Innate immune function predicts the development of nosocomial infection in critically injured children.

BACKGROUND: Critical injury has been associated with reduction in innate immune function in adults, with infection risk being related to degree of immune suppression. This relationship has not been reported in critically injured children. HYPOTHESIS: Innate immune function will be reduced in critically injured children, and the degree of reduction will predict the subsequent development of nosocomial infection. METHODS: Children (≤18 years old) were enrolled in this longitudinal, prospective, observational, single-center study after admission to the pediatric intensive care unit following critical injury, along with a cohort of outpatient controls. Serial blood sampling was performed to evaluate plasma cytokine levels and innate immune function as measured by ex vivo lipopolysaccharide-induced tumor necrosis factor α (TNF-α) production capacity. RESULTS: Seventy-six critically injured children (and 21 outpatient controls) were enrolled. Sixteen critically injured subjects developed nosocomial infection. Those subjects had higher plasma interleukin 6 and interleukin 10 levels on posttrauma days 1-2 compared with those who recovered without infection and outpatient controls. Ex vivo lipopolysaccharide-induced TNF-α production capacity was lower on posttrauma days 1-2 (P = 0.006) and over the first week following injury (P = 0.04) in those who went on to develop infection. A TNF-α response of less than 520 pg/mL at any time in the first week after injury was highly associated with infection risk by univariate and multivariate analysis. Among transfused children, longer red blood cell storage age, not transfusion volume, was associated with lower innate immune function (P < 0.0001). Trauma-induced innate immune suppression was reversible ex vivo via coculture of whole blood with granulocyte-macrophage colony-stimulating factor. CONCLUSIONS: Trauma-induced innate immune suppression is common in critically injured children and is associated with increased risks for the development of nosocomial infection. Potential exacerbating factors, including red blood cell transfusion, and potential therapies for pediatric trauma-induced innate immune suppression are deserving of further study.

Intestinal phenotype in mice overexpressing a heparin-binding EGF-like growth factor transgene in enterocytes.

PRIMARY OBJECTIVE: Heparin-binding EGF-like growth factor (HB-EGF) protects the intestine from damage in animals. Future clinical trials of HB-EGF may involve administration of repeated doses of HB-EGF. Since HB-EGF activates EGF receptors which have been implicated in tumor development, we examined the effects of HB-EGF overexpression in the intestine. RESEARCH DESIGN: We generated transgenic (TG) mice in which the human HB-EGF gene is driven by the villin promoter to overexpress HB-EGF along the crypt-villous axis from the duodenum to the colon. RESULTS: HB-EGF TG mice have increased enterocyte proliferation balanced by increased enterocyte apoptosis. Despite prolonged overexpression of HB-EGF, no evidence of intestinal hyperplasia or tumor formation occurs. Although HB-EGF TG mice have no significant phenotypic alterations under basal conditions, they have increased resistance to intestinal injury. CONCLUSIONS: Prolonged intestinal HB-EGF overexpression results in no significant phenotypic alterations under basal conditions, but confers protection against intestinal injury.

Heparin-binding epidermal growth factor-like growth factor gene disruption is associated with delayed intestinal restitution, impaired angiogenesis, and poor survival after intestinal ischemia in mice.

PURPOSE: We have demonstrated that administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) protects the intestines from injury. The aim of the current study was to evaluate the effect of HB-EGF gene disruption on intestinal restitution, angiogenesis, and long-term survival after intestinal ischemia/reperfusion (I/R) injury. METHODS: HB-EGF (-/-) and wild-type HB-EGF (+/+) littermate mice were subjected to 45 minutes of superior mesenteric artery occlusion followed by reperfusion. Functional recovery of the gut permeability barrier was evaluated with Ussing chamber studies, and microvessel density was evaluated immunohistochemically. Animal survival was evaluated using the Kaplan-Meier method. RESULTS: Histologic damage after ischemia was significantly higher in HB-EGF (-/-) mice compared with HB-EGF (+/+) mice, associated with a significantly higher number of incompetent (nonhealed, nonresurfaced) villi indicative of delayed structural healing by restitution. HB-EGF (-/-) mice had increased intestinal permeability after intestinal I/R. HB-EGF (-/-) mice had significantly lower microvessel density at 3 and 7 days after I/R, indicating that HB-EGF gene deletion resulted in delayed onset of angiogenesis. Two-week mortality rates were significantly higher in HB-EGF (-/-) mice. CONCLUSIONS: Endogenous HB-EGF significantly enhances healing by restitution, prolongs survival, and enhances angiogenesis in mice subjected to intestinal I/R injury. These findings support our hypothesis that HB-EGF administration may improve outcome in patients with intestinal I/R injury, including necrotizing enterocolitis.

Heparin-binding EGF-like growth factor preserves mesenteric microcirculatory blood flow and protects against intestinal injury in rats subjected to hemorrhagic shock and resuscitation.

BACKGROUND: The gut is highly susceptible to injury after hemorrhagic shock and resuscitation (HS/R) because of progressive mesenteric hypoperfusion. The aim of the current study was to evaluate the effect of heparin-binding EGF-like growth factor (HB-EGF) on mesenteric microcirculatory blood flow and intestinal injury in rats subjected to HS/R. METHODS: HS/R was induced in adult rats, with some rats receiving HB-EGF (600 mug/kg) IV at the onset of resuscitation (HS/R+HB-EGF) and others receiving vehicle only (HS/R). FITC-dextran was administered intra-arterially to evaluate mesenteric microcirculation, and intestinal damage and restitution were evaluated histologically. Data were expressed as mean +/- SE, with P < .05 considered statistically significant. RESULTS: Microcirculatory blood flow was significantly reduced 1 hour after HS/R. HS/R+HB-EGF rats had significantly increased microcirculatory flow compared with HS/R rats at 1 hour (4.5 +/- 0.43 vs 2.64 +/- 0.46, P < .05) and 3 hours (8.04 +/- 1.58 vs 2.89 +/- 0.63, P < .05) after HS/R. HS/R+HB-EGF rats had significantly less intestinal damage compared with HS/R rats 3 hours after resuscitation (2.04 +/- 0.5 vs 3.08 +/- 0.5, P < .05), along with significantly fewer incompetent (nonresurfaced, nonhealed) villi, which is indicative of improved restitution. CONCLUSIONS: HB-EGF significantly improved postresuscitation microcirculatory blood flow in rats subjected to HS/R, associated with significantly decreased intestinal damage and increased restitution. These results suggest that HB-EGF may be a useful therapeutic agent that improves intestinal blood flow in patients with intestinal injury secondary to hemorrhagic shock.

Cannabinoid-2 receptor mediates protection against hepatic ischemia/reperfusion injury.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

Heparin-binding epidermal growth factor-like growth factor reduces intestinal apoptosis in neonatal rats with necrotizing enterocolitis.

PURPOSE: We have previously demonstrated that enterally administered heparin-binding epidermal growth factor-like growth factor (HB-EGF) decreases the incidence and severity of necrotizing enterocolitis (NEC) in a neonatal rat model. Because apoptosis contributes to gut barrier failure in this model, the aim of this study was to investigate the effect of HB-EGF on apoptosis during the development of NEC. METHODS: NEC was induced in neonatal rats by exposure to hypoxia, hypothermia, hypertonic formula feeding (HHHTF) plus enteral administration of lipopolysaccharide (LPS). Fifty-one neonatal rats were randomly divided into the following groups: (1) breast-fed (BF), (2) HHHTF + LPS, and (3) HHHTF + LPS with HB-EGF (600 microg/kg) added to the formula. NEC was evaluated using a standard histological scoring system. Apoptotic cells in intestinal tissues were detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) and by active caspase 3 immunohistochemical staining. RESULTS: The incidence of NEC in the HHHTF + LPS group was higher than that in the BF group (65% vs 0%, P < .05). With administration of HB-EGF, the incidence of NEC significantly decreased to 23.8% (P < .05). The median TUNEL and active caspase 3 scores in the HHHTF + LPS group were higher than those in the BF group (1.9 vs 0.9 and 1.75 vs 0.6, respectively, P < .05). The median TUNEL and active caspase 3 scores were significantly decreased in the HHHTF + LPS + HB-EGF group compared with the HHHTF + LPS group (1.24 vs 1.9 and 1.0 vs 1.75, respectively, P < .05). CONCLUSION: HB-EGF reduces the incidence of NEC in a neonatal rat model in part by decreasing apoptosis. These results support the use of HB-EGF-based clinical regimens for the treatment of NEC.

Heparin-binding epidermal growth factor-like growth factor decreases the incidence of necrotizing enterocolitis in neonatal rats.

PURPOSE: We have previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent intestinal cytoprotective agent. The aim of this study was to determine the effect of enterally administered HB-EGF on the incidence of necrotizing enterocolitis (NEC) in neonatal rats. METHODS: Necrotizing enterocolitis was induced in neonatal rats delivered by C-section on day 21 of gestation by exposure to repeated cycles of hypoxia and hypothermia plus administration of hypertonic formula feeding (HHHTF) plus enteral administration of lipopolysaccharide (LPS) (2 mg/kg). Neonatal rats were randomly assigned to breast-feeding, hypertonic formula feeding, HHHTF + LPS, and HHHTF + LPS with HB-EGF (600 mug/kg) supplementation in the formula. Animals were monitored until 96 hours of life and assessed for death, histological NEC, and intestinal mucosal permeability. RESULTS: The incidence of NEC in the HHHTF group was higher than that in the breast-feeding or hypertonic formula feeding groups. With administration of HB-EGF, the incidence and severity of NEC were significantly decreased. Administration of HB-EGF also increased rat pup survival rate and extended survival time. In addition, treatment with HB-EGF significantly decreased intestinal permeability to fluorescein isothiocyanate-dextran. CONCLUSIONS: We conclude that HB-EGF reduces the incidence and severity of NEC in a neonatal rat model, with simultaneous preservation of gut barrier integrity. These results support our contention that HB-EGF administration may represent a useful therapeutic and prophylactic therapy for the treatment of NEC.

Tissue distribution and plasma clearance of heparin-binding EGF-like growth factor (HB-EGF) in adult and newborn rats.

Heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells from various forms of injury in vitro and attenuate intestinal ischemia/reperfusion damage in vivo. With the goal of eventual clinical use of HB-EGF to protect the intestines from injury in neonates, children, and adults, the pharmacokinetics and biodistribution of 125I-labeled HB-EGF were investigated. After intravenous bolus, HB-EGF had a distribution half-life of 0.8 min and an elimination half-life of 26.67 min. After gastric administration, the bioavailability was 7.8%, with a 2.38 h half-life in the absorption phase and an 11.13 h half-life in the elimination phase. After intravenous dosing, most radioactivity was found in the plasma, liver, kidneys, bile, and urine, whereas it was mainly distributed in the gastrointestinal tract after intragastric administration. The degradation of 125I-HB-EGF in plasma from newborn rats was lower than that in adult rats after gastric administration. This supports the feasibility of enteral administration of HB-EGF in the treatment of gastrointestinal diseases, including newborns afflicted with necrotizing enterocolitis.

HB-EGF enhances restitution after intestinal ischemia/reperfusion via PI3K/Akt and MEK/ERK1/2 activation.

BACKGROUND & AIMS: Early recovery of intestinal function after injury occurs by restitution, a complex process with a poorly understood molecular basis. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a potent chemotactic factor that is induced during ischemia/reperfusion in vivo and intestinal wounding in vitro. The role of HB-EGF in intestinal restitution and the underlying intracellular signaling pathways involved were investigated. METHODS: Adult rats were subjected to intestinal ischemia, with histologic and biochemical damage assessed during the first 3 hours of reperfusion. The effect of recombinant HB-EGF (rHB-EGF) on structural and functional recovery of the intestine by restitution was evaluated in vivo. Scrape wounding of intestinal epithelial cell monolayers was used to elucidate the mechanisms of intrinsic and rHB-EGF-induced restitution. RESULTS: Early structural recovery occurred within 3 hours of reperfusion and was attributed to restitution rather than proliferation. HB-EGF treatment significantly improved structural recovery and accelerated functional recovery of the gut barrier. In vivo restitution was preceded by activation of Akt and extracellular signal-regulated kinase (ERK) 1/2, which were accelerated and enhanced by HB-EGF treatment. Blocking of ErbB-1, phosphatidylinositol 3-kinase (PI3K)/Akt, or mitogen-activated protein kinase/ERK kinase (MEK)/ERK activity resulted in significant reduction in intrinsic and HB-EGF-induced restitution in vitro. Endogenous HB-EGF was shown to play an essential role in wound-induced ErbB-1 and ERK1/2 activation and in intrinsic restitution. CONCLUSIONS: Endogenous HB-EGF, ErbB-1, PI3K/Akt, and MEK/ERK are involved in intrinsic restitution. rHB-EGF enhances restitution in vivo and in vitro in a PI3K/Akt- and MEK/ERK1/2-dependent fashion.

Heparin-binding EGF-like growth factor (HB-EGF) and necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease that occurs predominantly in premature infants. Despite various advances in management, the mortality of this disease remains high. During the last decade, studies from our laboratory have shown that heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, can protect intestinal epithelial cells (IEC) from various forms of injury in vitro. Furthermore, we have used both an intestinal I/R injury model in adult rats, and a neonatal rat pup model of NEC, to show that HB-EGF can protect the intestines from injury. On administration of HB-EGF in the neonatal rat model, the incidence of NEC is reduced from 65% to 27.3% (P < 0.05), and the histological injury score is decreased from 2 to 1.1 (P < 0.05). In addition, the survival rate is increased from 25% to 63.6% and the survival time extended from 59 hours to 73 hours (P < 0.05). In addition, using human specimens from newborns undergoing bowel resection for NEC, we found that the expression of endogenous HB-EGF mRNA in normal areas of the intestine at the resection margins was higher than that of the intestine afflicted with acute NEC. Endogenous HB-EGF may be involved in epithelial cell repair, proliferation, and regeneration during recovery from injury. Exogenous administration of HB-EGF potentiates recovery from intestinal injury in vitro and in vivo. Taken together, these results support a potential therapeutic role for HB-EGF in the treatment of NEC in the future.

Overexpression of macrophage migration inhibitory factor induces angiogenesis and deteriorates prognosis after radical resection for hepatocellular carcinoma.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pivotal cytokine that regulates inflammatory and immune responses. Recently, many investigators reported that MIF is expressed highly in several tumors, including hepatocellular carcinoma (HCC). However, the role of MIF in tumor angiogenesis and patient prognosis has not been examined in patients with HCC. METHODS: The authors evaluated MIF expression in 56 samples of HCC by Western blot analysis, and the results were correlated with clinicopathologic factors and patient prognosis. MIF localization was determined by immunohistochemical methods, and the results were compared with tumor microvessel density (MVD), as assessed by anti-CD34 antibody. Furthermore, to validate the role of MIF in angiogenesis, both MIF expression during culture of HCC cells (using the Hep3B, HepG2, and Huh7 cell lines) under hypoxic condition and the angiogenic potential of recombinant MIF in an in vitro angiogenic model were examined. RESULTS: Tumors with high MIF expression had high alpha-fetoprotein levels (P = 0.049) and frequent intrahepatic recurrence (P = 0.043). Immunohistochemical MIF scores had a significant correlation with MVD (P = 0.007). Patients who had tumors with high MIF expression levels had a significantly worse (P = 0.025) disease-free survival, and this finding remained significant as an independent prognostic factor in the multivariate analysis. Hep3B cells had high expression of MIF at 6 hours and 12 hours after hypoxic stress and exogenous MIF stimulated endothelial tube formation in in vitro angiogenesis. CONCLUSIONS: The current findings suggest that MIF expression may play a pivotal role in the dismal prognosis of patients with HCC that may be attributable to the modulation of angiogenesis.

Expression of gap junction protein connexin32 in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.

BACKGROUND: The gap junction (GJ) plays important roles in the maintenance of tissue homeostasis, the control of cell growth and differentiation, and the prevention of experimental hepatocarcinogenesis. In this study, we examined the relationship between the expression of the GJ protein connexin (Cx) 32 in 24 human hepatocellular carcinomas (HCCs) and 29 non-carcinomatous liver specimens (NCLs) of 31 patients. METHODS: An immunohistochemical study of Cx32 was done in 24 HCCs and 29 NCLs from 31 patients who had undergone hepatic resection. RESULTS: The Cx32 expression decreased gradually as the disease progressed to cirrhosis and HCC. In all Cx32 positive HCCs, the expression was mostly recognized in cytoplasm, not only on the cell membrane. This internalization of Cx32 was also recognized in liver specimens showing hepatitis and cirrhosis, although it was less frequent than in the HCCs. CONCLUSIONS: These findings suggest the possibility that changes in both the amount and the distribution of Cx32 may be implicated in human hepatocarcinogenesis.

Heparin-binding epidermal growth factor-like growth factor and intestinal ischemia-reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury affects patients of different ages, especially premature babies and the elderly. The outcome after intestinal I/R is often dismal, which may be attributed to loss of the barrier and immune functions of the intestines, as well as development of secondary injury in remote organs. The available treatment for advanced gut ischemia mandates extensive resection, which may cause growth retardation in infants and nutritional problems in the elderly. Throughout the past decade we have been investigating the potential therapeutic role of heparin-binding epidermal growth factor-like factor (HB-EGF) in intestinal I/R. The mitogenic and chemoattractant functions of HB-EGF formed the initial rationale for our investigations. In addition, HB-EGF is a potent antiapoptotic protein that enables cells and tissues exposed to different apoptotic stimuli to survive hypoxic, oxidative, and nutritional stresses. HB-EGF is known to have a vital role in wound healing and postischemic regeneration in different organs. In the current review, we summarize the results of our findings of the beneficial effects of HB-EGF in intestinal I/R, supported by additional evidence from the literature and an explanation of different possible mechanisms of its actions. Collectively, the data strongly suggest a potential therapeutic role for the use of HB-EGF to treat intestinal ischemic diseases such as I/R and necrotizing enterocolitis.

IL-6-deficient mice are susceptible to ethanol-induced hepatic steatosis: IL-6 protects against ethanol-induced oxidative stress and mitochondrial permeability transition in the liver.

Interleukin-6 (IL-6)-deficient mice are prone to ethanol-induced apoptosis and steatosis in the liver; however, the underlying mechanism is not fully understood. Mitochondrial dysfunction caused by oxidative stress is an early event that plays an important role in the pathogenesis of alcoholic liver disease. Therefore, we hypothesize that the protective role of IL-6 in ethanol-induced liver injury is mediated via suppression of ethanol-induced oxidative stress and mitochondrial dysfunction. To test this hypothesis, we examined the effects of IL-6 on ethanol-induced oxidative stress, mitochondrial injury, and energy depletion in the livers of IL-6 (-/-) mice and hepatocytes from ethanol-fed rats. Ethanol consumption leads to stronger induction of malondialdehyde (MDA) in IL-6 (-/-) mice compared to wild-type control mice, which can be corrected by administration of IL-6. In vitro, IL-6 treatment prevents ethanol-mediated induction of reactive oxygen species (ROS), MDA, mitochondrial permeability transition (MPT), and ethanol-mediated depletion of adenosine triphosphate (ATP) in hepatocytes from ethanol-fed rats. Administration of IL-6 in vivo also reverses ethanol-induced MDA and ATP depletion in hepatocytes. Finally, IL-6 treatment induces metallothionein protein expression, but not superoxide dismutase and glutathione peroxidase in cultured hepatocytes. In conclusion, IL-6 protects against ethanol-induced oxidative stress and mitochondrial dysfunction in hepatocytes via induction of metallothionein protein expression, which may account for the protective role of IL-6 in alcoholic liver disease.

Sequential changes of connexin32 and connexin26 in ischemia-reperfusion of the liver in rats.

Serial changes in expression of hepatic gap junction components, connexin32 and connexin26 expressions during ischemia (60 min)-reperfusion injury of the liver were evaluated by immunofluorescence staining and reverse transcription-polymerase chain reaction in rats. Hepatic tissue calcium content and liver enzymes (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase), were also examined. Connexin expressions were down-regulated during ischemia and steeply increased during the early reperfusion period. This upsurge in connexin was coincided with the augmentation in tissue calcium content level. And the mRNA levels of connexin changed in parallel with the connexin protein level until 60 min after reperfusion. Since it is known that the changes in intracellular Ca(2+) concentration controls the intercellular communication via gap junction, these findings suggest the possibility that gap junction may play a definitive role in reperfusion injury of the liver. Further studies may be necessary to clarify the exact role of connexins in hepatic ischemia-reperfusion injury.

In vitro interleukin-6 treatment prevents mortality associated with fatty liver transplants in rats.

BACKGROUND & AIMS: Orthotopic liver transplantation is currently the only curative therapy for chronic end-stage liver disease and acute liver failure. However, a scarcity of cadaveric donors has led to a critical shortage of organs available for transplant. This is further complicated by the prevalence of steatosis in about 13%-50% of donor livers, which is associated with a high risk of dysfunction and primary nonfunction. METHODS: Steatotic Zucker rat livers and livers from alcohol-fed rats were transplanted into lean control rats. Liver injury, activation of survival signals, and hepatic microcirculation were compared in nontreated and interleukin-6 (IL-6)-treated steatotic isografts. RESULTS: IL-6 pretreatment of steatotic Zucker rat liver isografts dramatically reduces mortality and liver injury following transplantation. Reperfusion after transplantation induces significant sinusoidal endothelial cell necrapoptosis in steatotic Zucker rat liver isografts, which is prevented by in vitro IL-6 pretreatment. IL-6 treatment activates cell survival signal transducer and activator of transcription factor 3 (STAT3) in hepatocytes and sinusoidal endothelial cells. Laser Doppler imaging and microsphere analyses demonstrate that IL-6 treatment markedly improves hepatic microcirculation, which is impaired in steatotic Zucker rat liver transplants. Finally, in vitro IL-6 treatment of donor livers also markedly reduces mortality associated with fatty liver transplants from alcohol-fed rats. CONCLUSIONS: IL-6 induces hepatoprotection of steatotic liver isografts via preventing sinusoidal endothelial cell necrapoptosis and consequent amelioration of hepatic microcirculation, and protecting against hepatocyte death. IL-6 pretreatment of steatotic livers may render such allografts useable for clinical transplantation.

Opposing roles of STAT1 and STAT3 in T cell-mediated hepatitis: regulation by SOCS.

T cell-mediated fulminant hepatitis is a life-threatening event for which the underlying mechanism is not fully understood. Injection of concanavalin A (Con A) into mice recapitulates the histological and pathological sequelae of T cell-mediated hepatitis. In this model, both signal transducer and activator of transcription factor 1 (STAT1) and STAT3 are activated in the liver. Disruption of the STAT1 gene by way of genetic knockout attenuates liver injury, suppresses CD4(+) and NK T cell activation, and downregulates expression of proapoptotic interferon regulatory factor-1 protein and suppressor of cytokine signaling-1 (SOCS1), but enhances STAT3 activation and STAT3-controlled antiapoptotic signals. Studies from IFN-gamma-deficient mice indicate that IFN-gamma not only is the major cytokine responsible for STAT1 activation but also partially accounts for STAT3 activation. Moreover, downregulation of STAT3 activation in IL-6-deficient mice is associated with decreased STAT3-controlled antiapoptotic signals and expression of SOCS3, but upregulation of STAT1 activation and STAT1-induced proapoptotic signals and exacerbation of liver injury. Taken together, these findings suggest that STAT1 plays a harmful role in Con A-mediated hepatitis by activation of CD4(+) and NK T cells and directly inducing hepatocyte death, whereas STAT3 protects against liver injury by suppression of IFN-gamma signaling and induction of antiapoptotic protein Bcl-X(L). STAT1 and STAT3 in hepatocytes also negatively regulate one another through the induction of SOCS.

Interferon-alpha activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes.

BACKGROUND & AIMS: Interferon (IFN)-alpha therapy is currently the primary choice for viral hepatitis and a promising treatment for hepatocellular carcinoma (HCC). Primary mouse and rat hepatocytes respond poorly to IFN-alpha stimulation. Thus, it is very important to examine the IFN-alpha signal pathway in primary human hepatocytes. METHODS: The IFN-alpha-activated signals and genes in primary human hepatocytes and hepatoma cells were examined by Western blotting and microarray analyses. RESULTS: Primary human hepatocytes respond very well to IFN-alpha stimulation as shown by activation of multiple signal transducer and activator of transcription factor (STAT) 1, 2, 3, 5, and multiple genes. The differential response to IFN-alpha stimulation in primary human and mouse hepatocytes may be caused by expression of predominant functional IFN-alpha receptor 2c (IFNAR2c) in primary human hepatocytes vs. expression of predominant inhibitory IFNAR2a in mouse hepatocytes. Microarray analyses of primary human hepatocytes show that IFN-alpha up-regulates about 44 genes by over 2-fold and down-regulates about 9 genes by 50%. The up-regulated genes include a variety of antiviral and tumor suppressors/proapoptotic genes. The down-regulated genes include c-myc and c-Met, the hepatocyte growth factor (HGF) receptor. Down-regulation of c-Met is caused by IFN-alpha suppression of the c-Met promoter through down-regulation of Sp1 binding and results in attenuation of HGF-induced signals and cell proliferation. CONCLUSIONS: IFN-alpha directly targets human hepatocytes, followed by activation of multiple STATs and regulation of a wide variety of genes, which may contribute to the antiviral and antitumor activities of IFN-alpha in human liver.