Synergistic effect of antibodies to human leukocyte antigens and defensins in pathogenesis of bronchiolitis obliterans syndrome after human lung transplantation

Abstract

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.

Figure 1. Defensin levels are higher in DSA+ BOS+ (11/21) patients

ELISA was done to measure HNP (1-3) for BOS+ LTx recipients. HNP (1-3) levels were higher in the BOS+ recipients who also had DSA compared to BOS+ recipients who did not develop DSA, (**) p<0.05.

Figure 2. Increased HBD2 levels in response to HNP1 or HNP2 treatment compared to untreated cells

SAEC were treated with HNP1 or HNP2 for 24 hrs. HBD2 production was measured by ELISA in the culture supernatant.

Figure 3. Increased HBD2 levels in response to various concentrations of anti-HLA class I Ab as compared to isotype control Ab

SAEC were treated with various concentrations of anti-HLA class I Ab as mentioned in methods for 24 hrs. HBD2 production was measured by ELISA in the culture supernatant.

Figure 4. Increased neutrophil activity (MPO assay) in lung lysates of mice treated with anti-MHC Abs

Balb/c mice were administered anti-MHC (H2kd) Abs endobronchially as described in methods. Neutrophil activity at day 3, 5, 9, 11 and 15 post-Ab administration was measured in lung lysates of mice treated with anti-MHC Abs using MPO assay and compared to mice treated with isotype control Abs.

Figure 5. Increased neutrophils (MPO staining) in lungs of mice treated with anti-MHC Abs

Frozen sections of lungs from mice treated with anti-MHC Abs at day 5 were stained for MPO and neutrophil infiltration was compared to mice administered isotype control Abs.

Figure 6. α and β-defensin levels are higher in BAL of mice administered anti-MHC Abs

BAL fluid of mice administered with anti-MHC Abs was tested for α and β-defensins by western blotting and compared to mice treated with isotype control Abs.