Myocardial Nuclear Factor-Kappa B Activity And Nitric Oxide Production In Rejecting Cardiac Allografts

Background. Nuclear factor-kappa B (NF-kappa B) 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-kappa B DNA binding activity and nitric oxide ( NO) production in untreated allografts and whether inhibition of NF-kappa B suppresses NO production and prolongs graft survival.Methods. In allograft recipients and isograft controls, NF-kappa B 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-kappa B, 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-kappa B was per formed to identify the cell type responsible for NF-kappa B activity.Results. A time-dependent increase in myocardial NF-kappa B activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-kappa B 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-kappa B 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-kappa B activity with peak infiltrate on posttransplant day 4, PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-kappa B to the infiltrating mononuclear cells on posttransplant day 5.Conclusion. These data support a role for NF-kappa B in allograft rejection.