ALLOIMMUNE-MEDIATED APOPTOSIS

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Abstract
The purpose of the present study was (1) to compare apoptotic activity in models of acute and chronic rejection and (2) to study the cellular distribution of parenchymal versus inflammatory cell apoptosis. Heterotopic cardiac mouse transplantation (CBA into C57BL/6) was used to produce allografts undergoing acute (day 7, untreated recipients, n=6) or chronic (day 55, anti-CD4/8 for 28 days, n=6) rejection. As references, we used 55-day isograft controls (n=5) and native hearts (n=6). To assess apoptotic activity, we quantified DNA laddering (32P incorporation), DNA fragmentation (antinucleosome ELISA), and caspase-1 transcript levels (32P-reverse transcriptase-polymerase chain reaction). To localize apoptosis, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. DNA laddering and nucleosome levels were increased in allografts undergoing acute or chronic rejection when compared with both controls. Both parameters were twofold higher in acutely compared with chronically rejecting hearts. Caspase-1 transcript levels were increased in acutely (P<0.0001) and chronically rejecting hearts (P=0.004). Acutely rejecting grafts had more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive nuclei (53+/-3 nuclei/high-powered field) than chronically rejecting grafts (9+/-1 nuclei/high-powered field, P<0.0001), but the distribution between graft-infiltrating inflammatory cells and myocytes was similar. Vascular cells undergoing apoptosis were infrequent in both forms. Using four separate indices, apoptotic activity is more pronounced in cardiac allografts undergoing acute compared with chronic rejection. This reflects, in part, the degree of alloimmune response. However, we speculate that the contributions of apoptosis to various forms of rejection are multifactorial. The long-term outcome to the graft may depend upon the magnitude, timing, and target of programmed cell death.