Eiji Akizuki

Immunosuppressants Decrease Neutrophil Chemoattractant and Attenuate Ischemia/Reperfusion Injury of the Liver in Rats

BACKGROUND Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver. METHODS Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver. RESULTS Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion. CONCLUSION The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.

Neutrophil elastase enhances intercellular adhesion molecule-1 expression

BACKGROUND Elastase released from activated neutrophils is an important mediator of inflammatory tissue damage. We investigated the effect of human neutrophil elastase (NE) inhibitor (ONO-5046) on reperfusion injury after pancreaticoduodenal transplantation in rats by measuring the expression of intercellular adhesion molecule-1 (ICAM-1). Additional in vitro experiments were conducted to investigate the effect of NE on ICAM-1 mRNA transcription in a rat endothelial cell line (WK-5) and human umbilical vein endothelial cells (HUVEC). METHODS In an in vivo experiment, male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. An NE inhibitor, ONO-5046, was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. ICAM-1 expression was determined by immunostaining and Northern analysis. In in vitro experiments, the effects of NE and chemical agents on ICAM-1 mRNA transcripts were determined in WK-5 cells and HUVEC. RESULTS Pretreatment with ONO-5046 decreased ICAM-1 immunostaining in the pancreatic graft and inhibited the increase in ICAM-1 mRNA levels in grafts after reperfusion. ICAM-1 mRNA levels in WK-5 cells and HUVEC showed stimulation by NE, while ONO-5046 inhibited this increase. Calcium ionophore (A23187) augmented NE stimulation of ICAM-1 mRNA levels in these cells. In contrast, a phospholipase C inhibitor (U73122) blunted NE induction of ICAM-1 mRNA, and either calcium chelator (TMB-8) or a nuclear factor-kappa B inhibitor (pyrrolidine dithiocarbamate) completely inhibited induction. CONCLUSION These results indicate that NE stimulates ICAM-1 expression in pancreatic grafts via intracellular Ca2+ influx and a phospholipase C signal transduction.

Phenotype And Localization Of Macrophages Expressing Inducible Nitric Oxide Synthase In Rat Hepatic Allograft Rejection

BACKGROUND We investigated the phenotype and localization of macrophages expressing inducible nitric oxide synthase (iNOS) in rat hepatic allografts, using double immunostaining with anti-macrophage iNOS (macNOS) and rat anti-macrophage (ED1 or ED2) monoclonal antibodies. METHODS The animals were divided into three experimental groups: group 1, isografts; group 2, untreated hepatic allografts; and group 3, hepatic allografts treated with FK506. RESULTS Plasma nitrite/nitrate concentrations in group 2 increased on day 3, peaked on day 5, and decreased thereafter. In contrast, the plasma nitrite/nitrate concentrations in group 1 increased slightly on day 3, but decreased gradually thereafter. The plasma concentrations of nitrite/nitrate did not vary in group 3. The peak nitrite/nitrate values in group 2 were significantly greater than those in groups 1 and 3. The number of macNOS+ cells peaked on day 5 in group 2. In contrast, a few macNOS+ cells were seen in the liver grafts of groups 1 and 3. Double immunostaining revealed that the macNOS+ cells consisted of macNOS+ ED1+ (80%) and macNOS+ ED2+ (40%) in the untreated hepatic allografts on day 5. In addition, a number of macNOS+ cells also were seen in the red pulp of the recipient spleen in the untreated hepatic allografts. CONCLUSIONS These results suggest that the intense iNOS expression by the monocyte/macrophage lineage among the hepatic infiltrates and by the splenic macrophages after transplantation supports a role for nitric oxide in the immunomodulation of allogeneic responses in local and remote organs, and possibly serves as a mediator of cytotoxic graft damage.

Neutrophil elastase inhibitor (ONO-5046) decreases cytokine-induced neutrophil chemoattractant after reperfusion of pancreaticoduodenal transplantation in rats.

The protective effects of a neutrophil elastase inhibitor (ONO-5046) on reperfusion injury following pancreaticoduodenal transplantation in rats were studied by measuring serum concentrations of cytokine-induced neutrophil chemoattractant (CINC). Male Wistar rats were transplanted with syngeneic pancreaticoduodenal grafts. ONO-5046 was injected intravenously 5 min before vascular clamping and immediately after reperfusion at a dose of 10 mg/kg. No significant differences were observed in the peak serum concentrations of amylase between the groups treated with and treated without ONO-5046. The serum lipase concentrations in the untreated animals increased and peaked 3 hr after reperfusion. ONO-5046 significantly decreased the peak serum lipase concentration. The serum CINC concentrations, which were determined by enzyme-linked immunosorbent assay, increased and peaked 3 hr after reperfusion, decreasing gradually thereafter. However, pretreatment with ONO-5046 significantly inhibited the rise in serum CINC concentrations after reperfusion. Expression of CICN transcripts in the pancrease grafts was evaluated by Northern blot analysis and peaked 3 hr after reperfusion in untreated animals. Pretreatment with ONO-5046 also significantly inhibited the expression of CINC mRNA transcripts in the graft. ONO-5046 significantly decreased the number of neutrophils accumulated in the pancreas graft 24 hr after transplantation. In vitro CINC production by peritoneal macrophages was increased by neutrophil elastase in dose-dependent fashion. However, ONO-5046 decreased CINC production by peritoneal macrophages in response to neutrophil elastase. These results suggest that ONO-5046 prevents early neutrophil accumulation in the pancreas following ischemia/reperfusion of pancreaticoduodenal transplantation.

Calcium-channel blocker attenuates Kupffer cell production of cytokine-induced neutrophil chemoattractant following ischemia-reperfusion in rat liver.

We investigated the effects of the calcium-channel blocker verapamil hydrochloride on the production of cytokine-induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 min by portal vein occlusion. Animals were pretreated with intravenous injection of verapamil hydrochloride (2.5 mg/kg) 5 min before vascular clamp. Verapamil hydrochloride limited increases in the chemoattractant compared with nonpretreated rats. Most cells immunostained for chemoattractant were ED2-positive macrophages in sinusoids. In vitro chemoattractant production by Kupffer cells isolated from animals pretreated with verapamil hydrochloride was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hr after reperfusion in nonpretreated animals, while pretreatment with verapamil hydrochloride significantly decreased chemoattractant mRNA levels. In vitro chemoattractant production could be induced in naive Kupffer cells after stimulation with oxygen radicals generated by hypoxanthine and xanthine oxidase, but verapamil hydrochloride prevented these increases. We concluded that the calcium-channel blocker verapamil hydrochloride significantly attenuates chemoattractant release by Kupffer cells after ischemia–reperfusion in the rat liver.

The Novel Carboxamide Derivative IS-741 Reduces Neutrophil Chemoattractant Production by Bronchoalveolar Macrophages in Rats with Cerulein-Induced Pancreatitis Complicated by Sepsis

Background/Aims: The priming mechanism of macrophages to secrete cytokines in acute pancreatitis is important for remote organ failure following septic complication. The effects of novel carboxamide derivative, IS-741, on neutrophil chemoattractant production by bronchoalveolar macrophages were studied in rats with cerulein-induced pancreatitis complicated by sepsis. Methods: Pancreatitis was induced by four intramuscular injections of cerulein (50 µg/kg at 1-hour intervals). Pancreatitis rats were injected intraperitoneally with 10 mg/kg of lipopolysaccharide (LPS) 6 h following the first cerulein injection as a septic challenge. Pancreatitis rats received a continuous intravenous injection of IS-741 (3 mg/kg/h) 30 min before the septic challenge. Results: Intense mononuclear cell infiltration and lung hemorrhage occurred in untreated pancreatitis rats complicated with sepsis, but hemorrhage was not seen in septic pancreatitis rats receiving a continuous intravenous injection of IS-741 shortly before sepsis induction. The IS-741-treated rats had lower serum concentrations of cytokine-induced neutrophil chemoattractant (CINC), as well as fewer the pulmonary neutrophils and infiltrates immunoreactive for CINC or Mac-1 (CD11b/CD18). Conclusion: The novel carboxamide derivative IS-741 reduced CINC production by bronchoalveolar macrophages and effectively prevented pancreatitis-associated lung injury following the septic challenge.

Reduced Interleukin-12, Interleukin-18, and Interferon-γ Production with Prolonged Rat Hepatic Allograft Survival After Donor-Specific Blood Transfusion

Interferon-γ is a key immunoregulatory cytokine involved in acute graft rejection. Immunologic unresponsiveness to organ allografts has been induced by pretransplantation donor-specific blood transfusion, both experimentally and clinically. We investigated interferon-γ production and intragraft gene expression of type-1 T-helper cytokines such as interleukin-12 and -18 and type-2 T-helper cytokines such as interleukin-10 and transforming growth factor-β in rats receiving hepatic allografts after such transfusions. The animals were divided into four groups: group I received isografts; group II received allografts; group III received allografts after donor-specific transfusion; and group IV received allografts and was treated with FK 506. Donor blood given seven days prior to transplantation significantly prolonged allograft survival. The serum interferon-γ concentrations in group II increased, peaking on day 5 and then decreasing. Serum interferon-γ concentrations in groups I, III, and IV were significantly lower than those observed in group II, as were levels of interleukin-12 and interleukin-18 mRNA in the graft. Transforming growth factor-β and interleukin-10 mRNA levels in grafts in transfused animals were significantly greater than those in the untreated allograft group. Interleukin-12 and -18 mRNA transcripts in an allogeneic mixed lymphocyte reaction were inhibited by interleukin-10 and transforming growth factor-β. These results suggest that interleukin-12 and -18 expression in hepatic allografts is inhibited in the immunologically unresponsive state induced by donor-specific transfusion.

Novel carboxamide derivative (IS-741) attenuates lung injury in rats with cerulein-induced pancreatitis complicated by endotoxemia.

The therapeutic effects of an intravenouslyinjected carboxamide derivative (IS-741) on lung injurywere studied in rats with cerulein-induced pancreatitiscomplicated by endotoxemia. Pancreatitis was induced by four intramuscular injections of cerulein(50 μg/kg at 1-hr intervals). Pancreatitis rats wereinjected intraperitoneally with 10 mg/kg oflipopolysaccharide (LPS) 6 hr following the firstcerulein injection as a challenge of endotoxemia. Ratswere divided into four groups: group I, pancreatitiswith LPS; group II, pancreatitis with LPS treated witha continuous intravenous injection of IS-741 at 0.03 mg/kg/hr); group III, pancreatitis withLPS treated with a continuous intravenous injection ofIS-741 at 0.3 mg/kg/hr); and group IV, pancreatitis withLPS treated with a continuous intravenous injection of IS-741 at 3 mg/kg/hr). IS-741 wasadministered 30 min before the endotoxemia challenge.Intense mononuclear cell infiltration and lunghemorrhage occurred in untreated pancreatitis rats withLPS (group I), but hemorrhage was not seen in group IVrats receiving a continuous injection of IS-741 shortlybefore the induction of endotoxemia. The IS-741- treatedrats (groups II, III, and IV) had lower serum concentrations of cytokine-induced neutrophilchemoattractant (CINC), as well as fewer pulmonaryinfiltrates immunoreactive for CINC or Mac-1(CD11b/CD18). The number of neutrophils infiltrating thelung in groups II, III, and IV was significantlylower than that of group I. Conversely, CINC productionby bronchoalveolar macrophages in vitro were stimulatedby LPS but were reduced by the presence of IS-741. The carboxamide derivative IS-741 effectivelyprevented pancreatitisassociated lung injury followingthe challenge of endotoxemia.

Tumor necrosis factor-beta is associated with thymic apoptosis during acute rejection.

BACKGROUND Intrathymic events undergoing allograft rejection remain undefined. The present study investigated the role of tumor necrosis factor-beta on acute thymic involution in rat hepatic allograft recipients during rejection. METHODS Apoptosis and cellular phenotypic changes in the thymus were studied after hepatic transplantation. RESULTS Thymocytes in both the medulla and cortex were sparse during acute rejection. Phenotypically, CD4+CD8+ T cells decreased significantly, whereas there were relative increases in CD4-CD8-, CD4+CD8-, and CD4-CD8+ T cells in untreated allograft recipients. Additionally, thymic apoptosis was found by in situ DNA end labeling and electron microscopy. Apoptotic cells were predominantly distributed in the cortex. Biologic lymphotoxin (tumor necrosis factor-beta)/tumor necrosis factor-alpha cytotoxic activity in the serum was significantly increased in untreated hepatic allograft recipients. Tumor necrosis factor-beta mRNA was detected in untreated allograft livers, and intraperitoneal administration of recombinant human tumor necrosis factor-beta induced extensive apoptosis of thymocytes in vivo. In contrast, no significant thymic involution was observed in donor-specific blood transfusion-treated allograft and isograft recipients. Intraperitoneal administration of rabbit anti-human tumor necrosis factor-beta polyclonal antibody or recombinant human interleukin-10 inhibited thymic apoptosis in untreated hepatic allograft recipients. CONCLUSIONS Allograft rejection, but not donor-specific transfusion-induced immunologic unresponsiveness, is associated with thymic involution, a process that may be mediated by tumor necrosis factor-beta.

Prolonged survival of rat hepatic allografts treated with a pretransplant donor-specific blood transfusion is associated with reduced cytokine-induced neutrophil chemoattractant expression

BACKGROUND One intravenous injection of freshly heparinized donor blood 7 days before transplantation significantly prolongs hepatic allograft survival from ACI (RT1a) to LEW [RT1(1)] rats. The aim of this study was to investigate hepatic allograft expression of neutrophil chemoattractant and tumor necrosis factor in immunologic unresponsiveness. METHODS AND RESULTS Cytokine-induced neutrophil chemoattractant levels in untreated hepatic allografts were significantly higher than in allografts treated with donor-specific transfusion. Additionally, more neutrophils infiltrated untreated than transfusion-treated hepatic allografts. The number of chemoattractant-positive cells was significantly lower in donor-specific transfused allografts than in untreated hepatic allografts. The number of ED1+ mononuclear cells infiltrating portal areas of untreated allografts increased over time and expressed abundant cytokine-induced neutrophil chemoattractant mRNA during acute rejection. This correlated with significantly higher levels of chemoattractant mRNA in untreated allograft livers on postoperative day 5 as compared with transfusion-treated allografts. Serum concentrations of tumor necrosis factor-alpha in untreated hepatic allograft recipients increased over time and peaked on day 7, while those in transfused allografts were maintained at lower levels. Moreover, in vitro chemoattractant production by peritoneal macrophages responded in a dose-dependent manner to tumor necrosis factor-alpha. CONCLUSION Donor-specific transfusion treatment decreases tumor necrosis factor-alpha and chemoattractant expression as well as neutrophil accumulation in hepatic allografts.