.0)‡,§ | < 0.001 |
| Lymphocytes, % | 1.5 (0.5–2.2) | 0.4 (0.0–1.3)† | 0.0 (0.0–1.6)† | 0.012 |
| Columnar epithelial cells, % | 1.5 (0.5–6.0) | 1.8 (0.5–4.0) | 0.76 (0.0–6.9) | 0.298 |
| Squamous cells, % | 8.5 (3.8–26.1) | 5.5 (2.1–20.4) | 1.8 (0.5–6.9)†,‖ | 0.011 |
Figure 2. Induced sputum concentrations of 8-iso-prostaglandin F2α (PGF2α) in healthy control subjects (n = 29), subjects with stable asthma (n = 71), and subjects with bronchiectasis (n = 23). *p < 0.029 versus healthy control subjects; †p = 0.0001 versus healthy control subjects and subjects with stable asthma.
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Figure 3. Induced sputum concentrations of 8-iso-PGF2α in patients with stable asthma with intermittent (n = 7), mild persistent (n = 6), moderate persistent (n = 25), and severe persistent (n = 33) disease pattern. Subgroup analysis indicated that 8-iso-PGF2α increased as asthma pattern worsened. *p = 0.01.
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Figure 4. Induced sputum concentrations of 8-iso-PGF2α during and 4 to 6 weeks after asthma exacerbation (n = 39). *p < 0.001 versus during exacerbation.
[More] [Minimize]Induced sputum inflammatory cell profiles for each disease type are presented in Table 2. Subjects with stable asthma and bronchiectasis had a higher total cell count and percentage of eosinophils than healthy control subjects. Subjects with bronchiectasis also had elevated percentages of neutrophils compared with healthy control subjects and subjects with stable asthma.
Multiple regression analysis of data from healthy control subjects, subjects with stable asthma, subjects with acute asthma, and subjects with bronchiectasis identified disease group, age, sex, and percent predicted FEV1 as significant determinants of induced sputum log108-iso-PGF2α (Table 3)
R2 | F | p Value | |
|---|---|---|---|
| Overall Model | 0.3263 | 10.03 | < 0.0001 |
| Variable | Coefficient | 95% Confidence Interval | |
| Stable asthma | 0.11704 | −0.17302–0.40711 | 0.426 |
| Acute asthma | 0.40471 | 0.10918–0.70025 | 0.008 |
| Bronchiectasis | 0.62941 | 0.28764–0.97118 | 0.000 |
| Age | −0.00746 | −0.01260–0.00231 | 0.005 |
| Sex | −0.17695 | −0.35352–−0.00037 | 0.050 |
| ICS | −0.04377 | −0.02514–0.17760 | 0.697 |
| FEV1, % | −0.00673 | −0.01097–0.00248 | 0.002 |
| Constant | 3.08469 | 2.49567–3.67372 | 0.000 |
Figure 5. Log108-iso-PGF2α versus percent predicted FEV1 (r = −0.382, p < 0.0001) in healthy control subjects, in subjects with stable and acute asthma, and in subjects with bronchiectasis.
[More] [Minimize]To our knowledge, this is the first study to use induced sputum samples to directly investigate lipid peroxidation in airway disease. The data show that induced sputum concentrations of 8-iso-PGF2α were elevated in subjects with stable asthma and in subjects with bronchiectasis compared with healthy control subjects. Within the subgroup of patients with stable asthma, induced sputum concentrations of 8-iso-PGF2α were related to clinical pattern. Furthermore, the data indicate that induced sputum 8-iso-PGF2α concentrations significantly decreased after treatment of acute asthma exacerbation. Thus, airway oxidant stress, measured by induced sputum 8-iso-PGF2α, is related to respiratory disease type, pattern, and activity.
The elevation of 8-iso-PGF2α concentrations in subjects with stable asthma and in subjects with bronchiectasis demonstrates that lipid peroxidation is involved in the pathophysiology of these inflammatory airway diseases. In patients with stable asthma, sputum eosinophils were significantly higher than in control subjects, confirming eosinophilic inflammation as a key feature of asthma (28). Subjects with bronchiectasis had an elevated proportion of eosinophils and neutrophils compared with healthy control subjects. Bacterial infections may be facilitating the recruitment and activation of neutrophils in bronchiectasis (30, 34). Thus, recruitment and activation of inflammatory cells, and the resultant production of an excess of free radicals, may contribute to elevated oxidative stress in stable asthma and bronchiectasis. Note that, in bronchiectasis, there was no difference in 8-iso-PGF2α concentrations in ex-smokers compared with never-smokers, indicating that the significant smoking history of these subjects has not worsened oxidative stress.
For the asthma subgroup, this study provides direct evidence for the involvement of oxidative stress in the pathophysiology of asthma by linking biochemical and clinical markers. Within the stable asthma group, the relationship between 8-iso-PGF2α and disease pattern (Figure 3) agrees with previous reports of associations between 8-iso-PGF2α and asthma severity in plasma (12) and breath condensate (13). Further evidence that relates oxidative stress to clinical status in asthma is our observation that 8-iso-PGF2α concentrations decrease after treatment for acute asthma exacerbations (Figure 4). Other researchers have also observed changes in oxidative stress during acute asthma exacerbations, including increased serum/plasma thiobarbituric acid reactive substances (TBARS) (35, 36), decreased plasma β-carotene concentrations, increased exhaled pentane levels (37), decreased plasma Trolox equivalent antioxidant capacity (35), increased production of the hydroxyl and superoxide radicals by blood cells (36), and increased exhaled breath 8-iso-PGF2α (38) in acute versus stable asthma. 8-iso-PGF2α concentrations have also been shown to increase in urine and bronchoalveolar lavage fluid in patients with asthma after an experimental allergen challenge (14) and in aspirin-induced asthma (39). We have extended these observations by investigating the importance of clinical parameters in determining 8-iso-PGF2α concentration in airway disease. Clinical asthma pattern was associated with 8-iso-PGF2α, and in a multiple regression analysis, disease group (healthy control subjects, stable asthma, acute asthma, and bronchiectasis), age, and percent predicted FEV1 were each significant predictors of induced sputum 8-iso-PGF2α concentrations. Although these data relating oxidative stress to clinical markers do not necessarily imply a causative link, it is possible that oxidative stress may be directly contributing to asthma symptoms. Further research is needed to establish whether oxidative stress has an active role in the pathogenesis of asthma, or whether oxidative stress might be an epiphenomenon of the disease.
Although the inflammatory phenotype of stable asthma is different to acute asthma (29), the clinical expression represents a gradation with more severe airflow obstruction in acute asthma. Thus, isoprostane results parallel the clinical expression (Figure 3). Although the source of free-radical generation in asthma remains to be established with certainty, it is likely that oxidative stress occurs as the result of inflammatory cell activation. Our data suggest that this is irrespective of inflammatory cell type. That is, whether asthma is mediated via a predominantly neutrophilic or eosinophilic pathway, isoprostanes may form part of the common end pathway that leads to airflow obstruction (7). This possibility is supported by the weak association observed between 8-iso-PGF2α and %FEV1 (Table 3; Figure 5). Future studies are needed to determine whether the relationship between oxidative stress and airway obstruction exists in individual patients. If this proves true, then monitoring airway 8-iso-PGF2α levels may provide useful information regarding clinical status in inflammatory airway diseases.
In recent years, a method has been developed to measure biomarkers in exhaled breath condensate. This method is potentially another useful means of deriving a sample directly from the airways, in a noninvasive manner (40). Several groups have investigated airway oxidative stress using exhaled breath condensate and have reported increased 8-iso-PGF2α in chronic obstructive pulmonary disease (16), cystic fibrosis (41), and asthma (13), with 8-iso-PGF2α increasing with asthma severity (13). However, this method has proven to be technically difficult, with some concerns being raised over reproducibility (42). Recently, a radioimmunoassay for 8-iso-PGF2α in expired air has been developed. The specificity, sensitivity, and reproducibility of this assay may be important in progressing 8-iso-PGF2α research. We have carefully evaluated the measurement issues (33) associated with induced sputum 8-iso-PGF2α and found the measurement to be valid and reproducible and to correlate with GC-MS measurement of F2-isoprostanes (see online data supplement).
The extent of the biological activity of the isoprostanes remains to be determined because of limitations in the availability of individual isomers. However, the 8-iso-PGF2α isomer is a potent constrictor of smooth muscle in vitro (8). 8-iso-PGF2α has also been shown to elicit airway hyperresponsiveness (43) and to cause airway obstruction and airway plasma exudation (44) in animal models. These data invite speculation about the contribution of isoprostanes to the airway narrowing that is characteristic of asthma. The situation in humans in vivo may be different, and it is unknown whether the concentrations of 8-iso-PGF2α reached in the airways are biologically relevant; thus, the full impact of elevated isoprostane levels on pulmonary function remains to be established. Considering the relationship between 8-iso-PGF2α and clinical status, further work is needed to investigate the role of 8-iso-PGF2α, not just as a biomarker but also as a potentiator of asthma and other respiratory diseases.
In conclusion, 8-iso-PGF2α is involved in the pathophysiology of inflammatory airway disease, being related to disease type, asthma pattern, and asthma activity. Furthermore, analysis of 8-iso-PGF2α concentrations in induced sputum samples, which is reproducible and noninvasive, may provide a useful tool for monitoring oxidative stress and investigating strategies aimed at reducing oxidative stress in respiratory disease.
Assistance with statistical analysis was received from Heather Powell. Assistance with collection of samples and clinical data was received from Naomi Timmins, Rebecca Oldham, Joanne Smart, Kellie Fakes, Noreen Bell, and Philippa Talbot from the Department of Respiratory and Sleep Medicine, Hunter Medical Research Institute, John Hunter Hospital, Newcastle, NSW, Australia.
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