β Scribed by Yasuo Yamaguchi; Kazutoshi Okabe; Fujio Matsumura; Eiji Akizuki; Teishi Matsuda; Hajime Ohshiro; Jian Liang; Shinwa Yamada; Katsutaka Mori; Michio Ogawa
No coin nor oath required. For personal study only.
The role of nitric oxide (NO) on tissue injury of hepatic allografts during rejection remains controversial. We investigated inducible nitric oxide synthase (iNOS) expression and formation of peroxynitrite in ACI rat liver grafts implanted in recipients. Animals were divided into four experimental groups: group I, isografts; group II, untreated hepatic allografts; group III, allografts treated with FK506; and group IV, allografts pretreated with donor-specific blood transfusion (DST). Serum nitrite/nitrate, interferon-β₯ (IFN-β₯), and tumor necrosis factor-β£ (TNF-β£) concentrations increased significantly in group II rats after transplantation but were significantly lower in groups I, III, and IV. The numbers of macrophages that reacted with an antimacrophage iNOS monoclonal antibody as well as iNOS messenger RNA (mRNA) levels in liver specimens were also much lower in groups I, III, and IV as compared with group II. Immunostaining and Western blot analysis showed prominent tissue nitrotyrosine expression in untreated hepatic allografts, but not in allografts treated with FK506 or donor-specific blood. These results suggest that one of the mechanisms by which production of NO results in injury in rat hepatic allografts may be because of its reaction with superoxide to form peroxynitrite. (HEPATOLOGY 1999;29:777-784.)
Nitric oxide (NO) has been detected during organ transplant rejection 1,2 and plays a role in allogeneic responses. 3 NO is synthesized from the amino acid L-arginine by a family of enzymes, nitric oxide synthases, and is involved in diverse physiological and pathophysiological processes. In experimental organ graft rejection, elevated serum concentrations of nitrite and nitrate can be normalized by immunosuppressive treatment. 1 Several different experimental approaches suggest that the rejected organ is itself the source of increased end-products of L-arginine-NO synthesis. In a study of rat cardiac allograft rejection, nitrosyl iron hemoglobin signals were detected in the grafts but not in other recipient organs. 3 Similarly, a sponge matrix allograft showed increased nitrite and nitrate concentrations in the sponge fluids. 4 Despite many recent advances in immune research, the regulatory and effector mechanisms by which NO affect the rejection process remain incompletely understood. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide, which produces peroxynitrite. 5 Activated cellular infiltrates produce high levels of both superoxide and NO. NO reacts with superoxide to form peroxynitrite. 6,7 Beckman et al. [8][9][10] suggested that peroxynitrite may be an important mediator of free radical-dependent toxicity because of its strong oxidizing properties. Subsequently, several studies have shown that peroxynitrite mediates oxidative injury toward a variety of biomolecules including protein and nonprotein thiols, 11 deoxyribose, 12 and membrane phospholipids. 11,13 Thus, peroxynitrite may play an important role in the cellular damage associated with the overproduction of NO, but there are very limited data regarding its in vivo formation during organ allograft rejection.
Because of its reactivity, peroxynitrite cannot be measured directly in vivo. 14 Proteins are targets of reactive oxygen species, and detection of oxidatively modified proteins is often used as an index of oxidative stress. In this regard, peroxynitrite has the potential to nitrate tyrosine residues in proteins 7,15 forming nitrotyrosine, which leaves a footprint and can be detected by immunohistochemical means. 6,7 Nitration is catalyzed by superoxide dismutase. 14 Thus, tyrosine nitration is a marker of oxidants formed in vivo from NO with formation of peroxynitrite being the most facile nitrating reaction to occur. In fact, antibodies to nitrotyrosine have shown nitration in human atherosclerosis, myocardial ischemia, septic and distressed lung, and inflammatory bowel disease. 16 The detection of nitrotyrosine in hepatic allografts increases the possibility that NO, by virtue of its ability to form reactive nitrogen intermediates, may promote tissue injury during acute rejection. In the present study, we assessed peroxynitrite-mediated tissue damage in rat hepatic allograft rejection by examining the expression of inducible nitric oxide synthase (iNOS) and nitrotyrosine.
MATERIALS AND METHODS
Male LEW (RT1 l ) rats were used as recipients and ACI (RT1 a ) rats as donors. These rats were obtained from the Central Institute for Experimental Animals, Kawasaki, Japan. This strain combination is fully allogeneic and results in the acute rejection of liver transplants. 17 A LEW-to-LEW combination was used for control isografts. All animals were maintained under standard conditions and received water and rodent chow ad libitum. The animals weighed 225 to 250 g.
π SIMILAR VOLUMES
The kinetics of messenger RNA (mRNA) and protein levels pounds. 1 Thus, neutrophils were thought to be relevant only of cytokine-induced neutrophil chemoattractant (CINC) in rat to the efferent limb of the immune response. However, it has hepatic allografts during acute rejection were investigated.
W e used a model of rat hepatic allograft rejection to evaluate levels of procoagulant activity and tumor necrosis factor during acute cellular rejection. ACI livers were transplanted into Lewis rats, and Lewis-to-Lewis isografts and unoperated animals served as controls. Animals were killed on days