Volume 29, Issue 12 , Pages 1330-1336, December 2010
Synergistic effect of antibodies to human leukocyte antigens and defensins in pathogenesis of bronchiolitis obliterans syndrome after human lung transplantation
Article Outline
- Abstract
- Methods
- Results
- Human LTx recipients with anti-HLA DSA and BOS diagnosis have higher defensin levels
- Stimulation of AECs by anti-HLA class I or HNPs induces HBD production
- Endobronchial administration of anti-MHC class I Abs leads to increased neutrophil activity (MPO assay) in lungs
- Levels of α and β-defensin are higher in BAL of mice administered with anti-MHC Abs
- Discussion
- Disclosure statement
- References
- Copyright
Background
This study aims to determine the role of antibodies to donor-mismatched human leukocyte antigen (HLA) developed during the post-transplant period in inducing defensins and their synergistic role in the pathogenesis of chronic rejection, bronchiolitis obliterans syndrome (BOS), after human lung transplantation (LTx).
Methods
Bronchoalveolar lavage (BAL) and serum from 21 BOS+ LTx patients were assayed for β-defensins human neutrophil peptides (HNP) 1-3 (enzyme-linked immunosorbent assay [ELISA]) and anti-HLA antibodies (Luminex, Luminex Corp, Austin, TX). Human airway epithelial cells (AEC) were treated with anti-HLA antibodies, HNP-1/2, or both, and the levels of β-defensin were measured by ELISA. Using a mouse model of obliterative airway disease induced by anti-major histocompatibility (MHC) class-I antibodies, we quantitatively and qualitatively determined neutrophil infiltration by myeloperoxidase (MPO) staining and activity by MPO assay, and defensin levels in the BAL.
Results
In human LTx patients, higher defensin levels correlated with presence of circulating anti-HLA antibodies (p < 0.05). AEC treated with anti-HLA antibodies or HNP-1/2, produced β-defensin with synergistic effects in combination (612 ± 06 vs 520 ± 23 pg/ml anti-HLA antibody, or 590 ± 10 pg/ml for HNP treatment; p < 0.05). Neutrophil numbers (6-fold) and activity (5.5-fold) were higher in the lungs of mice treated with anti-MHC antibodies vs control. A 2-fold increase in α-defensin and β-defensin levels was also present in BAL on Day 5 after anti-MHC administrations.
Conclusions
Anti-HLA antibodies developed during the post-transplant period and α-defensins stimulated β-defensin production by epithelial cells, leading to increased cellular infiltration and inflammation. Chronic stimulation of epithelium by antibodies to MHC and resulting increased levels of defensins induce growth factor production and epithelial proliferation contributing to the development of chronic rejection after LTx.
Keywords: human neutrophil peptide, bronchiolitis obliterans syndrome, bronchoalveolar lavage, human β-defensin 2, airway epithelial cells
Chronic allograft rejection after human lung transplantation (LTx) manifesting as bronchiolitis obliterans syndrome (BOS) is the primary reason for adverse long-term survival outcomes in LTx recipients.1 Although immunologic and non-immunologic causes of BOS pathogenesis are postulated, mechanisms leading to BOS are still elusive. Alloimmune responses to mismatched donor histocompatibility antigens are important in the pathogenesis of BOS. Antibodies (Abs) specific for donor HLA class I have been shown to precede the development of BOS2 and can stimulate growth factor production, proliferation, and apoptosis of epithelial cells (ECs).3, 4
Defensins are anti-microbial peptides produced by neutrophils (α-defensins) and ECs (β-defensins) that have been implicated in immune modulation, inflammation, and wound healing. Nelsestuen et al,5 using proteomic approach, have reported increased levels of human neutrophil α-defensins in the bronchoalveolar lavage (BAL) of LTX recipients with chronic rejection. Human β-defensins (HBD) produced by the epithelium act as chemokines by chemokine (C-C motif) receptor (CCR) 6, providing a link between innate and adaptive immunity.6
In this study we tested the hypothesis that specific immune responses against donor HLA can induce production of not only Abs to HLA but also defensins and that both synergistically lead to the epithelial changes seen during chronic lung allograft rejection. Towards this, we determined the development of donor-specific Abs (DSA) to donor HLA and quantitated the levels of defensins in BAL and sera of BOS+ LTx recipients. In addition, using a mouse model of anti-MHC class I–induced obliterative airway disease (OAD), we determined the role of neutrophils infiltrating the lung and its production of α- and β-defensin in development of OAD. Our results with human LTx recipients with BOS and an animal model of OAD demonstrated that Abs to HLA as well as α-defensins stimulate airway epithelial cells (AEC) to produce HBD2 and induce morphologic changes in the epithelium. Therefore, anti-HLA Abs developed after LTx stimulate ECs to augment the production of defensins. DSA as well as defensins synergistically activates ECs, leading to sustained production of growth factors resulting in EC proliferation, fibrosis, and remodeling, the cardinal features of BOS.
Methods
All animal experiments were performed in compliance with guidelines of the Institutional Laboratory Animal Care and Use Committee of Washington University School of Medicine (Protocol 20070121). The protocol for the human study was approved by the Institutional Review Board.
Human participants
LTx patients at Washington University Medical Center/Barnes-Jewish Hospital were enrolled in this study with informed consent. Standard immunosuppression consisted of cyclosporine, azathioprine, and prednisone. After BOS diagnosis, immunotherapy was modified to FK506 (tacrolimus), mycophenolate mofetil, and prednisone. BOS diagnosis was according to International Society for Heart and Lung Transplantation (ISHLT) standard criteria.7 Forced expiratory volume in 1 second (FEV1) was measured at < 80% of baseline established in a stable post-operative period, or there was histologic evidence of BOS. Serum and BAL samples from 21 BOS+ patients and 9 BOS– patients were collected at 6 ± 2.3 months after the clinical diagnosis for BOS+, processed on the day of collection, and stored at −70°C.
Anti-HLA testing
Anti-HLA Abs and their donor specificity were detected in patient sera by a solid phase assay (Luminex, Luminex Corp, Austin, TX) using reagents from One Lambda, Canoga Park, CA.
Human neutrophil peptide 1-3 and HBD2 enzyme-linked immunosorbent assayLevels of human neutrophil peptide (HNP) 1-3 and HBD2 levels were determined using enzyme-linked immunosorbent assay (ELISA) test kits purchased from Cell Sciences (Canton, MA) and Phoenix Pharmaceuticals (Burlingame, CA), respectively.
Cell linesSAECs were cultured in small airway growth medium (Cambrex BioScience, Rockland, ME). Normal human bronchial epithelial cells (BEC) were obtained from American Type Culture Collection (Manassas, VA) and cultured in bronchial epithelial growth medium (Cambrex Bio Science, Rockland, ME).
Treatment of AECs with anti-HLA Abs (W6/32)To test the effect of anti-HLA Abs on defensin production, AEC/BEC were serum-starved for 16 hours and then incubated for 24 hours with various concentrations (2.5, 5, or 10 μg/ml) of anti-HLA class I Ab, W6/32. The supernatants from the treated cells were tested for HBD2.
Treatment of AECs with HNPsTo determine the mechanism by which increased defensin levels affect ECs, human AEC/BEC were serum-starved for 16 hours and treated for 24 hours with HNP1/HNP2 (Bachem, Torrance, CA) at 10-μg/ml concentrations. The supernatants from the treated cells were tested for HBD2.
Intrabronchial administration of anti-MHC Abs into murine lungsMurine monoclonal Abs (mAb; immunoglobulin G2a) or its isotype control C1.18.4 with no detectable endotoxin (limulus amoebocyte lysate assay) was given at a dose of 200 μg per administration intrabronchially into the lungs of H2Kb (BALB/c) test and control mice, respectively, as detailed in our previous publication.8
Staining for myeloperoxidase in mouse lungs administered with anti-MHC AbsFrozen lung samples were embedded in freeze tissue matrix (optimal cutting temperature compound), and sections were cut at 5-μm thickness. The sections were fixed in cold alcohol for 2 minutes (−20°C) and air-dried. The presence of positive cells was detected with the myeloperoxidase (MPO) kit (Sigma, St. Louis, MO), counter-stained with hematoxylin, and examined using a light microscope. Positive cells were counted by random sampling.
MPO assay
Lung extracts were prepared from 100 mg of frozen mouse lung samples by sonication and freeze-thaw in K-phosphate buffer. MPO standard (Sigma) was used at various dilutions. To all wells, MPO reaction mixture containing 0.167 mg/ml O-dianisidine dihydrochloride in K-phosphate buffer with 0.05% H2O2 was added to make 150 μl of total reaction mixture. The reaction was stopped after 5 minutes with 25 μl of 1% sodium azide prepared in K-phosphate buffer and read on ELISA reader at 460 nm.
Statistical analysis
Data are expressed as means ± standard deviation. The minimum number of replicates for all measurements was at least 3. For ELISA and Luminex differences between control and samples were compared by t test. Significance was assigned at p < 0.05.
Results
Human LTx recipients with anti-HLA DSA and BOS diagnosis have higher defensin levels
HNP1-3 levels were measured by ELISA in the BAL fluid of LTx recipients who developed BOS. Development of anti-HLA Abs DSA was determined in serum using Luminex assay. DSA was present in 12 of 21 BOS+ patients. When compared with separate cohorts (9 patients in each cohort) of BOS+ and BOS− patients without DSA, HNP 1-3 levels were higher in the DSA+ patients (630 ± 98 vs 460 ± 60 and 410 ± 76 pg/ml in BOS+ DSA− and BOS− DSA−, respectively) as shown in Figure 1. These results demonstrate that defensins levels are significantly increased in BOS+ LTx recipients (p < 0.05) and correlate strongly with development of DSA.
Figure 1.
Defensin levels are higher in donor specific antibody (DSA)+ bronchiolitis obliterans syndrome (BOS)+ patients (11 of 21). Enzyme-linked immunosorbent assay was done to measure human neutrophil peptides (HNP) 1-3 for BOS+ lung transplant recipients. HNP (1-3) levels were higher in the BOS+ recipients who also had DSA compared with BOS+ recipients who did not develop DSA. Data are shown with the standard deviation. **p < 0.05.
Stimulation of AECs by anti-HLA class I or HNPs induces HBD production
Supernatants from small airway epithelial cells (SAEC) incubated with HNP 1/2 for 24 hours were tested for HBD2 production by ELISA. Figure 2 demonstrates significant increases in HBD2 levels in response to HNP1 or HNP2 treatment (590 ± 10 for HNP-1 and 540 ± 8 for HNP-2 treatment vs 420 ± 12 for untreated cells, p < 0.05). To test the effect of anti-HLA class I on defensin production by AECs, SAECs were treated with various concentrations of W6/32 (anti-HLA class I mAb for 24 hours). Results, presented in Figure 3, demonstrate increase in HBD2 levels in response to W6/32 treatment compared with isotype control (509 ± 8 for 5 μg/ml and 520 ± 23 for 10 μg/ml W6/32 Ab treatment vs 420 ± 12 for untreated cells, p < 0.05). Further, SAECs treated with monoclonal and polyclonal anti-keratin Abs produced a similar response as isotype control (data not shown). Anti-HLA Ab treatment of AECs in combination with defensins (HNP1/2 or HBD2) showed a further increase in HBD2 production (612 ± 6 vs 520 ± 23 and 590 ± 10 for anti-HLA or HNP alone, p < 0.05; Table 1) demonstrating a synergistic effect. Because treatment of SAEC with HNP 1 or 2 and/or anti-HLA class I results in significant increase in HBD2 production, we propose that higher production of α-defensins by neutrophils may result in higher β-defensin production from ECs.
Figure 2.
Increased human β-defensin (HBD) 2 levels in response to human neutrophil peptides (HNP)1 or HNP2 treatment compared with untreated cells. Small airway epithelial cells (SAECs) were treated with HNP1 or HNP2 for 24 hours. HBD2 production was measured by enzyme-linked immunosorbent assay in the culture supernatant. Data are shown with the standard deviation.
Figure 3.
Increased human β-defensin (HBD) 2 levels in response to various concentrations of anti-human leukocyte antigen (HLA) class I antibody compared with isotype control antibody. Small airway epithelial cells (SAECs) were treated with various concentrations of anti-HLA class I antibody (see Methods) for 24 hours. HBD2 production was measured by enzyme-linked immunosorbent assay in the culture supernatant. Data are shown with the standard deviation.
Table 1. Human β-Defensin 2 Production From Small Airway Endothelial Cells Increases synergistically After Treatment With Both Anti-Human Leukocyte Antigen Antibody and Human Neutrophil Peptides
| Small airway endothelial cells | Human β-defensin 2 (pg/ml) |
|---|---|
| Untreated | 420 ±12 |
| With anti-HLA Ab (W6/32) | 520±23 |
| With α-defensins (HNP1/2) | 590±10 |
| With anti-HLA Ab (W6/32) α-defensins (HNP1/2) | 612±06 |
Endobronchial administration of anti-MHC class I Abs leads to increased neutrophil activity (MPO assay) in lungs
To determine that anti-HLA Abs can induce defensin production from lung epithelium, a mouse model of OAD was used wherein anti-HLA class I Abs were administered endobronchially to native lungs. Neutrophil activity was investigated by MPO assay in the lung lysate after administration of anti-MHC Abs/isotype control. As shown in Figure 4, lungs of mice treated with anti-MHC class I show 6-fold higher neutrophil activity at Day 5 (p < 0.01) and remained elevated at least 4-fold up to Day 15 after Ab administration compared with lungs of mice administered isotype control.
Figure 4.
Increased neutrophil activity by myeloperoxidase (MPO) assay in lung lysates of mice treated with anti-major histocompatibility complex (MHC) antibodies (Abs). BALB/c mice were administered anti-MHC (H2kd) antibodies endobronchially (see Methods). Neutrophil activity at Days 3, 5, 9, 11, and 15 after antibody administration was measured in lung lysates of mice treated with anti-MHC antibodies using MPO assay and compared with mice treated with isotype control antibodies. Data are shown with the standard deviation.
To determine neutrophil infiltration in the lungs of mice administered with anti-MHC Abs, MPO staining was done on lung sections after the administration of anti-MHC Abs/isotype control. As shown in Figure 5, lungs of mice treated with anti-MHC Abs showed a 5.5-fold increase in neutrophil numbers at Day 5 (p < 0.01) compared with lungs of mice administered isotype control.
Figure 5.
Increased neutrophils by myeloperoxidase (MPO) staining in lungs of mice treated with anti-major histocompatibility complex antibodies (MHC Abs). (A) Frozen sections of lungs from mice treated with anti-MHC antibodies at Day 5 were stained for MPO, and (B) neutrophil infiltration was compared with mice administered isotype control antibodies. Data are shown with the standard deviation (Original magnification ×40).
Levels of α and β-defensin are higher in BAL of mice administered with anti-MHC Abs
Levels of α-defensin and β-defensin were measured by Western blotting using anti-mouse α-defensin and β-defensin Abs (Santa Cruz Biotechnology, Santa Cruz, CA) in BAL of mice treated with anti-MHC Abs/isotype control. Levels of α-defensin and β-defensin in BAL fluid of mice administered with anti-MHC Abs were 1.7- to 2.5-fold higher than in BAL of mice treated with isotype control (Figure 6). These results demonstrate that chronic stimulation of lung AECs both by defensins and anti-HLA class I can lead to increased cellular infiltration in the lungs and secretion of cytokines and growth factors contributing to the fibroproliferative changes seen in BOS.
Figure 6.
Levels of α- and β-defensin are higher in bronchoalveolar lavage (BAL) of mice administered anti-anti-major histocompatibility complex antibodies (MHC Abs). BAL fluid of mice administered with anti-MHC antibodies was tested for α and β-defensins by Western blotting and compared with mice treated with isotype control antibodies.
Discussion
Improvements in surgical procedure and peri-operative and post-operative management have led to significant improvement in quality of life after LTx, but long-term outcomes remain poor due to chronic rejection manifesting as BOS. An important role for immune response to mismatched donor HLA antigens in the pathogenesis of BOS has been demonstrated in earlier studies from our laboratory9, 10 and others.11, 12 Development of Abs to donor HLA mismatches,13, 14 as well as increased precursor frequency of CD4+ T-cells specific for the mismatched HLA class I and II antigens,2, 9, 15 have been demonstrated to be important risk factors for BOS. Development of anti-HLA Abs precedes the development of BOS2, 4, 15 giving rise to its possible pathogenic role in the development of BOS. HLA Abs that develop after LTx have been shown to result in complement-mediated cell death and proliferation of AEC leading to apoptosis of AEC as well as production of growth factors.3, 4
BAL obtained from normal lungs show that the predominant cell is alveolar macrophage (85%), followed by lymphocytes (7% to 12%). Eosinophils, basophils (1%), and neutrophils (1% to 2%) are minimal in normal lung BAL.16 In contrast, significantly elevated numbers of neutrophils have been reported in the BAL of LTx recipients with BOS and are described as a hallmark of obliterative bronchiolitis.17, 18, 19 Defensins, produced by neutrophils, are a part of the innate immune system.20 These are small molecules released at the site of injury that promote inflammation and resistance to infections. They also affect various immune functions and can mediate wound repair. Exposure to neutrophil defensins results in more chemotaxis and proliferation of inflammatory and EC and fibroblasts.21 Hence, defensins functions as a link between innate and adaptive immune mechanisms.22, 23
Studies have shown that the levels of defensins are higher in serum and BAL fluids of BOS+ LTx recipients compared with the BOS– patients.24 Studies have reported that higher HNP levels can predate the clinical onset of disease up to 15 months.5 In our study, BAL samples collected from patients after LTx demonstrated significantly higher levels of α and β-defensins, and the development of DSA correlated with higher defensins levels in BOS+ LTx patients (Figure 1).
The mechanisms involved in recruitment and activation of neutrophils in the airways of patients with BOS is not fully understood. The finding that neutrophilia independent of infection may be involved in the pathogenesis of BOS25, 26 has been reported. Data presented here, using human LTx recipients with chronic rejection (Figure 1) as well as the animal model of OAD induced by anti-MHC class I, demonstrate that anti-HLA Abs produced after LTx can activate AECs to produce β-defensins, an important chemoattractant for neutrophils and macrophages. This can further increase production of defensins (HNP1-3 by the infiltrating neutrophils as well as HBD2 from ECs). We have also shown the production of HBD2 in vitro by AECs in response to anti-HLA Ab (W6/32; Figure 3). It is significant that in vitro treatment of AEC with HNP and anti-HLA Ab further increased HBD2 expression (Table 1).
All of these results support our hypothesis that an immune response to mismatched donor HLA can activate AEC directly by ligation of HLA molecules and can also induce activation indirectly by production of defensins. Most of the Abs to donor HLA may also have the capacity to activate complement, which can induce infiltration of neutrophils and its activation.27, 28 In addition, studies from our laboratory8 and others18, 29 have shown that interleukin (IL)-17 may be important in the pathogenesis of BOS. It is likely that IL-17 produced after alloimmune responses can also play an important role in attracting neutrophils to the transplanted lungs.
The contribution of pro-inflammatory cytokines towards LTx rejection has been reported by us30, 31 and others.32, 33 HBD2 with CD14 can complex with Toll-like receptors in the bronchiolar epithelium and can activate the nuclear factor-κB pathway, leading to an increase in cytokine gene expression. HBD2 has been shown to induce recruitment of immature CD34+ dendritic cells and memory (CD4+/CD45RO+) T lymphocytes through CCR6.24 DiGiovine et al34 first reported increased levels of IL-8 in conjunction with elevated neutrophil numbers in BAL from patients with BOS and found that the IL-8 in BAL of patients with BOS was biologically active as a neutrophil chemoattractant. Thus, release of IL-8 by bronchial ECs locally may also significantly contribute in attracting and activating neutrophils in the allograft.
MPO activity is an indicator of oxidative stress due to neutrophil activation. MPO was significantly elevated in patients with BOS compared with patients after LTx without BOS and healthy controls.35 Riise et al36 observed the increase of MPO in BAL fluid preceding the clinical diagnosis of BOS by several months. Neutrophil MPO activity also induces the generation of reactive nitrogen species using nitrite and hydrogen peroxide or hypochlorite.37 This is in agreement with our results presented in Figure 4 demonstrating higher MPO activity in the lungs of mice treated with anti-MHC Abs.
In conclusion, results presented here show that anti-HLA Abs that develop after LTx can result in the activation of α-defensins from neutrophils, which can stimulate β-defensin production by ECs leading to cellular infiltration and inflammation. Chronic stimulation of ECs, both by defensins and anti-HLA that develops after LTx, can synergistically increase MPO activity, reactive oxygen and nitrogen species, pro-inflammatory cytokines, and growth factor production contributing toward BOS.
Disclosure statement
The authors thank Billie Glasscock for her assistance in preparing and submitting this manuscript.
None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
This work was supported in part by National Institutes of Health/National Heart, Lung and Blood Institute American Recovery and Reinvestment Act Award HL056643 to Thalachallour Mohanakumar, and by a ISHLT Research Fellowship to Deepti Saini.
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PII: S1053-2498(10)00423-7
doi:10.1016/j.healun.2010.05.036
© 2010 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.
Volume 29, Issue 12 , Pages 1330-1336, December 2010
