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548: Donor Brain Death Prevents Tolerance Induction in Miniature Swine Recipients of MHC-Disparate Lung Allografts
The Journal of Heart and Lung Transplantation Volume 27, Number 2S
epithelial cells or in circulating WBCs between controls and treated animals. In TLR-4 siRNA pretreated rats, there was marked protection from injury as demonstrated by a marked reduction in vascular permeability (⬎90% reduction, p⬍0.001). Additionally, p38 and JNK phosphorylation was significantly attenuated in siRNA pretreated animals. Conclusions: Not only do these studies demonstrate the central role of TLR-4 in the initiation of injury in a model of lung ischemia reperfusion injury, but additionally the methods developed allow for specific molecular deletion in the alveolar macrophage in vivo, providing an excellent tool for understanding how the macrophage promotes injury with lung ischemia reperfusion. 546 The Contribution of Toll-Like Receptors (TLR) to Brain Death-Induced Donor Lung Inflammation A.J. Rostron,1 V.S. Avlonitis,1 D.M.W. Cork,1 J.H. Dark,1 J.A. Kirby,1 1 Applied Immunology and Transplant Research Group, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom Purpose: Brain death (BD) triggers an innate immune response prior to the recovery of lungs from the donor. This response is characterized by neutrophil activation and release of proinflammatory mediators. Although the magnitude of this response correlates with primary graft dysfunction, the causal mechanism is not well understood. In this study, TLR2 and 4 were desensitized by chronic ligand administration and the consequences for lung inflammation were studied after the induction of BD. Methods and Materials: Wistar rats (9 per group) received daily tolerogenic doses of either a TLR4 ligand (LPS) or TLR2 ligand (FSL1) by intraperitoneal injection; control animals received saline. After the induction of anesthesia, BD was induced in 6 rats per group by intracranial balloon inflation and the lungs were recovered from all animals. Neutrophil activation was assessed by flow cytometric quantification of CD11b and CD18 expression, ELISAs were used to measure cytokines in both serum and bronchoalveolar lavage (BAL) and a targeted qRT-PCR was used to quantify changes in expression of TLR-regulated genes. Results: A significant elevation in the cell surface expression of both CD11b and CD18 was observed after BD. However, after 3 and 5h this increase was significantly smaller in the LPS treated group (p⬍0.001); only the increase in CD18 was modulated in the group treated with FSL1 (p⬍0.001). The level of TNF␣ and CINC-1 in BAL and serum were also significantly lower in ligand-treated than control animals (p⬍0.05); this difference was more marked in those animals treated with LPS than FSL1. LPS pre-treatment prevented the upregulation of genes involved in both the MyD88-dependent and independent signaling pathways, whilst FSL1 did not down-regulate MyD88-independent genes such as IFN␥-induced protein 10 (CXCL10). Conclusions: BD can produce inflammation through TLR-mediated activation of both MyD88-dependent and independent signaling pathways. In order to optimize donor lung function it may be necessary to target proinflammatory molecules induced by both of these pathways. 547 Sensitization to Cardiac Myosin Induces Rejection of Cardiac Allografts in Miniature Swine M.J. Weiss,1 A.J. Meltzer,1 H. Sahara,1 B.R. Rosengard,1 M.E. Cochrane,1 J.K. Sayre,1 J.S. Allan,1 S.L. Houser,1 D.H. Sachs,1 G. Benichou,1 J.C. Madsen,1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA
Abstracts
S257
Purpose: We have previously shown that swine rejecting allogeneic cardiac allografts develop autoimmunity to cardiac myosin and develop circulating cardiac myosin-specific T-cells. In the present study, we evaluated the in vivo effects of cardiac myosin immunization on cardiac allograft survival in MHC-inbred miniature swine. Methods and Materials: All recipients underwent heterotopic cardiac transplants across a minor mismatch (MHC-identical) immunologic barrier, followed by 12 days of high-dose cyclosporine. Animals in the experimental group (n⫽3) were immunized with swine cardiac myosin on days – 42 and –21. Sensitization was evaluated by in vivo DTH and peptide proliferation assays. Animals were followed by daily palpation of cardiac grafts, weekly electrocardiography, and monthly biopsies. Results: Unimmunized control animals (n⫽4) all had viable, beating grafts when sacrificed approximately 6 months after surgery. In contrast, the two experimental animals that became sensitized to cardiac myosin following preoperative immunization rejected their grafts by 125 and 133 days. The third experimental recipient did not become sensitized to cardiac myosin and was sacrificed with a viable, beating graft at 6 months, similar to the controls. Conclusions: Sensitization to cardiac myosin promoted rejection in 2 of 3 cardiac allografts. These results support the hypothesis that indirect allorecognition of an organ-specific, autologous protein can promote the rejection of a vascularized organ, presumably through an autoimmune mechanism. 548 Donor Brain Death Prevents Tolerance Induction in Miniature Swine Recipients of MHC-Disparate Lung Allografts A.J. Meltzer,1 G.R. Veillette,1 A. Aoyama,1 M.E. Cochrane,1 S.L. Houser,1 J.C. Wain,1 J.C. Madsen,1 D.H. Sachs,1 J.S. Allan,1 B.R. Rosengard,1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA Purpose: A short course of high-dose tacrolimus induces tolerance to fully mismatched lung allografts procured from healthy MHC-inbred miniature swine. Here we investigate whether donor brain death affects tolerance induction. Methods and Materials: Transplants were performed across a fully disparate MHC barrier with 12 days of high-dose tacrolimus. Donor brain death was induced by intracranial inflation of a Foley catheter. Five hours later, an orthotopic left lung transplant was performed. Animals were followed with daily CBCs, weekly radiographs, and scheduled biopsies. In vitro studies (CML, MLR, ELISA, and qPCR) were performed. Results: We have previously reported that five of six tacrolimustreated recipients accepted MHC-disparate lung transplants for over a year. In stark contrast, two of the three recipients transplanted with lung allografts from brain dead donors rejected their grafts by POD 30. One of these animals also had a severe necrotizing pneumonia. The third animal has no evidence of rejection at POD 35. In vitro studies show persistent response to donor antigen by MLR. Serum ELISA demonstrated a significant increase in concentrations of IL-1 (p⫽0.041), TNF-alpha (p⫽0.0005), and IL-10 (p⫽0.0132) after 5 hours of brain death as compared to baseline. IL-4, TGF-beta, and IFN-gamma concentrations were unchanged. Quantitative PCR of graft lung-derived cDNA demonstrated a two-fold upregulation of IFN-gamma expression after brain death. IL-1 expression in lung also increased significantly (6 to 20 fold). However, there was no change in IL-4, TNF-alpha, or MHC class I expression after brain death induction. Conclusions: Donor brain death appears to abrogate tolerance induction in a swine model of orthotopic lung transplantation and is associated with upregulation of IL-1 and IFN-gamma in the graft. Serum cytokine concentrations, including IL-1, TNF-alpha, and IL-10,
S258
Abstracts
are also increased in the brain dead donor. Understanding this phenomenon may help facilitate tolerance induction in human transplantation 549 The Role of TLR2 in Pulmonary Endothelial and Epithelial Cells in Lung Ischemia Reperfusion Injury P.S. Wolf,1 H.E. Merry,1 J. Keech,1 M.S. Mulligan,1 1Cardiothoracic Surgery, University of Washington, Seattle, WA Purpose: TLR4 dependent signaling has been implicated in mediating ischemia reperfusion injury (IRI) in several vascular beds.We have shown that TLR4 deletion in pulmonary endothelial cells (PAEC) and epithelial cells (T2P) did not alter their inflammatory response to oxidative stress.An alternative innate immune receptor is likely responsible.In lung IRI,ERK1/2,but not SAPK,is activated in PAEC and T2P. Conversely,SAPK activation localizes only to the alveolar macrophage (AM) where ERK1/2 activation does not occur.A receptor responsive to oxidative stress and proximal to ERK1/2 activation has yet to be identified.Studies of renal IRI have indicated a role for TLR2 signaling in endothelial cells.We hypothesized that signaling in non-AM cell types is TLR2 dependent,and that
The Journal of Heart and Lung Transplantation February 2008
prestimulation of TLR2 in these cells prior to lung IR would be protective,similar to that seen with TLR4 mediated preconditioning with LPS. Methods and Materials: Rats underwent left lung hilar occlusion for 90 minutes with up to 4 hours of reperfusion.Experimental animals were pretreated 24 hours prior with a TLR2 agonist,Pam(3)Cys.Injury was assessed via lung vascular permeability and leukocyte content.Western blotting assessed MAPK activation.Cultured PAEC and T2P had TLR2 deleted with siRNA and were subjected to 90 minutes of hypoxia and up to 4 hours of reoxygenation.Injury was assessed by cytokine production and MAPK activation. Results: Pam(3)Cys pretreated animals demonstrated reductions in all injury parameters.TLR2 mediated preconditioning correlated with a reduction in ERK1/2,but not SAPK, phosphorylation.TLR2 deletion in vitro reduced ERK1/2 phosphorylation and cytokine secretion. Conclusions: TLR2 deletion eliminated inflammatory signaling in PAEC and T2P in response to oxidative stress.TLR2 mediated preconditioning was protective from subsequent IRI.This appears to occur via modulation of signaling in non-AM cells as ERK1/2,but not SAPK,phosphorylation was reduced.This provides evidence that TLR2 signaling is responsible for the inflammatory response of non-AM cells in LIRI.
epithelial cells or in circulating WBCs between controls and treated animals. In TLR-4 siRNA pretreated rats, there was marked protection from injury as demonstrated by a marked reduction in vascular permeability (⬎90% reduction, p⬍0.001). Additionally, p38 and JNK phosphorylation was significantly attenuated in siRNA pretreated animals. Conclusions: Not only do these studies demonstrate the central role of TLR-4 in the initiation of injury in a model of lung ischemia reperfusion injury, but additionally the methods developed allow for specific molecular deletion in the alveolar macrophage in vivo, providing an excellent tool for understanding how the macrophage promotes injury with lung ischemia reperfusion. 546 The Contribution of Toll-Like Receptors (TLR) to Brain Death-Induced Donor Lung Inflammation A.J. Rostron,1 V.S. Avlonitis,1 D.M.W. Cork,1 J.H. Dark,1 J.A. Kirby,1 1 Applied Immunology and Transplant Research Group, Newcastle University, Newcastle upon Tyne, Tyne and Wear, United Kingdom Purpose: Brain death (BD) triggers an innate immune response prior to the recovery of lungs from the donor. This response is characterized by neutrophil activation and release of proinflammatory mediators. Although the magnitude of this response correlates with primary graft dysfunction, the causal mechanism is not well understood. In this study, TLR2 and 4 were desensitized by chronic ligand administration and the consequences for lung inflammation were studied after the induction of BD. Methods and Materials: Wistar rats (9 per group) received daily tolerogenic doses of either a TLR4 ligand (LPS) or TLR2 ligand (FSL1) by intraperitoneal injection; control animals received saline. After the induction of anesthesia, BD was induced in 6 rats per group by intracranial balloon inflation and the lungs were recovered from all animals. Neutrophil activation was assessed by flow cytometric quantification of CD11b and CD18 expression, ELISAs were used to measure cytokines in both serum and bronchoalveolar lavage (BAL) and a targeted qRT-PCR was used to quantify changes in expression of TLR-regulated genes. Results: A significant elevation in the cell surface expression of both CD11b and CD18 was observed after BD. However, after 3 and 5h this increase was significantly smaller in the LPS treated group (p⬍0.001); only the increase in CD18 was modulated in the group treated with FSL1 (p⬍0.001). The level of TNF␣ and CINC-1 in BAL and serum were also significantly lower in ligand-treated than control animals (p⬍0.05); this difference was more marked in those animals treated with LPS than FSL1. LPS pre-treatment prevented the upregulation of genes involved in both the MyD88-dependent and independent signaling pathways, whilst FSL1 did not down-regulate MyD88-independent genes such as IFN␥-induced protein 10 (CXCL10). Conclusions: BD can produce inflammation through TLR-mediated activation of both MyD88-dependent and independent signaling pathways. In order to optimize donor lung function it may be necessary to target proinflammatory molecules induced by both of these pathways. 547 Sensitization to Cardiac Myosin Induces Rejection of Cardiac Allografts in Miniature Swine M.J. Weiss,1 A.J. Meltzer,1 H. Sahara,1 B.R. Rosengard,1 M.E. Cochrane,1 J.K. Sayre,1 J.S. Allan,1 S.L. Houser,1 D.H. Sachs,1 G. Benichou,1 J.C. Madsen,1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA
Abstracts
S257
Purpose: We have previously shown that swine rejecting allogeneic cardiac allografts develop autoimmunity to cardiac myosin and develop circulating cardiac myosin-specific T-cells. In the present study, we evaluated the in vivo effects of cardiac myosin immunization on cardiac allograft survival in MHC-inbred miniature swine. Methods and Materials: All recipients underwent heterotopic cardiac transplants across a minor mismatch (MHC-identical) immunologic barrier, followed by 12 days of high-dose cyclosporine. Animals in the experimental group (n⫽3) were immunized with swine cardiac myosin on days – 42 and –21. Sensitization was evaluated by in vivo DTH and peptide proliferation assays. Animals were followed by daily palpation of cardiac grafts, weekly electrocardiography, and monthly biopsies. Results: Unimmunized control animals (n⫽4) all had viable, beating grafts when sacrificed approximately 6 months after surgery. In contrast, the two experimental animals that became sensitized to cardiac myosin following preoperative immunization rejected their grafts by 125 and 133 days. The third experimental recipient did not become sensitized to cardiac myosin and was sacrificed with a viable, beating graft at 6 months, similar to the controls. Conclusions: Sensitization to cardiac myosin promoted rejection in 2 of 3 cardiac allografts. These results support the hypothesis that indirect allorecognition of an organ-specific, autologous protein can promote the rejection of a vascularized organ, presumably through an autoimmune mechanism. 548 Donor Brain Death Prevents Tolerance Induction in Miniature Swine Recipients of MHC-Disparate Lung Allografts A.J. Meltzer,1 G.R. Veillette,1 A. Aoyama,1 M.E. Cochrane,1 S.L. Houser,1 J.C. Wain,1 J.C. Madsen,1 D.H. Sachs,1 J.S. Allan,1 B.R. Rosengard,1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA Purpose: A short course of high-dose tacrolimus induces tolerance to fully mismatched lung allografts procured from healthy MHC-inbred miniature swine. Here we investigate whether donor brain death affects tolerance induction. Methods and Materials: Transplants were performed across a fully disparate MHC barrier with 12 days of high-dose tacrolimus. Donor brain death was induced by intracranial inflation of a Foley catheter. Five hours later, an orthotopic left lung transplant was performed. Animals were followed with daily CBCs, weekly radiographs, and scheduled biopsies. In vitro studies (CML, MLR, ELISA, and qPCR) were performed. Results: We have previously reported that five of six tacrolimustreated recipients accepted MHC-disparate lung transplants for over a year. In stark contrast, two of the three recipients transplanted with lung allografts from brain dead donors rejected their grafts by POD 30. One of these animals also had a severe necrotizing pneumonia. The third animal has no evidence of rejection at POD 35. In vitro studies show persistent response to donor antigen by MLR. Serum ELISA demonstrated a significant increase in concentrations of IL-1 (p⫽0.041), TNF-alpha (p⫽0.0005), and IL-10 (p⫽0.0132) after 5 hours of brain death as compared to baseline. IL-4, TGF-beta, and IFN-gamma concentrations were unchanged. Quantitative PCR of graft lung-derived cDNA demonstrated a two-fold upregulation of IFN-gamma expression after brain death. IL-1 expression in lung also increased significantly (6 to 20 fold). However, there was no change in IL-4, TNF-alpha, or MHC class I expression after brain death induction. Conclusions: Donor brain death appears to abrogate tolerance induction in a swine model of orthotopic lung transplantation and is associated with upregulation of IL-1 and IFN-gamma in the graft. Serum cytokine concentrations, including IL-1, TNF-alpha, and IL-10,
S258
Abstracts
are also increased in the brain dead donor. Understanding this phenomenon may help facilitate tolerance induction in human transplantation 549 The Role of TLR2 in Pulmonary Endothelial and Epithelial Cells in Lung Ischemia Reperfusion Injury P.S. Wolf,1 H.E. Merry,1 J. Keech,1 M.S. Mulligan,1 1Cardiothoracic Surgery, University of Washington, Seattle, WA Purpose: TLR4 dependent signaling has been implicated in mediating ischemia reperfusion injury (IRI) in several vascular beds.We have shown that TLR4 deletion in pulmonary endothelial cells (PAEC) and epithelial cells (T2P) did not alter their inflammatory response to oxidative stress.An alternative innate immune receptor is likely responsible.In lung IRI,ERK1/2,but not SAPK,is activated in PAEC and T2P. Conversely,SAPK activation localizes only to the alveolar macrophage (AM) where ERK1/2 activation does not occur.A receptor responsive to oxidative stress and proximal to ERK1/2 activation has yet to be identified.Studies of renal IRI have indicated a role for TLR2 signaling in endothelial cells.We hypothesized that signaling in non-AM cell types is TLR2 dependent,and that
The Journal of Heart and Lung Transplantation February 2008
prestimulation of TLR2 in these cells prior to lung IR would be protective,similar to that seen with TLR4 mediated preconditioning with LPS. Methods and Materials: Rats underwent left lung hilar occlusion for 90 minutes with up to 4 hours of reperfusion.Experimental animals were pretreated 24 hours prior with a TLR2 agonist,Pam(3)Cys.Injury was assessed via lung vascular permeability and leukocyte content.Western blotting assessed MAPK activation.Cultured PAEC and T2P had TLR2 deleted with siRNA and were subjected to 90 minutes of hypoxia and up to 4 hours of reoxygenation.Injury was assessed by cytokine production and MAPK activation. Results: Pam(3)Cys pretreated animals demonstrated reductions in all injury parameters.TLR2 mediated preconditioning correlated with a reduction in ERK1/2,but not SAPK, phosphorylation.TLR2 deletion in vitro reduced ERK1/2 phosphorylation and cytokine secretion. Conclusions: TLR2 deletion eliminated inflammatory signaling in PAEC and T2P in response to oxidative stress.TLR2 mediated preconditioning was protective from subsequent IRI.This appears to occur via modulation of signaling in non-AM cells as ERK1/2,but not SAPK,phosphorylation was reduced.This provides evidence that TLR2 signaling is responsible for the inflammatory response of non-AM cells in LIRI.