Intrinsic Photosensitivity Enhances Motility of T Lymphocytes.

Sunlight has important biological effects in human skin. Ultraviolet (UV) light striking the epidermis catalyzes the synthesis of Vitamin D and triggers melanin production. Although a causative element in skin cancers, sunlight is also associated with positive health outcomes including reduced incidences of autoimmune diseases and cancers. The mechanisms, however, by which light affects immune function remain unclear. Here we describe direct photon sensing in human and mouse T lymphocytes, a cell-type highly abundant in skin. Blue light irradiation at low doses (<300 mJ cm-2) triggers synthesis of hydrogen peroxide (H2O2) in T cells revealed by the genetically encoded reporter HyPerRed. In turn, H2O2 activates a Src kinase/phospholipase C-γ1 (PLC-γ1) signaling pathway and Ca2+ mobilization. Pharmacologic inhibition or genetic disruption of Lck kinase, PLC-γ1 or the T cell receptor complex inhibits light-evoked Ca2+ transients. Notably, both light and H2O2 enhance T-cell motility in a Lck-dependent manner. Thus, T lymphocytes possess intrinsic photosensitivity and this property may enhance their motility in skin.

Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners.

BACKGROUND & AIMS: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. METHODS: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon gamma. RESULTS: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon gamma gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon gamma enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor-related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor-related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid-activated natural killer cells against activated hepatic stellate cells. CONCLUSIONS: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanisms, thereby ameliorating liver fibrosis.

IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: regulation by SOCS3.

The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3+/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3+/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration.

Hepatitis B virus X protein sensitizes primary mouse hepatocytes to ethanol- and TNF-alpha-induced apoptosis by a caspase-3-dependent mechanism.

It is well-documented that alcohol drinking together with hepatitis viral infection accelerates liver injury; however the underlying mechanisms remain unknown. In this paper, we demonstrated that primary hepatocytes from transgenic mice overexpressing hepatitis B virus X protein (HBX) were more susceptible to ethanol- and TNF-alpha-induced apoptotic killing. Compared to normal control mouse hepatocytes, ethanol and/or TNF-alpha treatment led to a significant increase in reactive oxygen species, mitochondrial permeability transition, cytochrome C release, caspase-3 activity, and poly (ADP-ribose) polymerase degradation in hepatocytes from HBX transgenic mice. Blocking caspase-3 activity antagonized ethanol- and TNF-alpha-induced apoptosis in primary hepatocytes from HBX transgenic mice. Taken together, our findings suggest that HBX sensitizes primary mouse hepatocytes to ethanol- and TNF-alpha-induced apoptosis by a caspase-3-dependent mechanism, which may partly explain the synergistic effects of alcohol consumption and hepatitis B virus infection on liver injury.

Interferon-gamma inhibits interferon-alpha signalling in hepatic cells: evidence for the involvement of STAT1 induction and hyperexpression of STAT1 in chronic hepatitis C.

IFN-gamma (interferon-gamma) modulates IFN-alpha therapy in chronic hepatitis C infection; however, the underlying mechanism remains unclear. Here we demonstrate that long-term (3-6 days) but not short-term (up to 1 day) IFN-gamma treatment of human hepatoma Hep3B cells attenuates IFN-alpha activation of STAT1 (signal transducers and activators of transcription factor 1), STAT2 and STAT3, but enhances IFN-gamma and interleukin 6 activation of STATs. Prolonged exposure to IFN-gamma also significantly induces STAT1 protein expression without affecting STAT2, STAT3 and ERK (extracellular-signal-regulated kinase) 1/2 protein expression. To determine the role of STAT1 protein overexpression in regulation of IFN-alpha signalling, Hep3B cells were stably transfected with wild-type STAT1. Overexpression of STAT1 via stable transfection enhances IFN-gamma activation of STAT1, but surprisingly attenuates IFN-alpha activation of STAT1, STAT2 and STAT3 without affecting Janus kinase activation. This STAT1-mediated inhibition does not require STAT1 tyrosine phosphorylation because overexpression of dominant-negative STAT1 with a mutation on tyrosine residue 701 also blocks IFN-alpha activation of STAT1, STAT2 and STAT3. Moreover, overexpression of STAT1 blocks IFN-alpha-activated STAT2 translocation from IFN-alpha receptor 2 to IFN-alpha receptor 1, a critical step in IFN-alpha signalling activation. Finally, significantly higher levels of STAT1 protein expression, which is probably induced by IFN-gamma, are detected in the majority of hepatitis C virus-infected livers compared with healthy controls. In conclusion, long-term IFN-gamma treatment inhibits IFN-alpha-activated signals most probably, at least in part, through the induction of STAT1 protein expression, which could partly contribute to IFN-alpha treatment failure in hepatitis C patients.

Crucial role of IL-4/STAT6 in T cell-mediated hepatitis: up-regulating eotaxins and IL-5 and recruiting leukocytes.

T cell-mediated immune responses are implicated in the pathogenesis of a variety of liver disorders; however, the underlying mechanism remains obscure. Con A injection is a widely accepted mouse model to study T cell-mediated liver injury, in which STAT6 is rapidly activated. Disruption of the IL-4 and STAT6 gene by way of genetic knockout abolishes Con A-mediated liver injury without affecting IFN-gamma/STAT1, IL-6/STAT3, or TNF-alpha/NF-kappaB signaling or affecting NKT cell activation. Infiltration of neutrophils and eosinophils in Con A-induced hepatitis is markedly suppressed in IL-4 (-/-) and STAT6(-/-) mice compared with wild-type mice. IL-4 treatment induces expression of eotaxins in hepatocytes and sinusoidal endothelial cells isolated from wild-type mice but not from STAT6(-/-) mice. Con A injection induces expression of eotaxins in the liver and elevates serum levels of IL-5 and eotaxins; such induction is markedly attenuated in IL-4(-/-) and STAT6(-/-) mice. Finally, eotaxin blockade attenuates Con A-induced liver injury and leukocyte infiltration. Taken together, these findings suggest that IL-4/STAT6 plays a critical role in Con A-induced hepatitis, via enhancing expression of eotaxins in hepatocytes and sinusoidal endothelial cells, and induces IL-5 expression, thereby facilitating recruitment of eosinophils and neutrophils into the liver and resulting in hepatitis.

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.

STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury.

Interferon-gamma (IFN-gamma) has been implicated in liver damage in animal models and chronic hepatitis C infection; however, the underlying mechanism is not clear. Here we examined the role of STAT1, a key signaling molecule for IFN-gamma, in a model of murine hepatitis induced by the injection of LPS/D-galactosamine and in human hepatoma Hep3B cells. STAT1 is rapidly activated and highly induced after injection of LPS/D-galactosamine. Both overexpression of STAT1 and hepatocellular damage are located in the same pericentral region. Disruption of the STAT1 gene abolishes LPS/D-galactosamine-induced liver injury. Studies from IFN-gamma-deficient mice indicate that IFN-gamma is the major cytokine responsible for activation and hyperexpression of STAT1 in LPS/D-galactosamine-induced hepatitis. Hep3B cells overexpressing dominant negative STAT1 are resistant to IFN-gamma and IFN-gamma + TNF-alpha-induced cell death, whereas Hep3B cells overexpressing wild-type STAT1 are more susceptible to cell death. Taken together, these findings suggest that STAT1 plays an essential role in LPS/D-galactosamine-induced liver apoptosis and injury.

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.

Supplementation with antioxidant vitamins prevents oxidative modification of DNA in lymphocytes of HIV-infected patients.

There is evidence suggesting that patients infected with human immunodeficiency virus (HIV) are under chronic oxidative stress. In the present study, the level of oxidatively modified bases in lymphocyte DNA and some other parameters of oxidative stress were measured in HIV-infected patients (n = 30), as well as in control groups (10 healthy volunteers and 15 HIV-seronegative injected drug users). Additional experiments were conducted using lymphocyte DNA samples from asymptomatic seropositive, HIV-infected patients who were supplemented with antioxidant vitamins A, C, and E or received placebo. Significant increases in the amount of the modified DNA bases were observed in HIV-infected patients when compared with the control group. The concentration of thiobarbituric acid reactive substances (TBARS) was higher and activities of antioxidant enzymes (superoxide dismutase and catalase) were lower in the group of HIV-infected patients in comparison to the control group. Vitamin supplementation resulted in the significant decrease in the levels of all modified DNA bases when compared to the patients who received placebo. The reduction of TBARS and the restoration of the activity of the enzymes were also observed. Our data suggest that people infected with HIV can benefit from treatment with antioxidant vitamins.

Elevated interleukin-6 during ethanol consumption acts as a potential endogenous protective cytokine against ethanol-induced apoptosis in the liver: involvement of induction of Bcl-2 and Bcl-x(L) proteins.

Elevation of serum interleukin-6 (IL-6) levels is always associated with alcoholic liver disease (ALD), but the significance of such elevation is not clear. Here we show that chronic ethanol consumption induces significant apoptosis in the liver of IL-6 (-/-) mice but not IL-6 (+/+) mice. IL-6 (-/-) hepatocytes are more susceptible to ethanol- and tumor necrosis factor alpha- (TNFalpha-) induced apoptotic killing, which can be corrected by IL-6. Expression of both anti-apoptotic (such as Bcl-2 and Bcl-x(L)) and proapoptotic (such as Bax) proteins is markedly elevated in the liver of human ALD and chronically ethanol-fed IL-6 (+/+) mice. On the contrary, induction of Bcl-2 and Bcl-x(L) is not observed in the liver of chronically ethanol-fed IL-6 (-/-) mice, whereas expression of Bax protein remains elevated. Injection of IL-6 markedly induces expression of Bcl-2 and Bcl-x(L) but not Bax in the liver. Finally, high concentrations of ethanol inhibit IL-6-activated anti-apoptotic signal, but increasing the concentrations of IL-6 is able to overcome such inhibitory effect. These findings suggest that elevated serum IL-6 levels in ALD may overcome the inhibitory effect of ethanol on IL-6-mediated anti-apoptotic signals and prevent alcohol-induced hepatic apoptosis by induction of Bcl-2 and Bcl-x(L).