Intragraft mrna expression of the novel cytokine IL-21 during acute rejection after clinical heart transplantation

The Journal of Heart and Lung Transplantation Volume 21, Number 1 cells significantly, but resulted in a significant increase in macrophage (CD11b⫹) infiltration and significantly elevated intragraft mRNA expression of iNOS. During the development of transplant arteriosclerosis (TxA) PECAM-1-/- donor endothelial cells were replaced by recipient PECAM-1⫹/⫹ endothelial cells, a process that occurred only in the allogeneic situation. Endothelial replacement commenced 14 days after transplantation and was complete by day 30. Conclusion: PECAM-1 expression by donor endothelial cells may regulate macrophage infiltration into the graft thereby controlling the development of TxA. Furthermore, these data suggest that the alloantigen specificity of the effector cells that mediate endothelial cell destruction may change as the rejection process progresses. Initially cells responding to donor antigens via the direct pathway of allorecognition will play a key role, but later in the response, indirect pathway T cells will become increasingly important. 306 INTRAGRAFT mRNA EXPRESSION OF THE NOVEL CYTOKINE IL-21 DURING ACUTE REJECTION AFTER CLINICAL HEART TRANSPLANTATION H.A. de Groot-Kruseman,1 M. Klepper,1 W.M. Mol,1 H.G.M. Niesters,2 T. van Gelder,1 A.P.W.M. Maat,3 A. Balk,4 W. Weimar,1 C. Baan,1 1Internal Medicine; 2Diagnostic Institute of Molecular Biology; 3Thoracic Surgery; 4Cardiology, University Hospital Rotterdam, Netherlands Immune responses after transplantation are largely mediated by cytokines of the interleukin (IL)-2 family (IL-2, IL-4, IL-7, IL-9 and IL-15). These T-cell growth factors bind to the common ␥-chain of the IL-2 receptor complex, thereby exhibiting cytokine redundancy. IL-21 is a recently described member of this family and is almost exclusively produced by activated CD4⫹ T-cells. The unique biological function of IL-21 is its ability to increase the cytotoxic activity of mature NK-cells. To investigate the clinical relevance of this novel cytokine in the transplant setting, we studied the intragraft expression pattern of IL-21 in association with acute cellular rejection after heart transplantation. Therefore, we quantified IL-21 mRNA expression levels by real time PCR in serial endomyocardial biopsies (n⫽21) sampled before, during, and after the first acute rejection episode (defined as ISHLT rejection grade ⬎2). Maintenance immunosuppressive therapy in these patients consisted of cyclosporine, MMF, and low dose steroids. Our results show that IL-21 mRNA expression levels are significantly upregulated in acute rejection biopsies compared to the levels before and after acute rejection (median IL-21/GAPDH mRNA ratio before, during, and after acute rejection was respectively 135, 259, and 91; p⫽0.029, Friedman’s repeated measures ANOVA). These data indicate that intragraft IL-21 plays a role in the allogeneic response after clinical transplantation. IL-21 may enhance the cytotoxic effector functions of NK-cells in the graft, leading to myocyte injury characteristic for acute cardiac allograft rejection. 307 A RAPID ASSAY TO MONITOR CELL-MEDIATED IMMUNITY IN TRANSPLANT POPULATIONS R.J. Kowalski, D.R. Post, J.B. Woodcock, M.C. Schneider, J. Britz, Cyles, Inc., Columbia, MD Purpose: The CD4 in vitro CMI assay platform technology directly measures the functional response of CD4⫹ lymphocytes. The assay, performed on whole blood or peripheral blood mono-

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nuclear cells (PBMCs), measures the increase in ATP following activation of the lymphocytes by mitogenic or antigenic stimulation. Procedure: After 4-18 hours incubation with stimulant, CD4⫹ lymphocytes were selected, washed and lysed to release intracellular ATP. The ATP levels were then quantified using a firefly luciferin/luciferase bioluminescent readout. To monitor druginduced immunosuppression, populations of transplant recipients were compared to apparently healthy adults. Transplant populations included heart, kidney, liver and pancreas recipients. They were assessed for their response to mitogens (e.g. Phytohemagglutinin) and recall antigens in vitro. Results: Compared to apparently healthy adults, immune responses were significantly lower in transplant recipients. Responses were also compared by organ transplanted and by type of immunosuppressive therapy. Organ recipients were also found to be non-reactive to memory antigens. Conclusion: The in vitro CMI assay provides a useful measurement of immune response, which can help determine the balance between prevention of rejection of a transplanted organ by immunosuppressants, and over-suppression of the organ recipient. 308 EFFECT OF A NOVEL ANTI-OXIDANT AND ANTIINFLAMMATORY COMPOUND ON RODENT ALLOGRAFT ARTERIOSCLEROSIS S. Murata,1 M.A. Lijkwan,1 P. Hammainen,1 C. Coleman,1 C. York,1 C.L. Sundell,2 R.E. Morris,1 R.C. Robbins,1 1Stanford University, Palo Alto, CA; 2AtheroGenics, Inc. Background: The long term success of cardiac transplantation continues to be limited by the development of graft coronary artery disease. AGI-1096 is a newly developed antioxidant and anti-inflammatory compound that inhibits the inducible expression of VCAM-1 and MCP-1 in endothelial cells (IC50⫽11␮M and 5␮M, respectively) as well as smooth muscle cell proliferation (IC50 ⫽5.5␮M), giving it the potential to prevent chronic allograft arteriosclerosis. In this study, we evaluated the efficacy of this compound using a rodent aortic transplantation model. Methods: Donor descending aortas from ACI rats were heterotopically transplanted into Lewis rat abdomens in end-to-end fashion with minimal ischemic time. Animals were assigned to five groups as follows: AGI-1096 0 mg/kg/day(mkd) (vehicle, N⫽10), 10 mkd (N⫽10), 20 mkd (N⫽10), positive control (cyclosporine A (CsA) 10mkd by oral gavage, N⫽10), and isograft negative control (Lewis-to-Lewis, N⫽5). AGI-1096 was administrated subcutaneously to recipient animals three days prior to the surgery and for 90 days thereafter. On day 90, the allograft segment was fixed in situ with 10% buffered formalin. Paraffin-embedded sections were stained with hematoxylin and eosin (H&E) and von Giesson’s elastic stain (VG) and intima/ media (IM) ratio and luminal narrowing (%LN) assessed by digital morphometry. Results: Treatment with AGI-1096 was well tolerated and all animals regained weight postsurgery. Recipients treated with AGI-1096 demonstrated dose-dependent lowering of the I/M ratio and %LN when compared to vehicle controls that was statistically significantly different at the 20 mkd dose. (p⬍0.05, ANOVA and Dunnetts post hoc test). Conclusion: This is the first study to show that treatment of allograft recipients with AGI-1096 decreases the incidence of arteriosclerosis when compared to grafts treated with vehicle.