Lung transplantation is the only therapeutic option for patients with end-stage lung disease. However, long-term success of lung transplantation is hindered by the development of bronchiolitis obliterans syndrome (BOS), a fibroproliferative process that leads to airway obstruction. Bronchiolitis obliterans syndrome is characterized by bronchiolar inflammation, airway epithelial damage, aberrant epithelial repair, tissue remodeling, and bronchiolar fibrosis. Clinically, recipients with BOS develop nonreversible obstructive lung disease with a progressive decline in forced expiratory volume in 1 second (FEV1) of more than 20% from baseline, resulting in severe shortness of breath and dry cough.1 Histopathological features include extracellular matrix (ECM) degradation, airway epithelial damage, tissue remodeling, and fibrosis of the bronchioles along with inflammatory infiltrates of the small airways in particular lymphocytes and more abundant neutrophilic granulocytes.2-5 The mechanism responsible for the development of BOS is complex and still poorly understood.
Matrix metalloproteinases (MMPs) belong to the family of metal-dependent endopeptidases and are known for their capacity to degrade components of the ECM, facilitate cell migration, cleave cytokines from the surface, process growth factors, and activate defensins. Excess MMP activity may contribute to immunopathology resulting from uncontrolled ECM turnover, epithelial damage, fibrosis, tissue remodeling, and inflammation, which are all major risks for the development of BOS.6-9 Accordingly, increased expression of MMPs and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), have been observed in BOS recipients, suggesting a role for MMPs in the observed tissue remodeling and pathogenesis of BOS.10-13 Furthermore, multiple studies have shown that MMP-8 and ‐9 levels correlate with neutrophil numbers in the bronchoalveolar lavage (BAL) fluid from recipients with BOS.4,12,14,15 However, little is known about the proteolytic activity state of MMPs in the lungs of BOS recipients. Matrix metalloproteinases are synthesized as catalytically inactive zymogens with a propeptide domain.16 The conversion of zymogens to active MMPs is achieved by proteolysis of the prodomain,16 on which their activity can be counterbalanced by binding to TIMPs.
Aberrant activity of MMPs may reflect an imbalance between their production or activity and the production of their endogenous inhibitors. Advanced understanding of the regulation of MMP activity in lung transplant recipients may therefore help us to identify the relationship between their dysregulation and development of BOS. Moreover, profiling of active MMPs may serve as an early predictor of BOS. Using a novel method for activity-based MMP extraction, we investigated the levels of MMPs, their endogenous inhibitors, and their activity in BAL fluid from lung transplantation recipients with early stage BOS, who eventually all developed stage III, and recipients with good outcome.
MATERIALS AND METHODS
Approximately 30 lung transplantations are performed annually in the University Medical Centre Groningen, the prevalence of BOS at 2 years being approximately 7%. For the purpose of this retrospective case–control study, BAL samples were analyzed from 20 lung transplant recipients diagnosed with early- stage BOS (stage I), who eventually all reached stage III. The diagnosis of BOS was according to criteria, as formulated by the International Society of Heart and Lung Transplantation,1 with 65% to 80% of baseline FEV1 after transplantation. Within our program, bronchoscopy, including BAL, is performed routinely every 6 months during the first 2 postoperative years. Additionally, BAL is performed or on clinical indication, usually unexplained graft dysfunction. The control group consisted of 20 sex- and age-matched transplant recipients, who remained persistently free of BOS and acute rejection for at least 5 years after transplantation. Again, BAL samples were taken during the first 2 postoperative years. For both groups, immunosuppression was induced by basiliximab, accompanied and followed by maintenance with tacrolimus (long-term target trough levels of 6–10 μg/L), mycophenolate mofetil 1000 mg twice daily, and prednisolone 0.1 mg/kg. Postoperative prophylaxis was applied by broad spectrum antibiotics during the first week, cytomegalovirus prophylaxis during the first 3 months, and lifelong pneumocystis prophylaxis. See Table 1 for patient characteristics. The BAL samples were excluded for analysis when bacterial, fungal, CMV, or other viral infections was present at the time of the BAL procedure.
Surveillance of viral, bacterial, and fungal complications was compatible with published guidelines,17 that is, on a routine basis during the first 3 postoperative months, later on by clinical indication and in all bronchoscopy procedures.
BAL and Bronchoscopy
The BAL was performed according to the protocol as described previously18,19 for routine diagnosis within the scope of the University Medical Center Groningen lung transplant program, which was subject to medical ethics review at the start in 1990. All recipients gave their informed written consent for use of their data. The first fraction (20 ml) was isolated as bronchial fraction. The next 3 aliquots (50 mL) were pooled and isolated as the alveolar fraction. Cells from this fraction were pelleted by centrifugation and supernatants were stored at −80°C.
Differential cell counts in BAL were performed as described previously.18
Cytokine Levels in BAL Fluid
Levels of interleukin (IL)-6 and IL-8 were measured using the Immulite system (Siemens, Den Haag, The Netherlands) according to the manufacturer's instructions.
In Situ Determination of Total Matrix Metalloproteinase Activity
Active MMPs were extracted from alveolar fractions using the broad spectrum MMP inhibitor tumor necrosis factor protease inhibitor-2 (TAPI-2) immobilized to beads as described before.20 Ammonia-activated Sepharose beads (GE Healthcare, Uppsala, Sweden) were washed with ice-cold HCl (1 mM) and coupling buffer (0.2 M K2HPO4, pH 7.5), incubated overnight at 4 °C with TAPI-2 (5 mM, Calbiochem, San Diego, CA) in coupling buffer and blocked with ethanolamine (0.5 M, Sigma-Aldrich, Zwijndrecht, the Netherlands) for 1 hour (RT). Control beads were immediately blocked with ethanolamine. To validate for extraction of active MMPs, trypsin-activated full length MMP-7 (R&D systems, Abingdon, United Kingdom) was added to a physiological salt solution containing CaCl2 (10 mM) and treated with TAPI-2 or control beads (20 μL per 500 μL sample) for 40 minutes during rotation (RT). Beads were removed by centrifugation (1000 × g for 2 minutes), and the solution was analyzed for MMPs using Luminex according to the manufacturer's instructions (R&D Systems, Abingdon, United Kingdom). The MMP activity was analyzed by fluorescence resonance energy transfer peptide Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 (5 μM; Bachem California, CA) at 37°C in 50 μL MMP reaction buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35, pH 7,5) and 50 μL TAPI-2 or control-treated fractions. Fluorescence intensity was measured for 15 minutes at 37°C (excitation wavelength 340-nm through a 420-nm bandpass filter) in a FL600 fluorescent plate reader (Bio-Tek Instruments, the Netherlands).
Total MMP and TIMP Levels in BAL Fluid
Total MMP (Pro-MMP, MMP, and TIMP/MMP) levels of MMP-1, ‐2, ‐3, ‐7, ‐8, ‐9, ‐12, and ‐13 were determined using a Luminex Performance human MMP panel according to the manufacturer's instructions (R&D Systems), using Luminex technology (Luminex Corporation, Austin, TX). Total TIMP (TIMP and TIMP/MMP) levels of TIMP-1, ‐2, ‐3, and ‐4 were assessed using a Milliplex Map human TIMP panel according to the manufacturer’s instructions (Millipore, Amsterdam, The Netherlands).
TIMP/MMP Levels in BAL Fluid
The TIMP-bound MMP levels were measured using a sandwich Luminex approach where TIMP-immobilized beads from the Milliplex Map human TIMP panel were combined with biotinylated detection antibody of MMPs from Luminex Performance human MMP panel. The TIMP/MMP fluorescence intensity levels were measured using Luminex technology.
Comparisons between different subject groups were performed using the Mann–Whitney U test and comparisons between different conditions within groups were performed by the Wilcoxon signed-rank test (MMP levels in BAL) or t test (recombinant human MMP-7). The Spearman rank correlation coefficient with Bonferroni correction was used to study correlations between BAL neutrophils and MMP levels in BAL fluid. P less than 0.05 was considered significant.
Total and Active MMP Levels in the Alveolar Fraction From BOS and Good Outcome Patients
Because BOS is a disease of the small airways, we analyzed the alveolar BAL fraction, reflecting the peripheral airways.21 Population characteristics were not significantly different between BOS and good outcome recipients with regard to age, sex, unilateral or bilateral lung transplantation, and diagnosis for lung transplantation (Table 1). Furthermore, total cell numbers in the alveolar fraction were not significantly different between the 2 groups (data not shown), whereas the percentage of neutrophils and lymphocytes and the levels of IL-8 were significantly higher in BOS compared to good outcome recipients (Table 1).
Detection of total MMP levels in the alveolar fraction showed significantly higher MMP-2, ‐3, ‐7, ‐8, and ‐9 levels in BOS recipients than in good outcome recipients (MMP-2: 178.8 vs 18.8 pg/mL, P < 0.04; MMP-3: 14.4 vs 4.4 pg/mL, P < 0.01; MMP-7: 877 vs 449 pg/mL, P < 0.01; MMP-8: 937 vs 84 pg/ml, P < 0.0001; MMP-9: 3450 vs 378 pg/ml, P < 0.0001; Figure 1). Levels of MMP-1,-12, and ‐13 were not detectable in the alveolar fraction from recipients with or without BOS.
We used a novel method to investigate total activity of MMPs, where BAL fluid was treated with the broad spectrum MMP inhibitor TAPI-2 immobilized to beads to extract active MMPs. Subsequently, levels of MMPs were measured by Luminex ELISA. To validate our assay, we first measured levels of recombinant human MMP-7 in a physiological salt solution. Extraction with TAPI-2-labeled beads resulted in a strong and significant decrease in MMP-7 levels compared to the use of control beads (Figure S1A, SDC,https://links.lww.com/TP/B116). Similar results were observed when MMP-7 was spiked into pooled BAL fluid (data not shown). To confirm that we extracted active MMP-7, a fluorescence resonance energy transfer peptide was used as a generic MMP activity indicator, showing a strong and significant inhibition in activity on TAPI-2 extraction of MMP-7 (Figure S1B, SDC,https://links.lww.com/TP/B116). Next, we studied the effect of TAPI-2 extraction in BAL samples from recipients with good outcome and BOS. Using fluorescence resonance energy transfer peptide, we observed a significant decrease in MMP activity in BAL from good outcome recipients on extraction with TAPI-2 beads, indicating the presence of active/non–TIMP-bound MMPs (Figure 2A). In contrast, TAPI-2 treatment failed to significantly alter MMP activity in BAL fluid from BOS recipients, indicating that these recipients do not contain active MMPs in their lungs, in contrast to good outcome recipients (Figure 2A). Additionally, total and inactive levels of specific MMPs were determined in the alveolar fraction using TAPI-2 beads using Luminex. The MMP-2,-3, -8, and ‐9 levels in the alveolar fraction from both good outcome and BOS recipients did not significantly decrease on TAPI-2 extraction, indicating that all detected MMPs were inactive (Table 2). In contrast, a significant decrease in MMP-7 levels was observed on TAPI-2 treatment in BAL from good outcome patients, but not the BOS group, indicating that only good outcome recipients display active MMP-7 in their lungs (Table 2). Together, these results indicate that no active MMPs are present in BAL fluid of recipients with good outcome and BOS, except for MMP-7 in recipients with good outcome.
Total TIMP and TIMP/MMP Levels in the Alveolar Fraction
To gain further insight into the regulation of MMP activity in BOS, we studied TIMP levels and TIMP-bound MMP levels in the alveolar fraction of BOS and good outcome recipients. TIMP-1 and ‐2 were significantly higher in the BOS group than in the good outcome group (TIMP-1: 1312 vs 992 pg/mL, P < 0.007; TIMP-2: 830 vs 207 pg/mL, P < 0.004; Figure 2B). Levels of TIMP-3 and ‐4 were not detectable in the alveolar fractions of recipients with or without BOS.
The alveolar fraction of BOS recipients showed significantly higher levels of TIMP-1-bound MMP-7, ‐8, and ‐9 and TIMP-2-bound MMP-8 and ‐9 than in good outcome recipients (TIMP-1/MMP-7/: 67.0 vs 4.0 relative fluorescent units (RFU), P < 0.0005; /TIMP-1/MMP-8: 73.0 versus 1.0 RFU, P < 0.0005; TIMP-1/MMP-9/: 260.1 vs 21.1 RFU, P < 0.0005; TIMP-2/MMP-8/: 56.0 vs 3.0 RFU, P < 0.0005; TIMP-2/MMP-9-: 70.3 vs 1.0 RFU, P < 0.0005; Figure 2C-E), whereas values of TIMP-2–bound MMP-7 were not significantly different (Figure 2C). The presence of MMP-bound TIMPs suggests their previous activity.
Correlation Between MMP Levels and Neutrophils
Neutrophils are able to express TIMP-1, TIMP-2, and MMP-3, ‐7, ‐8 and ‐9 and store these in their granules,22,23 whereas epithelial cells may be the main producers of MMP-2.24 Of interest, we found that the levels of MMP-3, -7, -8, -9, but not MMP-2, significantly correlate with the numbers of neutrophils in BAL fluid from BOS recipients (Table 3), indicating that neutrophils may be an important source for MMP-3, ‐8, and ‐9 in BOS. Moreover, we observed a significant correlation between TIMP-bound MMP-8 and ‐9 and the numbers of neutrophils, indicating that the presence of these TIMP-bound, previously active MMPs may also be related to the development of neutrophilic inflammation on lung transplantation.
We present a novel approach to profile levels of active and inactive MMPs, including proenzymes and TIMP-bound MMPs, in BAL fluid of lung transplant recipients with and without BOS, using activity-based TAPI-2 extraction in combination with a multiplex immunoassay. We show for the first time that, in addition to total levels of MMP-2, ‐3, ‐7, ‐8, and ‐9, TIMP-bound levels of MMP-7, ‐8, and ‐9 are significantly increased in recipients with BOS compared to recipients with good outcome. Surprisingly, active MMP-7 was present in BAL fluid of stable transplant recipients, but not in BOS recipients, while we were unable to detect active MMP-2, ‐3, ‐8, and ‐9 in any of the BAL samples. Nevertheless, the increased levels of TIMP-bound MMP-7, ‐8, and ‐9 in BOS patients indicates previous activity of these MMPs.
In contrast to MMP-2, ‐3, ‐8, and ‐9, levels of MMP-7 were slightly, but significantly, decreased on TAPI-2 extraction in BAL fluid of good outcome recipients, suggesting that MMP-7 accounts for the majority of total MMP activity observed in BAL samples of these recipients. TAPI-2 extraction did not reveal the presence of active MMP-7 in the lungs of BOS recipients, indicating that MMP-7 activity relates with a better outcome of lung transplantation. This is in line with previous findings from Kastelijn and co-workers,25 showing that genetic polymorphisms of MMP-7 predispose to the development of BOS. Here, lung transplant recipients carrying the risk alleles expressed lower serum levels of MMP-7. Accordingly, MMP-7 may promote cell migration and proliferation,26 initial steps in repair of the airway epithelium, which is the primary target for injury on lung transplantation. Insufficient MMP-7 levels may thus account for impaired re-epithelialization and lung repair, contributing to the pathogenesis of BOS.11 On the other hand, epithelial adhesion molecule E-cadherin is a substrate of MMP-7, and MMP-7 activity may disrupt epithelial cell-cell contacts by cleavage of E-cadherin. Despite the fact that we were not able to detect active MMP-7 in BAL fluid of BOS recipients, total and TIMP-bound MMP-7 levels were significantly higher in transplant recipients with BOS. This discrepancy with the findings of Kastelijn et al27,28 possibly stems from the fact that we studied levels in BAL instead of systemic levels. Furthermore, binding to TIMP-1 suggests previous activity of MMP-7 because TIMPs are thought to bind exclusively to active MMPs to counterbalance their activity on cleavage of the prodomain. Nevertheless, our data suggest that specifically active MMP-7 may be a predictor for good outcome on lung transplantation.
Similar to MMP-7, we observed that TIMP-bound MMP-8 and MMP-9 levels are substantially higher in BAL fluid of recipients with than recipients without BOS. In line, it has previously been reported that MMP-8 and ‐9 are higher in BOS and are significant predictors of BOS development.4,14,24,29,30 Our current data on the TIMP binding indicate that a substantial part of these MMPs has indeed been active because TIMPs bind active MMPs. Matrix metalloproteinase-8 is a collagenase, while MMP-9 (together with MMP-2) belongs to the family of gelatinases that are also able to degrade collagen. Thus, both MMP-8 and MMP-9 may contribute to matrix degradation in BOS, leading to tissue remodeling and airway obstruction. Accordingly, sputum concentrations of MMP-9 correlate negatively with FEV1 in lung transplant recipients.10 Furthermore, MMP-9 contributes to inflammatory cell migration, and studies in MMP-9–deficient mice show that MMP-9 plays a crucial role in the pathogenesis of obliterative airway disease by the regulation of mononuclear cell influx and activation of alloreactive T cells.31 Importantly, neutrophil-derived MMP-9 has been implicated in disruption of epithelial junctions in the retina,32 and MMP-9 has been shown to disrupt airway epithelial junctions.33 This effect of MMP-9 may also contribute to the development of BOS because epithelial injury is thought to play an important role in obliteration of the small airways.
In line with previous studies, we observed that levels of MMP-8 and ‐9 significantly correlate with neutrophils in BAL. Neutrophils have been implicated in the pathogenesis of BOS, and neutrophil numbers in BAL negatively correlate with lung function.3 Increased numbers of neutrophils are present in the airway walls of stable transplant recipients as well as BOS recipients,2 with higher BAL neutrophils in BOS compared to both stable lung transplant recipients and healthy controls.3 We anticipate that neutrophils contribute to airway obstruction in BOS by their production of MMPs. Patients in the BOS group, but not the good outcome group were treated with azithromycin. Azithromycin has previously been described to reduce MMP-9 levels in lung transplant patients and to decrease MMP-9 mRNA expression in airway epithelial cells,34,35 whereas to the best of our knowledge, no effects of azithromycin on MMP-7 and ‐8 expressions have been reported. Therefore, we do not consider it likely that the observed increase in MMP levels in BOS patients is a consequence of treatment.
In addition to the role of MMPs in matrix tissue remodeling processes, MMPs may act to perpetuate neutrophilic inflammation in BOS in a self-augmenting loop. MMP-8 and MMP-9 mediate cleavage of collagen, resulting in generation of the proline-glycine-proline fragment, which exerts chemotactic effects on neutrophils via CXCR1 and CXCR2.36,37 Importantly, increased levels of proline-glycine-proline have been observed in BAL fluid of BOS recipients compared to stable transplant recipients5 and this correlated positively to MMP-9 activity. Here, MMP activity was assessed by zymography, a nonselective semiquantitative method that has been shown to activate pro-MMPs during the required denaturation-renaturation step, during which TIMP-MMP complexes are also dissociated.20 This method therefore does not distinguish properly between active and inactive MMPs, and an ELISA-based activity assay, which is limited by the fact that it does not allow to measure multiple MMPs at once. The advantage of our newly developed method is that it is quantitative and specific for the profiling of MMP activity, in contrast to the conventional zymography and collagenase assays. The latter technique uses [1-14C]-collagen as a substrate, which is rather nonspecific as multiple MMPs as well as non-MMP proteases can degrade collagen.
Together, we show that levels of TIMP-bound MMP-7,-8, and ‐9 are increased in BAL fluid of lung transplant recipients with BOS, while active MMP-7 is only present in recipients with good outcome. Although we were not able to detect MMP activity in BAL fluid of BOS recipients, TIMP binding indicates previous activity in the lungs. Our novel method may lead to advanced opportunities for the screening of MMP activity as early predictors of BOS or good outcome on lung transplantation and increased insight in new candidates for therapeutic intervention in BOS. Further studies on MMP-8 and ‐9 are warranted to explore their potential as novel therapeutic targets.
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