American Journal of Respiratory and Critical Care Medicine

We have obtained endobronchial biopsies (EBB), bronchoalveolar lavage (BAL), and transbronchial biopsies (TBB) in 17 stable lung transplant recipients (sLTR), 8 subjects with physiologic evidence of chronic rejection (BOS), and 9 normal subjects. A striking finding was the marked neutrophilia in BAL samples from patients with BOS, in the carefully screened absence of infection. A statistically higher neutrophil count was also present in the sLTR group relative to the normal group. Median BAL neutrophil count in BOS was 100 × 103/ml, range 13–1,661 103/ml (p < 0.001 relative to normal subjects and sLTR). Median BAL neutrophil count in sLTR was 7 × 103/ml, range 1–81 103/ml (p < 0.01 relative to normal subjects). Normal subjects had a median BAL neutrophil count of 3 × 103/ml, range 1–7 103/ml. There was evidence of a predominance of CD8 lymphocytes in BAL from sLTR and BOS with a lower CD4/CD8 ratio in both compared to normal subjects (p < 0.05). EBB mononuclear cell counts, class II major histocompatibility complex expression, and T-cell activation markers were normal in BOS, in contrast to the sLTR group. Our data may be consistent with BOS, representing a relative resolution of an active mononuclear cell chronic inflammation, perhaps at the expense of airway fibrosis. The relevance of the BAL neutrophilia and its role in BOS pathogenesis need further longitudinal investigation.

Lung transplantation is an established and increasingly available therapeutic intervention for end-stage pulmonary and pulmonary vascular disease (1). Following transplantation, chronic rejection in the form of bronchiolitis obliterans syndrome (BOS) (2, 3) is the most common cause of morbidity and mortality in lung transplant survivors beyond 3 mo. The reported prevalence in lung transplant populations is up to 54% (4), with a mortality rate of 50% (5). Despite this high prevalence and its serious outcomes, there is limited knowledge about the immunopathology of BOS, and current understanding of the disease is inadequate (1).

The use of tissue sampling methods using fiberoptic bronchoscopy offers potential insights into the pathogenesis of BOS, since they directly sample the affected organ. Studies to date have centered principally on the techniques of bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB) (6).

BAL, with poor reported sensitivity and specificity, has generally been regarded as of little value in providing specific information relating to rejection, and instead has been mainly used in the detection of infection (6). This may change, however, especially with the advent of more sophisticated analyses of BAL cells (7, 8). In addition, recent work has shown that the emphasis of previous BAL studies on the pathologic role of mononuclear cells in chronic rejection may have lead to a relative neglect of the role of the neutrophil (9), a cell type that has been shown to be important in a number of lung pathologies, including airway (10) and interstitial lung diseases (11).

TBB is, at present, regarded as the standard way of defining acute and chronic lung rejection, with an accepted pathologic grading scheme (6). TBB has some disadvantages, however; large numbers of specimens need to be taken (12), complications occur in up to 10% of patients (13), and deaths can occur (14).

In addition, the pathologic changes occurring during chronic rejection are likely to involve airway changes (2). The yield of useful bronchiolar tissue from TBB is variable, and in a recent study we showed that bronchiolar material was detected in only 78% of surveillance TBB procedures performed according to the current guidelines. Furthermore, the very small samples of bronchiolar tissue may not reflect airway inflammatory changes (15). This means that any comment on airway changes using TBB are censored, since not all of the procedures provide the necessary biological sample.

Given the prognostic importance of BOS and the fact that airway pathology is liable to be vitally important, we have been evaluating the use of endobronchial biopsy (EBB) as an adjunct to existing sampling methods used at surveillance bronchoscopy. The available data relating to EBB findings in lung transplant recipients are scant, emanating from a limited number of transplant centers (15-17). We believe that the data published to date indicate that further study of chronic inflammation and fibrosis in lung transplantation is warranted, and that there may be a possible role for EBB in surveillance bronchoscopies of lung allograft recipients (15, 17, 18), at least as an adjunct to the sampling methods that are currently used, i.e., TBB and BAL.

This study, for the first time, compares the immunopathologic findings of BAL and EBB in a cross-sectional study of clinically stable lung transplant recipients, lung transplant recipients who have a clinical diagnosis of BOS, and normal volunteers. This is a novel extension of our previous studies relating to EBB findings in lung transplant patients (17, 18) and the comparison of airway sampling methods in such patients (15).

BAL, EBB, and TBB were taken from eight allograft recipients with a clinical diagnosis of BOS (3), in which FEV1 was less than 64% of the best post-transplant values. Three BOS subjects were bilateral sequential lung transplant recipients, three were heart–lung recipients, and two were single-lung recipients. The same samples were obtained from 17 clinically well and stable allograft recipients at routine follow-up, 2–36 mo post-transplantation. All of these patients had an FEV1 greater than 90% of the best postoperative value at the time of bronchoscopy. The stable patients included nine bilateral sequential lung transplant recipients, six heart–lung recipients, and two single lung recipients. Stable patients who qualified for inclusion in this study had well-preserved lung function, both at the time of bronchoscopy as well as 3 mo before and after the time of bronchoscopic assessment. The BOS patients who qualified for inclusion had obstructed lung function, in the context of an inexorable deterioration in lung function over at least 6 mo with no improvement following the procedure.

Nonatopic, asymptomatic, nonsmoking volunteers provided a normal range for EBB and BAL parameters in the study. This group had a median age of 22 yr (range 19–37 yr) and were nonasthmatic with a negative methacholine challenge.

TBB were not taken from the normal volunteers due to ethical considerations.

Study Exclusion Criteria

Patients were excluded if they had overt intercurrent lung infection or acute lung rejection or if their status (stable versus BOS) was in doubt. The study was approved by the Alfred Hospital Ethics Committee. All subjects gave informed consent. Donor selection and donor/recipient matching protocols were as previously described (17).

Immunosuppression and Surveillance

Full clinical information and details about immunosuppressive therapy in the transplant patients at the time of investigation are given in Table 1 (stable lung transplant recipients) and Table 2 (patients with BOS).


No.SexAgeOriginal DiseaseDays Post-tx% Max FEV1 Rejection Grade* BAL MicroCsA Level (μg/L)Pred Dose (mg)Aza Dose (mg)
 1F32CF  67 95A0B0Nil36015 75
 2F23CF 102100A0B0Nil25715 25
 3F41PPH 200100A1B2CMV, Sa19515125
 4M36CF  75100A1B2Pa, ASP82020 75
 5F34E 190100A0B0CMV26415 75
 6F35E 419100A0B0Nil21012.5 75
 7F42E 542100A1BXNil134 7.5 50
 8M22CF 186100A1BxCMV432 7.5 50
 9F31CF  58100A1B2CMV71020 50
10M33E1301100A0B0Sp265 7.5100
11M46S 146100A0B2CMV31117.5 25
12F46B 107100A1BXSa30715 50
13F40B 189100A1B0Nil22512.5 50
14M20E 732 99A0B0Nil243 7.5100
15M39CF 547 91A1B0Pa, CMV493 7.5 50
16F50O 376 92A1BXCMV40215 75
17M55O 545 99A1B3Pa, CMV301 7.5100

Definition of abbreviations: Post-tx = post-transplantation; Micro = microbiology; BAL = bronchoalveolar lavage; B = bronchiectasis; CF = cystic fibrosis; O = emphysema; S = sarcoid; E = Eisenmenger's syndrome; PPH = primary pulmonary hypertension; ASP = aspergillus; Sp = Streptococcus pneumoniae; Sa = Staphylococcus aureus; Pa = Pseudomonas aeruginosa; CMV = cytomegalovirus (on viral immunofluorescence); CsA = cyclosporine blood level; Pred = prednisone; Aza = azathioprine.

*A = acute rejection, 0–4; B = bronchiolar inflammation, 0–4; X = ungradable on TBB.


No.SexAgeOriginal DiseaseDays Post-tx% Max FEV1 Rejection Grade* BAL MicroCsA Level (μg/L)Pred Dose (mg)Aza Dose (mg)
1F25PPH 70158A1BXNil297 15100
2M26CF 50855A0BXPa499 15 50
3M23CF1,09564A0B0Pa212 7.5 75
4M24CF1,14452A1B0Nil168 15100
5F47O 80628A1B2CMV238 15 50
6F26E1,00042A1B1Nil208 10 75
7F31PPH 77221A0B1Pa284 15  0
8F54O 82149A1B2Sa 7212.5  0

Definition of abbreviations: Post-tx = post-transplantation; Micro = microbiology; BAL = bronchoalveolar lavage; CF = cystic fibrosis; O = emphysema; E = Eisenmenger's syndrome; PPH = primary pulmonary hypertension; Sa = Staphylococcus aureus; Pa = Pseudomonas aeruginosa; CMV = cytomegalovirus (viral immunofluorescence); CsA = cyclosporine blood level; Pred = prednisone; Aza = azathioprine.

* A = acute rejection; 0–4; B = bronchiolar inflammation, 0–4, X = ungradable on TBB.

Immunosuppressive protocols were similar to those reported by other centers. All patients began triple therapy immediately postoperatively. Maintenance therapy included cyclosporine (to achieve a blood level of 200–350 μg/L; EMIT assay, parent drug only; Syva, CA), azathioprine (1.0–2 mg/kg/d), and prednisone (0.15–0.25 mg/kg/d). Azathioprine doses were also titrated to maintain a minimum white cell count of 5 × 109/L.

Exclusion of Infection

Because of the potential confounding influence of airway infection on the parameters measured, care was taken to minimize this possibility. Only patients clinically free of infection on physical examination, who were afebrile, with no new chest signs, with unchanged leukocyte count, and with unchanged chest radiograph were studied. This assessment was made by an experienced clinician, blinded to the study's cellular end points. The airways on inspection at bronchoscopy had to be free of acute bronchitis. Organisms revealed by BAL culture and immunofluorescence (Tables 1 and 2) had to be considered microbiologically as colonizers, e.g., cytomegalovirus (CMV), or commensals. No patients had CMV cellular inclusions detected in the BAL or biopsies, and gram stains for bacteria were negative in all cases. These clinical and microbiological features would all suggest a very low load of organisms, not consistent with active infection.

Pulmonary Function Testing

FEV1 was measured 1 wk prior to bronchoscopy (SensorMedics 922 rolling seal spirometer; Yorba Linda, CA). These were compared to the recipient's best value post-transplant to ascertain the percentage of maximal FEV1.

Fiberoptic Bronchoscopy with BAL and Biopsies

Bronchoscopy was carried out with intravenous sedation with midozalam (Roche, Basel, Switzerland), with pulse oximetry performed throughout the procedure. The upper and lower airways were anesthetized with topical 1.5% lidocaine. The study used rigorously standardized protocols for specimen sampling and handling. A laboratory histologist attended each bronchoscopic procedure to assess the adequacy of the specimens and to transport all the samples to the laboratory as soon as they were taken. BAL of the middle lobe or lingula segment was carried out after wedging the bronchoscope in a suitable subsegment, with three 60-ml aliquots of phosphate-buffered saline (PBS) warmed to 37° C, instilled via a hand-operated syringe. The fluid was then immediately aspirated into a siliconized container at a negative pressure of approximately −80 mm Hg, and a representative 10-ml aliquot was retained for microbiological testing. The BAL fluid was immediately transported to the laboratory at 4° C for processing and analysis. BAL was followed by EBB, then TBB. All recipients underwent venepuncture for cyclosporin assay immediately before the procedure.

BAL Cell Processing

Total cell counts were performed on the unfiltered BAL fluid using a modified Neubauer hemocytometer. Duplicate cytocentrifuge preparations were made using 200 μl of unfiltered BAL aspirate (Shandon Cytospin III, Runcorn, UK; 82 g, 10 min) and cell counts were performed by counting 500 cells on each slide, with the results meaned. BAL fluid was then filtered through a 200-μm nylon mesh and cells pelleted at 100 g for 15 min for subsequent analysis of lymphocyte subsets by flow cytometry.

Flow Cytometry

BAL cell pellets were resuspended to a cell concentration of 4 × 106/ ml in PBS, and 100-μl aliquots were then incubated with 30 μl of monoclonal antibodies, directly conjugated to either phycoerythrin (PE, red fluorochrome), fluorescein isothiocyanate (FITC, green fluorochrome), or PERCP (orange fluorochrome) as a third color. The antibody combinations used were CD45/CD14 (leukogate, allowing the identification of lymphocytes), CD4/CD8/CD3 (helper/inducer, suppressor/cytotoxic, and total T cells), CD4/CD25/CD3, CD3/CD4/HLADR (CD25 and HLADR on T lymphocytes as markers of activation), and an isotype control (all Becton-Dickinson antibodies, Mountain View, CA). Following staining for 20 min at 4° C, red blood cells were lysed for 10 min, using a commercially available lysing buffer (Ortho Mune; Ortho Diagnostic Systems, Raritan, NJ). Cells were then washed in 2 ml of PBS and centrifuged (15 min, 100 g), and analyzed by a flow cytometer equipped with an argon ion laser (FACScan; Becton-Dickinson). Lymphocytes were gated using the leukogate tube on forward and side light scatter, verified using the characteristic staining pattern of lymphocytes with CD45 (common leukocyte antigen), and lack of staining with CD14 (a monocyte/macrophage marker). Lysis II software was used for analysis (Becton-Dickinson). In order to correct results for the variable number of lymphocytes in each sample, results were expressed as a percentage of lymphocytes that stained for the respective markers using the characteristic staining of CD45 as a denominator (19).

Endobronchial Biopsies

Four specimens were taken from lower lobe subcarinae using alligator forceps (Olympus, FB 15C; Tokyo, Japan) and placed into chilled saline. Biopsies embedded in OCT (a glycerol-based freezing matrix; Sigma, St. Louis, MO) were snap frozen in a liquid N2-chilled isopentane slurry, then sectioned on a cryostat immediately or after storage at −70° C.

Frozen tissue sections of 7 μm were fixed in a paraformaldehyde-based fixative (PLP) for 15 min at 4° C prior to staining. The staining panel (DAKO, Gloserup, Denmark) for lymphocyte typing included anti-CD3, -CD4, and -CD8. Anti-CD25 was used as a T-cell activation marker. Anti-CD68 was used as a marker of macrophages. Anti-HLADR was used as an index of immunologic activation potentially related to several cell types present in airway biopsies, including macrophages, epithelial cells, endothelial cells, and T lymphocytes. Staining for cell subsets and activation markers was undertaken using a standard three-layer immunoperoxidase stain (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) using DAB substrate (DAKO). Irrelevant antibodies, including immunoglobin G1 (IgG1) and IgG2a (DAKO), were used as negative controls. Human nasal polyp tissue was used as a positive control, as appropriate.

Quantification of Cells and Antigen in EBB

A computerized color image analysis system (Video Pro 32; Leading Edge, Sydney, Australia) was used for quantification. Two sections, 30 μm apart, were examined by a single experienced observer blinded to the results of the clinical status, BAL, and TBB data. The submucosa was analyzed in five nonoverlapping high-power fields to a depth of 150 μm, with the epithelium excluded from quantitative analysis due to potential sampling bias as a result of variable epithelial denudation. Only nucleated stained cells were counted as positive and cells within vascular spaces were excluded. Counts were expressed per millimeter of basement membrane.

Transbronchial Lung Biopsies

Five to seven TBB were taken using alligator forceps (Olympus, FB 15C) and stained with hematoxylin and eosin for routine clinical assessment of acute or chronic rejection according to standard criteria. The assessment, performed by an expert specialist transplant pathologist, was made blind with respect to the other investigations and yielded a standard grade of inflammation (2).

Statistical Analysis

Nonparametric statistical methods that do not assume any underlying distribution for the data were mainly used, except where stated. The Kruskal-Wallis test for differences in location between groups was performed for each outcome measure. Where there was evidence of a statistically significant difference between groups (p < 0.05), the Kruskal-Wallis test was followed by pairwise Mann-Whitney tests. All tests performed are detailed in Results.

We realized that the possible effect of infection on cell counts and, in particular, the neutrophil data, had to be evaluated as a potential confounder (although care had been taken to exclude infection on clinical grounds). This was done by performing a two-way analysis of variance to evaluate simultaneously the effect of transplant status and bacterial infection status on neutrophil counts. For this analysis the data were logarithmically transformed to satisfy the assumptions of equal variance across groups and for normality.

Minitab for Windows software was used for analyses (release 11; Minitab Inc., State College, PA). TBB data were restricted to the conventional semi-quantitative scoring used in routine clinical work (2).

EBB Findings in Subjects with BOS

EBB data for patients with BOS are summarized in Table 3. Reference ranges for sLTR and normal subjects which provide context for the BOS findings, were presented in a previous publication (17). In contrast to sLTR, subjects with BOS had normalized EBB counts with no differences (or trends) from normal for the mononuclear cell markers evaluated (Figure 1).


BOS69171     33     55  27
n = 821–2823–770–13 10–8715–231 1–130
Stable4528153* 96* 111*
19–1073–122 0–416–21627–43221–364
Normal43192     27     6826
 9–1154–80 0–4 8–15527–117 3–119

Values are for absolute cell counts per mm of basement membrane, given as medians and ranges. Data adapted from Reference 17.

*p Value < 0.05 compared with normal.

BAL Findings in Subjects with BOS

BAL data are summarized in Table 4. Unfortunately, only data for six patients with BOS were evaluable due to oxygen desaturation in two subjects, which led to termination of the bronchoscopy on clinical grounds. BAL returns were significantly different between the three groups. The median return for the BOS group was 54 ml, range 22–95 (p < 0.001 relative to normals and p < 0.05 to sLTR). The median return in stable patients was 90 ml, range 40–112 (p < 0.05 relative to normals), and the median return in normal subjects was 120 ml, range 80–145.


Return (ml )TCC (× 103/ml )AM (%)LYM (%)PMN (%)EOS (%)EP (%)
BOS,           54, 180    40*  1038*       0.75
n = 622–95 70–1,900 6–552–4116–870–3   0–11
Stable,     90* 2807122 4*      02
n = 1740–112100–1,70020–913–780.4–180–3   0–15
Normal,12017078171  0.32
n = 980–145 90–27061–945–351–50.1–60.2–7

Definition of abbreviations: Return = volume return in ml from a 180-ml bronchoalveolar lavage; TCC = total cell count (× 103/ml); AM = alveolar macrophages; LYM = lymphocytes; PMN = neutrophils; EOS = eosinophils; EP = epithelial cells. All results are median counts with ranges. Values are given as medians and ranges.

*  p Value < 0.05 compared with normal.

  p Value < 0.001 compared with normal.

 p Value < 0.05 stable compared with BOS.

A striking finding was the frank neutrophilia in samples from patients with BOS, although a statistically higher neutrophil count relative to normal subjects was also present in the stable group (Figure 2). The median neutrophil count in BOS was 100 × 103/ml, range 13–1,661 × 103/ml (p < 0.001 relative to normals and sLTR). The median neutrophil count in stable patients was 7 × 103/ml, range 1–81 × 103/ml (p < 0.01 relative to normals), and the median neutrophil count in normal subjects was 3 × 103/ml, range 1–7 × 103/ml. In addition, there was a reciprocally lower number of macrophages compared with normal in the samples from patients with BOS (median count 40 × 103/ml, range 12–610 × 103/ml versus median 130 × 103/ml, range 68–240 × 103/ml in the normal subjects; p < 0.05).

These results were no different when the presence of cultured organisms were taken into account in our analysis of the neutrophil counts. A two-way analysis of variance on the log-transformed neutrophil count data found no evidence of a difference in average counts between patients with and without infection (p = 0.8), but there was a significant difference between groups (p < 0.001).

BAL Flow Cytometry

BAL flow cytometry results are summarized in Table 5. There was an overall trend for higher levels of CD8-positive lymphocytes in both groups of transplant patients compared with normal subjects. This was statistically significant for the comparison of the percentage of CD8-positive cells (BOS: median 57%, range 20–71%, p < 0.05 relative to control; sLTR: median 60%, range 23–91%, p < 0.05 relative to normals; normals: median 43%, range 16–75%).


Bos,2357*  8450.40*
 n = 612–7520–714–1418–630.31–3.73
Stable,2560*  9430.45*
 n = 1710–5223–912–1726–760.15–2.21
Normal,53 431339       1.26
 n = 913–7016–753–2817–700.17–4.25

Markers are expressed as percent of lymphocytes, with lymphocytes being identified by a characteristic pattern of CD45 staining. Values are given as medians and ranges.

*  p Value < 0.05 compared with normal.

In addition, the CD4/CD8 ratio was lower in both the BOS and sLTR groups compared with the normal group for both percentages and absolute numbers (Table 5).

Salient EBB and BAL findings are presented graphically in Figures 1 and 2.

Comparison of TBB Findings

All the sLTR were lung rejection grade A0 or A1 by standard grading techniques (2). For the patients with BOS, lung rejection grades ranged between A0 and A1.

Airway tissue was absent in TBB from 4 of the 17 stable patients (24%) and 2 of the 8 patients with BOS (25%). In addition, two subjects with a clinical diagnosis of BOS, in whom bronchiolar tissue was present, had no evidence of inflammation or fibrosis suggestive pathologically of obliterative bronchiolitis (OB). This meant that, overall, TBB were unable to detect OB in 50% of the patients with BOS.

To our knowledge, this is the first formal study comparing and contrasting EBB, BAL, and TBB information in stable lung transplant recipients and subjects with physiologic evidence of BOS, which is a manifestation of chronic rejection after lung transplantation.

In a previous study using EBB in physiologically and clinically stable lung transplant recipients, we found that these patients had significantly increased numbers of CD8-positive (suppressor/cytotoxic) lymphocytes and CD68-positive macrophages, and an increased level of class II major histocompatibility complex expression, as denoted by staining with HLADR in the airway wall, compared with normal subjects (17). In contrast, subjects with BOS in the current study had normalized EBB counts with no differences (or trends) for CD8-positve lymphocytes or CD68-positive macrophages and no increased staining with HLADR, compared with normal control subjects.

In conjuction with our previous work showing altered scar collagen deposition in airway biopsies in BOS (18), our current cellular data may be consistent with BOS, representing an end-stage fibrotic pathology, when damage occurs as part of the resolution of a previous active mononuclear cell infiltrate. Such progression from chronic inflammation to fibrosis would be complex, but preliminary work in lung allografts suggests that it could possibly be orchestrated through the effects of growth factors and cytokines such as platelet-derived growth factor (PDGF), and the transforming growth factor beta (TGF-β) family (1), which have important effects on the dynamics of fibrosis. Such a hypothesis would be supported by the larger body of evidence available from other solid-organ transplants (20) and the established literature on fibrosing lung diseases (21).

The complexity of managing the human transplant patient is perhaps underlined by evidence that cyclosporine induces TGF-β, which in turn inhibits interleukin 2 (IL-2)–driven T-cell proliferation (22). Such a scenario might underlie the paradoxical epidemiology of lung and other solid-organ transplantation, which is both promising and frustrating, i.e., that the short-term prognosis for the allograft has improved considerably over the last 10 years, but that chronic rejection characterized by organ fibrosis, as well as mortality rates, remains essentially unaltered (1).

BAL samples have traditionally been thought to be less useful than TBB in monitoring the lung allograft because of the confounding effect of infection (1). This may change, however, especially with the advent of more sophisticated analyses of BAL cells. Recent work has shown that analysis of T-cell restriction may provide insight into inflammation associated with rejection, with lung allograft rejection, but not infection, being associated with the presence of T cells expressing a limited number of T-cell receptor V Beta families (7). Other work has shown the potential importance of the balance of cytokine and growth factor production (8) in the lungs of allograft recipients. These studies are becoming practicable using BAL samples because of their yield of large numbers of relevant cells in suspension.

In this study, BAL returns were significantly different between the three investigated groups. Lowest returns were from patients with BOS, with sLTR having an intermediate return compared with the BOS and normal groups. A decreased BAL return is a common finding in many diseases of the airway (23) and lung (24). The decreased BAL returns observed in the transplant groups might therefore represent a manifestation of pathologic change, detectable in sLTR before measurable airflow limitation as denoted by change in FEV1, but more pronounced following the physiologic deterioration in BOS.

A striking finding in our BAL samples was a marked neutrophilia in samples from patients with BOS. There was also a statistically higher neutrophil count present in the sLTR relative to normal subjects, but with a very wide range. Chronic bone marrow suppression is a feature of the immunosuppression used in transplant recipients. It is of some interest to us, therefore, that we observed BAL neutrophilia, despite systemic suppression of white cell numbers.

The number of patients with BOS in this study were limited because we found this group difficult to recruit. Because of their severe airflow obstruction, they often desaturate during bronchoscopy. If this occurs, the procedure time is always minimized by the clinician, and the samples taken entirely restricted to the clinically standard samples (TBB) rather than prolonging the bronchoscopy for the research protocol. However, the main messages of the study, in particular the neutrophil changes, are robust enough that the sample sizes were quite adequate.

As with any transplant study, we have to consider the question: are the pathologic findings due to the biology of the allograft or infection? This is a problem faced by many of the BAL studies that have been performed in transplantation. We collected material only in the prospectively screened absence of overt infection, as assessed by standard clinical and microbiological criteria. All BAL samples were negative on gram stain and did not have CMV inclusions. This can be regarded as a reasonable cut-off for the objective definition of infection. Nevertheless, as is our experience, organisms were still grown, although these were considered as colonizers or commensals and not part of an active infection. These findings were included in an analysis of the neutrophil data, which took into account both the transplant status and presence of bacterial culture. This analysis confirmed that the higher neutrophil count was related to the transplant status.

It is of interest to note the approach taken by another recent BAL study in allowing for the problem of infection. DiGiovine and colleagues (9) excluded subjects from analysis on the basis of clinical judgment about infection status, but also on the basis of laboratory tests, although the number excluded on this basis alone was not stated. In total they excluded 45 BALs from 223 bronchoscopies. If we had adopted this approach in our study, with our sensitive laboratory-based methodology for the detection of organisms, we would have excluded 11 out of 17 stable subjects and 5 out of 8 patients with BOS. We feel that the discrepancy in test positivity between the two studies may be due to a difference in laboratory methodology and interpretation, with our results being skewed in favor of sensitivity for reporting of organisms. Overall, we feel that we dealt with the possible confounding effect of infection as far as is practicable in this patient group. It is of interest that our cellular findings are consistent with, and further support, the above study (9), and that both indicate that BAL neutrophilia is associated with BOS after lung transplantation. Overall, these results indicate that the emphasis of studies to date on the pathologic role of mononuclear cells in chronic rejection may have led to a relative neglect of the role of the neutrophil in this process. Careful reading of other studies indicates that neutrophils have been higher in lung transplant recipients, even in studies concentrating on lymphocyte subset and activation marker analyses (19, 25). These current results are particularly interesting because a BAL differential cell count for neutrophils is a relatively simple procedure. There is already established work in the interstitial lung diseases (26) and vasculitides (27) showing that increased numbers of BAL polymorphonuclear cells may be associated with a relatively aggressive fibrosing disease course (11). This study was unfortunately unable to comment on the presence of neutrophils in the biopsy specimens of lung transplant recipients, although this is being actively pursued.

This study also found an overall trend for higher levels of CD8-positive lymphocytes in BAL samples from both the stable and BOS transplant groups compared with normal subjects with a decreased CD4/CD8 ratio also present. BAL findings are therefore concordant with our EBB findings in stable lung transplant recipients but not for the patients with BOS. These results again indicate the importance of studying the cell populations of both compartments in future studies, as they may be giving different but complementary signals. Some disparity between BAL and EBB results is not surprising. BAL represents a mixed sample of the somewhat theoretical epithelial lining fluid from both large and distal airways as well as lung alveoli. This yields a liquid suspension, containing cells and solutes, with some of the cells representing populations that have undergone trafficking through the bronchial wall. In contrast, EBB are taken focally from the major airways, yielding cells that may well have different functional and phenotypic characteristics to those in BAL. Even within BAL there are heterogeneous populations of alveolar macrophages that have been isolated and partially characterized (28).

This study was inherently limited by its cross-sectional nature. The stable group was recruited, on average, earlier after transplant than the subjects with BOS, and it is therefore not possible to absolutely exclude a temporal effect post-transplant on our findings. However, we did not find any relationship between BAL neutrophil or CD8 numbers and time post-transplant, indicating that our findings are unlikely to be merely related to temporal changes, and are most likely a reflection of the biology of chronic rejection. Such questions can only be satisfactorily answered by a prospective longitudinal study.

In summary, we compared EBB, BAL, and TBB in a cross-sectional study of stable lung transplant recipients and subjects with BOS. TBB from the BOS group either failed to yield bronchiolar material or to show evidence of airway inflammatory or fibrotic changes in half the subjects. This confirms our previous data, indicating that TBB have fundamental drawbacks in commenting on the airway pathology of chronic rejection after lung transplantation (15). We also showed that EBB are practicable in BOS and, when considered with our earlier work (16-18), that EBB offer useful information in this situation. Our BAL data indicated that neutrophils, as well as mononuclear cells, should be monitored in the longitudinal studies that are now required to detect early markers of incipient BOS. Such studies are necessary to try and elucidate potential relationships between early pathologic changes in the allograft and the clinical development of BOS and the mechanism of this pathologic change. From such initial studies, targeted intervention protocols may become feasible. Given the huge financial and human investment in lung transplant programs throughout the world and the limited outcomes imposed by chronic allograft rejection, such basic and applied research is overdue.

The writers gratefully acknowledge Professor John Dowling, Alfred Hospital Pathology Department, for expert transbronchial biopsy assessments; and Vicky Ryan, MSc, University of Melbourne Statistical Consulting Centre, for statistical advice.

Supported by a grant from the Alfred Foundation and Glaxo Wellcome Australia.

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Correspondence and requests for reprints should be addressed to Professor E. H. Walters, Department of Respiratory Medicine, Alfred Hospital, Prahran, Melbourne, Victoria, 3181, Australia.


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