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127
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Abstracts
T-cells, p⫽0.025 vs C3H ⫹ pfp⫺/⫺ CD4⫹ T-cells, and p⫽0.025 vs C3Hlpr ⫹ CD4⫹ T-cells). Mixed lymphocyte reactions demonstrated equal proliferation of Wt Bl/6 and Bl/6 pfp⫺/⫺ CD4 T-cells to both Wt C3H and Fas-deficient C3Hlpr stimulators. Conclusions: Results demonstrate that in vivo CD4 T-cell mediated cardiac rejection is dependent on both donor Fas and CD4 effector T-cell perforin expression in a parallel fashion. This strongly implicates a cytotoxic phenotype for the CD4 effector T-cell in cardiac allograft rejection. Additionally, equal proliferation of pfp⫺/⫺ CD4 T-cells to both Wt C3H and C3Hlpr stimulators strongly suggests that perforin and Fas have their in vivo effects at the level of the T-cell effector mechanism and not at the level of recognition and/or costimulation. 127 GENETIC CONTROL OF CD44 EXPRESSION BY INTRAGRAFT FIBROBLASTS G.D. Wu,1 M.L. Barr,2 H. Zhu,1 J.-I. Woo,1 Y. He,1 A.S. Klein,1 1 Comprehensive Transplant Center at Cedars-Sinai Medical Center; 2Department of Cardiothoracic Surgery, University of Southern California, Los Angeles, CA Background: We have previously demonstrated that fibroblast progenitors actively migrate to cardiac allografts and contribute to graft fibrosis. The cellular/molecular mechanisms responsible for fibroblast migration to the allograft remain poorly understood. Methods: A series of experiments were designed to quantify the role that adhesion molecule CD44 plays in fibroblastic cell migration. A LEW-to-F344 rat chronic cardiac rejection model was used. Patterns and intensity of the extracellular matrix hyaluronic acid (HA) in the allografts were evaluated with immunochemistry. Intragraft fibroblastic cells were isolated from the allografts and analyzed for CD44 gene expression using quantitative real time PCR. Competencies of CD44⫺/⫺ or CD44⫹/⫹ fibroblastic cells to adhere to and to migrate through HA were examined in cell adhesion and cell migration assays, respectively. Opposing effects on fibroblast migration by PDGF up-regulation and siRNA down-regulation of CD44 expression were studied in a mesenchymal stem cell line Ap8C3 which contained fibroblast progenitors. Results: Allografts undergoing chronic rejection exhibited massive deposition of extracellular HA. Metamorphic scanning of HA binding protein-stained tissue sections demonstrated a significant increase in HA accumulation in allografts when compared with isografts (p ⬍0.05). Fibroblastic cells expressing CD44 were in close contact with extracellular matrix HA. Fibroblastic cells recovered from chronically rejecting allografts displayed a 37-fold increase in CD44 expression when compared with that of fibroblasts isolated from native hearts of the recipients (p ⬍0.0001). CD44 mRNA expressed by intragraft fibroblasts were predominantly encoded by variant exons with precursor frequencies of v6 (18%), v7 (17%), v9 (17%), v4 (13%), v1 (12%), v8 (12%), v5 (11%), v2 (6%) and v10 (5%). CD44 deficient fibroblastic cells exhibited impairment of binding to and migration through extracellular HA. PDGF up-regulated CD44 expression by Ap8c3 cells at both mRNA and protein levels. Increases in CD44 expression by the target cells were accompanied by significantly increased ability of the cells to bind to (p ⬍0.01) and migrate through (p ⬍0.01) HA gel. siRNA treatment had potent inhibitory effects on CD44 expression by Ap8c3, which lasted for up to 72 h. The inhibition of cell adhesion to HA by siRNA was comparable to the effects of neutralizing anti-CD44 antibodies. CD44 siRNA also significantly inhibited cell migration through HA-coated migration chambers (p⫽0.05). Conclusion: Development of chronic rejection in cardiac allografts was accompanied by deposition of extracellular HA, which served as a ligand for hyaluronan receptor CD44. CD44 expressed by migrating
The Journal of Heart and Lung Transplantation February 2006
fibroblasts interacts with extracellular HA and plays a primary role in recruitment of fibroblast progenitors from the circulation to the allografts. CD44 expression by the majority of intragraft fibroblasts is restricted to variant isoforms. Fibroblast migration can be promoted by fibrogenic cytokine PDGF which up-regulates CD44 expression and can be suppressed by siRNA CD44 which down-regulates CD44 expression. Therapeutic down-regulation of CD44 by siRNA in combination with PDGF antagonism may effectively inhibit fibroblast migration. 128 IL-21: THE PRIMARY PLAYER IN REJECTION PROCESSES C.C. Baan,1 A.H.M.M. Balk,2 S.S. Korevaar,1 A.M.A. Peeters,1 W. Weimar,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Cardiology, Erasmus MC, University Medical Center, Rotterdam, Netherlands IL-21 is the most recently described cytokine that signals via the common IL-2 cytokine receptor (␥c), is produced by activated CD4⫹ T-cells, and regulates CD8⫹ T-cell expansion and their effector function. To explore the actions of IL-21 and how it integrates its signals with other ␥c dependent cytokines in anti-donor responses, we studied the mRNA expression levels of IL-21 and its receptor (IL-21R␣) after allogeneic stimulation of T-cells in the absence and presence of anti-IL-21R␣ (0.01–1 g/ml ), IL-21 (1–100 ng/ml), anti-IL-2 (1 g/ml), anti-IL2R␣: 0.5 g/ml and immunosuppressive agents that block IL-2 production (CsA: 100 ng/nl, Tac: 10 ng/ml), or interfere with IL-2R signaling (Rapa: 10 ng/ml). Additionally, IL-21 and IL-21R␣ mRNA levels were measured in endomyocardial biopsies of heart transplant patients. Proliferation of allo-activated T-cells was inhibited by anti-IL-21R␣ in a dose-dependent fashion (max 70%), while addition of IL-21 to the cultures increased the proliferated response (range 20 –50%). AntiIL-2, anti-IL-2R␣ and the immunosuppressive agents also inhibited the proliferative response (⬎80%). However, IL-21 could not abrogate the inhibitory effects of the IL-2 blockade. Analysis of gene expression levels of allo-stimulated T-cells showed a 20-fold induction of both IL-21 and IL-21R␣. Interestingly, induction of IL-21 was highly dependent on endogenous IL-2 production as in the presence of anti-IL-2 , anti-IL-2R␣, and immunosuppressive drugs that block IL-2 production (CsA, Tac) or interfere in IL-2R signaling (Rapa) almost completely inhibited the transcription of IL-21 (range 80 –95%). After heart transplantation intragraft IL-21 and IL-21R␣ expression levels were associated with acute rejection. Compared to non-rejection biopsies (N⫽14), rejection specimens (N⫽11) expressed the highest amounts of IL-21 and IL-21R␣ mRNA (3 fold, p⫽0.03). In conclusion, the growth factor IL-21 is the primary non-redundant player in the cascade leading to acute cardiac rejection via an IL-2 dependent mechanism. 129 ANTI-VIMENTIN ANTIBODIES CAUSE ACCELERATED REJECTION OF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) IDENTICAL MURINE CARDIAC ALLOGRAFTS B. Mahesh,1 P. Sarathchandra,1 A.M. McCormack,1 A.L. Holder,1 M. Jacovides,1 J.D. Smith,1 M.L. Rose,1 1Transplant Immunology, Harefield Hospital, Harefield, Middlesex, United Kingdom Purpose: There is increasing evidence that non-MHC antibodies reduce graft survival. Among these, anti-vimentin antibodies (AVA) are significantly associated with human cardiac allograft vasculopathy. Here we examine mechanisms of damage caused by AVA in a murine model.
Abstracts
T-cells, p⫽0.025 vs C3H ⫹ pfp⫺/⫺ CD4⫹ T-cells, and p⫽0.025 vs C3Hlpr ⫹ CD4⫹ T-cells). Mixed lymphocyte reactions demonstrated equal proliferation of Wt Bl/6 and Bl/6 pfp⫺/⫺ CD4 T-cells to both Wt C3H and Fas-deficient C3Hlpr stimulators. Conclusions: Results demonstrate that in vivo CD4 T-cell mediated cardiac rejection is dependent on both donor Fas and CD4 effector T-cell perforin expression in a parallel fashion. This strongly implicates a cytotoxic phenotype for the CD4 effector T-cell in cardiac allograft rejection. Additionally, equal proliferation of pfp⫺/⫺ CD4 T-cells to both Wt C3H and C3Hlpr stimulators strongly suggests that perforin and Fas have their in vivo effects at the level of the T-cell effector mechanism and not at the level of recognition and/or costimulation. 127 GENETIC CONTROL OF CD44 EXPRESSION BY INTRAGRAFT FIBROBLASTS G.D. Wu,1 M.L. Barr,2 H. Zhu,1 J.-I. Woo,1 Y. He,1 A.S. Klein,1 1 Comprehensive Transplant Center at Cedars-Sinai Medical Center; 2Department of Cardiothoracic Surgery, University of Southern California, Los Angeles, CA Background: We have previously demonstrated that fibroblast progenitors actively migrate to cardiac allografts and contribute to graft fibrosis. The cellular/molecular mechanisms responsible for fibroblast migration to the allograft remain poorly understood. Methods: A series of experiments were designed to quantify the role that adhesion molecule CD44 plays in fibroblastic cell migration. A LEW-to-F344 rat chronic cardiac rejection model was used. Patterns and intensity of the extracellular matrix hyaluronic acid (HA) in the allografts were evaluated with immunochemistry. Intragraft fibroblastic cells were isolated from the allografts and analyzed for CD44 gene expression using quantitative real time PCR. Competencies of CD44⫺/⫺ or CD44⫹/⫹ fibroblastic cells to adhere to and to migrate through HA were examined in cell adhesion and cell migration assays, respectively. Opposing effects on fibroblast migration by PDGF up-regulation and siRNA down-regulation of CD44 expression were studied in a mesenchymal stem cell line Ap8C3 which contained fibroblast progenitors. Results: Allografts undergoing chronic rejection exhibited massive deposition of extracellular HA. Metamorphic scanning of HA binding protein-stained tissue sections demonstrated a significant increase in HA accumulation in allografts when compared with isografts (p ⬍0.05). Fibroblastic cells expressing CD44 were in close contact with extracellular matrix HA. Fibroblastic cells recovered from chronically rejecting allografts displayed a 37-fold increase in CD44 expression when compared with that of fibroblasts isolated from native hearts of the recipients (p ⬍0.0001). CD44 mRNA expressed by intragraft fibroblasts were predominantly encoded by variant exons with precursor frequencies of v6 (18%), v7 (17%), v9 (17%), v4 (13%), v1 (12%), v8 (12%), v5 (11%), v2 (6%) and v10 (5%). CD44 deficient fibroblastic cells exhibited impairment of binding to and migration through extracellular HA. PDGF up-regulated CD44 expression by Ap8c3 cells at both mRNA and protein levels. Increases in CD44 expression by the target cells were accompanied by significantly increased ability of the cells to bind to (p ⬍0.01) and migrate through (p ⬍0.01) HA gel. siRNA treatment had potent inhibitory effects on CD44 expression by Ap8c3, which lasted for up to 72 h. The inhibition of cell adhesion to HA by siRNA was comparable to the effects of neutralizing anti-CD44 antibodies. CD44 siRNA also significantly inhibited cell migration through HA-coated migration chambers (p⫽0.05). Conclusion: Development of chronic rejection in cardiac allografts was accompanied by deposition of extracellular HA, which served as a ligand for hyaluronan receptor CD44. CD44 expressed by migrating
The Journal of Heart and Lung Transplantation February 2006
fibroblasts interacts with extracellular HA and plays a primary role in recruitment of fibroblast progenitors from the circulation to the allografts. CD44 expression by the majority of intragraft fibroblasts is restricted to variant isoforms. Fibroblast migration can be promoted by fibrogenic cytokine PDGF which up-regulates CD44 expression and can be suppressed by siRNA CD44 which down-regulates CD44 expression. Therapeutic down-regulation of CD44 by siRNA in combination with PDGF antagonism may effectively inhibit fibroblast migration. 128 IL-21: THE PRIMARY PLAYER IN REJECTION PROCESSES C.C. Baan,1 A.H.M.M. Balk,2 S.S. Korevaar,1 A.M.A. Peeters,1 W. Weimar,1 1Internal Medicine, Erasmus MC, Rotterdam, Netherlands; 2Cardiology, Erasmus MC, University Medical Center, Rotterdam, Netherlands IL-21 is the most recently described cytokine that signals via the common IL-2 cytokine receptor (␥c), is produced by activated CD4⫹ T-cells, and regulates CD8⫹ T-cell expansion and their effector function. To explore the actions of IL-21 and how it integrates its signals with other ␥c dependent cytokines in anti-donor responses, we studied the mRNA expression levels of IL-21 and its receptor (IL-21R␣) after allogeneic stimulation of T-cells in the absence and presence of anti-IL-21R␣ (0.01–1 g/ml ), IL-21 (1–100 ng/ml), anti-IL-2 (1 g/ml), anti-IL2R␣: 0.5 g/ml and immunosuppressive agents that block IL-2 production (CsA: 100 ng/nl, Tac: 10 ng/ml), or interfere with IL-2R signaling (Rapa: 10 ng/ml). Additionally, IL-21 and IL-21R␣ mRNA levels were measured in endomyocardial biopsies of heart transplant patients. Proliferation of allo-activated T-cells was inhibited by anti-IL-21R␣ in a dose-dependent fashion (max 70%), while addition of IL-21 to the cultures increased the proliferated response (range 20 –50%). AntiIL-2, anti-IL-2R␣ and the immunosuppressive agents also inhibited the proliferative response (⬎80%). However, IL-21 could not abrogate the inhibitory effects of the IL-2 blockade. Analysis of gene expression levels of allo-stimulated T-cells showed a 20-fold induction of both IL-21 and IL-21R␣. Interestingly, induction of IL-21 was highly dependent on endogenous IL-2 production as in the presence of anti-IL-2 , anti-IL-2R␣, and immunosuppressive drugs that block IL-2 production (CsA, Tac) or interfere in IL-2R signaling (Rapa) almost completely inhibited the transcription of IL-21 (range 80 –95%). After heart transplantation intragraft IL-21 and IL-21R␣ expression levels were associated with acute rejection. Compared to non-rejection biopsies (N⫽14), rejection specimens (N⫽11) expressed the highest amounts of IL-21 and IL-21R␣ mRNA (3 fold, p⫽0.03). In conclusion, the growth factor IL-21 is the primary non-redundant player in the cascade leading to acute cardiac rejection via an IL-2 dependent mechanism. 129 ANTI-VIMENTIN ANTIBODIES CAUSE ACCELERATED REJECTION OF MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) IDENTICAL MURINE CARDIAC ALLOGRAFTS B. Mahesh,1 P. Sarathchandra,1 A.M. McCormack,1 A.L. Holder,1 M. Jacovides,1 J.D. Smith,1 M.L. Rose,1 1Transplant Immunology, Harefield Hospital, Harefield, Middlesex, United Kingdom Purpose: There is increasing evidence that non-MHC antibodies reduce graft survival. Among these, anti-vimentin antibodies (AVA) are significantly associated with human cardiac allograft vasculopathy. Here we examine mechanisms of damage caused by AVA in a murine model.