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CXCL9 neutralization attenuates chronic rejection of transplanted hearts
S154
Abstracts
provided the strongest correlation with 6-minute walk (r ⫽ -0.63, p ⬍ 0.0001) and WHO class (r ⫽ 0.50, p ⬍ 0.0001). Receiver operator characteristic (ROC) plots for norepinephrine and 6 minute walk distance were created using a cutoff of 350 meters to define serious exercise limitation. The AUC of the ROC curve was 0.854 and the NE threshold value that minimized error was 443 pg/ml, with a sensitivity and specificity of 82% at this value. Conclusions: Activation of multiple neurohumoral systems occurs in PAH, and is proportional to disease severity. Of the markers studied, BNP correlates best with hemodynamic parameters, while norepinephrine, a marker of activation of the sympathetic nervous system, correlates best with exercise capacity and functional class. These markers may be useful in the noninvasive assessment of prognosis and response to therapy in patients with PAH.
247 MIG/CXCL9 NEUTRALIZATION ATTENUATES CHRONIC REJECTION OF TRANSPLANTED HEARTS D. Whiting,1 S. Chun,1 H. Laks,1 J. Yun,1 X. Gan,2 W. Yao,1 B. Bonavida,2 A. Ardehali,1 1Division of Cardiothoracic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA Introduction: Chronic rejection in heart transplantation or cardiac allograft vasculopathy (CAV) remains the leading cause of late death in heart transplant recipients. We sought to determine if MIG/CXCL9, a CXC chemokine which targets T-lymphocytes, NK cells, and B-cells, participates in the development of CAV. Methods: B6.CH2bm12 donor hearts were transplanted heterotopically into C57Bl/6 mice. In this model, the donor hearts reproducibly develop CAV within 24 days of transplantation. In the anti-MIG/CXCL9 group (n⫽6), the recipient mice were treated with a goat anti-murine MIG/ CXCL9 antibody every other day. In the control group, (n⫽5), the recipient mice were treated with a control goat serum using a similar protocol. The donor hearts were harvested at 24 days post-transplantation. Intimal lesions were measured by morphometric analyses. Graft infiltrating cells were quantitated by flow cytometry and then analyzed for IFN-␥ production via intracellular cytokine assay. Results: The donor hearts in the control allografts developed severe intimal lesions (36.5⫾5.9%), as expected. Neutralization of MIG/ CXCL9 resulted in significant reduction in the severity of lesions (6.8⫾2%; p⬍0.05 when compared to control allografts). Furthermore, MIG/CXCL9 antagonism also yielded a significant reduction in the number of graft infiltrating CD4 lymphocytes (2.8⫾1.9X104 vs. control allografts 71.7⫾29.6X104; p⬍0.05). Intracellular cytokine assay of graft infiltrating cells also showed that MIG/CXCL9 neutralization caused a marked reduction in the number of IFN-␥ producing CD4 and CD8 lymphocytes. Conclusion: This study suggests that MIG/CXCL9 participates in the development of CAV via a)direct T-lymphocyte trafficking into the graft, and b)promoting T-lymphocyte IFN-␥ production. Strategies that block MIG/CXCL9 biologic function may control the development of chronic rejection in heart transplantation. All results presented as mean⫾SEM.
248 NATURAL KILLER CELLS CAN MEDIATE CARDIAC ALLOGRAFT VASCULOPATHY IN MURINE ALLOGRAFTS
The Journal of Heart and Lung Transplantation January 2003 H.S. Rose,1 W.H. Kitchens,1 C.M. Chase,1 R.B. Colvin,2 P.S. Russell,1 J.C. Madsen,1 1Department of Surgery, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA Purpose: Current dogma suggests that the antidonor responses of the adaptive immune system are the primary immunological determinants of cardiac allograft vasculopathy (CAV). However, recent results from our laboratory demonstrated that CAV develops in recipient mice devoid of an adaptive immune system. Here, we asked whether innate immune responses, specifically natural killer (NK) cells, can mediate CAV development. Procedures: Mouse hearts were heterotopically transplanted from parental donors to F1 hybrid recipients (P3 F1) and examined 8 weeks later for CAV in the proximal coronary arteries. In a P3 F1 transplant, the host anti-donor T cell response is absent, but the host anti-donor NK response remains. Initial experiments were conducted with C57BL/6 (B6) parental donors and BALB/c x B6 F1 hybrid (CB6F1) recipients. B6 isografts were used as controls. To confirm a lack of T cell involvement, additional transplants from B6 background RAG1 knockout mice (B6.RAG) to B6CF1 background RAG1 knockout mice (B6CF1.RAG) were performed. Finally, to validate the necessity of NK cells for CAV development, another set of B6.RAG to B6CF1.RAG transplants was performed in recipients depleted of NK cells by weekly injections of anti-NK1.1 mAb (PK136). Results: 9/9 B63 CB6F1 transplants developed CAV as compared to 2/29 B6 isografts (p⬍.001). Similarly, 6/6 B6.RAG3 B6CF1.RAG transplants developed CAV. In contrast, only 1/4 (p⫽.03) PK136treated B6.RAG3 B6CF1.RAG transplants developed CAV. Furthermore, the CAV found in the single PK136-treated animal was much less severe (by percent occlusion) than that observed in the untreated group. Conclusions: These data suggest that NK cells can mediate the development of CAV. This represents a previously unidentified pathway of CAV. Since this pathway would be functional in immunocompetent recipients, but would not be targeted by current immunosuppressive therapy, further analysis of its mechanisms is of clinical importance. 249 GAMMA-INTERFERON-DEPENDENT T CELL-MEDIATED INJURY AND REMODELING OF HUMAN CORONARY ARTERIES Y. Wang, W. Burns, P. Tang, Y. Bai, T. Yi, J.S. Schechner, M.I. Lorber, J.S. Pober, G. Tellides, Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, CT Changes in vessel diameter rather than intimal expansion is the most important predictor of luminal loss in graft arteriosclerosis, yet little is known about its immune mechanisms. We investigated the role of IFN-␥ in vascular remodeling by transplanting adjacent segments of human coronary arteries to paired SCID/beige mice, one receiving allogeneic human PBMCs and the other left untreated. The grafts (n⫽60) were analyzed after 2, 4, or 6 weeks by immunohistochemistry, morphometry, and quantitative RT-PCR. Vessels from PBMC-treated hosts showed progressive inflammation and enlargement of the intima and adventitia. The rejecting arteries more than doubled in size and the outward remodeling preserved luminal area at 2 weeks. By 4 and 6 weeks, a ten-fold increase in intimal thickness compromised and even obliterated the lumen. The graft infiltrating cells were almost all CD3⫹, CD45RO⫹ T lymphocytes with approximately twice as many CD4⫹ as CD8⫹ T cells. Transcripts encoding IFN-␥, CD3⑀ (pan-T cell marker), and CXCR3 (chemokine receptor expressed primarily on IFN-␥-
Abstracts
provided the strongest correlation with 6-minute walk (r ⫽ -0.63, p ⬍ 0.0001) and WHO class (r ⫽ 0.50, p ⬍ 0.0001). Receiver operator characteristic (ROC) plots for norepinephrine and 6 minute walk distance were created using a cutoff of 350 meters to define serious exercise limitation. The AUC of the ROC curve was 0.854 and the NE threshold value that minimized error was 443 pg/ml, with a sensitivity and specificity of 82% at this value. Conclusions: Activation of multiple neurohumoral systems occurs in PAH, and is proportional to disease severity. Of the markers studied, BNP correlates best with hemodynamic parameters, while norepinephrine, a marker of activation of the sympathetic nervous system, correlates best with exercise capacity and functional class. These markers may be useful in the noninvasive assessment of prognosis and response to therapy in patients with PAH.
247 MIG/CXCL9 NEUTRALIZATION ATTENUATES CHRONIC REJECTION OF TRANSPLANTED HEARTS D. Whiting,1 S. Chun,1 H. Laks,1 J. Yun,1 X. Gan,2 W. Yao,1 B. Bonavida,2 A. Ardehali,1 1Division of Cardiothoracic Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA Introduction: Chronic rejection in heart transplantation or cardiac allograft vasculopathy (CAV) remains the leading cause of late death in heart transplant recipients. We sought to determine if MIG/CXCL9, a CXC chemokine which targets T-lymphocytes, NK cells, and B-cells, participates in the development of CAV. Methods: B6.CH2bm12 donor hearts were transplanted heterotopically into C57Bl/6 mice. In this model, the donor hearts reproducibly develop CAV within 24 days of transplantation. In the anti-MIG/CXCL9 group (n⫽6), the recipient mice were treated with a goat anti-murine MIG/ CXCL9 antibody every other day. In the control group, (n⫽5), the recipient mice were treated with a control goat serum using a similar protocol. The donor hearts were harvested at 24 days post-transplantation. Intimal lesions were measured by morphometric analyses. Graft infiltrating cells were quantitated by flow cytometry and then analyzed for IFN-␥ production via intracellular cytokine assay. Results: The donor hearts in the control allografts developed severe intimal lesions (36.5⫾5.9%), as expected. Neutralization of MIG/ CXCL9 resulted in significant reduction in the severity of lesions (6.8⫾2%; p⬍0.05 when compared to control allografts). Furthermore, MIG/CXCL9 antagonism also yielded a significant reduction in the number of graft infiltrating CD4 lymphocytes (2.8⫾1.9X104 vs. control allografts 71.7⫾29.6X104; p⬍0.05). Intracellular cytokine assay of graft infiltrating cells also showed that MIG/CXCL9 neutralization caused a marked reduction in the number of IFN-␥ producing CD4 and CD8 lymphocytes. Conclusion: This study suggests that MIG/CXCL9 participates in the development of CAV via a)direct T-lymphocyte trafficking into the graft, and b)promoting T-lymphocyte IFN-␥ production. Strategies that block MIG/CXCL9 biologic function may control the development of chronic rejection in heart transplantation. All results presented as mean⫾SEM.
248 NATURAL KILLER CELLS CAN MEDIATE CARDIAC ALLOGRAFT VASCULOPATHY IN MURINE ALLOGRAFTS
The Journal of Heart and Lung Transplantation January 2003 H.S. Rose,1 W.H. Kitchens,1 C.M. Chase,1 R.B. Colvin,2 P.S. Russell,1 J.C. Madsen,1 1Department of Surgery, Massachusetts General Hospital, Boston, MA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA Purpose: Current dogma suggests that the antidonor responses of the adaptive immune system are the primary immunological determinants of cardiac allograft vasculopathy (CAV). However, recent results from our laboratory demonstrated that CAV develops in recipient mice devoid of an adaptive immune system. Here, we asked whether innate immune responses, specifically natural killer (NK) cells, can mediate CAV development. Procedures: Mouse hearts were heterotopically transplanted from parental donors to F1 hybrid recipients (P3 F1) and examined 8 weeks later for CAV in the proximal coronary arteries. In a P3 F1 transplant, the host anti-donor T cell response is absent, but the host anti-donor NK response remains. Initial experiments were conducted with C57BL/6 (B6) parental donors and BALB/c x B6 F1 hybrid (CB6F1) recipients. B6 isografts were used as controls. To confirm a lack of T cell involvement, additional transplants from B6 background RAG1 knockout mice (B6.RAG) to B6CF1 background RAG1 knockout mice (B6CF1.RAG) were performed. Finally, to validate the necessity of NK cells for CAV development, another set of B6.RAG to B6CF1.RAG transplants was performed in recipients depleted of NK cells by weekly injections of anti-NK1.1 mAb (PK136). Results: 9/9 B63 CB6F1 transplants developed CAV as compared to 2/29 B6 isografts (p⬍.001). Similarly, 6/6 B6.RAG3 B6CF1.RAG transplants developed CAV. In contrast, only 1/4 (p⫽.03) PK136treated B6.RAG3 B6CF1.RAG transplants developed CAV. Furthermore, the CAV found in the single PK136-treated animal was much less severe (by percent occlusion) than that observed in the untreated group. Conclusions: These data suggest that NK cells can mediate the development of CAV. This represents a previously unidentified pathway of CAV. Since this pathway would be functional in immunocompetent recipients, but would not be targeted by current immunosuppressive therapy, further analysis of its mechanisms is of clinical importance. 249 GAMMA-INTERFERON-DEPENDENT T CELL-MEDIATED INJURY AND REMODELING OF HUMAN CORONARY ARTERIES Y. Wang, W. Burns, P. Tang, Y. Bai, T. Yi, J.S. Schechner, M.I. Lorber, J.S. Pober, G. Tellides, Interdepartmental Program in Vascular Biology and Transplantation, Yale University School of Medicine, New Haven, CT Changes in vessel diameter rather than intimal expansion is the most important predictor of luminal loss in graft arteriosclerosis, yet little is known about its immune mechanisms. We investigated the role of IFN-␥ in vascular remodeling by transplanting adjacent segments of human coronary arteries to paired SCID/beige mice, one receiving allogeneic human PBMCs and the other left untreated. The grafts (n⫽60) were analyzed after 2, 4, or 6 weeks by immunohistochemistry, morphometry, and quantitative RT-PCR. Vessels from PBMC-treated hosts showed progressive inflammation and enlargement of the intima and adventitia. The rejecting arteries more than doubled in size and the outward remodeling preserved luminal area at 2 weeks. By 4 and 6 weeks, a ten-fold increase in intimal thickness compromised and even obliterated the lumen. The graft infiltrating cells were almost all CD3⫹, CD45RO⫹ T lymphocytes with approximately twice as many CD4⫹ as CD8⫹ T cells. Transcripts encoding IFN-␥, CD3⑀ (pan-T cell marker), and CXCR3 (chemokine receptor expressed primarily on IFN-␥-