Abstract
Rationale: Bronchiolitis obliterans syndrome (BOS) remains the leading cause of death after lung transplantation. Treatment is difficult, although azithromycin has recently been shown to improve FEV1. The exact mechanism of action is unclear.
Hypotheses: (1) Azithromycin reduces airway neutrophilia and interleukin (IL)-8 and (2) airway neutrophilia predicts the improvement in FEV1.
Methods: Fourteen lung transplant patients with BOS (between BOS 0-p and BOS 3) were treated with azithromycin, in addition to their current immunosuppressive treatment. Before and 3 mo after azithromycin was introduced, bronchoscopy with bronchoalveolar lavage (BAL) was performed for cell differentiation and to measure IL-8 and IL-17 mRNA ratios.
Results: The FEV1 increased from 2.36 (± 0.82 L) to 2.67 L (± 0.85 L; p = 0.007), whereas the percentage of BAL neutrophilia decreased from 35.1 (± 35.7%) to 5.7% (± 6.5%; p = 0.0024). There were six responders to azithromycin (with an FEV1 increase of > 10%) and eight nonresponders. Using categorical univariate linear regression analysis, the main significant differences in characteristics between responders and nonresponders were the initial BAL neutrophilia (p < 0.0001), IL-8 mRNA ratio (p = 0.0009), and the postoperative day at which azithromycin was started (p = 0.036). There was a significant correlation between the initial percentage of BAL neutrophilia and the changes in FEV1 after 3 mo (r = 0.79, p = 0.0019).
Conclusion: Azithromycin significantly reduces airway neutrophilia and IL-8 mRNA in patients with BOS. Responders have a significantly higher BAL neutrophilia and IL-8 compared with nonresponders and had commenced treatment earlier after transplantation. BAL neutrophilia can be used as a predictor for the FEV1 response to azithromycin.
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 FEV1, 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 FEV1 (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 FEV1 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 FEV1 response.
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.
Age, yr | 47.7 ± 12.5 |
| M/F | 9/5 |
| Type of transplantation | 10 SS/3 S/1 HL |
| Underlying disease | 6 E/4 PF/3 CF/1 PAH |
| FK506/CsA | 13/1 |
| MMF/AZA | 5/9 |
| FEV1, L | 2.36 ± 0.82 |
| BOS stage | 3 stage 0-p, 8 stage 1, 2 stage 2, 1 stage 3 |
| Postoperative day | 936 ± 878 (range, 164 – 2,904) |
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 105 cells/ml in a Shandon cytocentrifuge (Techgen, Zellik, Belgium). The cytospins were colored with May-Grünwald-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 β-actin mRNA (14).
FEV1 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 FEV1 according to American Thoracic Society criteria (15).
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.
The mean FEV1 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) × 106/ml to 0.0046 (± 0.0064) × 106/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] × 103 to 3.5 [± 11.3] × 103; 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).
Figure 1. (A) Evolution of FEV1 from the best postoperative value obtained (BEST), to 3 mo before the initiation of azithromycin (−3m), at the start of azithromycin (AZI start), and after 3 mo of treatment (+3m). There is a significant increase of the FEV1 after 3 mo of therapy with azithromycin (p = 0.007; n = 14), which is mainly due to the increase in FEV1 in the responders group (p = 0.03; n = 6). (B) There is a significant decrease in bronchoalveolar lavage (BAL) neutrophilia 3 mo after the addition of azithromycin (p = 0.0024; n = 14), also mainly due to the effect in the responders group (p = 0.03; n = 6). (C) There is also a significant decrease in interleukin (IL)-8/β-actin ratio in BAL after 3 mo of treatment with azithromycin (p = 0.04; n = 14). Solid circles = responders; solid squares = nonresponders.
[More] [Minimize]Of the 14 included patients, six had an FEV1 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 FEV1 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] × 103 to 7.9 [± 17.5] × 103, 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 FEV1, 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 R2 = 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.
Responders | Nonresponders | R2 Value | p Value | |
|---|---|---|---|---|
| Age, yr | 48.8 ± 15.0 | 46.3 ± 12.5 | 0.004 | 0.83 |
| Sex | 4 M/2 F | 5 M/3 F | 0.002 | 0.88 |
| POD | 438 ± 493 | 1,309 ± 942 | 0.26 | 0.036 |
| FK506/CsA | 5/1 | 8/0 | 0.10 | 0.26 |
| Type of transplant | 4 SS/2S | 6 SS/1 S/1 HL | 0.008 | 0.76 |
| Diagnosis | 2 E/3 PF/1 CF | 4 E/1 PF/2 CF/1 PAH | 0.034 | 0.53 |
| FEV1 at inclusion, L | 2.20 ± 0.56 | 2.5 ± 1.03 | 0.036 | 0.52 |
| BOS stage, n | 0-p (2),1 (2), 2 (2) | 0-p (1), 1 (6), 3 (1) | 0.006 | 0.9 |
| BAL neutrophilia, % | 69.6 ± 24.3 | 9.2 ± 12.4 | 0.74 | < 0.0001 |
| BAL neutrophils, ×106/ml | 1.394 ± 0.005 | 0.008 ± 0.009 | 0.84 | < 0.0001 |
| IL-8mRNA ratio, ×103 | 44.9 ± 69.9 | 1.2 ± 2.6 | 0.69 | 0.0009 |
| IL-17mRNA ratio, ×103 | 3.51 ± 6.28 | 0.57 ± 1.17 | 0.13 | 0.35 |
| Pseudomonas in BAL | 2 | 2 | 0.01 | 0.73 |
A BAL neutrophilia of more than 15% had a positive predictive value of 85% for a significant FEV1 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 FEV1 after 3 mo of treatment with azithromycin (r = 0.74, p = 0.0026; Figure 2).
Figure 2. Spearman rank correlation (with 95% confidence intervals calculated by univariate regression model) between initial BAL neutrophilia (in %) and changes in FEV1 (in % from baseline) after 3 mo of treatment with azithromycin in the whole group (r = 0.74, p = 0.0026).
[More] [Minimize]In the present study, we have confirmed that azithromycin significantly improves the FEV1 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 FEV1.
Azithromycin represents the only medical therapy that improves the FEV1 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 FEV1 by a mean of 17% (6). In our previous study, the mean increase in FEV1 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 FEV1 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 FEV1 did not improve (9). In the present study, the mean improvement in FEV1 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 FEV1, which was evidenced by the significant correlation between the initial BAL neutrophilia and the changes in FEV1. 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 nonresponders, 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 FEV1 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).
The authors thank Tim Nawrot for his statistical advice.
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