Abstract
In a prospective cohort study, we assessed whether changes in total cell counts and differentiation and interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) concentrations in bronchoalveolar lavage fluid (BALF) are associated with a higher risk to develop obliterative bronchiolitis (OB). We investigated 60 lung transplant patients (follow-up of 2 to 8 yr) with either histologic evidence of OB within 1 yr after lung transplantation (n = 19) or no pathology, good outcome (GO) for at least 24 mo and well-preserved lung function, i.e., FEV ⩾ 80% of baseline (n = 41). Median time between lung transplantation and the first BAL was 42 d for the GO group and 41 d for the OB group (p > 0.05). In the bronchial fraction, median total cell counts (0.06 × 103/ml versus 0.04 × 103/ml), lymphocyte (9 × 103/ml versus 2 × 103/ml), and eosinophilic granulocyte counts (1 × 103/ml versus 0) were significantly higher in the OB group than in the GO group (p < 0.05). In the alveolar fraction, this was the case for the median value of neutrophilic granulocyte counts (19 × 103/ml versus 4 × 103/ml), respectively. Median values of IL-6 and IL-8 concentrations in both bronchial (IL-6: 23 versus 6 pg/ml, IL-8: 744 versus 102 pg/ml) and alveolar fractions (IL-6: 13 versus 3 pg/ml, IL-8: 110 versus 30 pg/ml) of the BALF were significantly higher in the OB group than in the GO group. By means of logistic regression, we showed that higher total cell, neutrophilic granulocyte, and lymphocyte counts, the presence of eosinophilic granulocytes, and higher concentrations of IL-6 and IL-8 were significantly associated with an increased risk to develop OB. We conclude that monitoring cell counts, neutrophilic and eosinophilic granulocytes, IL-6, and IL-8 in BALF within 2 mo after lung transplantation in addition to the transbronchial lung biopsy (TBB) pathology will contribute to a better identification and management of the group of patients at risk for developing OB within a year.
Lung transplantation is generally accepted as therapy for several end-stage pulmonary diseases. Long-term survival of lung transplant patients is limited by the development of chronic transplant dysfunction. This condition affects at least 30 to 40% of the patients within the first years after lung transplantation, is nearly always irreversible, and is the most common cause of death 1 yr after lung transplantation (1). Clinically, chronic transplant dysfunction is associated with bronchiolitis obliterans syndrome (BOS). BOS is defined as a 20% or larger decrease in the forced expiratory volume in one second (FEV1) from the baseline FEV1 of the recipient, whereas other possible causes of deterioration of graft function, such as infection, have to be excluded (2). Histologically, chronic transplant dysfunction is represented by obliterative bronchiolitis (OB) and vasculopathy in lung tissue. OB is characterized by submucosal scarring that causes partial or total obliteration of membranous and respiratory bronchioles (3).
Usually, when chronic transplant dysfunction is suspected, a bronchoscopy is performed to exclude other causes of the deterioration of graft function, such as infection, problems with anastomoses, or acute rejection. The sensitivity of transbronchial lung biopsy (TBB) in detecting OB varies but is generally low (4-6). A more definitive diagnosis can be made by the use of open lung biopsy, but the drawback of this sensitive method is the supposed increased risk of associated morbidity. Bronchoalveolar lavage (BAL), a less invasive technique than TBB, is commonly accepted for detecting infections. Several studies have investigated the usefulness of BAL in providing specific information related to chronic transplant dysfunction but did not focus on finding early predictors for the development of OB. At present, the exact pathophysiology mechanisms of chronic transplant dysfunction and the development of OB are unknown, and diagnostic markers for predicting the development of OB at an early timepoint are poor. Accumulating evidence suggests that airway neutrophilia observed in the lungs plays an important role in the development of OB (7-9). Moreover, the neutrophilic granulocyte is the only cell that correlates with OB development and is associated with interleukin-8 (IL-8) concentrations, where IL-8 seems to act as neutrophil chemoattractant (10, 11). Because OB may be the result of dysbalance between tissue injury and repair leading to inflammation and fibrosis of the lungs, we postulate that also other proinflammatory and profibrotic cytokines such as IL-6 and monocyte chemoattractant protein-1 (MCP-1) may be involved. IL-6 has been shown to possess profibrotic properties in relation with tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) (12), and IL-6 production by epithelial cells and alveolar macrophages is one of the mechanisms by which local pulmonary inflammatory processes like acute rejection and infectious lung diseases are stimulated (13, 14). The CC-chemokine MCP-1 is a potent monocyte chemoattractant, which is expressed by airway epithelial cells. Accelerated MCP-1 release is seen in chronic inflammatory pulmonary diseases such as idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Monocytes may stimulate fibrinogenic mediator release which enhances tissue remodeling. Recently, MCP-1 has been described as a potential inflammatory mediator in OB (15).
It may be proposed that some of the patients are more prone to an enhanced inflammatory response after lung transplantation, ultimately resulting in OB. Therefore, in this prospective cohort study, we investigated the early relation between changes in leukocyte and differential counts, IL-6, IL-8, and MCP-1 concentations in BAL fluid (BALF) and the development of OB in 60 lung transplant patients with a follow-up of 2 to 8 yr.
A prospective cohort study including 60 patients who underwent lung transplantation between November 1990 and September 1996 was carried out using BALF samples. Informed consent was obtained from the patients. The study group consisted of patients who had histologic evidence of OB within 1 yr after lung transplantation (n = 19). The diagnosis of OB was based on a classification and grading of pulmonary transplant dysfunction promulgated by the International Society for Heart and Lung Transplantation (3). Control patients with good outcome (GO) were patients who did not develop OB or other pathology for at least 24 mo (43 mo median; range: 24 to 96 mo) after lung transplantation and had well-preserved lung function, i.e., FEV1 ⩾ 80% of baseline value (n = 41). Single and bilateral lung transplantations were performed according to established techniques and criteria described earlier (16).
Graft function was determined by formal spirometry (at least twice weekly during hospitalization and at every outpatient visit) and by extensive pulmonary function assessment (volume–flow measurements, diffusion capacity, pulmonary exercise testing), i.e., before discharge and every 6 mo after lung transplantation. In addition, daily spirometry with a pocket spirometer (17) was carried out at home. Acute allograft rejection was diagnosed clinically in case of deteriorating pulmonary function without infection and with a positive response on high-dose methylprednisolone. The histologic diagnosis was defined according to Yousem and coworkers (3). Cytomegalovirus (CMV) infection was monitored by CMV serology (18) and testing for CMV antigenemia (19) as described before. Active infection was defined as the presence of CMV antigenemia or when a significant rise of CMV-specific antibodies occurred.
Immunosuppression induction included up to five gifts of antithymocyte globulins (ATG) in the first 10 d after transplantation. The maintenance immunosuppressive regimen consisted of cyclosporine, azathioprine (1 to 3 mg/kg/d), and prednisolone (0.1 to 0.2 mg/kg/d). Cyclosporine administration was aimed at a trough level of 400 ng/ml at the start, as determined by high-performance liquid chromatography, which was tapered in 3 wk to 150 ng/ml. Acute rejection was treated with a 3-d course of 500 to 1,000 mg methylprednisolone intravenously daily. In case of persistent transplant dysfunction or when OB was diagnosed, cytolytic therapy with rATG was started. During this study, the newer immunosuppressive drugs, tacrolimus and mycophenolate mofetil, were not yet available. All patients received aciclovir 4 daily doses of 200 mg orally for herpes prophylaxis, and co-trimoxazole 960 mg orally on alternative days for Pneumocystis carinii prophylaxis.
BAL and bronchoscopy were carried out according to our protocol (20) at the same intervals as the pulmonary function measurements. This protocol was approved by the Medical Ethical Committee. BAL and bronchoscopy were carried out, 1 mo and every 6 mo after lung transplantation, and in the event of clinical indication. A fiberoptic bronchoscope was placed in wedge position in one of the segments of usually the right middle lobe, and 2 aliquots of 20 ml and 3 aliquots of 50 ml prewarmed phosphate-buffered saline (PBS) were instilled. The first 20-ml portion was investigated for viruses, bacteria, and fungi. The second 20-ml portion (bronchial fraction) was isolated and studied separately, whereas the other 50-ml fractions were pooled (alveolar fraction) (21, 22). Bronchial and alveolar fraction were immediately placed on ice (4° C), filtered through a nylon gauze, and centrifuged (Heraeus-Sepatech, Osterode, Germany) for 5 min at 400 g and 4° C to remove cells and debris. BALF was decanted and stored in small portions at −80° C. BAL cells were washed two times with PBS/ 0.1% (wt/vol) glucose (Merck, Amsterdam, The Netherlands). Viability was tested by incubation of 106 cells/ml with trypan blue solution (1:1) (vol:vol) (Gibco, Life Technology, Breda, The Netherlands) for 5 min. Viability was measured by determining the percentage of unstained cells in duplicate.
TBB (usually 5 to 10 at each procedure) were taken under fluoroscopic control using forceps No. 21 and/or 15c (“alligator”; Olympus, Tokyo, Japan). TBB were evaluated histopathologically.
Cytocentrifuge slides, precoated with PBS/0.5% bovine serum albumin (BSA) (wt/vol) (Boseral 20T; Organon Teknika B.V., Boxtel, The Netherlands), were prepared using 100 μl BAL cell suspension (0.3 × 106/ml) with a cytocentrifuge (cytospin 3; Shandon, Zeist, The Netherlands) by centrifugation for 5 min at 550 rpm. Slides were stained with May-Grünwald-Giemsa. Percentage of alveolar macrophages (AM), lymphocytes, and neutrophilic and eosinophilic granulocytes were determined by counting 200 cells on two slides each.
IL-6 (23), IL-8 (24) and MCP-1 were measured in duplicate in unconcentrated bronchial and alveolar fractions with an enzyme-linked immunosorbent assay (IL-6 kit [sensitivity: 1.5 pg/ml] from CLB, Amsterdam, The Netherlands; IL-8 [sensitivity: 30 pg/ml] and MCP-1 kits [sensitivity: 15 ng/ml] from R&D Systems, Minneapolis, MN). Levels were not adjusted for total protein as dilution effects resulting from the BAL procedure were comparable between GO and OB groups. (Recovery of BALF was not significantly different between both groups as shown in Figure 1.)
Fig. 1. Recovery of bronchoalveolar lavage fluid (BALF) of lung transplant patients who developed OB or not (GO). GO = good outcome group; OB = obliterative bronchiolitis group. Dash = median value.
[More] [Minimize]Data were analyzed using SPSS/PC+ software (SPSS Benelux b.v., Gorinchem, The Netherlands). Demographic categorical data were compared with the chi-square test. Total cell and differential cell counts, IL-6, IL-8, and MCP-1 concentrations were compared using the nonparametric Mann-Whitney U test. P values ⩽ 0.05 were considered significant. The effect of IL-6, IL-8, MCP-1, leukocytes, and differentiation on the risk to develop OB was estimated using multiple logistic regression. All regressions were performed separately, with simultaneous adjustment for age, sex, time after lung transplantation, occurrence of CMV infection, and acute rejection at the moment of BAL. Eosinophilic granulocyte counts were transformed in dichotomous data (absence/presence of eosinophilic granulocytes in the BALF). IL-6, IL-8, MCP-1, leukocytes, differentiation, and the dichotomous eosinophilic data were compared after logarithmic transformation (25).
Population characteristics (Table 1) were not significantly different between the OB and GO groups with regard to age, sex, diagnosis for lung transplantation, and unilateral or bilateral lung transplantation. There were no significant differences in the prevalence of acute rejection and infections at the time of BAL between the OB and GO groups (Table 2). The time lag between lung transplantation and the first BAL [median values and (ranges) of GO versus OB] was 42 (20 to 300) d versus 41 (27 to 124) d. In the GO group 76% of the patients and in the OB group 90% of the patients underwent BAL within 2 mo after transplantation. Recovery of BALF (median value GO versus OB, 69% versus 74%) (Figure 1) was not significantly different between the OB and GO groups. The median time of histologic evidence of OB was 198 days after transplantation with a range of 80 to 354 d.
| GO | OB | |||
|---|---|---|---|---|
| Patients | 41 | 19 | ||
| Age, yr (SD) | 41.5 (1.8) | 39.3 (3.2) | ||
| Sex, M/F | 20/21 | 10/9 | ||
| Diagnosis | ||||
| Emphysema/COPD | 25 | 7 | ||
| CF | 6 | 6 | ||
| PPH | 6 | 4 | ||
| Fibrosis | 0 | 1 | ||
| Bronchiectasis | 3 | 1 | ||
| Miscellaneous | 1 | 0 | ||
| Unilateral/bilateral lung transplantation | 6/35 | 5/14 |
| GO% | OB% | |||
|---|---|---|---|---|
| Acute rejection | 34 | 17 | ||
| CMV | 42 | 42 | ||
| Fungi | 13 | 6 | ||
| Bacterial | 21 | 29 | ||
| Viral other than CMV | 5 | 0 |
The bronchial fraction of the OB patient group had significantly higher numbers of cells/ml in BALF compared with the GO patient group (0.06 × 103/ml versus 0.04 × 103/ml) (Figure 2). The differentiation of cells in the bronchial fraction showed significantly higher numbers of lymphocytes (9 × 103/ml versus 2 × 103/ml) and eosinophilic granulocytes (1 × 103/ml versus 0) in the OB group than in the GO group (Table 3). Neutrophilic granulocyte counts tended to be higher in the OB group. In the alveolar fraction, the neutrophilic granulocyte counts were significantly higher in the OB group (19 × 103/ml versus 4 × 103/ml) (Table 3), and the eosinophilic granulocyte counts tended to be higher in the OB group.
Fig. 2. Total cell counts in BALF of lung transplant patients who developed OB or not (GO). Dash = median value. *Significant difference (p < 0.05) between GO and OB groups.
[More] [Minimize]| Cell Type | GO | n | OB | n | ||||
|---|---|---|---|---|---|---|---|---|
| Bronchial fraction | ||||||||
| AM | 29 (0–229) | 29 | 36 (13–233) | 12 | ||||
| Lymphocytes | 2 (0–41) | 29 | 9 (1–64)† | 12 | ||||
| Neutrophils | 1 (0–128) | 29 | 9 (0–2,328)‡ | 12 | ||||
| Eosinophils | 0 (0–2) | 29 | 1 (0–14)† | 12 | ||||
| Alveolar fraction | ||||||||
| AM | 206 (11–534) | 37 | 211 (2–1,001) | 19 | ||||
| Lymphocytes | 9 (0–59) | 37 | 13 (0–55) | 19 | ||||
| Neutrophils | 4 (0–77) | 37 | 19 (1–3,265)† | 19 | ||||
| Eosinophils | 0 (0–9) | 37 | 0 (0–44)‡ | 19 |
IL-6 and IL-8 concentrations were significantly higher in the OB group than in the GO group both in bronchial (IL-6: 23 versus 6 pg/ml, IL-8: 744 versus 102 pg/ml) and in alveolar fractions (IL-6: 13 versus 3 pg/ml, IL-8: 110 versus 30 pg/ml) (Figure 3B). This was not the case for MCP-1 concentrations, although there was a tendency of higher levels in the alveolar fraction of the OB group (p = 0.09).
Fig. 3. Cytokine concentrations in BALF of lung transplant patients who developed OB or not (GO). Dash = median value. *Significant difference (p < 0.05) between GO and OB groups. †Tendency toward difference (p < 0.1) between GO and OB groups.
[More] [Minimize]The association between the cellular and soluble cytokine markers of BALF with the development of OB after lung transplantation was investigated using multiple logistic regression analyses (Table 4). Higher numbers of total cell, neutrophilic granulocyte and lymphocyte counts and the presence of eosinophilic granulocytes in the bronchial fraction, as well as higher IL-6 and IL-8 concentrations, were associated with an increased risk to develop OB (odds ratios of 3.1 to 22.0). In the alveolar fraction higher numbers of total cell and neutrophilic granulocyte counts and higher IL-6, IL-8, and MCP-1 levels were significantly associated with an increased risk to develop OB (odds ratios of 3.8 to 20.5). When comparing IL-6 in presence or absence of CMV infection in GO and OB no significant differences were found (GO bronchial fraction: p = 0.99; alveolar fraction: p = 0.24; OB bronchial fraction: p = 0.740; alveolar fraction: p = 0.57). Similar calculations for IL-8 and MCP-1 also showed no differences between GO and OB.
| Odds Ratio (CI) | ||||
|---|---|---|---|---|
| Bronchial Fraction | Alveolar Fraction | |||
| Cells | 7.7 (1.7–35.2)* | 11.1 (1.1–115.6)* | ||
| AM | 1.6 (0.2–12.3) | 1.1 (0.3–5.1) | ||
| Neutrophils | 4.5 (1.2–16.6)* | 5.5 (1.7–18.4)* | ||
| Lymphocytes | 11.8 (1.3–101.5)* | 3.1 (0.7–13.1) | ||
| Eosinophils | 22.0 (19.8–24.2)* | 3.9 (2.3–5.4)† | ||
| IL-6 | 18.2 (2.8–119.5)* | 20.5 (3.2–131.3)* | ||
| IL-8 | 3.1 (1.2–8.2)* | 3.8 (1.3–11.5)* | ||
| MCP-1 | 3.7 (0.9–14.0)† | 3.9 (0.3–55.5)* | ||
The results reported in this prospective cohort study show that early leukocytosis, neutrophilia, presence of eosinophils, and higher concentrations of IL-6 and IL-8 in BALF are associated with the development of OB after lung transplantation. These parameters were studied in both bronchial and alveolar fraction. Differences between the GO and OB group were most often found for cell types in the bronchial fraction. This finding is in agreement with the fact that the bronchial fraction is assumed to be sampled from the proximal airways (21) and OB is a term restricted to membranous and respiratory bronchioles (3, 4) that are part of the proximal airways.
Leukocytosis, especially the influx of neutrophilic granulocytes and lymphocytes, in relation to OB has been reported by several other investigators (8-10, 26). DiGiovine and coworkers (10) investigated lung transplant patients at the time when OB was diagnosed and also a year before the diagnosis was set. In addition to cell types in the BALF they also evaluated the role of IL-8. They exclusively investigated the alveolar fraction of the lavage fluid and found a trend toward higher concentrations of neutrophilic granulocytes and IL-8 in the so-called “future OB patients” as compared with those not developing OB. We confirm and extend their findings in that we found significantly higher numbers of neutrophilic granulocytes and concentrations of IL-6 and IL-8 in the alveolar fraction of the BALF of the “future OB patients” compared with those of the GO patients. The highly significant differences of neutrophilic granulocytes and IL-8 between the OB and GO groups in our study are probably a result of the restrictive inclusion of the OB group according to Cooper and coworkers (2) and Yousem and coworkers (3). Therefore, IL-8 and neutrophil activity do not only seem to play a role in active OB, but also in developing OB, probably by acting as a profibrotic agent. DiGiovine and coworkers included patients without histologic evidence of OB as well and thereby probably found only trends toward the same direction. Holland and coworkers (27) reported increased lymphocyte counts in BALF of OB patients, in particular CD8+ lymphocytes. In our study, lymphocyte counts were increased in the bronchial fraction, but not in the alveolar fraction.
To our knowledge, eosinophilia in relation to OB has not yet been reported by others. Riise and coworkers (8) and other investigators (9, 10, 26) evaluated the influx of leukocytes and its differentation in association with BOS. They found no relation between eosinophilic counts and BOS. It is important to mention that eosinophils are only found in some of the patients with OB and cannot be used as a general marker of OB but may reflect the severeness of the inflammatory response in the development of OB. Eosinophilic granulocytes are known to be able to release potent cytotoxic granule products associated with the cellular damage seen in a variety of inflammatory diseases. It is unclear whether eosinophilic granulocytes are actively involved in the development of OB or merely present because of more pronouced activation of and attraction by other cells, for example, epithelial cells. We hypothesize that the presence of eosinophilic granulocytes in the bronchial fraction of the BAL is an early finding in some of the patients who develop OB. The specificity and association with severeness of OB remain to be determined.
To our knowledge, we are the first to report increased IL-6 concentrations in BALF of “future OB patients.” Magnan and coworkers (28) reported data on in vitro cytokine production by alveolar macrophages isolated from OB patients, showing that the production of IL-6 was not elevated in OB patients, in contrast with the production of TGF-β. They suggested that during OB tissue repair dominated over tissue injury. In our study, patients were monitored shortly after their lung transplantation, with a median value of 42 and 41 d in the GO and OB groups respectively. None of the patients was at that moment classified as having OB. As IL-6 is associated with tissue injury, we hypothesize that patients with elevated IL-6 levels in BALF are the patients at high risk for developing OB and other pathology. Our hypothesis is supported by the finding that in Magnan's study IL-6 concentrations are elevated during acute rejection accompanied by low TGF-β levels. In this study TGF-β was first secreted when IL-6 returned to normal values (28). So IL-6 seems to play an important role in the early stages of developing OB.
Humbert and coworkers also showed elevated IL-6 production in BALF of patients with CMV pneumonia after lung transplantation and to a lesser extent in allograft rejection, suggesting a possible role in OB (13). Because in our study CMV infections were equally divided between the GO and OB groups and because no significant difference was found between GO and OB in IL-6 and other cytokines in the presence or absence of CMV infection, IL-6 seems to play a role in the inflammatory mechanisms of early development of OB.
The elevated levels of the proinflammatory cytokine IL-6, IL-8, MCP-1, chemoattractants, and numbers of leukocytes, (e.g., lymphocytes, neutrophilic and eosinophilic granulocytes) in BALF of patients developing OB after lung transplantation support the indication that OB is caused by an immunologically mediated injury directed against epithelial cells (29). Based upon the study by Borger and coworkers, we hypothesize that the epithelial cell itself may play an important role in the release of IL-6, IL-8, and MCP-1 possibly in part induced by immunosuppressive agents commonly used in lung transplantation (14).
The value of MCP-1 in the pathogenesis of OB has yet to be determined. In our study, a tendency toward higher concentrations in OB was found, together with a significant odds ratio of 3.9 in the alveolar fraction, suggesting additional involvement in the early proces of tissue remodeling. This suggestion is confirmed by preliminary data of Belperio and coworkers (30) who showed a direct relationship between mononuclear cell recruitment and elevation of MCP-1 levels together with CCR2 messenger RNA (mRNA), which is its major receptor.
The early recognition of the patients at risk to develop OB suggests the possible intervention of this process by anti-inflammatory or more specific medication such as mofetil, FK506, or rapamycine as an inhibitor of cytokine-driven lymphocyte proliferation. Early adjustment of the therapeutic regimen of patients according to their BALF cell count and cytokines profile may contribute to better prevention of OB, but further studies are needed to attain a better defined risk stratification and to gain more insight into the underlying mechanisms contributing to the development of OB.
We conclude that monitoring total and differential cell counts, IL-6, IL-8, and MCP-1 in BALF within 2 mo after lung transplantation in addition to the TBB pathology will contribute to a better identification and management of groups of patients at risk to develop OB within a year.
The authors thank Minze Hamstra, Henk Meekers, and Mathilda Wind for assistance in the processing of BAL and determination of IL-6, IL-8, and MCP-1 and André Timmerman and Kristine Traanberg for assistance with preparing the figures.
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Correspondence and requests for reprints should be addressed to J. Scholma, University Hospital Groningen, Department of Pulmonary Diseases, Hanzeplein 1, P.O. Box 30.001, 9700 RB Groningen, The Netherlands. E-mail: j.
