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

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.

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.

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.

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.

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 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.