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Keywords:

  • Bronchiolitis obliterans;
  • ELISA;
  • lung transplantation;
  • marker;
  • matrix metalloproteases

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Bronchiolitis obliterans (BO) is a survival-limiting factor in lung transplantation. There are no common BO markers in use. Since BO is associated with extracellular matrix remodeling, we asked whether matrix metalloproteases (MMPs) and their tissue inhibitors (TIMPs) could serve as BO markers. In 72 lung transplant patients (34 BO syndrome (BOS) 0, 15 BOS 0-p, and 23 BOS 1) serum and broncho-alveolar lavage (BAL) MMP and TIMP levels were examined by ELISA. The BAL cell counts were additionally analyzed. The serum MMP-2, MMP-8, MMP-9 and TIMP-2 levels were not different in all groups. In contrast, the BAL MMP-8, -9 and TIMP-1 levels were significantly elevated in BOS 0-p (p = 0.003; p = 0.007; p = 0.0003, respectively) and BOS 1 (p = 0.003; p = 0.001; p = 0.0004, respectively) as compared to BOS 0 patients. The BAL MMP-8, -9 and TIMP-1 levels were significant predictors of BOS 0-p (p = 0.01; p = 0.01; p = 0.01, respectively) and BOS-1 (p = 0.007; p = 0.01; p = 0.006, respectively) in receiver operating characteristic analysis. Except for BAL macrophages that were significantly decreased in BOS 0-p versus BOS 0 patients; other cell counts were not different between the groups. BAL MMP-8, -9 and TIMP-1 might be useful markers to detect BO in lung transplant patients.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Lung transplantation has gained widespread acceptance as a therapeutic option in patients with severe pulmonary function impairment and limited life expectancy. The outcome of this procedure, however, might be jeopardized by complications that result in constraints on long-term preservation of graft function (1). One major complication in lung transplant patients is bronchiolitis obliterans (BO) that is characterized by progressive decline of pulmonary function, with a prevalence of 34–65% in long-term survivors (2,3). BO involves airway epithelial damage, followed by repair and remodeling processes of the lung graft (4–6). Currently, transbronchial biopsy (TBB) represents the golden standard in BO diagnosis. However, TBB is invasive and difficult to be performed repeatedly in patients with impaired pulmonary function. More importantly, obtaining representative BO tissue by TBB might be problematic; and false negative histology results occur despite declining pulmonary function.

The pathogenesis of BO has been related to ischemia-reperfusion injury, repeated acute rejection, infection and alloimmunologic reactions (7). Although, none of these hypotheses has been proved conclusively, the remodeling of the pulmonary tissue and the bronchial fibrosis seem to be the final common pathway in BO (8). Increasing evidence suggests that MMPs play a major role in the process of extracellular matrix remodeling (9,10). Ample evidence suggests that MMPs increase following lung transplantation and that the ratio of MMP-9 to TIMP-1 is elevated in chronic lung graft failure (10). Whether MMPs could serve as useful BO markers, remains unclear.

This prospective cross-sectional study was designed to elucidate serum and BAL MMP and TIMP levels in lung transplant patients with unimpaired pulmonary function and clinically diagnosed BOS according to the published guidelines (8). Our results indicate that BAL MMP-8, -9 and TIMP-1 levels are significant predictors of BOS 0-p and BOS 1.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Patients

Between November 1989 and July 2002 a total of 385 lung transplantations have been performed at the Department of Cardio-Thoracic Surgery, Medical University of Vienna. In a prospective cross-sectional study approved by the Ethics Committee at the Medical University of Vienna, between June 2002 and June 2004, 72 double lung transplant patients in surveillance, who gave informed consent to be enrolled, were studied. Patients were eligible for this study if they survived at least 6 months after transplantation. Thirty-four of the study patients, mean age 45.8 ± 14 (19–64 years), had no evidence of infection at sampling, acute rejection (see below) or BOS (BOS 0). Fifteen patients had BOS 0-p, mean age 54 ± 11 (28–65 years) and the remaining 23 patients, mean age 49.6 ± 12 (20–66 years), had BOS 1 without evidence of infection or acute rejection at sampling. These patients were enrolled immediately after the diagnosis BOS was made during the surveillance tests and before the BOS therapy (see below) was initiated. The pulmonary diseases leading to lung transplantation and the number of patients with these diagnoses were: chronic obstructive pulmonary disease (COPD: 14 BOS 0, 7 BOS 0-p, 10 BOS 1), idiopathic pulmonary fibrosis (IPF: 8 BOS 0, 5 BOS 0-p, 6 BOS 1), primary pulmonary hypertension (PPH: 6 BOS 0, 1 BOS 0-p, 3 BOS 1), cystic fibrosis (CF: 5 BOS 0, 1 BOS 0-p, 4 BOS 1) and others (1 BOS 0, 1 BOS 0-p).

Diagnosis of BOS

Pulmonary function measurements in the first 6 months after transplantation were used to establish baseline FEV1 and FEF25–75. Subsequently BOS stages were defined relative to this baseline, following the published guidelines (8). According to this classification, BOS 0 was defined by FEV1≥ 90% and FEF25–75≥ 75% of the baseline. In patients with FEV1 ranging from 81% to 89% of the baseline, BOS 0-p was defined. BOS 1 was defined in patients with FEV1 of 66–80% of the baseline. None of the patients with the clinical diagnosis of BOS had histologic evidence of chronic rejection (C grading) in TBB.

Surveillance and screening of rejection and infection

All patients were routinely followed according to a protocol including clinical observation weekly for the first 3 weeks, monthly in the first year, and every 2 months from the second year. The surveillance protocol involved the history, the physical status, the pulmonary function and the bronchoscopy. TBBs and BAL were obtained in the first year and also thereafter when either infection or rejection was suspected.

Acute rejection, cytomegalovirus (CMV) and other infections at the time of sampling were diagnosed as follows; and patients with infections and acute rejection at sampling were excluded from the study. All TBBs underwent A, B and C grading. Rejection was diagnosed histologically if the samples showed rejection grade A2 or B2 or higher according to the scoring of the International Society for Heart and Lung Transplantation (11,12). Rejection was diagnosed clinically if patients had new radiographic pulmonary opacification and deterioration of blood gas and pulmonary function without evidence of infection at a TBB rejection score of A or B0–1. These patients underwent steroid pulse therapy at 1 g/d for 3 d. If the patient was resistant to the steroid pulse therapy, OKT-3 was administered at 5 mg/d for 7 d (12). Patients included in this study did not have acute rejection grade A1 or B1 or higher.

An episode of infection was defined clinically and approved using microbiologic, serologic or histologic tests. CMV screening was performed by measurement of CMV serology (quantitative polymerase chain reaction) and detection of CMV early antigen in blood, urine, throat smear, and BAL. Bacterial and fungal cultures as well as PCR for toxoplasmosis and pneumocystis carinii were performed in each BAL. Transbronchial biopsies were also histologically screened for CMV, pneumocystis carinii, toxoplasmosis, and bacterial or invasive fungal infection (12). Colonization was defined if the BAL microbiology was positive without histological or clinical evidence of infection. Seven patients included in this study had bronchial colonization without histological or clinical evidence of infection.

Serum and BAL sampling, BAL cell count

All BOS 0-p and BOS 1 patients who matched the criteria to be included in the study, as well as control patients (BOS 0) were enrolled after giving their informed consent and BAL fluid was obtained. Hundred milliliters of pre-warmed physiologic saline solution were instilled intrabronchially; the first 20-mL portion was considered as bronchial, the next 20-mL fraction was considered as the alveolar BAL. An alveolar BAL aliquot was used for assessment of the total BAL cell count. Then, the alveolar BAL fraction was centrifuged for 5 min at 500 rpm onto cytocentrifuge slides (1 mL BAL per slide) using a cytocentrifuge (Heraeus, Hanau, Germany). The obtained slides were stained with May-Grünwald-Giemsa. The percentage of different BAL cells was determined using light microscopy. On each slide, 200 cells were analyzed for differentiation; four slides were examined per patient. Cell free BAL and serum samples were snap frozen and stored in liquid nitrogen until further use.

Immunosuppression

Peri- and post-operative immunosuppression included 1g methylprednisolone intraoperatively, followed by 125 mg after 8, 16 and 24 h. Thereafter, prednisolone was administered at a dose of 1 mg/kg/d and tapered to 0.25 mg/kg within 3 months. All patients received rabbit-ATG (Thymoglobuline, Sero-Merieux, Lion, France) 2.5 mg/kg intravenously for the first four post-operative days. Mycophenolate mofetil (CellCept, Hofmannn-La Roche, Switzerland) at 3 g/d was given i.v. on the first post-operative day and then orally. Cyclosporine A (target level 350 ng/mL) or tacrolimus (target level 15 ± 3 ng/mL) was administered intravenously immediately after surgery, and was subsequently switched to oral administration (12).

After the exclusion of acute rejection and infection as described above, recipients with deteriorating lung function (i.e. BOS) received steroid bolus therapy at 1000 mg/d for 3 d. Then, the steroid was reduced to 1 mg/kg over 2 weeks. After the first BOS treatment the recipient underwent pulmonary function test and bronchoscopy. In case of repeated occurrence of BOS, a switch of immunosuppression followed: Cyclosporine A was switched to tacrolimus with a target level of 18 ± 2 ng/mL depending on kidney function. If the switch in immunosuppression was not effective, the steroid treatment was repeated. If this therapy failed, the patient received rabbit ATG (Thymoglobuline) or OKT-3 as described above.

ELISA

ELISA was performed to detect MMP-2, MMP-8, MMP-9, TIMP-1 and TIMP-2 in serum and BAL samples, using Quantikine kits (R&D Systems, Minneapolis, MN, USA). Assay diluent was added to each well of microplates pre-coated with specific antibodies. Afterwards, standard, serum or BAL samples were added to each well. After incubation for 2 h at room temperature and rinsing, 200 μL of specific conjugates (horseradish peroxidase-conjugated specific antibodies) were added, and incubated for 1 or 2 h at room temperature followed by rinsing. Then, 200 μL substrate solution, consisting of equal volumes of hydrogen peroxide and tetramethylbenzidine, was added to each well and incubated for 30 min at room temperature, protected against light. This reaction was stopped by adding 50 μl stop solution. The optical density was detected using a microplate reader (Anthos, Salzburg, Austria) at 450 nm.

Statistical analysis

At the end of the study, the clinical parameters as well as the protein concentration levels of all molecules measured in serum and BAL were compared between BOS 0, BOS 0-p and BOS 1 patients by analysis of variance with Bonferroni t-test. Data are expressed as mean value ± SD and statistical significance is set at p < 0.05. The predictive ability of MMP-8, MMP-9 and TIMP-1 BAL levels to identify BOS 0-p and BOS 1 was assessed by receiver operating characteristic analysis. The correlation between BAL neutrophil counts and BAL MMP-8 and -9 levels was analyzed by Spearman's rank method. Statistical analyses were performed using SAS version 8.2.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

In ELISA, there was no significant difference in total amount of serum MMP-8 between the three patient groups. We found, however, significantly higher BAL MMP-8 levels in BOS 1 (116.7 ± 81.3 ng/mL; p = 0.003) and BOS 0-p (29.7 ± 19.4 ng/mL; p = 0.003) versus BOS 0 (2.5 ± 2 ng/mL) patients. The BAL MMP-8 levels were not significantly different between BOS 0-p and BOS 1 patients (Figure 1A). BAL MMP-8 levels were a significant predictor for BOS 1 and BOS 0-p in the receiver operating characteristic analysis (p = 0.007, CI = 1.009–1.551; AUC = 0.926 for BOS 1; p = 0.01, CI = 0.998–1.341, AUC = 0.807 for BOS 0-p). Accordingly, BAL MMP-8 levels could not discriminate between BOS 1 and BOS 0-p in receiver operating characteristic analysis (p = 0.211, CI = 0.578–1.566, AUC = 0.694). The serum or BAL MMP-2 concentration was not significantly higher in BOS 1 and BOS 0-p as compared to BOS 0 group (Figure 1B). Similarly, there was no difference in serum MMP-9 levels between the three patient groups (Figure 1C). In contrast, BAL MMP-9 levels were markedly elevated in the BOS 1 (490.3 ± 356.9 ng/mL; p = 0.001) and in the BOS 0-p (153.4 ± 112.1 ng/mL; p = 0.007) as compared to the BOS 0 (0.3 ± 0.9 ng/mL) group (Figure 1C). MMP-9 BAL levels were also a significant predictor for BOS 1 and BOS 0-p in the receiver operating characteristic analysis (p = 0.01, CI = 0.661–1.850, AUC = 0.986 for BOS 1; p = 0.013, CI = 0.575–3.530, AUC = 0.832 for BOS 0-p). The receiver operating characteristic analysis did not discriminate between BAL MMP-9 levels in BOS 0-p and BOS 1 patients (p = 0.219, CI = 1.000–1.006, AUC = 0.694).

image

Figure 1. MMP serum and BAL concentrations measured by ELISA. (A) Serum MMP-8 levels are not changed in BOS 0 (n = 34) versus BOS 0-p (n = 15) and BOS 1 (n = 23) group. BAL MMP-8 concentration is significantly elevated in BOS 1 and BOS 0-p versus BOS 0 group. (B) Serum and BAL MMP-2 levels are not significantly changed in BOS 0 versus BOS 0-p and BOS 1 patients. (C) Serum MMP-9 level is not different in the three patient groups, although BAL MMP-9 concentration is significantly higher in BOS 1 and BOS 0-p versus BOS 0. Results are expressed as mean ± SD. *, significantly different from BOS 0 group (p = 0.988 [BOS 1, serum], p = 0.945 [BOS 0-p, serum], p = 0.003 [BOS 1, BAL], p = 0.003 [BOS 0-p, BAL], A; p = 0.408 [BOS 1, serum], p = 0.567 [BOS 0-p, serum], p = 0.114 [BOS 1, BAL], p = 0.246 [BOS 0-p, BAL], B; p = 0.989 [BOS 1, serum], p = 0.875 [BOS 0-p, serum], p = 0.001 [BOS 1, BAL], p = 0.007 [BOS 0-p, BAL], C).

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The serum level of the tissue inhibitor of MMPs, TIMP-1, was significantly elevated in BOS 1 (311.8 ± 37.5 ng/mL) versus BOS 0 (255.8 ± 35.1 ng/mL) patients (p = 0.043), but there was no significant difference between BOS 0-p (280.4 ± 51.3 ng/mL) patients and the other groups (Figure 2A). Likewise, the BAL TIMP-1 level was significantly higher in BOS 1 (48.7 ± 26.0 ng /mL; p = 0.0004) as compared to patients without evidence of BO (1.0 ± 1.4 ng/mL). TIMP-1 was also elevated in the BAL of BOS 0-p (18.9 ± 12.7 ng/mL; p = 0.0003) as compared to BOS 0 patients (Figure 2A). BAL TIMP-1 levels were a significant predictor for BOS 1 and BOS 0-p in the receiver operating characteristic analysis (p = 0.006, CI = 0.987–1.603, AUC = 0.932 for BOS 1; p = 0.01, CI = 1.001–1.550, AUC = 0.875 for BOS 0-p), however, the receiver operating characteristic analysis did not discriminate between BOS 0-p and BOS 1 TIMP-1 levels (p = 0.172, CI = 0.995–1.067, AUC = 0.653). The serum and BAL TIMP-2 levels in BOS 0 patients were not significantly changed as compared to BOS 1 and BOS 0-p patients (Figure 2B).

image

Figure 2. Serum and BAL TIMP concentration measured by ELISA. (A) Serum TIMP-1 level is significantly elevated in BOS 1 (n = 23) versus BOS 0 (n = 34) patients, and BAL TIMP-1 concentration is significantly higher in BOS 1 and BOS 0-p (n = 15) versus BOS 0. (B) TIMP-2 serum and BAL levels are not significantly changed in BOS 0 versus BOS 1 and BOS 0-p patients. Results are expressed as mean ± SD. *, significantly different from BOS 0 group (p = 0.043 [BOS 1, serum], p = 0.137 [BOS 0-p, serum], p = 0.0004 [BOS 1, BAL], p = 0.0003 [BOS 0-p, BAL], A; p = 0.195 [BOS 1, serum], p = 0.247 [BOS 0-p, serum], p = 0.111 [BOS 1, BAL], p = 0.139 [BOS 0-p, BAL], B).

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The results of BAL cell counts are shown in Table 1. Alveolar macrophages (AM) were significantly decreased in the BAL of BOS 0-p as compared to BOS 0 patients (p = 0.02), although the increase in percentage of neutrophils and lymphocytes in the BOS 0-p group was not significant versus BOS 0 group. BAL cell counts in BOS 1 were not significantly different as compared to those in BOS 0 as well as BOS 0-p patients. There was no significant correlation between BAL neutrophil counts and MMP levels in Spearman correlation analysis (MMP-8: r = 0.488, p = 0.266; MMP-9: r = 0.344, p = 0.405).

Table 1.  Cell counts in the alveolar BAL fluid fraction of patients
 BOS 0 (n = 34)BOS 0-p (n = 15)BOS 1 (n = 23)
  1. *Statistically significant versus BOS 0 (p = 0.02).

Cells 103/mL108 (10–450)130 (45–500)183 (65–1500)
AM (%)92.3 (88–96)86.5 (83–90)*89.4 (86–93.5)
Lymphocytes (%)6.2 (3–10)9.3 (8–13)8.3 (6–11)
Neutrophils (%)1.2 (0–3)3.7 (1–7)2.1 (0.5–5)
Eosinophils (%)0.3 (0–2)0.5 (0–1)0.3 (0–1)

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

Transplant failure due to chronic graft rejection, BOS, is characterized by decline of pulmonary function due to airflow limitation caused by inflammation, predominantly in small airways (13). Although the etiology of BOS is not clearly understood, it has been suggested that small airway inflammation may lead to matrix degradation and tissue fibrosis (10). MMPs are crucial for turnover of the extracellular matrix and comprise a large family of enzymes that share common structural features, particularly those involved in the regulation of proteolytic activity (14). MMP-8 is a collagenase and is predominantly secreted by neutrophils (15), while MMP-2 and MMP-9 belong to the family of gelatinases. MMP-2 can be produced by alveolar epithelial cells (15). MMP-9, TIMP-1 and TIMP-2 can be expressed by macrophages, endothelial cells and pneumocytes (16). MMP-9 is also produced by neutrophils (17). Three key findings underlied this study that aimed to examine MMPs as BO markers. First, macrophages are frequent habitants of lung tissue; second both macrophage and neutrophil density increases in pulmonary tissue of patients with higher BOS stages (10); and third the MMP expression correlates with sputum neutrophils in LTX patients (10). In our study MMP-8, MMP-9 and their inhibitor TIMP-1 were dramatically increased in BAL of LTX patients with BOS 0-p and BOS 1 as compared to those with no signs of BOS. More importantly, receiver operating characteristic analysis clearly indicated that MMP-8, -9 and TIMP-1 can serve as markers for both BOS 0-p and BOS 1. These findings may not be necessarily in contrast to the significant increase in number of BAL neutrophils in other studies for the following reasons. Patients included in our study belonged to BOS stage 1 or 0-p. Earlier studies (18) have reported that BAL neutrophil counts do not discriminate between BOS 1 and BOS 0; and in line with this, BAL neutrophils increase in higher BOS stages (10). Another explanation for the observed sensitivity of certain MMPs to indicate BOS might be the ability of ‘sensitized’ cells (primarily neutrophils) to express larger amounts of certain MMPs. The fact that the pulmonary tissue remodeling observed in BO involves specific MMPs or their tissue inhibitors, is in agreement with this hypothesis. The immunologic mechanism underlying this process, however, needs further study. The balance between MMPs and their tissue inhibitors, on the other hand, justifies the assumption that elevated TIMP-1 serum levels may indicate a compensation mechanism to counteract increased pulmonary MMP levels. In any case, BAL seems to be a more sensitive medium to measure MMPs in BOS patients as compared to serum.

Although the impact of acute graft rejection on the development of BOS remains uncertain (11), acute lung injury of different etiology (including infection or immunosuppression-induced) is now proven to induce MMP-2 as wells as MMP-9 overproduction (19). On the other hand, BOS patients may have not experienced one episode of rejection, although patients with several acute graft rejection episodes may never develop BOS (11). Patients with TBB-verified acute rejection were excluded from the present study. Thus, the current results will not help in distinguishing patients with BOS from acute rejection by BAL alone. However, our recent study clearly indicated that acute rejection can be diagnosed specifically and sensitively by measuring serum hepatocyte growth factor (HGF) levels (12). For these reasons, and because negative BO histopathology results are often present in patients with clinical BOS diagnosis, the combined knowledge of BAL MMP and serum HGF levels might be more useful to distinguish BOS from acute rejection in lung transplanted patients. This suggested protocol would bypass TBB.

In the present study, we were interested to learn whether MMP BAL or serum levels could serve as markers to diagnose BO. We demonstrate that BAL MMP-8, -9 and TIMP-1 can serve as specific markers to identify BOS in its early stages, that is 1 and even 0-p. Beside their ability to recognize BO, MMPs could be useful targets to counteract pulmonary tissue remodeling that is observed in BO. This hypothesis is supported by recent work, indicating that synthetic MMP blockers can inhibit collagenolytic activity that ameliorates experimentally induced pulmonary hypertension (20). This issue definitely deserves further study that is being performed in our center.

In summary, BOS seems to be a multi-etiology condition that involves, at least, two major components of the protease family that is gelatinases and collagenases and one of their tissue inhibitors, TIMP-1. The BAL concentration of MMP-8, -9 and TIMP-1 increases in BOS 0-p and BOS 1 patients, and outranges the concentrations seen in lung transplanted patients without BOS symptoms. BAL MMP-8 and -9 as well as TIMP-1 might be useful markers to detect BO.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References

This study was supported by a grant from the Austrian Heart Foundation to Seyedhossein Aharinejad.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. References
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