Alloimmune-mediated apoptosis during acute and chronic rejection of the transplanted heart

301 Apoptotic cells have been detected in transplanted organs suggesting potential roles for programmed cell death in allograft rejection. Purpose of this study was to compare indices of apoptosis in grafts undergoing acute and chronic rejection, Using a heterotopic cardiac mouse transplant model employing CBA donors and C57BL6 recipients, we produced allografts undergoing acute (day 7,untreated recipients, n=6) or chronic rejection (day 55, anti-CD4/8 for 28 days, n=6) and compared these to native hearts (n=6) and isografts (n=5) as controls. To assess apoptotic activity, we quantitated DNA laddering (32P incorporation), DNA fragmentation (anti-nucleosome ELISA) and ICE transcript levels (32P RT-PCR). DNA laddering and DNA fragmentation were markedly increased in allografts and were two-fold higher in acutely compared with chronically rejecting hearts. ICE levels show a concordant pattern with low baseline levels in native hearts (0.01±0.01 units) and isografts (0.07±0.03 units) and significantly increased levels in acutely (0.70±0.26 units, p<0.0001) and chronically rejecting hearts (0.27±0.13 units, p=0.004). To localize apoptotic activity within the grafts, we used TUNEL in representative sections of each group. While the majority of TUNEL positive nuclei localized to infiltrating, inflammatory cells, a less abundant subset was present in myocytes and endothelial cells. To identify potential triggers and regulators of apoptosis, we measured allograft-specific mean transcript levels (semiquantitative32 P RT-PCR) for FASLigand, its receptor FAS, death agonists (p53 and Bax) and death antagonists (Bcl-2 and Bcl-X1.) While FAS showed similar expression in both isografts and allografts (acute and chronic), transcript levels for FASLigand were significantly upregulated during acute and chronic rejection (p<0.0001). Allografts did not have increased levels of Bcl-X1, Bax and p53 arguing against alloimmune-specific regulation patterns. In contrast, the expression of the antiapoptotic gene Bcl-2 was significantly upregulated in acutely rejecting grafts (p<0.0001).Conclusion. Apoptosis is upregulated during both acute and chronic rejection but more in acutely rejecting hearts. Upregulation of FASLigand in allografts is a potential signalling mechanism that could induce apoptosis. Upregulation of Bcl-2 may be part of a counterregulatory response to limit alloimmune-stimulated apoptosis. Further studies are required to decifer the functional roles of apoptosis in various forms and phases of rejection. We speculate that apoptosis may contribute to grafts failure by promoting parenchymal damage during acute rejection and to graft vasculopathy by mediating endothelial injury during chronic rejection.