Myocardial nuclear factor-kappaB activity and nitric oxide production in rejecting cardiac allografts.
BACKGROUND
Nuclear factor-kappaB (NF-kappaB) 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-kappaB DNA binding activity and nitric oxide (.NO) production in untreated allografts and whether inhibition of NF-kappaB suppresses .NO production and prolongs graft survival.


METHODS
In allograft recipients and isograft controls, NF-kappaB 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-kappaB, 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-kappaB was per formed to identify the cell type responsible for NF-kappaB activity.


RESULTS
A time-dependent increase in myocardial NF-kappaB activity was seen in untreated allografts as compared with isografts as determined by PhosphorImage analysis. Peak NF-kappaB 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-kappaB 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-kappaB activity with peak infiltrate on posttransplant day 4. PDTC treatment had no effect on the extent of infiltrate. Immunohistochemical staining localized NF-kappaB to the infiltrating mononuclear cells on posttransplant day 5.


CONCLUSIONS
These data support a role for NF-kappaB in allograft rejection.
Cooper
M
M
Department of Transplant Surgery, Medical College of Wisconsin, Milwaukee 53226, USA.
Lindholm
P
P
Pieper
G
G
Seibel
R
R
Moore
G
G
Nakanishi
A
A
Dembny
K
K
Komorowski
R
R
Johnson
C
C
Adams
M
M
Roza
A
A
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D006084:Graft Rejection / Q000378:metabolism* / Q000473:pathology; D006085:Graft Survival / Q000187:drug effects; D016027:Heart Transplantation; D007150:Immunohistochemistry; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D011759:Pyrrolidines / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D011920:Rats, Inbred WF; D013859:Thiocarbamates / Q000494:pharmacology; D014184:Transplantation, Homologous; D014185:Transplantation, Isogeneic
D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D009569:Nitric Oxide