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INFLAMMATION AND IMMUNOPHARMACOLOGY

Treatment with -Phenyl-N-tert-butylnitrone, a Free Radical-Trapping Agent, Abrogates Inflammatory Cytokine Gene Expression during Alloimmune Activation in Rat Cardiac Allografts

Division of Transplant Surgery (G.M.P., V.N., C.P.J., X.Z., A.M.R., M.B.A., G.H.), the Free Radical Research Center (G.M.P.), the Cardiovascular Center (G.M.P., A.K.K.), Department of Medicine (A.K.K.) and the Department of Biophysics (C.C.F.), Medical College of Wisconsin, Milwaukee, Wisconsin

Spin-trapping nitrones such as -phenyl-N-tert-butylnitrone (PBN) have traditionally been used to trap and stabilize free radicals for detection by electron paramagnetic resonance (EPR) spectroscopy. Unlike classical antioxidants, these agents have never been evaluated therapeutically in allograft transplantation. In the present study, we examined potential mechanisms of action of treatment with PBN in a rat model of acute cardiac allograft transplantation. Graft rejection was determined by histological examination and graft function determined by in situ sonomicrometry. DNA binding for nuclear factor (NF)-B and activator protein (AP-1) were determined by gel shift assays. Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed for inducible nitric-oxide synthase (iNOS) and inflammatory cytokines. Histological rejection scores were elevated in untreated allografts and decreased by treatment with PBN. In situ sonomicrometry revealed decreased heart rate and distended end diastolic and end systolic segment lengths with rejection. Although PBN did not alter heart rate, it did normalize the distention of both diastolic and systolic cardiac dimension. EPR spectroscopy revealed nitrosylation of myocardial heme protein in untreated allografts that was decreased by treatment with PBN. PBN also decreased iNOS protein and iNOS mRNA. RT-PCR analysis revealed enhanced cytokine gene expression for interferon-, interleukin-6, and interleukin-10 in untreated allografts. Expression for these genes was potently inhibited or abolished in recipients treated with PBN. PBN treatment also decreased DNA binding of transcription factors, NF-B and AP-1. Thus, PBN retains significant anti-inflammatory properties through its action to down-regulate cytokine gene expression that contribute to protection against acute alloimmune activation in cardiac allografts.

Address correspondence to: Dr. Galen M. Pieper, Division of Transplant Surgery, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, WI 53226. E-mail: gmpieper{at}mcw.edu

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