Background. Nuclear factor-κB (NF-κB) is a rapid response transcription factor for genes whose products are critical for inflammation and immunity. In a rat model of heterotopic cardiac transplantation, we studied NF-κB DNA binding activity and nitric oxide (·NO) production in untreated allografts and whether inhibition of NF-κB suppresses ·NO production and prolongs graft survival. Methods. In allograft recipients and isograft controls, NF-κB was assayed by electrophoretic mobility shift assay, daily from transplant until rejection. Myocardial ·NO was directly detected in explanted allografts by electron spin resonance spectroscopy on day 6 after transplant. The potent inhibitor of NF-κB, pyrrolidine dithiocarbamate (PDTC; 250 mg/kg s.c.) was administered daily from transplant until day of rejection. The extent of graft lymphocytic infiltrate was assessed by routine hematoxylin and eosin staining. Immunohistochemical staining of NF-κB was per formed to identify the cell type responsible for NF-κB activity. Results. A time-dependent increase in myocardial NF-κB activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-κB activity occurred in allografts on day 4 with a ninefold increase as compared with isografts (24.0±3.7% vs. 2.7±0.5; P < 0.05). On posttransplant day 6, electron spin resonance spectroscopy analysis of allografts demonstrated ·NO identified by a triplet nitrogen signal centered at g=2.012 with hyperfine splitting of 17.5 Gauss, which is consistent with nitrosoheme formation and low-field signals at g=2.08 and g=2.03 consistent with nitrosomyoglobin. These signals were not seen in native hearts of allograft recipients. With PDTC administration, a threefold decrease in NF-κB activity within the transplanted heart was observed on posttransplant day 5 as compared with untreated allografts (9.7±1.6% vs. 23.5±2.5%; P < 0.01). PDTC prolonged graft survival as compared with untreated allografts (11.7±0.3 vs. 6.6±0.2 days; P < 0.05) and reduced the intensity of the nitrosoheme and nitrosomyoglobin signals. Allograft mononuclear cell infiltrate correlated with peak NF-κB activity with peak infiltrate on posttransplant day 4. PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-κB to the infiltrating mononuclear cells on posttransplant day 5. Conclusion. These data support a role for NF-κB in allograft rejection.
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