INTRODUCTION

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Keywords: bronchial hyperreactivity; airway obstruction; lung hyperinflation; dyspnea; bronchial challenge

Inhalation of hypertonic saline solution has been extensively used in patients with asthma as a method of bronchial challenge (1). It is also part of the procedure of sputum induction, which has gained much interest in patients with airway diseases including those with chronic obstructive pulmonary disease (COPD). Although prior inhalation of a ₂-agonist is mandatory in sputum induction, patients might still show a deterioration in lung function after saline inhalation (2).

The proper assessment of lung function responses is, however, difficult in patients with severe COPD owing to the intricacies of lung mechanics that characterize the disease involving a reduction in alveolar attachments (5) and changes in airway morphology and function (6). Until now, responses to hypertonic saline have been studied only by forced expiratory maneuvers, whereas the role of lung hyperinflation versus airway obstruction, the sensitivity of different indices in the detection of responses, and their relationship to dyspnea are currently unknown. This is particularly true in view of the evidence that inspiratory parameters might be superior to expiratory parameters in exploring airway tone and dyspnea in COPD (7, 8).

Analysis of the response to hypertonic saline might also be relevant for the assessment of nonspecific airway hyperresponsiveness, which is present in up to two-thirds of patients with COPD (9) and considered a predictor of the decline in lung function over time (10). The inhalation of saline solution during sputum induction is considered acceptable in the presence of moderate to severe airflow obstruction (2), in contrast to methacholine or histamine challenges (11). Furthermore, such indirect challenges could be of greater interest with regard to stimuli encountered in the patients' environment than pharmacological agonists.

In addition to its usefulness as a challenge, saline inhalation offers the advantage that the induced sputum might give clues on the mechanisms involved. This seems to be of particular interest in view of the fact that ₂-agonists are incapable of fully preventing the adverse response. The information gained might also have implications for future improvements regarding the safety of sputum induction.

We therefore analyzed the airway response to inhalation of hypertonic and isotonic saline in patients with moderate to severe COPD by monitoring different lung function measures including forced inspiratory and expiratory maneuvers. We additionally compared sputum composition between the challenges to obtain information on the mechanisms mediating the response.

	METHODS

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Patients

Twenty patients with stable COPD according to ATS guidelines (12) and FEV₁  60 %pred were studied (Table 1). The protocol was approved by the local Ethics Committee and all patients gave their written informed consent.

Study Protocol

The study comprised one screening and two study days, separated by  72 h and  10 d. Inhaled short(long)-acting bronchodilators were withheld for  6 (12) h, whereas the other medication was not changed. On the screening day medical history and clinical state, total immunoglobulin E (IgE), lung function, transfer factor for carbon monoxide (TL_co), inspiratory pressure after 0.1 s (P_0.1), and maximal inspiratory pressure (PI_max) were assessed.

On the study days patients underwent lung function testing, inhaled 200 µg salbutamol through a metered dose inhaler (MDI), and were measured again 15 min later. The change in dyspnea by visual analog scale (VAS) (13) was assessed immediately before lung function testing. Then patients inhaled, in a randomized double-blind manner, either isotonic (0.9%) or hypertonic (3%) saline from a ultrasonic nebulizer (NE-U12; Omron, Tokyo, Japan; output 1.7 ml/min [14]) for up to 4 × 5 min. Lung function and VAS were obtained after each of the 5-min inhalation periods. When FEV₁ fell  20% from the postbronchodilator value, the induction was stopped. If this occurred after the first period, sputum was collected after that period, otherwise after the second period. Fifteen min after the last inhalation, VAS and lung function were assessed again.

Inspiratory vital capacity (IVC), expiratory (SRaw_ex) and inspiratory (SRaw_in) specific airway resistance, total lung capacity (TLC), intrathoracic gas volume (ITGV), residual volume (RV), and inspiratory capacity (IC) were measured in a body plethysmograph (Masterlab; Jaeger, Würzburg, Germany) (15, 16). Then FEV₁, FVC, peak expiratory and peak inspiratory flow (PEF, PIF), and FIV₁ were determined from maximal expiratory and inspiratory flow volume curves (8). VAS ratings were used for the assessment of changes in dyspnea (13, 17) ranging from 100% ("very much worse") to +100% ("very much better").

Sputum was processed immediately after induction by selecting plugs (3, 14) and cell counts from coded Giemsa-stained cytospin slides were expressed as percent of nonsquamous cells. The concentrations of IL-8 (CLB, Amsterdam, Netherlands), histamine (IBL, Hamburg, Germany), and MMP-9 and TIMP-1 (Amersham Pharmacia, Freiburg, Germany) in supernatants were measured by ELISA. Tryptase levels were determined by FEIA (Pharmacia & Upjohn, Freiburg, Germany) after samples had been concentrated 4-fold. The concentration of leukotrienes C/D/E₄ was assessed by ELISA (Biotrak, Amersham Pharmacia) after extraction (18) and additional spiking with 0, 5, and 10 pg.

Statistical Analysis

Mean values and standard deviations (SD) (Table 1) or standard errors of the mean (SEM) (otherwise) were computed. Values measured before and at the end of an inhalation challenge were compared to each other using the paired t test. In an analogous manner, absolute and percent changes were compared between the two study days. Correlation analysis was performed using ANCOVA and linear correlation coefficients. Significance was assumed for p < 0.05.

	RESULTS

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Of the 20 patients studied, 9 showed values of FEV₁ of  50 %pred, and 7 of  35 %pred (Table 1). All patients were on inhaled ₂-agonists and anticholinergics; 9 of them additionally were on theophylline and 11 on inhaled corticosteroids.

Lung Function

Baseline lung function did not differ significantly between the two study days. Inhalation of salbutamol led to an increase in IVC, FVC, FEV₁, FIV₁, IC, PIF, and PEF, whereas RV, SRaw_ex, and SRaw_in decreased (p < 0.001, each; Table 2). No significant changes occurred in TLC and ITGV. Neither absolute values nor the absolute or percent changes induced by salbutamol differed between the two days. The same was true for VAS scores, which were different from zero on both days (p < 0.01, each).

The effects of inhaled saline on FEV₁ and FIV₁ are shown in Figure 1. During inhalation of 0.9% saline, 9 challenges were stopped due to a  20% fall in FEV₁. FIV₁ decreased by  20% in 10 patients. The mean time of inhalation was 16.5 min (11.3 min for those that were stopped). Compared with postbronchodilator values there was a decrease in VC, FVC, FEV₁, FIV₁, PEF, PIF, and IC (p < 0.05, each) and an increase in ITGV, RV, SRaw_in, and SRaw_ex (p < 0.01, each; Table 2). On average, FEV₁ had decreased by 202 ml when the challenge was stopped, and this value was smaller (p < 0.001) than the corresponding decrease in FIV₁ by 606 ml.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Time course of FEV1 (A) and FIV1 (B) versus the consecutive 5-min periods of inhalation of 0.9% saline. Data obtained with 3% saline are given in C and D. The challenge was stopped when FEV1 had fallen by  20% or the subjects reported intolerable symptoms. Data obtained 15 min after pretreatment with salbutamol (indicated by 2) were calculated as percent changes relative to baseline (indicated by B). Data obtained during saline inhalation were expressed as percent changes relative to the value 15 min after pretreatment with salbutamol. To facilitate the graphic comparison, salbutamol values are chosen as a common reference.

During inhalation of 3% saline, challenges had to be terminated in all but two patients because of a  20% fall in FEV₁. These patients were not able to continue the challenge after 10 min because of severe dyspnea, although their decline in FEV₁ was < 20%. All patients showed a 20% fall in FIV₁. Mean inhalation time was 8.8 min. VC, FVC, FEV₁, FIV₁, PEF, PIF, and IC decreased and ITGV, RV, SRaw_in, and SRaw_ex increased during inhalation (p < 0.01, each; Table 2). At the end of the challenge, FEV₁ had fallen by 363 ml, which was less (p = 0.006) than the corresponding change in FIV₁ by 1005 ml (Table 2). In addition, mean VAS scores were lower than zero (p = 0.01).

Changes in FEV₁, FIV₁, PEF (p < 0.001, each), PIF, IC, ITGV, RV (p < 0.05, each), and SRaw_in and SRaw_ex (p < 0.01, both) were greater after inhalation of 3% as compared with 0.9% saline. Furthermore, VAS scores (p < 0.001) and the time of inhalation (p < 0.01) were lower after 3% saline.

To assess whether the set-up used for inhalation via mouthpiece caused airway responses by itself, a subgroup of 6 patients (6 m; FEV₁, 39 ± 4 %pred) additionally performed a simulated inhalation for 20 min, with the nebulizer filled but switched off. Lung function and VAS did not change, mean (± SEM) FEV₁ being 1.43 ± 0.21 L before and 1.44 ± 0.22 L after the test, FIV₁ being 3.48 ± 0.40 and 3.40 ± 0.37 L, and VAS 1.83 ± 1.83.

Correlation between Changes in Lung Function and Dyspnea

Salbutamol inhalation led to a significant correlation between VAS and the absolute changes in FIV₁ (r = 0.64, p = 0.021; see Figure 2B), while there was no correlation between VAS and the changes in FEV₁ (r = 0.19; Figure 2A) or IC (r = 0.18; Figure 2C). These analyses were performed by ANCOVA using the data from both study days. Conversely, when pooling the data obtained after inhalation of 0.9 and 3% saline by ANCOVA, correlations with VAS were r = 0.62 for FEV₁ (p < 0.0001; Figure 2D), r = 0.73 for FIV₁ (p < 0.0001; Figure 2E), and r = 0.67 for IC (p < 0.0001; Figure 2F). The respective correlations between the percent changes and VAS were r = 0.70 (p < 0.0001), r = 0.78 (p < 0.0001), and r = 0.69 (p < 0.0001). Similar data as for IC were obtained for ITGV.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Relationship between VAS and percent changes in FEV1 (A), FIV1 (B), and IC (C) after inhalation of salbutamol. The results obtained before inhalation of 0.9% saline are indicated by open squares, those before 3% saline by closed squares. The lower panels illustrate the analogous relationships after saline inhalation, whereby the percent changes represent FEV1 (D), FIV1 (E), and IC (F) at the end of the challenge. The open circles refer to 0.9% and the closed circles to 3% saline.

Induced Sputum

No significant differences in total cell count and the percentages of macrophages, neutrophils, eosinophils, and lymphocytes were found when comparing 0.9% saline and 3% saline (Table 3). Furthermore, there were no significant differences regarding the soluble-phase concentrations of IL-8, MMP-9, TIMP-1, the ratio MMP-9/TIMP-1, and leukotrienes C/D/E₄. However, histamine concentrations were elevated after 3% as compared with 0.9% saline inhalation (p = 0.007, Wilcoxon matched-pairs signed-ranks test). Tryptase was detectable in 6 patients after at least one of the two inhalations; in 5 of 6 patients its concentration was higher after 3% as compared with 0.9% saline (p = 0.09, Fisher's exact test, one-tailed). In addition, there were no significant correlations between the levels of soluble mediators and baseline lung function or its changes.

	DISCUSSION

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In the present study we observed a significant decrease in lung function through saline inhalation in patients with COPD. Responses were stronger after 3% as compared with 0.9% saline and involved an increase in airway obstruction as well as lung hyperinflation. Changes in FIV₁ were greater than those in FEV₁. Furthermore, the response to hypertonic saline seemed to involve mast cell activation in moderate to severe COPD as indicated by the rise in histamine levels in induced sputum.

Our study aimed to assess the pattern of lung function responses after saline inhalation, a stimulus regularly used in sputum induction. According to current recommendations patients inhaled salbutamol before the challenges (2). Similar to previous data (8), the concomitant bronchodilator response showed a reduction in dyspnea that was associated with the improvement in FIV₁ but not in FEV₁, IC, or ITGV. This finding supports the assertion that forced inspiration is indeed informative in delineating the effects of bronchodilators in COPD.

Previous studies already reported on a fall in FEV₁ after inhalation of isotonic (3, 4) or hypertonic (4) saline in patients with severe COPD, despite pretreatment with a ₂-agonist. According to our data, 0.9% saline led to a 20% fall in FEV₁ in nearly 50% of patients; after 3% saline challenges had to be terminated at less than 20 min inhalation time in all patients. Irrespective of that difference, the pattern of lung function responses in terms of airway obstruction and lung hyperinflation appeared to be the same with 0.9 and 3% saline.

Owing to the intricate relationship between bronchoconstriction and lung hyperinflation in COPD that renders the interpretation of functional parameters difficult, we assessed a panel of indices allowing both hyperinflation and obstruction to be identified. As a prerequisite, the trial involving the switched-off nebulizer demonstrated that it was not a change in breathing pattern through the apparatus that caused the functional changes.

TLC was not altered by inhalation of either salbutamol or saline. We conclude that patients probably performed breathing maneuvers with the same maximal effort in the presence of either bronchodilation or bronchoconstriction. The reduction in IC and the rise in ITGV and RV indicated an increase in lung hyperinflation that was most pronounced after hypertonic saline inhalation. Furthermore, changes in ITGV and IC were of similar magnitude, indicating the consistency of measurements. Changes in IVC and RV were also opposite to each other, the slight discrepancies being probably due to different selection criteria for final values as formulated in the recommendations for lung function measurement (15).

The rise in specific airway resistance, both inspiratory and expiratory, suggested an increase in bronchoconstriction, in particular as specific airway resistance is thought to be largely independent of lung volume. When taking into account the constant TLC, the fall in FEV₁ and PEF additionally supported the conclusion that saline caused bronchoconstriction.

Interestingly, changes in FIV₁ were similar to those in IVC, indicating a major role for hyperinflation during saline inhalation. Although there was a deterioration in both FEV₁ and FIV₁, FIV₁ seems to have the advantage over FEV₁ by being less affected by airway collapse (8). Although it is difficult to quantify the impact of bronchoconstriction on the reduction of FIV₁ in the presence of increased lung hyperinflation, it is likely that FIV₁ reflected both obstruction and hyperinflation. With bronchodilation, changes in lung function and dyspnea were smaller than with saline but changes in FIV₁ were also similar to those in IVC.

We also analyzed the relationship between changes in lung function and dyspnea during acute saline-induced bronchoconstriction, as opposed to bronchodilation (8). A previous study found only a weak correlation between changes in FEV₁ and dyspnea as quantified by the Borg scale, when adenosine 5'-monophosphate and methacholine were used as bronchoconstrictors (19). We chose the VAS, as this scale has been reported to be more sensitive than the Borg scale under resting conditions (17, 20). The acute saline-induced bronchoconstriction was reflected in significant and similar correlations of VAS versus FEV₁, FIV₁, and IC (Figure 1D-1F). Correlations appeared to be strongest for FIV₁ and IC. These findings are in agreement with those of Noseda and colleagues, who also studied both bronchoconstriction and bronchodilation; they found that the relationship between VAS and the changes in inspiratory parameters, such as IVC, MIF₅₀, and SRaw_in, is stronger than the relationship between VAS and FEV₁ (7).

Inhalation of hypertonic saline is well introduced for the assessment of airway hyperresponsiveness in asthma (1). The major causative factor is thought to be the rate of change in osmolarity, whereby the volume of aerosol provoking a 20% fall in FEV₁ decreases with increasing concentration (1). As a consequence of mediator release from epithelial cells, sensory nerves, and mast cells (21), smooth muscle contraction and airway edema occur. The response to hypertonic saline in asthma could be blocked with histamine antagonists (22, 23), and human lung mast cells in vitro demonstrated histamine release within seconds that was maintained after removal of the stimulus (24). Leukotrienes, prostaglandins, and sensory neuropeptides might also be involved (25).

Indeed, as compared with nonsmokers, patients with chronic bronchitis show higher numbers of mast cells in large (26) and distal airways (27), and the number of mast cells increases with the degree of airway obstruction (28). Furthermore, histamine levels were found to be elevated in sputum (29) and urine (30). The present study revealed increased histamine levels in sputum after inhalation of 3% compared with 0.9% saline; in addition, tryptase levels tended to be raised. This suggests that mast cells are involved in the response to hypertonic saline in COPD. Mast cells are also a potent source of cysteinyl leukotrienes, which were, however, not elevated within the, on average, 8.8-min period of hypertonic saline inhalation.

A role for mast cells in COPD is further supported by the finding that bronchoconstriction by AMP, which is thought to be mediated via mast cell activation (31), can be blocked through an antihistamine but not an anticholinergic (32). Alternatively, activation of sensory nerve endings by AMP has been proposed (33). Nerve endings could also be involved, as substance P levels in sputum were raised through inhalation of hypertonic saline (34).

The bronchoconstriction induced by both isotonic and hypertonic saline might be considered as a sequel of the fluid inhaled and of water moving into the airway lumen. However, assuming a pure volume effect one would have to expect a decrease in cell numbers and concentrations of fluid phase parameters with the stronger response. In contrast, cell numbers and cell differentials remained constant and the levels of histamine and tryptase even increased. Therefore, whatever the (osmotic) mechanism of the response to hypertonic saline might be, it seems to involve the release of inflammatory mediators. The presence of "true" airway hyperresponsiveness is also suggested by the high percentage of positive bronchodilator responses where osmotic and/or volume effects do not play a role.

It might be argued that the pretreatment with 200 µg inhaled salbutamol has biased our findings, as salbutamol inhalation tended to result in lower histamine levels in the sputum of patients with asthma (35). In our study salbutamol neither prevented the difference in histamine concentrations nor supplied full protection as demonstrated by the adverse responses after saline inhalation.

In all patients included the analysis of cell differentials supported the diagnosis of COPD, showing a high number of neutrophils and a low number of eosinophils. Values were not altered by the challenges, similar to the concentrations of IL-8, MMP-9, TIMP-1, and leukotrienes. In COPD sputum composition seems not to change during consecutive induction periods (3), in contrast to healthy subjects (14). Although hypertonic stimuli can cause IL-8 release (36) and increase neutrophil chemotactic activity (37), the time involved in our protocol was seemingly too short to elicit differences in release or to induce synthesis. Most importantly, the data indicate that there was no appreciable difference in sputum dilution between the two saline inhalations, ensuring that the difference in histamine levels was not an artifact. In contrast to previous data (38), we found no relationship between MMP-9, TIMP-1, or their ratio and airflow obstruction at baseline, probably due to the fact that we included only patients with marked obstruction, resulting in a much narrower range of FEV₁.

In summary, the present data suggest that the lung function response to inhaled hypertonic saline in patients with moderate to severe COPD involves both bronchoconstriction and lung hyperinflation. The sensation of dyspnea elicited by the challenge was associated with changes in forced inspiratory and expiratory volumes as well as hyperinflation (IC, ITGV), in contrast to bronchodilator responses, where only forced inspiratory parameters (FIV₁) were important. The stronger response to hypertonic as compared to isotonic saline could be mediated, at least partially, through activation of mast cells.