竹中 政史

BACKGROUND: The goal of this study was to define the role of T-cell sub-sets in the pathogenesis of autoimmunity-induced obliterative airway disease by passive transfer of CD8+ or CD4+ T cells. METHODS: Antibodies to major histocompatibility complex (MHC) class I were administered intrabronchially into C57BL/6 animals. Lungs were analyzed by histopathology and immunohistochemistry. The CD8+ and CD4+ T-cell sub-sets were purified from the lung-infiltrating cells and intrabronchially transferred. Frequency of cells secreting interleukin-17, interferon-gamma, or interleukin-10 to self-antigens was enumerated by enzyme-linked immunospot assay. Myeloperoxidase and antibodies to self-antigens were determined by enzyme-linked immunosorbent assay. Cytokine and growth factor expression was determined by quantitative reverse-transcription polymerase chain reaction. RESULTS: Passive transfer of lung-infiltrating CD8 T cells isolated after anti-MHC class 1 administration, along with sub-optimal dose, induced significantly higher cellular infiltration (89.3% +/- 7.9% vs 62.8% +/- 10.1%, p < 0.05) vs the CD4 transfer group. Further, passive transfer of CD8 cells resulted in infiltration of neutrophils and macrophages, suggesting early injury response. In contrast, passive transfer of CD4+ T cells induced a significantly higher degree of luminal occlusion (29.3% +/- 5.6% vs 8.6 +/- 2.5%, p < 0.05) and fibrosis (54.4% +/- 9.3% vs 10.2% +/- 2.4%, p < 0.05) vs the CD8 group and B-cell infiltration, leading to immune responses to lung-associated self-antigens and fibrosis. CONCLUSION: Ligation of MHC molecules by its specific antibodies induced early injury with neutrophils, macrophages, and CD8 T cells, which leads to exposure of cryptic self-antigens and their presentation by the infiltrating CD4+ T cells and B cells, leading to the development of immune responses to self-antigens and culminating in obliterative airway disease. J Heart Lung Transplant 2013;32:714-722 (C) 2013 International Society for Heart and Lung Transplantation. All rights reserved.

Background. The IL-17 axis is implicated in pathogenesis of chronic rejection after human lung transplantation. Using a murine model of obliterative airway disease (OAD), we recently demonstrated that Abs to MHC class I antigens can induce immune responses to self-antigens that contributes to immunopathogenesis of chronic rejection. Using a murine model of OAD, we determined the role of IL-17 family members in induction of autoimmunity leading to OAD after ligation of MHC class I. Methods. Anti-MHC class I or control antibodies (Abs) were administered intrabronchially to wild-type (WT) and IL-17a knock out (IL-17A-/-) C57BL/6. Results. By day 30, anti-MHC I administered endobronchially in IL-17A-/- mice demonstrated significant reduction in cellular infiltration, a 36.8% reduction in CD4(+) T cells, 62.7% in CD11b(+) macrophages, 37.5% in degree of fibrosis, 1.94 fold and 2.17 fold decrease in anti-KAT and anti-Col-V, respectively, when compared with wild-type mice. Analysis of lung infiltrating cells in anti-MHC I WT revealed increase in IL-17A (KAT: 92+21, Col-V:103+19spm) and IL-17F (KAT:5.03%, Col-V:2.75%) secreting CD4+ T cells. However, administration of anti-MHC I in IL-17A-/- demonstrated increase only in IL-17F for KAT (13.70%) and Col-V (7.08%). Anti-IL-17(A-F) mAb administration after anti-MHC I abrogated OAD in both WT and IL-17A-/-. Conclusion. Our findings indicate that IL-17A and IL-17F secreted by CD4+Th17 cells specific to lung self-antigens are critical mediators of autoimmunity leading to the pathogenesis of OAD.

BACKGROUND: The role of non-complement activating antibodies (ncAbs) to mismatched donor human leukocyte antigen (HLA) in the pathogenesis of chronic lung rejection is not known. We used a murine model of obliterative airway disease (OAD) induced by Abs to major histocompatibility major histocompatibility complex (MHC) class I and serum from donor-specific Abs developed in human lung transplant (LTx) recipients to test the role of ncAbs in the development of OAD and bronchiolitis obliterans syndrome (BOS). METHODS: Anti-MHC ncAbs were administered intrabronchially in B.10 mice or in C3 knockout (C3KO) mice. Lungs were analyzed by histopathology. Lymphocytes secreting interleukin (IL)-17, interferon-gamma, or IL-10 to collagen V and K-alpha 1 tubulin (K alpha 1T) were enumerated by enzyme-linked immunospot assay. Serum antibodies to collagen V and K alpha 1T were determined by enzyme-linked immunosorbent assay. Cytokine and growth factor expression in lungs was determined by real-time polymerase chain reaction. Donor-specific Abs from patients with BOS and control BOS-negative LTx recipients were analyzed by C1q assay. RESULTS: Administration of ncAbs in B.10 mice or C3KO resulted in OAD lesions. There were significant increases in IL-17- and interferon-gamma-secreting cells to collagen V and K alpha 1T, along with serum Abs to these antigens. There was also augmented expression of monocyte chemotactic protein-1, IL-6, IL-1 beta, vascular endothelial growth factor, transforming growth factor-beta, and fibroblastic growth factor in mice administered ncAbs by Day 3. Among 5 LTx recipients with BOS, only 1 had C1q binding donor-specific Abs. CONCLUSION: Complement activation by Abs to MHC class I is not required for development of OAD and human BOS. Therefore, anti-MHC binding to epithelial and endothelial cells can directly activate pro-fibrotic and pro-inflammatory cascades leading to immune response to self-antigens and chronic rejection. J Heart Lung Transplant 2012; 31: 1214-1222 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.

The molecular mechanisms leading to the development of chronic lung allograft dysfunction following de novo development of antibodies to mismatched donor MHC remain undefined. We demonstrated that intrabronchial administration of antibodies to MHC class I resulted in induction of both innate and adaptive cellular immune responses characterized by a predominance of Th17 specific to lung associated self-antigens Ka1-tubulin and Collagen-V leading to the development of obliterative airway lesions (OAD), correlate of chronic rejection following human lung transplantation. To determine the role of regulatory T cells (Treg) in the pathogenesis of OAD, we administered anti-MHC class I to mice, in which Treg were depleted by conditional ablation of FoxP3+cells. Under this condition, we observed a threefold increase in pulmonary cellular infiltration, luminal occlusion and fibrous deposition when compared anti-MHC class I Ab administered mice maintaining FoxP3. OAD lesions were accompanied with enhanced accumulation of neutrophils along with self-antigen-specific Th17 and humoral responses. However, IL-17-blockade or adoptive transfer of Treg abrogated OAD. We conclude that Treg exerts a suppressive effect on anti-MHC induced IL-8-mediated neutrophil infiltration and innate immune responses that leads to inhibition of Th17 immune responses to lung associated self-antigens which is critical for development of OAD.

Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- alpha 1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from M Phi 1 (F4/80(+)CD11c(+)) to M Phi 2 (F4/80(+)CD206(+)) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naive animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from M Phi 1 (F4/80(+)CD11c(+)) to M Phi 2 (F4/80(+)CD206(+)) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.

Using a murine model, we demonstrated that endobronchial administration of antibodies (Abs) to major histocompatibility complex (MHC) class I results in cellular infiltration, epithelial metaplasia, fibrosis and obstruction of the small airways (obliterative airway disease [OAD]) mediated predominantly by Th17 responses to self-antigens. This resembles bronchiolitis obliterans syndrome developed following human lung transplantation. Since B cells play a crucial role in induction of autoimmune responses, we defined the role of B cells and its antigen presenting properties in induction of OAD in this study. Anti-MHC class I was administered endobronchially in B-/- and wild-type mice. In contrast to wild type, B-/- animals did not demonstrate cellular infiltration, epithelial metaplasia and obstruction of airways following anti-MHC. Frequency of K-a1 tubulin and CollagenV-specific IL-17 cells was significantly decreased in B-/- mice. As expected, Abs against self-antigens and germinal center formation were not developed in B-/- mice. Thus, we conclude that B cells and its antigen presenting capacity play an important role in induction of immune responses to self-antigens and immunopathogenesis of OAD following the administration of anti-MHC. Therefore, strategies to block B-cell and its antigen presenting functions should be considered for preventing the development of chronic rejection.

BACKGROUND: Alloimmune-induced immune responses to self-antigens are involved in the development of chronic lung allograft rejection. Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs) have been shown to modulate autoimmune diseases. This study investigated the effect of modulation of the renin angiotensin aldosterone system (RAAS) a murine model of obliterative airways disease (OAD). METHODS: Major histocompatibility complex (MHC) class I antibodies were administered intrabronchially to C57BI/6 mice on Days 1, 2, 3, and 6, and weekly thereafter. ACEI/ARB (10 mg/kg/day) were administered in water 5 days before antibody administration. Antibodies were analyzed by enzyme-linked immunosorbent assay, cytokines by Luminex, Th-frequency by enzyme-linked immunosorbent spot, and transcription factors by Western blotting and real-time polymerase chain reaction. RESULTS: Significant decreases (50%-70%) in airway lesions and fibrous deposition were noted in lungs at Day 30 in the animals administered ACEI and ARB vs controls. Antibody concentrations to self-antigens also decreased from 14 +/- 21 to 62 +/- 18 mu g/ml for collagen V and from 263 +/- 43 to 84 +/- 28 mu g/ml for K-alpha 1 tubulin. Th-precursor frequency and cytokine analysis showed increased interleukin (IL)-10 (3-fold increase) and decreased levels of IL-6 (3.4-fold) and IL-17 (4-fold decrease; p < 0.05) in ACEI and ARB groups. There was also messenger RNA level downregulation of tumor necrosis factor-alpha (8.6-fold) and p38/mitogen-activated protein (MAP)kinase (3.1-fold) in the treatment groups. CONCLUSIONS: Our results demonstrate that modulation of RAAS leads to downregulation of IL-17 through tumor necrosis factor-alpha dependant IL-6 through p38/MAPKinase pathway and thus abrogation of anti-MHC induced OAD. J Heart Lung Transplant 2012;31:419 26 (C) 2012 International Society for Heart and Lung Transplantation. All rights reserved.