Azithromycin Reduces Airway Neutrophilia and Interleukin-8 in Patients with Bronchiolitis Obliterans Syndrome

Lung transplantation has become an established treatment option for patients with different end-stage pulmonary diseases. The survival rates are still lower than in other organ transplantations (1), mainly due to the occurrence of chronic allograft dysfunction, pathologically denominated as obliterative bronchiolitis and clinically as bronchiolitis obliterans syndrome (BOS) (2). BOS leads to a progressive decline in FEV^sub 1^, as the result of an inflammatory response to the epithelium and an excessive repair mechanism (3, 4). Although its treatment has been disappointing (5), patients with BOS who were treated with azithromycin have recently shown an increase in FEV^sub 1^ (6). This has been corroborated by two further studies (7, 8); however, the most recently published study showed no benefit (9). Taken together, about half of the reported patients had a significant FEV^sub 1^ response to azithromycin.

Because BOS is characterized by increased airway neutrophilia and interleukin (IL)-8 (10), and because neomacrolides have been reported to decrease IL-8 production (11) and to reduce neutrophil recruitment into the airways of mice (12) as well as in humans (13), we hypothesized that azithromycin reduces airway neutrophilia and IL-8 and that airway neutrophilia is indeed essential to have a positive FEV^sub 1^ response.

METHODS

Fourteen lung transplant patients were included in this prospective 3-mo study. Azithromycin was added to their current immunosuppressive drug regimen consisting of tacrolimus/cyclosporin A, azathioprine/ mycophenolate mofetil, and corticosteroids, and was started at 250 mg/d for 5 d, followed by 250 mg 3 times/wk for 3 mo. There was no significant change in immunosuppressive medication during the 3-mo treatment period with azithromycin. None of the patients had any signs of infection at the time of inclusion in the study (no fever, no radiologic changes), although some were colonized with Pseudomonas.

Their characteristics at entry are summarized in Table 1. BOS was diagnosed according to established criteria (2). All patients gave written, informed consent. The study was approved by the local hospital's ethics committee.

Before starting and 3 mo after azithromycin had been added, all patients underwent bronchoscopy with bronchoalveolar lavage (BAL). Bronchoscopy was performed by only two investigators (G.M.V. or L.J.D.). BAL was performed by wedging the scope in a subsegmental branch of the right middle lobe or the lingula, after which 2 × 50 ml of saline at room temperature was instilled. After each 50-ml instillation, the fluid was returned by gentle suction. The two fractions were pooled and were put on ice before processing. Part of the returned fluid was cultured; the other part was transported to the laboratory for total cell count, cell differentiation, and cytokine analysis.

A cytospin was performed with 10^sup 5^ cells/ml in a Shandon cytocentrifuge (Techgen, Zellik, Belgium). The cytospins were colored with MayGrünwald-Giemsa stain. Differential cell counts were determined by counting at least 300 cells. Another fraction was immediately centrifuged at 500 g for 10 min at 4°C. The cell pellet was lysed and used for quantitative polymerase chain reaction (PCR). Classical methodology was used for extraction of total cellular RNA and reverse transcriptase-PCR for IL-17 and IL-8 as already described in previous studies from our group (14). All mRNA results were calculated as ratios of cytokine mRNA over ß-actin mRNA (14).

FEV^sub 1^ was measured with the Masterscreen spirometer (Jaeger, Hoechberg, Germany). The best of three attempts, with a variability of less than 10%, was retained for analysis, and results were expressed in absolute values of FEV^sub 1^ according to American Thoracic Society criteria (15).

Statistical Analysis

Where appropriate, results are given as mean ± SD. To test significance between the pre- and the postdata in the whole patient group, the Wilcoxon signed rank test was used. Categorical linear univariate regression analysis was used to test significant differences between responders and nonresponders. Correlations were calculated with the Spearman's rank test. A p value of less than 0.05 was considered significant.

RESULTS

The mean FEV^sub 1^ for the whole patient group increased by 13%, from 2.36 (± 0.82 L) to 2.67 L (± 0.85 L; p = 0.007). The absolute number of BAL neutrophils decreased from 0.602 (± 1.069) × 10^sup 6^/ml to 0.0046 (± 0.0064) × 10^sup 6^/ml (p = 0.0041), and the BAL neutrophilia from 35.1 (± 35.7%) to 5.7% (± 6.5%; p = 0.0024; Figures 1A and 1B). There was a significant decrease of the IL-8 mRNA ratio (from 19.4 [± 47.9] × 10^sup 3^ to 3.5 [± 11.3] × 10^sup 3^; p = 0.042) but not of IL-17 mRNA ratio (p = 0.13; Figure 1C). The mean tacrolimus trough level before initiation of azithromycin was 11.2 ± 4.5 and 10.1 ± 2.8 ng/ml 3 mo later (p = 0.62) and the oral dose of methylprednisolone was 10 ± 10 and 6 ± 3 mg, respectively (p = 0.12).

Of the 14 included patients, six had an FEV^sub 1^ increase of more than 10% (group R), whereas eight patients were nonresponders (group NR). In the R group, the percentage of BAL neutrophilia significantly decreased from 69.6 (± 24.6%) to 5.3% (± 7.7%; p = 0.03), whereas the FEV^sub 1^ increased from 2.20 (± 0.54 L) to 2.93 L (± 0.62 L; p = 0.03). Their BAL IL-8 mRNA and IL-17 mRNA ratios tended to decrease (from 44.9 [± 69.9] × 10^sup 3^ to 7.9 [± 17.5] × 10^sup 3^, p = 0.06, and from 5.41 [± 8.15] to 0.26 [± 0.32], p = 0.06, respectively). In the NR group, there were no significant changes in FEV^sub 1^, percentage of BAL neutrophilia, or IL-8 mRNA and IL-17 mRNA ratios (data not shown).

Categorical univariate linear regression analysis was used to detect significant differences between the R and the NR group. All tested variables are shown in Table 2. The percentage of BAL neutrophilia, the total number of BAL neutrophils, IL-8 mRNA ratio, and the postoperative day at inclusion were the only significant differences between the R and NR group (Table 2). The percentage of BAL neutrophilia and the total number of BAL neutrophils explained, respectively, 74 and 84% of the variance (partial R^sup 2^ = 0.74 and 0.84). Multivariate analysis could not be performed due to the strong correlation of some factors (e.g., IL-8 mRNA ratio and BAL neutrophilia) and the limited number of patients.

A BAL neutrophilia of more than 15% had a positive predictive value of 85% for a significant FEV^sub 1^ response to azithromycin, whereas a BAL neutrophilia of less than 15% had a negative predictive value of 100%. We found a significant correlation between the initial percentage of BAL neutrophilia and the changes in FEV^sub 1^ after 3 mo of treatment with azithromycin (r = 0.74, p = 0.0026; Figure 2).

DISCUSSION

In the present study, we have confirmed that azithromycin significantly improves the FEV^sub 1^ in patients with BOS; however, this improvement occurred in only 6 of 14 patients (43%). We also demonstrated that azithromycin significantly reduced the IL-8 mRNA ratio and the percentage of BAL neutrophilia, which correlated with the increase in the FEV^sub 1^.

Azithromycin represents the only medical therapy that improves the FEV^sub 1^ in patients with BOS, except perhaps for fundoplication (16), although the four studies published to date show variable results. In the initial study, five of the six treated patients improved their FEV^sub 1^ by a mean of 17% (6). In our previous study, the mean increase in FEV^sub 1^ was 18%, although in four of eight patients there was no improvement at all (7). In the study by Yates and colleagues, the mean increase in FEV^sub 1^ was 14%, with again only 10 of 20 patients responding (8). Finally, in the Shitrit and coworkers study, 11 patients were included in whom the FEV^sub 1^ did not improve (9). In the present study, the mean improvement in FEV^sub 1^ was 13%, which is in line with the other published data.

Until now, the mechanisms of action of azithromycin have been speculative, and it is unknown why only 40 to 45% of patients seem to benefit from azithromycin. An interaction between azithromycin and tacrolimus or cyclosporine, leading to an increased trough level of these agents and resulting in improved immunosuppression, has been suggested as one explanation. Although this has recently been described (17), we found no significant change in tacrolimus levels after 3 mo of treatment with azithromycin, as was also found in previous studies (6, 7).

Because macrolides are recognized as motiline agonists (18), they might improve gastroesophageal reflux. In the present study, we do not have data on the presence of gastroesophageal reflux in our patients, and therefore this potential mechanism needs further investigation.

The antiinflammatory effects of azithromycin might become very important, because our study suggests that neutrophils may be the prerequisite for azithromycin to have a positive effect on the FEV^sub 1^, which was evidenced by the significant correlation between the initial BAL neutrophilia and the changes in FEV^sub 1^. The significant difference in the postoperative day when azithromycin was started between the R and NR group suggests that earlier presentation of BOS (with neutrophilia) predicts a positive response to azithromycin.

Chronic colonization, for instance with Pseudomonas species, might be responsible for the neutrophilia and it has recently been shown that azithromycin blocks neutrophil recruitment in Pseudomonas endobronchial infection in mice (12). Our present study could not corroborate this hypothesis, because there was no difference in colonization rate between responders and non-responders, although colonization might not always be picked up by BAL culture, since even in stable, noninfected lung transplant patients there is an increase in quorum sensing signals (19), which may be inhibited by azithromycin (20). On the other hand, in the Shitrit and colleagues' study, 9 of 11 patients were colonized with Pseudomonas, but none responded to azithromycin (9). Persistent infection with Chlamydia pneumoniae can also contribute to BOS (21); however, none of our patients had a positive BAL PCR for Chlamydia pneumoniae.

One could also speculate on the activation status of the BAL neutrophils, although there is clear evidence from the literature that neutrophils in BOS are indeed activated because there is significant oxidative stress within the airways of these patients (22). Moreover, a correlation has been found between BAL neutrophilia and myeloperoxidase, which is regarded as a marker of activation of neutrophils (23). A positive correlation also has been found between BAL neutrophilia and BAL matrix metalloproteinase (MMP)-9, again pointing to the active role neutrophils may play in the pathogenesis of BOS (24).

BAL IL-8 mRNA ratio also significantly decreased, which is in line with previous findings in other patient populations (11, 13). On the other hand, the IL-17 mRNA ratio only showed a trend toward a decrease (as was also the case with IL-8 mRNA ratio) in the R group. IL-17 is regarded as an indirectly acting chemokine for neutrophil attraction (25), especially into the airways (26), via induction of IL-8 production in epithelial cells (27) and airway smooth muscle cells (28). We have recently shown that IL-17 is indeed up-regulated in BAL during acute (14) and chronic rejection after lung transplantation (29), and that azithromycin concentration-dependently inhibited the IL-17-induced IL-8 production from human airway smooth muscle cells in vitro, which provides further evidence for its antiinflammatory and antineutrophilic effect (30).

BOS is characterized by a neutrophilic airway inflammation (10). Although our present data support this finding, they also show that some patients with BOS do not have increased neutrophilia in the airways. This has already been demonstrated by Devouassoux and colleagues (31) who described low neutrophilia in patients who developed BOS more than 1 yr after lung transplantation. This is again in line with our present results, demonstrating that a later onset of BOS (weakly) correlated with no response to azithromycin. In the Shitrit and colleagues study, in which no patient responded to azithromycin, the mean time from transplantation to initiation of azithromycin was 33 mo, which is comparable to our own study (mean postoperative day at initiation of azithromycin was 936 d); however, they did not report on the airway neutrophilia in their patient population (9).

In conclusion, we have shown that azithromycin significantly reduces BAL neutrophilia and IL-8 mRNA ratio in patients with BOS after lung transplantation and that BAL neutrophilia is able to predict the FEV^sub 1^ response to azithromycin. This provides further evidence for the antiinflammatory effects of azithromycin in these patients.

Although these data may bring about hope for patients with BOS, it is clear that double-blind, placebo-controlled, randomized multicenter studies are needed to shed further light on the mechanisms of action of azithromycin in the treatment of established BOS, but also to determine whether early treatment with azithromycin is able to prevent BOS (32).

Conflict of Interest Statement: G.M.V. has been invited by several pharmaceutical companies (GlaxoSmithKline [GSK], AstraZeneca, Merck, Sharp & Dohme [MSD] Belgium) to international meetings (European Respiratory Society [ERS], American Thoracic Society [ATS]). He has also served on several local advisory boards for GSK, AstraZeneca, and MSD. He holds the GSK Chair in respiratory pharmacology at the Katholieke Universiteit Leuven, sponsored by GSK, Belgium. V.M.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. L.J.D. has been invited by several pharmaceutical companies (GSK, AstraZeneca, MSD Belgium) to international meetings (ERS, ATS). He has also served on the advisory board for AstraZeneca. D.E.V.R does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Acknowledgment: The authors thank Tim Nawrot for his statistical advice.