Clinical immunology
The effects of N-acetylcysteine and inhaled steroid on inflammatory and oxidative stress markers in chronic obstructive pulmonary disease (COPD)

Introduction

Chronic obstructive pulmonary disease (COPD) has been recognized for many years as a leading cause of mortality and morbidity. There was however a pessimistic approach to the possibilities of effective treatment. Recently however, some new interest in the pathogenesis and treatment emerged. Obviously the poor outcome of the disease and the lack of progress in the pharmacological therapy have forced many clinicians and scientists to renew their attention. Several consensus statements have been published during the last decade [1-4].
Damage to lung tissue as a result of an inflammatory response is usually accompanied by an influx of neutrophilic granulocytes. In patients with COPD an enhanced number of neutrophils was found both in the airways and in bronchoalveolar lavage fluid (BAL) [5-7]. The initial step in this process is the expression of adhesion molecules by vascular endothelial cells in the vicinity of the inflammatory spot, followed by an interaction between adhesion molecules of both endothelial cells and neutrophils. Next step is the action of infiltrating neutrophils upon other cells in the lung, with release of toxic metabolites as oxygen radicals and proteolytic enzymes [7, 8] leading to extension of the inflammation [9, 10]. The extent of inflammation may be reduced by use of anti-inflammatory drugs [11, 12]. In
a model of extra corporeal circulation during cardiac surgery with activation of neutrophils and complement, it was also shown that administration of steroids or of the radical scavenger N-acetylcysteine (NAC) could inhibit the production of pro-inflammatory cytokines in both peripheral blood and in the lung [10, 13]. It was also reported that soluble intercellular adhesion molecule (sICAM-1) in both serum and BAL fluid of COPD patients was enhanced in comparison with normal individuals, although in serum there existed some overlap with normal values [14]. Recently, elevated levels of cell bound ICAM-1 were reported
[15-17]. High concentrations of the cytokines interleukin-8 (IL-8) and tumor necrosis factor a (TNF-α) in induced sputum from patients with COPD have been reported [18], and these cytokines may be involved in the recruitment of neutrophils [19, 20]. Although the role of corticosteroids in the management of COPD is controversial [3, 12, 21, 22] cytokine gene expression is known to be reduced both in vitro [23] and in vivo [24] in response to corticosteroids. This has been postulated as the mechanism of action of corticosteroids in COPD [25]. If steroids do play a role in the treatment of COPD, it is reasonable to suggest that they act through down-regulation of cytokines and adhesion molecules. The latter may also be true for any reducing effect of NAC on cell migration and activation [13, 26-28].
The aim of the present study is to evaluate the mechanisms of inflammation in COPD by studying the release of inflammatory mediators in the airways of patients, and to evaluate the effects of treatment with inhaled corticosteroids or anti-oxidants on the inflammatory pathway.

Material and Methods

Twenty stable patients were recruited (16 males,
4 females) at the out-patient clinic of the National Institute of Tuberculosis and Lung Diseases, Warsaw, Poland. Inclusion criteria for entry were: smoker or ex-smoker, not fully reversible airway obstruction with 50%
Study design

The study consisted of 4 consecutive periods of 10 weeks (Figure 1), and involved at least five visits. At each scheduled visit, induced sputum and blood samples were collected and spirometry was performed. During the first and third period no medication was allowed, except for short-acting beta agonists and/or cholinergics (salbutamol or fenoterol and/or ipratropium bromide) which were allowed to use in case of necessity during the whole study period. During the second and fourth study period, patients were treated with either inhaled FP or oral NAC in a randomized and cross-over setting. A prolongation of the study period by two weeks was allowed in case of an occurring acute exacerbation of COPD which required treatment with antibiotics. Thus the respective study period ended 2 weeks after finishing antibiotic treatment of the patient. Patients were withdrawn from the study if they required treatment with either anti-inflammatory agents, oral steroids, theophylline, and/or oxygen and thus compromising the study protocol.

Sputum collection and processing

Sputum induction and processing were performed according to the guidelines of the Task Force Induced Sputum of the European Respiratory Society [29, 30]. The collected sputum was processed as soon as possible, but always within two hours after induction. Total cell count was performed manually using a Bürker counting chamber, and cell viability was determined by trypan blue exclusion method. The sputum supernatant was aspirated and stored at -80°C until assay. The cells were used for cytospin slides.

Laboratory assays

Trolox equivalent antioxidant capacity (TEAC) was measured by use of a spectrophotometrical method [31], glutathione peroxidase (GPx) in full blood was measured based on the method of Paglia and Valentine [32], and superoxide dismutase (SOD) was measured spectrophotometrically using a commercially available kit. Albumin was measured spectrophotometrically as described previously [33]. Alpha-tocopherol and retinol were measured by HPLC. Eosinophil Cationic Protein (ECP) and tryptase were analyzed in serum and in sputum through a fluoroenzyme immunoassay. Free neutrophil elastase activity in serum samples was determined using conversion of a substrate [34]. Soluble ICAM-1 and interleukins were assayed by use of commercially available enzyme-linked immunosorbent assays.

Statistical analysis

All data are presented as mean±standard error of the mean (SEM). Assuming Gaussian distribution, data were analyzed for differences by the Student’s t-test for paired samples. Data were compared between the baselines and the outcome of the treatment after 10 weeks. For non-parametric data (IL-10, tryptase, sputum albumin, and neutrophil elastase activity) the Wilcoxon test was used. At a P value <0.05, differences were considered significant.

Results

The results represent an analysis of 20 patients who provided adequate samples from each visit. Two patients dropped out after the fourth visit: one because of protocol violation and the other one due to prolonged exacerbation of COPD. Three patients (included in the analysis) experienced acute exacerbation of COPD during a wash-out period. One of them before FP treatment, the two other patients before NAC treatment. The acute exacerbation of COPD required treatment with antibiotics.
Three patients resumed smoking during the study period. One of them dropped already out because of
a prolonged exacerbation of COPD. One patient resumed smoking during the first wash-out period and was considered as active smoker. The third patient resumed smoking during FP treatment in the last study period. Resuming to smoke during FP treatment resulted in a strong enhancement of GPx and TEAC levels. However, the mean values and the resulting statistical outcome were not significantly influenced by this fact.
The characteristics of the patients are given in Table 1. Samples collected after the wash-out period before FP treatment were considered as baseline FP samples. The remaining samples collected during the same visit were considered as baseline NAC samples.
The total number of cells in the sputum was assessed, as was the percentage and absolute numbers of neutrophils. No significant differences in cell numbers were observed between the various visits and treatment modalities (Table 2). Also percentages of sputum macrophages, lymphocytes, and eosinophils did not differ between the sampling points. Differential cell counts and cell numbers in the circulation did not show any significant differences between the subsequent visits.

Inflammatory markers

NAC decreased significantly sputum ECP levels from 266±61 to 136±27 ng/ml (P<0.02, n=18), while FP showed only a non-significant trend towards decrease (352±113 vs. 247±56 ng/ml, n=14) (Figure 2). Treatment with NAC led also significant decreases in the sputum levels of IL-8 (403±65 vs. 326±62 ng/ml, P<0.05, n=17) and tryptase,
a mast cell protease (1.80±0.29 vs. 1.33±0.16 ng/ml, P<0.01, n=18). Also here no significant influence of FP treatment was observed. IL-8 changed from 603±225 to 466±64 ng/ml, while tryptase changed from 1.77±0.31 to 1.58±0.37 ng/ml respectively. For all other inflammatory parameters that were measured no significant differences were observed. The results of these measurements are presented in Table 3. The mediator values of the different baselines were never significantly different from each other.

Oxidative stress markers

The only oxidative stress markers that were significantly influenced by treatment were GPx and TEAC. Treatment
with FP significantly increased GPx from 47.5±5.3 to 57.6±7.8 U/g Hb (P<0.05, n=19) (Figure 3). Treatment
with NAC did not change the GPx levels: 58.1±6.8 vs. 53.6±6.0 U/g Hb (n=17). Although small in increase,
TEAC levels were significantly enhanced by FP treatment
(Figure 4). The determined levels rose from 1.41±0.05 to 1.50±0.06 mmol/l (P<0.05, n=14). NAC treatment tended to decrease levels of TEAC, but this effect was not significant (1.55±0.05 vs. 1.46±0.06 mmol/l, n=15). Levels of SOD and albumin did not change during the course of the study, as did not retinol and tocopherol. Also in case of the oxidative stress markers, no differences were observed between the different baseline values.

Discussion

The present study demonstrated that treatment of COPD patients with ICS during a period of 10 weeks did not significantly influence both local and systemic markers of inflammation. However, some enhancing effect of ICS was observed on the levels of GPx and TEAC in the circulation, which suggest a positive influence on antioxidant capacity. Treatment of the same patients with NAC demonstrated that NAC is able to inhibit some local markers of inflammation. Effects on systemic inflammatory markers and on any systemic marker of oxidative stress were not observed. The study was designed as a direct, head-to-head comparison of the effects of a high dose of FP with NAC, with an emphasis on the assessment of inflammatory markers and markers of antioxidant defense.
The inflammatory processes in the airways may serve as a reason for use of inhaled steroids as anti-inflammatory therapy in COPD. It was suggested, because the inflammation in COPD is neutrophilic, the treatment with steroids would be less successful. Systemic steroids are known to increase peripheral neutrophil counts, which may reflect a delayed apoptosis [35]. Although it has not been investigated if this is also the case for lung neutrophils, we did not observe any reduction in the numbers of neutrophils in sputum. Contrary to Thompson and coworkers, who could also not show any effect on neutrophil numbers, and we did not observe any effect on total cell counts [36], but others did [37]. Although the use of steroid treatment is controversial, even some patients can benefit from it [38]. However, from our study the benefit of steroid treatment was not found on the level of inflammatory markers assessed. One would expect some effect on these inflammatory markers as many in vitro studies suggest. Surprisingly, we found an effect on two markers of oxidative stress, albeit only marginal, but significant. However, this effect is limited to systemic members of the anti-oxidant system, GPx and TEAC, the latter a rather unspecific anti-oxidant system. On the other hand, the observed effects of steroids on the oxidant-anti-oxidant balance are not completely unexpected. A few publications associate estrogen, cortisol and 17b-estradiol with elevated levels of plasma and erythrocyte GPx activity during the menstrual cycle [39, 40]. In acute exacerbation of COPD an enhancement of TEAC and plasma sulfhydryl levels were described after steroid treatment [41]. In asthma it was demonstrated that ICS reduced inflammation, attenuating the release of oxidants by inflammatory cells, and suppressing proinflammatory cytokines production, which may lead to
a reduction in lipid peroxidation [42-44]. Typically the inflammatory processes in COPD are dominated by a neutrophilic inflammation, and because lack of suppressive effects of ICS on neutrophils was reported [42], the inability to see inhibitory effects of ICS in COPD could be suspected. This negative observation is supported by studies in COPD patients where exhaled NO and CO, which are in fact indirect markers of oxidative stress, are enhanced even if the patients were taking ICS. This may be explained by
a constant activation of NO synthase [45] and therefore reflect airway inflammation and failure of ICS to inhibit neutrophilic inflammation. It was also reported that a potent inhalation corticosteroid (FP) had an inhibitory effect on the production of reactive oxygen species from lavage cells obtained from smoking COPD patients. Heavy smoking impaired the ability of alveolar macrophages to produce reactive oxygen species, which was not further decreased by FP incubation [46]. However, in our study we did not observe any significant different values of oxidative stress related parameters between the current smokers and the ex-smokers. The in the literature described effects of steroid treatment on the level of exhaled gases may be the result of a complex interaction with enzymes and release of reactive oxygen species by inflammatory cells [47]. The benefit of steroid treatment in certain COPD patients, as reported in literature, may be only applicable to the mixed-type patient with asthma symptoms.
NAC affected some of the inflammatory markers in the sputum, and had no effect on systemic inflammatory markers nor markers of oxidative stress. It was reported that IL-8 levels are elevated in both sputum and lavage fluid from COPD patients as compared to normal individuals [48, 49]. NAC treatment is able to reduce IL-8 levels in plasma, but in the present study no such effect was observed [13]. Our present findings are in accordance with previous reports which showed NAC as an inhibitor of factors with chemotactic and priming activity for neutrophils [13, 50, 51]. It was also reported that NAC is able to inhibit
TNF-α-induced NF-kb activation and reactive oxygen species dependent IL-8 increase [52]. NAC demonstrated a similar potent inhibition on NF-kb activation in vascular endothelial cells [53]. Therefore, the suppression of NF-kb activation by NAC, which seems an important property of this antioxidant, may account for the observed decrease in IL-8 levels, and for described reduction in neutrophil influx, although we could not demonstrate this latter effect. Furthermore, the lower levels of IL-8 may lead to lower ECP levels. Despite the fact that ECP is of eosinophilic origin, no relationship was found in some studies between eosinophil number and ECP levels in induced sputum [54]. This fact suggests that ECP sputum concentrations are not merely
a function of eosinophil numbers. It has been suggested that ECP can be taken up and transported by neutrophils [55], for which IL-8 is a strong chemoattractant. Therefore, any changes in IL-8 levels would affect neutrophil activation status and thus influence the sputum level of ECP.
In conclusion, we might say that use of inhaled corticosteroids in stable COPD has only minor effects and these effects are probably indirect, leading to an enhancement of the redox balance. Something that was not observed for NAC. On the other hand, NAC demonstrated some anti-inflammatory actions.

This work was supported by funding in the framework of the Bilateral Scientific and Technological Cooperation Program between the Flemish Community (Belgium) and the Republic of Poland, project no. BIL99/27.


Acknowledgments

The authors want to acknowledge the valuable cooperation of MSc Zdral A and MSc Filewska M for superb technical support.

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