CLINICAL LUNG AND HEART/LUNG TRANSPLANTATION
Prognostic Value of Bronchoalveolar Lavage Neutrophilia in Stable Lung Transplant Recipients Claus Neurohr, MD,a Patrick Huppmann, MD,a Benedikt Samweber,a Stefan Leuschner,a Gregor Zimmermann, MD,a Hanno Leuchte, MD,a Rainer Baumgartner, MD,a Rudolf Hatz, MD,b Ludwig Frey, MD,c Peter Ueberfuhr, MD,d Iris Bittmann, MD,e and Juergen Behr, MD,a for the Munich Lung Transplant Group Background: Bronchoalveolar lavage (BAL) neutrophilia may identify patients prone to develop bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTx). This study assessed the predictive value of BAL neutrophilia in stable recipients. Methods: Evaluated were 63 consecutive recipients 3 to 12 months after LTx demonstrating no acute rejection (AR) and lymphocytic bronchitis (LB; B ⱕ 1 without infection; BOS, 0). Recipients were subdivided into never-BOS (follow-up ⱖ 12 months) and ever-BOS groups (i.e., BOS development ⱖ 1 after bronchoscopy). Results: The groups were statistically indistinguishable for demographic data and preceding AR and LB episodes. Onset of BOS was at a median of 232 days (range, 87–962) after bronchoscopy. The ever-BOS group (16 patients) demonstrated a significantly higher percentage of neutrophils compared with the never-BOS group (47 patients) at the time of bronchoscopy (33.6% ⫾ 2.1% vs 9.9% ⫾ 1.1%, p ⬍ 0.05). By Cox regression analysis, a BAL neutrophil percentage of ⱖ 20% remained a significant predictor for BOS ⱖ 1 (hazard ratio, 3.57; 95% confidence interval, 1.71– 8.40, p ⬍ 0.05) distinct from known potential BOS predictor variables. The positive and negative predictive value of BAL neutrophilia of ⱖ 20% for future BOS was 0.72 and 0.93, respectively (p ⬍ 0.05). Conclusion: BAL neutrophilia in stable recipients is of predictive value to identify recipients at risk for BOS. These data warrant prospective confirmation and further studies to evaluate the benefit of preemptive therapy for potential BOS patients. J Heart Lung Transplant 2009;28:468–74. Copyright © 2009 by the International Society for Heart and Lung Transplantation.
Lung transplantation (LTx) is an established therapeutic option for patients with end-stage lung disease.1 However, infections and bronchiolitis obliterans syndrome (BOS) remain the leading causes of limited survival rates.2 Acute rejection (AR) and lymphocytic bronchitis (LB) have clearly emerged as the primary risk factors for BOS.3,4 Transbronchial biopsy (TBB) and bronchoalveolar lavage (BAL) are the standard tools to evaluate recipients with functional deterioration or to detect asymptomatic minimal rejection.5,6 In this respect, BAL From the Departments of aInternal Medicine I, Division of Pulmonary Diseases, bSurgery and Thoracic Surgery, and cAnesthesiology, Klinikum Grosshadern; and dCardiac Surgery, Klinikum Grosshadern, and the eInstitute of Pathology, Ludwig-Maximilians University, Munich, Germany. Submitted October 26, 2008; revised January 9, 2009; accepted January 14, 2009. Reprint requests: Juergen Behr, MD, Department of Internal Medicine I, Division for Pulmonary Diseases, Klinikum Grosshadern, Ludwig-Maximilians University, Munich, Marchioninistrasse 15, 81377 Munich, Germany. Telephone: ⫹49-89-7095-3071. Fax: ⫹49-89-70958877. E-mail:
[email protected] Copyright © 2009 by the International Society for Heart and Lung Transplantation. 1053-2498/09/$–see front matter. doi:10.1016/ j.healun.2009.01.014
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has become the primary method for the assessment of infection and also provides a tool to analyze the cellular population of the lower respiratory tract. Previous studies have shown that granulocyte activation in BAL is a prominent feature not only of infection but also of BOS and allograft rejection.6 –9 BOS is thought to be the result of an excessive repair mechanism after alloimmunologic and non-alloimmunologic injuries directed against epithelial structures.10 It is characterized by an airway inflammatory response with increased interleukin (IL)-8 levels, and an alteration of the BAL cellular profile may actually precede the diagnosis of BOS.8,11,12 Secretory leukocyte protease inhibitor (SLPI) is thought be protective in host defence and autoimmune conditions.13 Reduced levels of SLPI have been shown in the BAL of patients with established BOS.14 Azithromycin, a macrolide antibiotic, demonstrated benefits in patients with established BOS in several small studies. A subgroup analysis identified patients with increased BAL neutrophilia and early-onset of BOS to be more likely to respond to treatment.15–17 The aim of this observational study was to evaluate the associations of BAL neutrophilia with allograft re-
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jection and BOS in a cohort of stable LTx patients. We hypothesized that after exclusion of confounding factors, airway neutrophilia would have predictive value to identify stable recipients who are at risk to develop BOS. METHODS Study Design This study retrospectively evaluated 184 consecutive patients who underwent LTx from January 1996 through December 2007 at the University of Munich. The study was performed in accordance with recommendations of the local board on medical ethics at Ludwig Maximilians University of Munich. Informed written consent was obtained from each patient. Follow-up data were collected through December 1, 2007. Demographic and clinical data were prospectively obtained from medical records and computerized databases. Human leukocyte antigen mismatches were determined in accordance with the European Federation of Immunogenetics.18 Patients received no induction therapy and were maintained on triple immunosuppression with corticosteroids, tacrolimus, and mycophenolate mofetil. In case of recurrent AR, toxicity, or BOS, patients were switched to an alternative immunosuppressive regimen based on a caseby-case decision. Patients received a viral prophylaxis with acyclovir for 3 months. In addition, preemptive therapy with ganciclovir and/or immune globulin was initiated based on positive cytomegalovirus polymerase chain reaction. Bronchoscopic Workup Bronchoscopic workup included assessment of the anastomosis, BAL, and TBB. The details of bronchoscopy have been previously described.19,20 Briefly, a flexible bronchoscope was wedged into a subsegmental bronchus (preferentially middle lobe). Five serial infusions, each comprising 20 ml of sterile saline (0.9% sodium chloride), and aspirations with a 20-second suction period were performed. BAL aliquots were pooled, and 15 ml of BAL were analyzed for evidence of viral, bacterial, and fungal infections. BAL total cell and differential cell counts were assessed on cytospin preparations, and BAL supernatants were then stored at ⫺80° C. Viability was measured using the trypan blue exclusion method. Scheduled bronchoscopy was performed at least twice during the first 3 months and at least once between 6 and 12 months after LTx. Clinically indicated bronchoscopies were also conducted for new respiratory symptoms, and follow-up bronchoscopies were performed to monitor for treatment response.
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Definitions The diagnosis of BOS was established using the International Society of Heart and Lung Transplantation (ISHLT) definition.4 AR was diagnosed using standard histologic criteria according to the Lung Rejection Study Group.21 Any biopsy specimen with AR A ⱖ 2 was considered positive and treated with methylprednisolone at a dose of 500 mg/day for 3 consecutive days. In case of AR A1, the decision to proceed with therapy was based on clinical status. Isolated LB (B grade) was not treated. Patients with an absence of pulmonary infection or merely colonization were defined as asymptomatic with either no secretion, white blood count and C-reactive protein within normal limits, negative Gram stain and negative bacterial, viral, and fungal culture or polymerase chain reaction, or positive microbiologic findings, but no treatment was initiated. Stable LTx Recipients Stable LTx recipients were defined as follows: BOS stage 0 at the time of scheduled bronchoscopy within 3 to 12 months after transplantation and biopsy specimen with exclusion of AR (ie, AR A0 and LB B ⱕ 1) with no signs of pulmonary infection. Stable recipients were further subdivided into 2 categories. The never-BOS group were patients who never developed BOS ⱖ 1 with a subsequent minimum of 12 months of follow-up after bronchoscopy, including patients that moved from BOS stage 0 to BOS stage 0-p but never developed BOS ⱖ 1. The ever-BOS group were patients who had no BOS at the time of initial bronchoscopy but in whom BOS ⱖ 1 was subsequently diagnosed during the time of the study. In case of 2 or more available bronchoscopies fulfilling the aforementioned criteria, the most recent data of each patient were used for analysis. We stipulated to combine no airway inflammation (LB B0) and minimal airway inflammation (LB B1) because of the high incidence of B1 biopsy specimens in our study population.22 IL-8 and Secretory Leukocyte Protease Inhibitor in BAL Secretory leukocyte protease inhibitor (SLPI) protein was measured in BAL with a commercially available enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN). IL-8 protein was assessed in BAL as described previously by ELISA using a monoclonal anti-IL-8 antibody (R&D Systems).17 Statistical Analysis The demographic data and outcomes between groups were compared using the 2-sided chi-square test or the 2-sided Fisher exact test (when the expected cell size was ⬍ 5) for categoric variables and the 2-sided Mann-
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Whitney U-test of independent samples for continuous variables. Data are presented as mean ⫾ standard error of the mean or as individual values and box and whisker plots displaying the median, the 25th and 75th percentiles and the smallest and largest value within 1.5 box lengths from the box. BOS-free survival was calculated using the Kaplan-Meier method, and groups were compared by means of log-rank testing. To evaluate for an association between potential risk factors and the development of BOS ⱖ 1, initial univariate Cox regression analysis was used. Identified variables were included in subsequent multivariate Cox regression models with restriction to 2 covariables due to limited sample size. Receiver operating characteristic (ROC) analysis was done to assess the discriminatory ability of BAL neutrophilia for the development of BOS. Results were considered statistically significant at p ⬍ 0.05. The statistical calculations were done using SPSS 15.0.2 software (SPSS Inc, Chicago, IL). RESULTS Patient Cohorts From 652 TBBs in 184 patients (median, 3 bronchoscopies/patient; range, 1–9) that were assessed, 469 biopsy specimens (72%) showed no AR (A0), 112 (17%) demonstrated minimal AR (A1), and in 71 (11%) showed AR of A ⱖ 2. No evidence of LB (B0) was found in 187 specimens (28%), LB grade B1 was confirmed in 306 (47%), and grade B ⱖ 2 in 163 (25%). Of a total of 184 patients, 63 (34.2%) fulfilled the criteria for stable LTx recipients and were entered in this analysis. Of these, 47 (74.6%) did not develop BOS ⱖ 1 during follow-up and were allocated to the never-BOS group; however, 7 recipients (14.9%) in this group moved on to BOS stage 0-p at a median of 697 days (range, 144 –1053 days) after bronchoscopy. BOS stage ⱖ 1 eventually developed in 16 patients (25.4%) at a median of 232 days (range, 87–962 days) after bronchoscopy, and they were allocated to the ever-BOS group. At the end of this study, BOS stage 1 was present in 7 patients (44%), BOS stage 2 in 5 (31%), and BOS stage 3 in 4 (25%). The demographic and clinical information were statistically indistinguishable (Tables 1 and 2). The frequency of preceding bronchoscopies (never-BOS group, 3.09 ⫾ 0.13; ever-BOS group, 3.44 ⫾ 0.30, p ⫽ 0.12) and preceding episodes of AR and LB demonstrated no significant difference. Moreover, there was no significant difference in the length of the interval between LTx and date of bronchoscopy. Regular use of proton pump inhibitors (PPI) was similar between the groups: 41 (87.2%) in the never-BOS group and 14 (87.5%) in the ever-BOS group (p ⫽ 0.67).
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Table 1. Basic Characteristics of Never-BOS and Ever-BOS Patients Group Never-BOS Ever-BOS p-value Total patients 47 16 Total follow-up, years ⫾ SEM 3.71 ⫾ 0.35 3.21 ⫾ 0.43 0.21 Baseline FEV1, % predicted ⫾ SEM 80.16 ⫾ 3.63 78.38 ⫾ 4.56 0.36 Female, No. (%) 24 (51) 7 (44) 0.67 Age, years ⫾ SEM 47.6 ⫾ 2.0 48.9 ⫾ 2.6 0.86 Pre-LTx diagnosis, n (%) COPD 16 (34) 5 (31) 0.19 CF/bronchiectasis 9 (19) 3 (18) IPF 12 (26) 4 (25) ␣1-Antitrypsin deficiency 3 (6) 2 (13) Other 7 (15) 2 (13) Ischemic time, min ⫾ SEM 362.8 ⫾ 16.9 336.3 ⫾ 17.9 0.38 CMV mismatch, No. (%) 9 (19) 2 (12) 0.16 Type of LTx, No. (%) Single LTx 23 (49) 8 (50) 0.72 Bilateral LTx 24 (51) 8 (50) HLA mismatches, No. (%) 0–2 5 (11) 2 (12) 3–4 22 (47) 6 (38) 0.26 5–6 20 (42) 8 (50) BOS, bronchiolitis obliterans syndrome; CF, cystic fibrosis; CMV mismatch, cytomegalovirus donor⫹/recipient⫺; COPD, chronic obstructive pulmonary disease; FEV1, forced expiratory volume in 1 second; HLA, human leukocyte antigen; IPF, idiopathic pulmonary fibrosis; LTx, lung transplantation; NS, not significant; SEM, standard error of the mean.
BAL Cellular Profile in Stable LTx Recipients Cell viability was not different between the groups. BAL fluid recovery expressed as volume of the 100 ml instilled showed a significant reduction in the ever-BOS group. BAL cellular profile analysis demonstrated a significant increased percentage of neutrophils and total cell count in the ever-BOS group at the initial bronchoscopy (Table 2, Figure 1). The percentages of macrophages were significantly reduced in the everBOS group by reason for compensating for the differences in neutrophils. Percentages of lymphocytes were similar between groups (Table 2). The subset of patients in the never-BOS group that developed BOS 0-p did not have an increased percentage of neutrophils (BOS 0, 9.30% ⫾ 1.31% vs BOS 0-p, 7.86% ⫾ 2.01%; p ⫽ 0.21). Airway colonization had no significant effect on BAL neutrophilia (never-BOS group: 13 (28%); ever-BOS group: 4 (25%), p ⫽ 0.49). There was no statistical difference in the number of B1-graded biopsy specimens, with 27 (57%) in the never-BOS group and 9 (56%) in the ever-BOS group (p ⫽ NS). However, ever-BOS patients demonstrated a significantly increased risk for subsequent AR and LB episodes detected with a significantly higher number of following bronchoscopies (never-BOS group, 1.85 ⫾ 0.15; ever-BOS group, 4.69 ⫾ 0.68; p ⫽ 0.01; Table 2).
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The first AR episode (A ⱖ 1) occurred at a median of 151 days (range, 65–323 days) in the ever-BOS group compared with 419 days (range, 192– 664 days) in the never-BOS group (p ⫽ 0.074). Further analysis revealed a strong trend towards an increased risk of death in the ever-BOS group, at 7 (43.7%) compared with 9 (19.1%) the never-BOS group (p ⫽ 0.056), with 5 BOS-related and 2 infection-related deaths in the ever-BOS-group at a median of 579 days (range, 139 –935 days) after the initial bronchoscopy. IL-8 and SLPI BAL Protein Levels in Stable LTx Recipients IL-8 protein levels were significantly elevated and SLPI protein levels significantly reduced in the ever-BOS group (Table 2, Figure 2). IL-8 concentration was positively correlated (r ⫽ 0.53, p ⫽ 0.01), whereas SLPI levels were negatively correlated with the relative number of neutrophils (r ⫽ ⫺0.69, p ⫽ 0.01). Moreover, there was a trend for a negative correlation between IL-8 and SLPI levels without the differences reaching statistical significance (r ⫽ ⫺0.42, p ⫽ 0.064). Risk Factors for BOS and Predictive Value of BAL Neutrophilia Cox regression analysis was performed to detect associations between potential predictor variables and BOS
Table 2. Bronchoalveolar Lavage Cellular Profile and Rejection Never-BOS and Ever-BOS Patients Variablea BAL: Days post-LTx Follow-up post-BAL, days Episodes/patient pre-BAL, No. AR A ⱖ 1 AR A ⱖ 2 LB B ⱖ 2 Epsiodes/patient post-BAL, No. AR A ⱖ 1 AR A ⱖ 2 LB B ⱖ 2 BAL: Recovery, ml Total cells, ⫻106/ml Viability, % Macrophages, % Lymphocytes, % Neutrophils, % SLPI, ng/ml IL-8, pg/ml
Never-BOS (n ⫽ 47) 283 ⫾ 51 924 ⫾ 129
Ever-BOS (n ⫽ 16) 240 ⫾ 18 793 ⫾ 87
p-value 0.31 0.09
0.69 ⫾ 0.08 0.23 ⫾ 0.09 1.51 ⫾ 0.18
0.49 ⫾ 1.2 0.10 ⫾ 0.06 1.28 ⫾ 0.14
0.11 0.14 0.43
0.3 ⫾ 0.06b 0.2 ⫾ 0.02b 0.73 ⫾ 0.10b 43.87 ⫾ 2.01b 12.66 ⫾ 2.35b 71.6 ⫾ 3.1 85.28 ⫾ 2.22b 8.93 ⫾ 2.28 9.9 ⫾ 1.1b 718.1 ⫾ 142.2b 237.7 ⫾ 51.2b
1.24 ⫾ 0.04b 0.89 ⫾ 0.12b 1.71 ⫾ 0.10b 35.69 ⫾ 2.66b 22.83 ⫾ 2.47b 68.0 ⫾ 3.6 46.06 ⫾ 6.86b 5.13 ⫾ 1.72 33.6 ⫾ 2.1b 164.6 ⫾ 47.1b 704.1 ⫾ 177.2b
0.02b 0.03b 0.01b 0.01b 0.034b 0.57 0.01b NS 0.01b 0.01b 0.01b
AR, acute rejection; BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; IL-8, Interleukin-8; LB, lymphocytic bronchitis; LTx, lung transplantation; SLPI, secretory leukoprotease inhibitor. a Continuous data are presented with the standard error of the mean. b Statistically significant.
Figure 1. Box and whisker plots show the comparison of bronchoalveolar lavage (BAL) neutrophils percentages in 47 stable recipients at the time of bronchoscopy who did not developed bronchiolitis obliterans syndrome (BOS; never-BOS) and 16 recipients who subsequently were diagnosed with BOS ⱖ 1 (ever-BOS). *p ⬍ 0.05. The horizontal line displays the median, the box edges show the 25th and 75th percentiles, and the whiskers show the smallest and largest value within 1.5 box lengths from the box.
stage ⱖ 1. Univariate analysis revealed that a neutrophil percentage of ⱖ 20% in stable LTx recipients and the number of subsequent AR episodes (A ⱖ 1) were significantly associated with BOS stage ⱖ 1 (Table 3). Figure 3 depicts the significant association of BAL neutrophilia with reduced BOS-free survival after LTx. To determine if BAL neutrophilia is a BOS risk factor separate from subsequent AR episodes, multivariate Cox regression analysis was used. It demonstrated that a neutrophil percentage of ⱖ 20% remained a significant predictor for BOS stage ⱖ 1, with a hazard ratio (HR) of 3.57 (95% confidence interval [CI], 1.71– 8.40; p ⬍ 0.05). To determine the ability of BAL neutrophilia to predict the development of BOS, we performed a ROC
Figure 2. Box and whisker plots show the comparison of (A) interleukin (IL)-8 and (B) secretory leukoprotease inhibitor (SLPI) bronchoalveolar lavage protein levels between 47 stable recipients at the time of bronchoscopy who did not develop bronchiolitis obliterans syndrome (never-BOS) and 16 who were diagnosed with BOS ⱖ 1 (ever-BOS) during this study. *p ⬍ 0.05. The horizontal line displays the median, the box edges show the 25th and 75th percentiles, and the whiskers show the smallest and largest value within 1.5 box lengths from the box.
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Table 3. Univariate Cox Regression for Bronchiolitis Obliterans Syndrome Stage ⱖ 1 and Risk Factors BOS ⱖ stage 1 Variable Age ⬍ 50 years COPD CF/bronchiectasis IPF Other Transplant type Single Bilateral HLA mismatches 3–4 5–6 CMV mismatch Preceding episodes, No. AR ⱖ 1 LB ⱖ 2 BAL neutrophilia ⱖ 20% Subsequent episodes, No. AR ⱖ 1 LB ⱖ 2
HR (95% CI) 0.93 (0.35–2.50) 1.81 (0.45–7.31) 3.22 (0.86–9.55) 1.01 (0.05–2.27) 1.07 (0.93–1.22)
p-value 0.88 0.41 0.35 0.33 0.35
1.00 0.88 (0.33–2.34)
0.79
1.00 1.45 (0.88–2.40) 0.61 (0.13–2.74)
0.15 0.52
1.65 (0.61–4.47) 0.44 (0.12–1.67) 4.40 (2.82–10.04)a
0.32 0.27 0.001a
2.17 (1.18–3.98)a 1.43 (0.87–2.36)a
0.012a 0.16
AR, acute rejection; BAL, bronchoalveolar lavage; BOS, bronchiolitis obliterans syndrome; CF, cystic fibrosis; CI, confidence interval; CMV mismatch, cytomegalovirus donor⫹/recipient⫺; COPD, chronic obstructive pulmonary disease; HLA, human leukocyte antigen; HR, hazard ratio; IPF, idiopathic pulmonary fibrosis; LB, lymphocytic bronchitis. a Statistically significant.
analysis. By applying a 20% neutrophilia cutoff value, we calculated a sensitivity of 0.81 and a specificity of 0.89 (area under the curve [AUC], 94.1; p ⬍ 0.001; Figure 4). The positive and negative predictive value was 0.72 and 0.93, respectively.
Figure 3. Bronchoalveolar lavage (BAL) neutrophilia and bronchiolitis obliterans syndrome (BOS)-free survival after lung transplantation stratified by the presence (dashed line, n ⫽ 18) or absence (solid line, n ⫽ 45) of BAL neutrophilia ⬎ 20%.
Figure 4. Receiver operating characteristic analysis shows the ability for bronchoalveolar lavage (BAL) neutrophilia to predict development of bronchiolitis obliterans syndrome (BOS) ⱖ 1 in stable lung transplantation recipients.
DISCUSSION Our results demonstrated that in stable LTx recipients, an increased percentage of BAL neutrophils in the absence of confounders constitutes an increased risk for AR and has a predictive value for BOS. Moreover, IL-8 BAL levels were positively and SLPI BAL levels were negatively correlated with the relative number of neutrophils, indicating a potentially important role in the pathogenesis of BOS. Several studies have investigated the usefulness of BAL analysis in providing information related to acute and chronic transplant dysfunction. Previous findings indicate that infections and BOS result in significantly increased percentages of neutrophils.23 Although not consistently reported, BAL neutrophilia are a common finding in acute rejection.8,24 An explanation for discrepant results may be the relatively new insight that peri-vascular AR and LB are both manifestations of AR and need to be assessed separately.25 Moreover, Tikkanen et al26 observed only increased percentages of lymphocytes in early AR episodes, whereas BAL neutrophilia was found in intermediate and late episodes (⬎30 days after LTx). Nevertheless, a subset of recipients seems to be more prone to an enhanced inflammatory response that ultimately results in BOS.10,27 In accordance with this hypothesis, Schloma et al28 demonstrated that BAL neutrophilia and elevated IL-8 levels in the absence of AR and infection can identify patients at risk for developing obliterative bronchitis within the first year after LTx. These findings were confirmed by Reynaud-Gaubert et al29 using BOS as the outcome parameter, sug-
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gesting a threshold of 24% for BAL neutrophilia to discriminate for future BOS. However, differences in the number of preceding AR and LB episodes were not part of their analysis.29 In contrast, Zheng et al30 did not detect any significant differences in BAL neutrophil percentages between never-BOS and ever-BOS in 28 stable LTx patients. Slebos et al31 proposed that these discrepant findings are explained by differences in immunosuppressive regimen, timing of the BAL, and laboratory protocols for BAL analysis. In addition, we speculate that various definitions of airway colonization and a lack of consideration of LB or minimal AR are important. For patients with a “good outcome” for a minimum of 2 years after LTx, Slebos et al31 reported neutrophil percentages within a range of 0% to 16%. The present report confirms and extends these findings, in that we found significantly higher percentages of neutrophils and IL-8 levels in stable LTx recipients with future BOS in the absence of confounding factors regardless of the number of preceding rejection episodes including LB. For IL-17, IL-13, IL-8, and the CD4:CD8 ratio, specific BAL changes related to acute rejection and BOS have been revealed.16,20,32,33 SLPI is constitutively expressed at many mucosal surfaces and has been shown to possess multifunctional activities, including anti-microbial, anti-oxidative, and immunomodulatory properties. Because it is available in a recombinant aerosol form, SLPI was proposed to be an attractive therapeutic candidate in chronic lung disease.13,34 Recently, Anderson et al35 confirmed a protease/antiprotease imbalance in BOS with excess free neutrophil elastase present that is not counteracted by SLPI. Our study, however, observed a reduction in SLPI in currently stable patients with future BOS. We clearly recognize the inherent limitations of our report with respect to our observational design, limited sample size, and a surveillance protocol with the risk of selection bias beyond 12 months after LTx. Nevertheless, we conclude that in stable recipients, a BAL neutrophil percentage of ⬎ 20% in the absence of confounding factors is of significant predictive value for an increased frequency of future AR episodes and the onset of BOS. Moreover, our data suggest that further evaluation of initially stable recipients may provide important clues about genuine factors contributing to the epithelial damage and the fibroproliferative response in BOS. Interestingly, BAL neutrophilia has been shown to correlate with exhaled nitric oxide (eNO) levels regardless of BOS status, and induced sputum neutrophilia allows discrimination of BOS from non-BOS recipients.36,37 This suggests that serial assessment of these markers may be helpful to non-invasively monitor for
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the development of overt airway inflammation and allow an earlier diagnosis of BOS.38 In accordance with the concept of dichotomy in BOS recently suggested by Vanudenaerde et al,39 some LTx recipients with future or established BOS did not have increased airway neutrophilia. However, it may be that in most recipients, AR, LB, and obliterative bronchitis only represent a highly variable continuum of inflammatory events triggered by multiple pathogenic factors resulting in epithelial injury. Preliminary data suggest that azithromycin therapy offers a safe option for the treatment of BOS patients with BAL neutrophilia regardless of BOS stage. From our findings we speculate that in the currently conducted trials with azithromycin as a preemptive strategy in non-BOS recipients, this therapy will be effective in preventing the development of BOS only in stable patients with accompanying airway inflammation. The authors thank A. Crispin, MD, Institut fuer Medizinische Informations-verarbeitung, Biometrie und Epidemiologie for his continuing support and his excellent statistical assistance.
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