Ingraft chimerism in lung transplantation--a study in a porcine model of obliterative bronchiolitis.

BACKGROUND: Bronchial epithelium is a target of the alloimmune response in lung transplantation, and intact epithelium may protect allografts from rejection and obliterative bronchiolitis (OB). Herein we study the influence of chimerism on bronchial epithelium and OB development in pigs. METHODS: A total of 54 immunosuppressed and unimmunosuppressed bronchial allografts were serially obtained 2-90 days after transplantation. Histology (H&E) was assessed and the fluorescence in situ hybridization (FISH) method for Y chromosomes using pig-specific DNA-label was used to detect recipient derived cells in graft epithelium and bronchial wall, and donor cell migration to recipient organs. Ingraft chimerism was studied by using male recipients with female donors, whereas donor cell migration to recipient organs was studied using female recipients with male donors. RESULTS: Early appearance of recipient-derived cells in the airway epithelium appeared predictive of epithelial destruction (R=0.610-0.671 and p<0.05) and of obliteration of the bronchial lumen (R=0.698 and p<0.01). All allografts with preserved epithelium showed epithelial chimerism throughout the follow-up. Antirejection medication did not prevent, but delayed the appearance of Y chromosome positive cells in the epithelium (p<0.05), or bronchial wall (p<0.05). CONCLUSIONS: In this study we demonstrate that early appearance of Y chromosomes in the airway epithelium predicts features characteristic of OB. Chimerism occurred in all allografts, including those without features of OB. Therefore we suggest that ingraft chimerism may be a mechanism involved in the repair of alloimmune-mediated tissue injury after transplantation.

Local C-reactive protein expression in obliterative lesions and the bronchial wall in posttransplant obliterative bronchiolitis.

The local immunoreactivity of C-reactive protein (CRP) was studied in a heterotopic porcine model of posttranplant obliterative bronchiolitis (OB). Bronchial allografts and control autografts were examined serially 2-28 days after subcutaneous transplantation. The autografts stayed patent. In the allografts, proliferation of inflammatory cells (P < .0001) and fibroblasts (P = .02) resulted in occlusion of the bronchial lumens (P < .01). Influx of CD4+ (P < .001) and CD8+ (P < .0001) cells demonstrated allograft immune response. CRP positivity simultaneously increased in the bronchial walls (P < .01), in macrophages, myofibroblasts, and endothelial cells. Local CRP was predictive of features characteristic of OB (R = 0.456-0.879, P < .05-P < .0001). Early obliterative lesions also showed CRP positivity, but not mature, collagen-rich obliterative plugs (P < .05). During OB development, CRP is localized in inflammatory cells, myofibroblasts and endothelial cells probably as a part of the local inflammatory response.

Epithelial tenascin predicts obliterative airway disease.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis and airway obliteration result in remodeling of terminal bronchi in post-transplant obliterative bronchiolitis. Tenascin as an extracellular matrix glycoprotein is expressed in several remodeling processes. METHODS: Heterotopic bronchial allografts of pigs were studied to assess tenascin expression during development of post-transplant obliterative bronchiolitis. A total of 157 allografts or autograft controls were serially obtained 2 to 28 days after transplantation and processed for histology and immunocytochemistry for tenascin, CD4, CD8 and macrophages. Epithelial tenascin index was calculated by multiplying the percentage of positive cells by the grade of tenascin intensity (1 to 3). RESULTS: Epithelial tenascin expression occurred during the initial ischemic damage to the respiratory epithelium. After partial recovery and before total epithelial loss and subsequent airway obliteration, tenascin expression peaked in allografts (p < 0.001). Epithelial tenascin index on Day 7 was predictive of subsequent epithelial damage, bronchial wall inflammation and the number of (CD4(+) and CD8(+)) cells, fibroproliferation, and obliteration of the bronchial lumen (R > or = 0.47, p < or = 0.01). Tenascin expression in the bronchial wall was more intense in allografts (p < 0.001), paralleling proliferation of fibroblasts and influx of inflammatory cells, and was predictive of inflammatory alterations also in the early obliterative lesions (R > or = 0.45, p < 0.05). Expression decreased during maturation of fibrosis (p < 0.05). CONCLUSIONS: Epithelial tenascin was predictive of features observed in post-transplant obliterative bronchiolitis, demonstrating a role for tenascin in the development of obliterative bronchiolitis. Tenascin may have relevant properties in serving as a clinical marker for early obliterative bronchiolitis.

Platelet-derived growth factor, transforming growth factor-beta, and connective tissue growth factor in a porcine bronchial model of obliterative bronchiolitis.

The expression of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, and connective tissue growth factor (CTGF) and the effect of imatinib, an agent inhibiting PDGF receptors, were assessed in a porcine bronchial transplantation model of obliterative bronchiolitis (OB). Up-regulation of PDGF-A, PDGF receptors alpha and beta, and TGF-beta expression occurred in allografts, whereas PDGF-B and CTGF expression was similar in allo- and autografts. Imatinib modified the inflammatory responses and expression patterns of PDGF-A and PDGF receptors. This study further confirms PDGF and TGF-beta as mediators of OB and supports the concept of the importance of the pathways signaled through PDGF receptors in post-transplant OB.

Immune cells in a heterotopic lamb-to-pig bronchial xenograft model.

We developed our porcine model to elucidate the cellular rejection mechanisms of xenografts. Bronchial segments from a donor lamb were implanted into domestic pigs. The immunosuppressive regimens consisted of no immusuppression, or of daily oral cyclosporine A (CsA) 15 mg/kg, or of everolimus, 1.5 mg/kg, or of both. Implants were serially harvested during 17 days. Epithelial damage and obliteration were graded histologically, followed by a count of CD4+, CD8+, MHC class II-expressing cells, and macrophages. Furthermore, we studied the pharmocokinetics of everolismus. Epithelial damage preceded luminal obliteration, which was eventually total, except when both drugs had been given. In xenografts, an influx of cells with CD8+ cells dominating peaked on day 9, thereafter declining, except in the combination drug group. There, the immunological reaction was delayed and blunted, with CD4+ cells dominating. More macrophages appeared in xenografts than in allografts except with the combination CsA and everolimus. A dose of 1.5 mg/kg everolimus yields adequate blood concentrations for porcine studies. In this xenograft model, chronic rejection appears to be caused by an immune response to the graft, but it is more short-lived than the response in allografts. The combination of CsA and everolimus was able to blunt the response and delay the subsequent obliteration.

Matrix metalloproteinase induction in post-transplant obliterative bronchiolitis.

BACKGROUND: Epithelial cell injury, inflammation, fibrosis, and airway obliteration are associated in post-transplant obliterative bronchiolitis. Fibrosis is a consequence of fibroblastic activity and of collagen deposition after disturbances in the balance of protein formation and degradation. Proteolytic enzymes such as the matrix metalloproteinases mediate degradation. To assess matrix metalloproteinases during obliterative bronchiolitis development, we studied porcine, heterotopic bronchial allografts. METHODS: A total of 119 allografts or autografts were harvested serially at 3 to 60 days after transplantation and processed for histology and in situ hybridization for matrix metalloproteinases 2 and 9. Immunocytochemistry for vimentin and alpha-smooth-muscle-cell actin was performed with specific antibodies. RESULTS: Implants had initial ischemic injury to airway epithelium and to the bronchial wall. Recovery was rapid in autografts and in immunosuppressed allografts. In matrix metalloproteinase-2 mRNA activity in fibroblasts, correlation with endothelial expression and expression in macrophages occurred during intense fibroproliferation. We observed intense matrix metalloproteinase-9 positivity during onset of inflammation and fibroproliferation in endothelial cells (p < 0.01), fibroblasts (p < 0.05), macrophages (p < 0.05), and lymphocytes (p < 0.05). Matrix metalloproteinase-9 mRNA activity in fibroblasts correlated with that in endothelial and inflammatory cells and also proved predictive of early obliteration. CONCLUSIONS: Matrix metalloproteinase-2, and especially matrix metalloproteinase-9, gene activity was associated with onset of inflammation and fibroblastic proliferation in allografts, predicting early obliteration. Although this may be the case in the model described, its role in human-allograft post-transplant obliterative bronchiolitis requires further supportive data.

Cyclooxygenase-2 expression in experimental post-transplant obliterative bronchiolitis.

Epithelial cell injury, inflammation, progressive fibrosis, and airway obliteration are histological features of post-transplant obliterative bronchiolitis (OB). Cyclooxygenase (COX)-2 is expressed in acute and chronic inflammatory responses. Our aim was to elucidate the possible role of COX-2 in post-transplant OB by using a heterotopic bronchial porcine model. Bronchial allografts from non-related donors were transplanted subcutaneously into 24 random-bred domestic pigs, each weighing about 20 kg. Groups studied had grafts, non-treated allografts, allografts given cyclosporine A (CsA), methylprednisolone (MP), and azathioprine (Aza), and allografts given CsA, MP, and everolimus. Grafts were serially harvested during a follow-up period of 21 days for histology (H&E) and immunohistochemistry. Immunostaining was performed with monoclonal IgG against human COX-2 peptide, and histological alterations and immunohistochemical positivity were graded on a scale from 0 to 5. Epithelial COX-2 index was calculated by multiplying the percentage of positive cells by grade of epithelial COX-2 intensity. Ischaemic epithelial loss, evident in all implants, recovered rapidly in autografts, and bronchi remained patent. Epithelial loss in non-treated allografts preceded fibroblast proliferation, resulting in total luminal obliteration. In CsA-, MP-, and Aza-treated allografts epithelial destruction and luminal obliteration were delayed, and these were prevented in CsA-, MP-, and everolimus-treated allografts. COX-2 expression due to operative ischaemia was evident in all implants on day 2. Thereafter, the epithelial COX-2 index preceded epithelial injury and obliteration. During the inflammatory response and fibroblast proliferation, COX-2 expression occurred in macrophages and fibroblasts. In conclusion, in the early stage of OB development, COX-2 induction occurred in airway epithelial cells prior to luminal obliteration. In addition, the observation that COX-2 expression in macrophages and fibroblasts paralleled the onset of inflammation and fibroblast proliferation indicates a role in OB development, but the causal relationships need further study.

Epithelial apoptosis in experimental obliterative airway disease after lung transplantation.

BACKGROUND: Epithelial damage is an important feature in the pathogenesis of obliterative airway disease. We investigated the extent of epithelial apoptosis in this process in pig bronchial allografts. METHODS: The bronchial grafts (total, n = 200) were placed subcutaneously into recipients. Three allograft groups were formed: the first group had no immunosuppression therapy; the second received triple therapy with 10 mg/kg/day cyclosporine, 2 mg/kg/day azathioprine, and 20 mg/day methylprednisolone; and the third was given triple therapy in which azathioprine was replaced with 1.5 mg/kg/day everolimus (40-O-[2-hydroxyethyl]-rapamycin). The fourth group, which had allograft and autograft implants, received only 1.5 mg/kg/day everolimus. The implants were serially removed during 3 months of follow-up. We evaluated graft histology and analyzed the apoptotic index percentage (apoptotic cells / total number of cells) of the bronchial epithelium using in situ 3'-end labeling of apoptotic DNA. RESULTS: Epithelial destruction and subsequent obliteration of the bronchial lumen were complete by Day 28 in non-treated allografts and in most allografts with inadequate immunosuppression to prevent these changes (those treated with cyclosporine, azathioprine, and methylprednisolone and those treated with everolimus only). The apoptotic indexes of the epithelium were high (>1% of the cells were apoptotic) and increased with concomitant epithelial destruction. In allografts with adequate immunosuppression to prevent epithelial destruction (those treated with cyclosporine, everolimus, and methylprednisolone) and in autografts, after initial damage, well-pre-served epithelium was maintained with low apoptotic indexes (<1% of the cells apoptotic). CONCLUSIONS: Apoptotic activity increased with progressing epithelial damage preceding bronchial obliteration. Our results give further evidence that apoptotic death of epithelial cells is an important mechanism in events that lead to graft deterioration in obliterative bronchiolitis after lung transplantation.

Tumor necrosis factor-alpha in a porcine bronchial model of obliterative bronchiolitis.

BACKGROUND: In posttransplant obliterative bronchiolitis (OB), the major pathologic features are inflammation, epithelial cell injury, fibrosis, and obliteration of the small airways. Tumor necrosis factor (TNF)-alpha is a cytokine known to mediate and augment the inflammatory reaction and to enhance fibroblast proliferation. We assessed the role of TNF-alpha in the development of OB in our heterotopic porcine bronchial transplantation model. METHODS: Three groups were formed: autografts, nontreated allografts, and allografts treated with preoperative anti-TNF-alpha monoclonal antibody (infliximab) infusion. The implants were harvested on days 2, 4, 7, 11, 14, 21, and 28 for histologic and immunohistochemical analysis. RESULTS: TNF-alpha inhibition reduced inflammation, rate of epithelial loss, fibrosis, and obliteration early in the development of OB. In the epithelium, the numbers of TNF-alpha-positive epithelial and inflammatory cells and macrophages were significantly lower in treated than in nontreated allografts on day 4; furthermore, in the epithelium and in the bronchial wall, invasion of CD8+ lymphocytes was significantly decreased during the first week. CONCLUSIONS: These results indicate that TNF-alpha promotes the development of OB, and inhibition of TNF-alpha may prove beneficial in a clinical setting.