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Intracoronary interferon-γ accellerates cardiac allograft rejection in miniature swine
The Journal of Heart and Lung Transplantation Volume 21, Number 1
Abstracts
188 INTRACORONARY INTERFERON-␥ ACCELLERATES CARDIAC ALLOGRAFT REJECTION IN MINIATURE SWINE L.C. Benjamin, J.S. Allan, J.D. Mezrich, S.L. Houser, D.R. Johnston, R.S. Lee, H.C. Amoah, L.G. Ledgerwood, D.H. Sachs, J.C. Madsen, Transplantation Biology Research Center, Department of Surgery, Massachusetts General Hospital, Boston, MA Purpose: The effect of interferon-␥ (IFN-␥) on allograft rejection remains controversial. Some have proposed that IFN-␥ has a protective effect on the microcirculation of allografts, while others have shown IFN-␥ to be deleterious. We investigated the effects of intracoronary IFN-␥ on cardiac rejection in a preclinical large animal model. Methods: MHC class I disparate hearts were heterotopically transplanted into swine treated with a 12-day course of cyclosporine (10-14mg/kg/day) (n⫽3). An Alzet osmotic mini-pump delivered IFN-␥ into the coronary vasculature at a rate of 100ng/day. Control recipients were treated with a similar course of cyclosporine but without IFN-␥ (n⫽3). Results: Swine treated with IFN-␥ developed an earlier onset of high grade acute rejection as compared to controls, and rejected their allografts in 19 days vs. 46 days for controls (p⫽0.02). Elastin staining of the cytokine treated allografts showed severe endothelialitis and intimal thickening as early as POD 13. Conclusion: The delivery of IFN-␥ into the coronary circulation of cardiac allografts appears to accelerate both acute and chronic rejection. Targeting IFN-␥ in antirejection regimens may ameliorate both of these processes. IFN-␥ treated swine
Histology (ISHLT Grade)
POD
Survival
14660
3a/4 4/4 3a/4 1a/4 4/4 3b/4 4/4
7 17 6 12 27 7 13
17d
1b/4 1b/4 1a/4 1a/4 0/4 0/4
7 17 7 17 7 14
35d
14630 14784 Control Swine 11065 11168 11749
27d 13d
47d 55d
189 IL-10 INHIBITS ENDOTHELIAL-INDUCED T CELL ACTIVATION THROUGH EFFECTS UPON COSTIMULATION BUT INDEPENDENT OF ANTIGEN PRESENTATION T.J. Dengler, R. Klingenberg, A. Zastrow, H. Ko ¨nig, N. Xia, M. Haass, Cardiology, University of Heidelberg, Heidelberg, Germany Interleukin-10 (IL-10), a predominantly immunosuppressive cytokine, has been shown to attenuate lymphocyte responses, also after organ transplantation, through a direct effect upon CD4 and CD8 T cells. In addition, IL-10 inhibits antigen uptake and presentation in antigen-presenting cells, including rodent endothelial cells (EC), but its actions upon human EC are less well defined. In the present study, IL-10 receptor expression on human umbilical vein EC (HUVEC) was demonstrated by flow cytometry, western blotting and rt-PCR. Effective IL-10 signaling
123
in EC was ascertained by phosphorylation of transduction factor STAT-1. Cytokine stimulation of EC with IL-1, TNF-␣, interferon-␥, IL-4 and LPS did not significantly modulate IL-10 receptor expression. IL-10 effects on EC-dependent T cell activation were investigated with a novel T cell proliferation assay utilizing HUVEC which were stably transduced with Fc␥ receptor type II (CD32). T lymphocytes were activated with mitogenic anti-CD3 antibody bound to CD32 on EC, allowing the study of endothelial costimulation independent of alloantigen. 24 h pretreatment of HUVEC with IL-10 resulted in a dose-dependent inhibition of CD4 T cell proliferation, reaching ⬃ 70% inhibition with 10 ng/ml of IL-10. Complete removal of IL-10 after the pretreatment effected similar levels of inhibition, while addition of IL-10 only at the beginning of T cell stimulation showed significantly less inhibition. Similar effects were seen with CD8 T cell stimulation by HUVEC. IL-10 pretreatment did not alter the binding of comitogenic CD3 antibody to HUVEC, also no change in the expression of costimulatory surface molecules ICAM-1, VCAM-1, E-selectin, CD40, CD58, OX40 ligand and ICOS ligand could be demonstrated. In addition to direct effects on T lymphocytes and interference with antigen presentation, IL-10 markedly reduces the T cell costimulatory potential of human EC. As EC exposure to IL-10 prior to T cell contact is required for maximum effect and the expression of major costimulator antigens remains unchanged, modulation of EC cytokine secretion profiles might account for this immunosuppressive effect. 190 CORTICOSTEROIDS BUT NOT CYCLOSPORINE A INHIBIT CYTOKINE-INDUCED UPREGULATION OF INDUCIBLE COSTIMULATOR LIGAND (ICOS-L) ON HUMAN ENDOTHELIAL CELLS R. Klingenberg, U. Heidl, W. Nottmeyer, N. Xia, T.J. Dengler, Cardiology/Transplant Research, University Hospital Heidelberg, Heidelberg, Germany The recently described accessory T cell receptor “inducible costimulator” (ICOS) mediates prolonged costimulation of human T-lymphocytes resulting in proliferation and cytokine production. The ICOS pathway contributes to chronic tissue inflammation; in transplantation, animal studies suggest a crucial role in acute allograft rejection and potentially in transplant vasculopathy. While expression of the costimulatory ligand for ICOS (ICOS-L) has been demonstrated on B cells and macrophages its expression on human endothelial cells has not been studied. In the present study, constitutive expression of ICOS-L was demonstrated on the surface of human umbilical vein endothelial cells (HUVEC) by flow cytometry and immunofluorescence staining using an ICOS-Ig fusion protein and confirmed by western blotting and reverse transcription (RT)-PCR. The expression of ICOS-L on HUVEC was further regulated by cytokines in a doseand time-dependent manner: Interleukin-1 (IL-1) and TNF-␣ rapidly induced a more than 3-fold upregulation of ICOS-L expression persisting for ⬎ 72h; IL-4 led to a similar increase in expression but peak expression was only reached after 72h. Stimulation with IFN-␥ caused a transient, 2-fold increase in expression whereas IL-10 had no effect. 12h preincubation of HUVEC with prednisolone caused a dose-dependent decrease in basal expression of ICOS ligand (maximum: -50% at 10 M). Prednisolone also completely abolished IL-4-induced upregulation of ICOS-L and inhibited the increase in expression after stimulation with TNF-␣ and IL-1 by more than 50%. In contrast,
Abstracts
188 INTRACORONARY INTERFERON-␥ ACCELLERATES CARDIAC ALLOGRAFT REJECTION IN MINIATURE SWINE L.C. Benjamin, J.S. Allan, J.D. Mezrich, S.L. Houser, D.R. Johnston, R.S. Lee, H.C. Amoah, L.G. Ledgerwood, D.H. Sachs, J.C. Madsen, Transplantation Biology Research Center, Department of Surgery, Massachusetts General Hospital, Boston, MA Purpose: The effect of interferon-␥ (IFN-␥) on allograft rejection remains controversial. Some have proposed that IFN-␥ has a protective effect on the microcirculation of allografts, while others have shown IFN-␥ to be deleterious. We investigated the effects of intracoronary IFN-␥ on cardiac rejection in a preclinical large animal model. Methods: MHC class I disparate hearts were heterotopically transplanted into swine treated with a 12-day course of cyclosporine (10-14mg/kg/day) (n⫽3). An Alzet osmotic mini-pump delivered IFN-␥ into the coronary vasculature at a rate of 100ng/day. Control recipients were treated with a similar course of cyclosporine but without IFN-␥ (n⫽3). Results: Swine treated with IFN-␥ developed an earlier onset of high grade acute rejection as compared to controls, and rejected their allografts in 19 days vs. 46 days for controls (p⫽0.02). Elastin staining of the cytokine treated allografts showed severe endothelialitis and intimal thickening as early as POD 13. Conclusion: The delivery of IFN-␥ into the coronary circulation of cardiac allografts appears to accelerate both acute and chronic rejection. Targeting IFN-␥ in antirejection regimens may ameliorate both of these processes. IFN-␥ treated swine
Histology (ISHLT Grade)
POD
Survival
14660
3a/4 4/4 3a/4 1a/4 4/4 3b/4 4/4
7 17 6 12 27 7 13
17d
1b/4 1b/4 1a/4 1a/4 0/4 0/4
7 17 7 17 7 14
35d
14630 14784 Control Swine 11065 11168 11749
27d 13d
47d 55d
189 IL-10 INHIBITS ENDOTHELIAL-INDUCED T CELL ACTIVATION THROUGH EFFECTS UPON COSTIMULATION BUT INDEPENDENT OF ANTIGEN PRESENTATION T.J. Dengler, R. Klingenberg, A. Zastrow, H. Ko ¨nig, N. Xia, M. Haass, Cardiology, University of Heidelberg, Heidelberg, Germany Interleukin-10 (IL-10), a predominantly immunosuppressive cytokine, has been shown to attenuate lymphocyte responses, also after organ transplantation, through a direct effect upon CD4 and CD8 T cells. In addition, IL-10 inhibits antigen uptake and presentation in antigen-presenting cells, including rodent endothelial cells (EC), but its actions upon human EC are less well defined. In the present study, IL-10 receptor expression on human umbilical vein EC (HUVEC) was demonstrated by flow cytometry, western blotting and rt-PCR. Effective IL-10 signaling
123
in EC was ascertained by phosphorylation of transduction factor STAT-1. Cytokine stimulation of EC with IL-1, TNF-␣, interferon-␥, IL-4 and LPS did not significantly modulate IL-10 receptor expression. IL-10 effects on EC-dependent T cell activation were investigated with a novel T cell proliferation assay utilizing HUVEC which were stably transduced with Fc␥ receptor type II (CD32). T lymphocytes were activated with mitogenic anti-CD3 antibody bound to CD32 on EC, allowing the study of endothelial costimulation independent of alloantigen. 24 h pretreatment of HUVEC with IL-10 resulted in a dose-dependent inhibition of CD4 T cell proliferation, reaching ⬃ 70% inhibition with 10 ng/ml of IL-10. Complete removal of IL-10 after the pretreatment effected similar levels of inhibition, while addition of IL-10 only at the beginning of T cell stimulation showed significantly less inhibition. Similar effects were seen with CD8 T cell stimulation by HUVEC. IL-10 pretreatment did not alter the binding of comitogenic CD3 antibody to HUVEC, also no change in the expression of costimulatory surface molecules ICAM-1, VCAM-1, E-selectin, CD40, CD58, OX40 ligand and ICOS ligand could be demonstrated. In addition to direct effects on T lymphocytes and interference with antigen presentation, IL-10 markedly reduces the T cell costimulatory potential of human EC. As EC exposure to IL-10 prior to T cell contact is required for maximum effect and the expression of major costimulator antigens remains unchanged, modulation of EC cytokine secretion profiles might account for this immunosuppressive effect. 190 CORTICOSTEROIDS BUT NOT CYCLOSPORINE A INHIBIT CYTOKINE-INDUCED UPREGULATION OF INDUCIBLE COSTIMULATOR LIGAND (ICOS-L) ON HUMAN ENDOTHELIAL CELLS R. Klingenberg, U. Heidl, W. Nottmeyer, N. Xia, T.J. Dengler, Cardiology/Transplant Research, University Hospital Heidelberg, Heidelberg, Germany The recently described accessory T cell receptor “inducible costimulator” (ICOS) mediates prolonged costimulation of human T-lymphocytes resulting in proliferation and cytokine production. The ICOS pathway contributes to chronic tissue inflammation; in transplantation, animal studies suggest a crucial role in acute allograft rejection and potentially in transplant vasculopathy. While expression of the costimulatory ligand for ICOS (ICOS-L) has been demonstrated on B cells and macrophages its expression on human endothelial cells has not been studied. In the present study, constitutive expression of ICOS-L was demonstrated on the surface of human umbilical vein endothelial cells (HUVEC) by flow cytometry and immunofluorescence staining using an ICOS-Ig fusion protein and confirmed by western blotting and reverse transcription (RT)-PCR. The expression of ICOS-L on HUVEC was further regulated by cytokines in a doseand time-dependent manner: Interleukin-1 (IL-1) and TNF-␣ rapidly induced a more than 3-fold upregulation of ICOS-L expression persisting for ⬎ 72h; IL-4 led to a similar increase in expression but peak expression was only reached after 72h. Stimulation with IFN-␥ caused a transient, 2-fold increase in expression whereas IL-10 had no effect. 12h preincubation of HUVEC with prednisolone caused a dose-dependent decrease in basal expression of ICOS ligand (maximum: -50% at 10 M). Prednisolone also completely abolished IL-4-induced upregulation of ICOS-L and inhibited the increase in expression after stimulation with TNF-␣ and IL-1 by more than 50%. In contrast,