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The impact of CD4 and CD8 transendothelial migration in the development of transplant arteriosclerosis following experimental cardiac transplantation
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Abstracts
230 THE IMPACT OF CD4 AND CD8 TRANSENDOTHELIAL MIGRATION IN THE DEVELOPMENT OF TRANSPLANT ARTERIOSCLEROSIS FOLLOWING EXPERIMENTAL CARDIAC TRANSPLANTATION M.H. Richter1, M. Krauss1, S. Zahn1, H.G. Olbrich1, R. Autschbach2, F.W. Mohr2; 1Experimental Heart Transplant Group Frankfurt, Parthenstein, Germany; 2Heart Center Leipzig, University of Leipzig, Leipzig, Germany Purpose: Chronic rejection after transplantation is characterized by vascular smooth muscle cell (VSMC) migration and proliferation in the subintimal space. This activation of VSMC could be mediated by transendothelial migration of CD4 and CD8 positive (⫹) leucocytes. The objective of this study was to test the ability of CSA, FK-506 and MMF to reduce the transendothelial migration of CD4⫹ and CD8⫹ leukocytes in correlation with the resulting neointimal proliferation. Methods: After heterotopic cardiac transplantation from Lewis to Fisher rats animals were divided into four therapygroups of therapy: CSA 3mg/kg/d (n⫽74), MMF 40 mg/kg/d (n⫽96), FK-506 0.03 mg/kg/d (n⫽96) and a control group receiving no immunosuppressive therapy (n⫽74). 3-4 animals of each group were sacrificed in intervals of 1-4 days up to day 60. Immunohistochemistry was performed using mAb against CD4 and CD8 and positive cells were analysed at the endothelium and in the perivascular space of intra (s)- and epicardial (L) arteries. Statistical analysis was performed using the areas under the curves over the time of 60 days by an extended t-test. Results: CSA, FK-506 and MMF reduced CD4 and CD8 positive leukocytes at the endothelium and in the perivascular space (PVS) of s and L arteries, as well as they reduced neointimal proliferation of the arteries, compared with the control group. MMF significantly reduced CD4⫹ cells in the PVS of s and L arteries compared with FK-506 and CSA treated groups (p⬍0.05). We found no significant difference between FK-506 and CSA, neither in the reduction of the CD4⫹ cells nor in the extent of chronic rejection. MMF significantly reduced neointimal proliferation compared with FK-506 and CSA. There was a significant correlation between the CD4⫹ cells in the PVS and the resulting neointima but no with the CD8⫹ cells in the PVS. Conclusion: MMF attenuates transendothelial migration of CD4⫹ leucocytes and reduced chronic rejection. Immunosuppression with MMF after cardiac transplantation seems to be an option to prevent chronic rejection.
231 INDUCIBLE EXPRESSION OF BASIC TRANSCRIPTION FACTOR BINDING PROTEIN 2 PLAYS A POTENTIAL ROLE IN THE DEVELOPMENT OF THE ALLOGRAFT VASCULAR DISEASE T. Ogata1, R. Nagai2, M. Kurabayashi1, Y. Hoshino1, K. Sekiguchi1, K. Kowase1, A. Akuzawa1, S. Ishikawa1, I. Takeyoshi1, Y. Morishita1; 1Gunma University School of Medicine, Maebashi, Gunma, Japan; 2Tokyo University School of Medicine, Tokyo, Japan A number of studies have demonstrated that various genes are differentially expressed between proliferating and quiescent
The Journal of Heart and Lung Transplantation February 2001 vascular smooth muscle cells (SMCs). However, transcription factors which play an important role in this process remain largely unknown. We have recently identified basic transcription factor binding protein 2 (BTEB2) as a transcription factor regulating the expression of the SMemb gene, which is involved in the phenotypic modulation of vascular SMCs. SMemb is a nonmuscle myosin heavy chain-B isoform that is expressed in proliferating SMCs. It is also expressed in the thickened intima and media of cardiac allograft coronary arteries. The aim of this study was to investigate the expression of BTEB2 in allograft vascular disease. [Methods] We firstly performed cardiac or aortic allo transplantation in rats (LEW-F344). All grafts were stained with antibodies against for BTEB2, SM-actin, macrophage and cdk4 for immunohistochemical study. BTEB2 mRNA expression in aortic allograft were evaluated by RT-PCR. We secondly performed Northern blot analysis and RT-PCR for detecting BTEB2 mRNA expression in cultured C2/2 cells (rabbit aortic SMCs) and THP-1 cells (monocytic leukemia cells) activated by growth factors. [Results] Significant intimal thickening was observed in cardiac and aortic allografts. BTEB2 expression was observed in proliferating SMCs and monocytes/macrophages, which constituted thickened intima. BTEB2 expression was closely associated with cdk4 expression. BTEB2 mRNA expression was observed in C2/2 cells, THP-1 cells and aortic allografts, and BTEB2 mRNA levels were increased in response to PMA or bFGF in cultured C2/2 cells and THP-1 cells. These data suggested that BTEB2 acted as an important transcription factor regulating phenotypic modulation of SMCs and activation of macrophage. [Conclusion] The induced expression of BTEB2 may play a potential role in the development of the allograft vascular disease. 232 CD8ⴙ LYMPHOCYTES PARTICIPATE IN THE DEVELOPMENT OF CHRONIC REJECTION M.P. Fischbein, J. Yun, H. Laks, M.C. Fishbein, Y. Irie, C. Wortham, B. Bonavida, A. Ardehali; University of California at Los Angeles, Los Angeles, CA, USA Purpose: The contribution of CD8⫹ lymphocytes to the pathogenesis of Cardiac Allograft Vasculopathy (CAV) or chronic rejection remains undefined. We utilized a non-immunosuppressed model of CAV to define the role of CD8⫹ lymphocytes. Methods: Donor hearts from B10.A mice were transplanted into the following B10.BR recipients (MHC class I mismatched): (a) wild type, (b) wild type ⫹ CD40L blocking antibody (MR-1), and (c) wild type ⫹ isotype control antibody (IgG2a). Donor hearts were harvested at 14 and 30 days post-transplantation, and (a) quantitated morphometrically, (b) stained immunohistochemically, or (c) digested for isolation of graft infiltrating cells. The cytotoxic phenotype of graft infiltrating CD8⫹ lymphocytes was determined with flow cytometry (CD62 low and CD44 high). Intracellular cytokine staining of CD8⫹ and CD4⫹ lymphocytes for IL-2, IFN-g, IL-4, and IL-10 was performed with two-color flow cytometry. Results: In wild type recipients, a greater number of CD8⫹ compared to CD4⫹ lymphocytes infiltrated the allograft. The CD8⫹ lymphocytes localized to perivascular areas and demonstrated markers of activation (IL-2Ra, CD62 low, CD44 high). The intracellular cytokine staining assay demonstrated that CD8⫹ lymphocytes were the primary source of allograft IL-2 and
Abstracts
230 THE IMPACT OF CD4 AND CD8 TRANSENDOTHELIAL MIGRATION IN THE DEVELOPMENT OF TRANSPLANT ARTERIOSCLEROSIS FOLLOWING EXPERIMENTAL CARDIAC TRANSPLANTATION M.H. Richter1, M. Krauss1, S. Zahn1, H.G. Olbrich1, R. Autschbach2, F.W. Mohr2; 1Experimental Heart Transplant Group Frankfurt, Parthenstein, Germany; 2Heart Center Leipzig, University of Leipzig, Leipzig, Germany Purpose: Chronic rejection after transplantation is characterized by vascular smooth muscle cell (VSMC) migration and proliferation in the subintimal space. This activation of VSMC could be mediated by transendothelial migration of CD4 and CD8 positive (⫹) leucocytes. The objective of this study was to test the ability of CSA, FK-506 and MMF to reduce the transendothelial migration of CD4⫹ and CD8⫹ leukocytes in correlation with the resulting neointimal proliferation. Methods: After heterotopic cardiac transplantation from Lewis to Fisher rats animals were divided into four therapygroups of therapy: CSA 3mg/kg/d (n⫽74), MMF 40 mg/kg/d (n⫽96), FK-506 0.03 mg/kg/d (n⫽96) and a control group receiving no immunosuppressive therapy (n⫽74). 3-4 animals of each group were sacrificed in intervals of 1-4 days up to day 60. Immunohistochemistry was performed using mAb against CD4 and CD8 and positive cells were analysed at the endothelium and in the perivascular space of intra (s)- and epicardial (L) arteries. Statistical analysis was performed using the areas under the curves over the time of 60 days by an extended t-test. Results: CSA, FK-506 and MMF reduced CD4 and CD8 positive leukocytes at the endothelium and in the perivascular space (PVS) of s and L arteries, as well as they reduced neointimal proliferation of the arteries, compared with the control group. MMF significantly reduced CD4⫹ cells in the PVS of s and L arteries compared with FK-506 and CSA treated groups (p⬍0.05). We found no significant difference between FK-506 and CSA, neither in the reduction of the CD4⫹ cells nor in the extent of chronic rejection. MMF significantly reduced neointimal proliferation compared with FK-506 and CSA. There was a significant correlation between the CD4⫹ cells in the PVS and the resulting neointima but no with the CD8⫹ cells in the PVS. Conclusion: MMF attenuates transendothelial migration of CD4⫹ leucocytes and reduced chronic rejection. Immunosuppression with MMF after cardiac transplantation seems to be an option to prevent chronic rejection.
231 INDUCIBLE EXPRESSION OF BASIC TRANSCRIPTION FACTOR BINDING PROTEIN 2 PLAYS A POTENTIAL ROLE IN THE DEVELOPMENT OF THE ALLOGRAFT VASCULAR DISEASE T. Ogata1, R. Nagai2, M. Kurabayashi1, Y. Hoshino1, K. Sekiguchi1, K. Kowase1, A. Akuzawa1, S. Ishikawa1, I. Takeyoshi1, Y. Morishita1; 1Gunma University School of Medicine, Maebashi, Gunma, Japan; 2Tokyo University School of Medicine, Tokyo, Japan A number of studies have demonstrated that various genes are differentially expressed between proliferating and quiescent
The Journal of Heart and Lung Transplantation February 2001 vascular smooth muscle cells (SMCs). However, transcription factors which play an important role in this process remain largely unknown. We have recently identified basic transcription factor binding protein 2 (BTEB2) as a transcription factor regulating the expression of the SMemb gene, which is involved in the phenotypic modulation of vascular SMCs. SMemb is a nonmuscle myosin heavy chain-B isoform that is expressed in proliferating SMCs. It is also expressed in the thickened intima and media of cardiac allograft coronary arteries. The aim of this study was to investigate the expression of BTEB2 in allograft vascular disease. [Methods] We firstly performed cardiac or aortic allo transplantation in rats (LEW-F344). All grafts were stained with antibodies against for BTEB2, SM-actin, macrophage and cdk4 for immunohistochemical study. BTEB2 mRNA expression in aortic allograft were evaluated by RT-PCR. We secondly performed Northern blot analysis and RT-PCR for detecting BTEB2 mRNA expression in cultured C2/2 cells (rabbit aortic SMCs) and THP-1 cells (monocytic leukemia cells) activated by growth factors. [Results] Significant intimal thickening was observed in cardiac and aortic allografts. BTEB2 expression was observed in proliferating SMCs and monocytes/macrophages, which constituted thickened intima. BTEB2 expression was closely associated with cdk4 expression. BTEB2 mRNA expression was observed in C2/2 cells, THP-1 cells and aortic allografts, and BTEB2 mRNA levels were increased in response to PMA or bFGF in cultured C2/2 cells and THP-1 cells. These data suggested that BTEB2 acted as an important transcription factor regulating phenotypic modulation of SMCs and activation of macrophage. [Conclusion] The induced expression of BTEB2 may play a potential role in the development of the allograft vascular disease. 232 CD8ⴙ LYMPHOCYTES PARTICIPATE IN THE DEVELOPMENT OF CHRONIC REJECTION M.P. Fischbein, J. Yun, H. Laks, M.C. Fishbein, Y. Irie, C. Wortham, B. Bonavida, A. Ardehali; University of California at Los Angeles, Los Angeles, CA, USA Purpose: The contribution of CD8⫹ lymphocytes to the pathogenesis of Cardiac Allograft Vasculopathy (CAV) or chronic rejection remains undefined. We utilized a non-immunosuppressed model of CAV to define the role of CD8⫹ lymphocytes. Methods: Donor hearts from B10.A mice were transplanted into the following B10.BR recipients (MHC class I mismatched): (a) wild type, (b) wild type ⫹ CD40L blocking antibody (MR-1), and (c) wild type ⫹ isotype control antibody (IgG2a). Donor hearts were harvested at 14 and 30 days post-transplantation, and (a) quantitated morphometrically, (b) stained immunohistochemically, or (c) digested for isolation of graft infiltrating cells. The cytotoxic phenotype of graft infiltrating CD8⫹ lymphocytes was determined with flow cytometry (CD62 low and CD44 high). Intracellular cytokine staining of CD8⫹ and CD4⫹ lymphocytes for IL-2, IFN-g, IL-4, and IL-10 was performed with two-color flow cytometry. Results: In wild type recipients, a greater number of CD8⫹ compared to CD4⫹ lymphocytes infiltrated the allograft. The CD8⫹ lymphocytes localized to perivascular areas and demonstrated markers of activation (IL-2Ra, CD62 low, CD44 high). The intracellular cytokine staining assay demonstrated that CD8⫹ lymphocytes were the primary source of allograft IL-2 and