NK cells suppress experimental cholestatic liver injury by an interleukin-6-mediated, Kupffer cell-dependent mechanism.

BACKGROUND & AIMS: Natural killer (NK) cells are innate immune effector cells first characterized by their ability to lyse susceptible tumor cells. Recent studies demonstrated their role in initiating and modulating adaptive immunity. NK cells represent a larger percentage of the lymphoid population in liver than other organs, suggesting that hepatic NK cells express some unique function. Here, we examined the response of NK cells to liver injury that occurs in a mouse model of biliary obstruction. METHODS: Bile duct ligations (BDL) were performed in mice previously depleted or not depleted of NK cells. NK cell activation, interleukin (IL)-6 mRNA expression and protein production by Kupffer cells, and the ability of exogenous IL-6 to ameliorate liver injury in NK cell-depleted mice, were determined. RESULTS: The number of activated hepatic NK cells increased markedly following BDL. Activation was suppressed in mice rendered Kupffer cell-depleted prior to ligation. Increased liver injury occurred in NK cell-depleted mice correlating with a reduction in IL-6 production. Purified Kupffer cells, obtained from NK cell-depleted or anti-interferon (IFN)-γ monoclonal antibody-pretreated mice following BDL, produced less IL-6 in culture than did Kupffer cells derived from control animals. In culture, hepatic NK cells derived from BDL mice stimulated IFN-γ-dependent IL-6 production by Kupffer cells; splenic NK cells obtained from the same animals had a negligible effect. Treatment with recombinant murine IL-6 reduced liver injury in BDL, NK cell-depleted mice. CONCLUSIONS: Hepatic NK cells suppress cholestatic liver injury by stimulating Kupffer cell-dependent IL-6 production.

Neutrophils sequestered in the liver suppress the proinflammatory response of Kupffer cells to systemic bacterial infection.

The liver plays a major role in clearing bacteria from the bloodstream. Rapid clearance is primarily the function of fixed tissue macrophages (Kupffer cells) that line the hepatic sinusoids. Although Kupffer cells play a critical role in blood clearance, the actual elimination of the bulk of bacteria taken up by the liver depends upon the accumulation of bactericidal neutrophils. Subsequent experiments demonstrating neutrophils inside Kupffer cells derived from infected animals prompted our speculation that neutrophils modulate the proinflammatory response of Kupffer cells to bacteria cleared from the bloodstream. Indeed, we report here that neutrophils accumulated in the liver sinusoids suppress cytokine and chemokine mRNA expression and protein production by Kupffer cells. Using listeriosis in mice as an experimental model, we found that IL-1beta, IL-6, IL-10, IL-12, TNF-alpha, MIP-1alpha, keratinocyte-derived chemokine, and MCP-1 mRNA levels were >or=10-fold more in the livers of Listeria-infected, relative to noninfected control, mice at 0.5-2 h after i.v. infection. Most message levels were sharply diminished thereafter, correlating inversely with increased neutrophil sequestration. Relative to intact animals, mice rendered neutrophil deficient exhibited marked increases in cytokine/chemokine mRNA expression and protein production in the liver subsequent to infection. Moreover, purified Kupffer cells derived from infected, neutrophil-depleted mice produced significantly more IL-6, IL-10, IL-12, TNF-alpha, keratinocyte-derived chemokine, and MCP-1 in culture. These findings document the critical role of neutrophils in moderating the proinflammatory response of Kupffer cells to bacteria taken up by the liver.

Invariant natural killer T cells suppress the neutrophil inflammatory response in a mouse model of cholestatic liver damage.

BACKGROUND & AIMS: NK1.1(+) TCRalphabeta(int) CD1-restricted T (NKT) cells are a unique subset of T lymphocytes that are believed to have an immunoregulatory role in a wide range of diseases. Most mouse NKT cells express a T-cell receptor that contains an invariant Valpha14Jalpha18 chain and recognizes antigenic glycolipids presented in association with major histocompatibility complex class Ib (CD1d) molecules. These invariant NKT (iNKT) cells have been implicated in cholestatic liver injury. METHODS: We examined the role of iNKT cells in liver injury associated with biliary obstruction in mice with ligations of the common bile duct. RESULTS: The number of activated iNKT cells increased markedly in the livers of mice following bile duct ligation. Plasma alanine aminotransferase levels, an indicator of liver injury, were significantly higher in iNKT cell-deficient (Jalpha18(-/-)) mice compared with wild-type mice following bile duct ligation. Photo image analysis of histologic sections confirmed that more damage was present in the livers of Jalpha18(-/-) mice; liver damage correlated with increases in keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2) production as well as neutrophil sequestration. Liver injury was significantly reduced in Jalpha18(-/-) mice treated with anti-KC and anti-MIP-2 or rendered neutrophil deficient before bile duct ligation. Similarly, Jalpha18(-/-) mice that were injected with iNKT cells before bile duct ligation exhibited significant decreases in neutrophil accumulation and liver damage. CONCLUSIONS: These data document the role of iNKT cells in suppressing the neutrophil proinflammatory response and neutrophil-dependent cholestatic liver damage.

Generation of immune responses against hepatitis C virus by dendritic cells containing NS5 protein-coated microparticles.

Dendritic cells (DCs) internalize and process antigens as well as activate cellular immune responses. The aim of this study was to determine the capacity of DCs that contain antigen-coated magnetic beads to induce immunity against the nonstructural hepatitis C virus (HCV) antigen 5 (NS5). Splenocytes derived from Fms-like tyrosine kinase receptor 3 (Flt3) ligand-pretreated BALB/c mice were incubated with magnetic beads coated with HCV NS5, lipopolysaccharide (LPS), and/or anti-CD40; purified; and used for immunization. Cellular immunity was measured using cytotoxic T-lymphocyte (CTL) and T-cell proliferation assays, intracellular cytokine staining, and a syngeneic tumor challenge using NS5-expressing SP2/0 myeloma cells in vivo. Splenocytes isolated from animals vaccinated with DCs containing beads coated with NS5, LPS, and anti-CD40 secreted elevated levels of interleukin-2 (IL-2) and gamma interferon in the presence of NS5. The numbers of CD4(+), IL-2-producing cells were increased >5-fold in the group immunized with DCs containing beads coated with NS5, LPS, and anti-CD40, paralleled by an enhanced splenocyte proliferative response. Immunization promoted antigen-specific CTL activity threefold compared to the level for control mice and significantly reduced the growth of NS5-expressing tumor cells in vivo. Thus, strategies that employ NS5-coated beads induce cellular immune responses in mice, which correlate well with the natural immune responses that occur in individuals who resolve HCV.

Low density contrast agents for x-ray phase contrast imaging: the use of ambient air for x-ray angiography of excised murine liver tissue.

We report a new preparative method for providing contrast through reduction in electron density that is uniquely suited for propagation-based differential x-ray phase contrast imaging. The method, which results in an air or fluid filled vasculature, makes possible visualization of the smallest microvessels, roughly down to 15 microm, in an excised murine liver, while preserving the tissue for subsequent histological workup. We show the utility of spatial frequency filtering for increasing the visibility of minute features characteristic of phase contrast imaging, and the capability of tomographic reconstruction to reveal microvessel structure and three-dimensional visualization of the sample. The effect of water evaporation from livers during x-ray imaging on the visibility of blood vessels is delineated. The deformed vascular tree in a cancerous murine liver is imaged.

Generation and characterization of an immunogenic dendritic cell population.

Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.

Chronic ethanol consumption impairs cellular immune responses against HCV NS5 protein due to dendritic cell dysfunction.

BACKGROUND & AIMS: Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. METHODS: Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. RESULTS: Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. CONCLUSIONS: Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Prevalence of autoantibodies and the risk of autoimmune thyroid disease in children with chronic hepatitis C virus infection treated with interferon-alpha.

AIM: To evaluate the prevalence of autoantibodies in chronic hepatitis C virus (HCV)-infected children focusing on thyroid autoimmunity. METHODS: We investigated the prevalence of auto-antibodies in 123 chronic HCV-infected children before, during and after monotherapy with interferon-alpha (IFN-alpha) or combined treatment with interferon-alpha or peginterferon-alpha and ribavirin. Besides antibodies against smooth muscle (SMA), nuclei (ANA), and liver/kidney microsomes (LKM), the incidence of anti-thyroid peroxidase antibodies as well as thyroid function parameters (TSH, FT3 and FT4) were determined. RESULTS: We found that 8% of children had autoantibodies before treatment. During treatment, 18% of children were found positive for at least one autoantibody; 15.5% of children developed pathologic thyroid values during IFN-alpha treatment compared to only one child before therapy. Six children had to be substituted while developing laboratory signs of hypothyroidism. CONCLUSION: Our data indicate a strong correlation between interferon-alpha treatment and autoimmune phenomena, notably the emergence of thyroid antibodies. The fact that some children required hormone replacement underlines the need of close monitoring in particularly those who respond to therapy and have to be treated for more than 6 mo.

Relationship between viral load and genotypes of hepatitis B virus in children with chronic hepatitis B.

OBJECTIVE: We evaluated the prevalence and clinical significance of hepatitis B virus (HBV) genotypes in children with chronic hepatitis B. METHODS: Hepatitis B virus genomes of 249 hepatitis Be antigen-positive chronic hepatitis B surface antigen carriers were genotyped based on restriction fragment length polymorphism. Genotypes were correlated with corresponding values for alanine aminotransferase levels, quantitative HBV DNA and histological findings. RESULTS: One hundred and sixty-two boys and 87 girls (mean age, 7.2 years) were studied. Ninety-six percent were attributed to HBV genotypes A (32.5%) or D (63.5%). The remaining were classified as genotypes B, C, E and F. There was no significant difference in both alanine aminotransferase levels and histological findings among different genotypes. However, there was a clear association between very high HBV DNA levels and individuals with genotype D (P = 0.006). Mean time follow-up of 3.6 years showed later anti-HBe seroconversion in patients with genotype D than in those with genotype A (58% vs 37%). CONCLUSIONS: Compared with children with genotype A, children with genotype D showed a significantly higher viral load. Inasmuch as a relationship exists between viral load and response to treatment in children infected through vertical transmission, children with genotype D have to be carefully monitored. Long-term studies are needed to determine whether these patients are at risk of a poorer outcome.

Prevalence of hepatitis B virus precore stop codon mutations in chronically infected children.

AIM: To find out whether there is a significant difference in the prevalence of the precore stop codon mutation between HBeAg positive and anti-HBe positive children. METHODS: We investigated a large pediatric population of 155 European children (mean age 10.9 years) with chronic hepatitis B by PCR and direct sequencing. Ninety were HBeAg positive and 65 had seroconversion to anti-HBe. Additionally genotyping was performed. RESULTS: Seventy-four (48%) of the sequenced HBV strains were attributed to genotype D and 81 (52%) to genotype A. In the group of 90 HBeAg positive patients, 2 (2.2%) 1896-G-to-A transitions leading to precore stop codon mutation were found, and in the group of 65 anti-HBe positive children, 5 (7.7%) were identified harbouring HBeAg-minus mutants. The difference was not statistically significant (P = 0.13). CONCLUSIONS: HBeAg minus variants as predominant viral HB strains play a minor role in the course of chronic hepatitis B in European children.

Prevalence of hepatitis C virus infection in children admitted to an urban hospital.

BACKGROUND AND AIM: Hepatitis C infection is a global health problem affecting about 3% of the world's population. However, very little data exists on the prevalence of hepatitis C virus infection in childhood. METHODS: We performed a cross-sectional study in 2000 children and adolescents who were treated as in- or out patients in our hospital. Blood samples were collected between February 2002 and June 2004 and were tested for HCV antibodies (anti-HCV). Positive samples were further investigated by HCV specific PCR and Western blot assay. RESULTS: Mean age of children was 8.1 years. 908 (45%) were female and 1092 (55%) male. One thousand eight hundred and ninety-eight were Caucasian, 37 African, and 65 Asian. 16 (0.8%) tested positive for anti-HCV. HCV-RNA was detectable in one child (0.05%), and three were positive in the Western blot assay (0.15%). The HCV viremic child had received multiple blood transfusions after cardiac surgery. She was asymptomatic with normal transaminases. Seroprevalence of HCV antibodies were equally distributed among boys and girls. CONCLUSIONS: The prevalence of persistent hepatitis C in children from an urban hospital in Germany is low. Most patients with HCV antibodies are not infected. Therefore, although universal screening is not warranted, it should always be performed in risk groups such as transfused children because HCV infection in childhood is usually asymptomatic.

Complete hepatitis B virus genome analysis in chronically infected children before and during lamivudine treatment.

It is well established that during lamivudine treatment, mutations emerge within the polymerase gene but there is little information about the selection of other mutations in the whole hepatitis B virus (HBV) genome. The aim of this study was to investigate whether mutations outside this region are selected during lamivudine treatment. The complete HBV genomes of six HBsAg positive chronically infected children were isolated from the children's sera before, at the beginning and during lamivudine treatment, amplified and sequenced directly. A change in the mutation rate and type in periods with and without treatment for one and the same patient were examined longitudinally because blood samples were taken long before treatment started. During the testing period before treatment, 12 mutations occurred within 11.7 +/- 8.1 months in all genomes, resulting in a mutation rate of 1.1 x 10(-3) substitutions per site per year. During treatment with lamivudine, 20 mutations occurred in all patients within an average period of 7.6 +/- 1.2 months, giving an average mutation rate of 1.8 x 10(-3) substitutions per site per year. The 20 mutations observed during the treatment period occurred in only 4 of the patients and only 3 patients experienced nonsense mutations during lamivudine treatment. The mutations were spread across the entire genome with a non-significant cluster during treatment in the P-ORF (18 mutations vs. 7, P = 0.073) and S-ORF (11 vs. 2, P = 0.063). Mutations causing drug resistance did not emerge. This study describes the changes in the complete HBV genome in the spontaneous course of infection and during lamivudine treatment. An increased mutation rate and the occurrence of specific mutations could not be proven for the early phase of lamivudine treatment.

Peginterferon alfa-2b plus ribavirin treatment in children and adolescents with chronic hepatitis C.

Peginterferon plus ribavirin is standard therapy for adults with chronic hepatitis C. As no data are available for children, the aim of the study was to evaluate the efficacy and tolerability of peginterferon alfa-2b in combination with ribavirin in chronically infected children. Genotypes, alanine aminotransferase levels, and different routes of viral transmission were considered. In an open-labeled, uncontrolled pilot study, 62 children and adolescents (range, 2-17 years) were treated with subcutaneous peginterferon alfa-2b at a dose of 1.5 microg/kg body weight once per week plus oral ribavirin (15 mg/kg x day) for 48 weeks. Sixty-one patients completed the study. Twenty-three children discontinued therapy after 6 months according to study protocol. Sustained viral response was documented in 22 (47.8%)of 46 patients with genotype 1, in 13 (100%) of 13 with genotype 2 or 3, in 1 of 2 with genotype 4, in 19 (70.4%) of 27 children with parenteral, in 12 (48%) of 25 with vertical, and in 5 of 9 with unknown route of infection. Overall, treatment was well tolerated. Nevertheless, some side effects were present in all treated patients. Eighty-three percent had leucopenia, but only 3 individuals required dose reduction and 10.3% developed thyroid autoantibodies and thyroid dysfunction. In conclusion, combination treatment of peginterferon alfa-2b with ribavirin showed encouraging results and was well tolerated in children and adolescents with chronic hepatitis C. Weekly dosing of peginterferon alfa-2b is a considerable advance for this age group. The treatment is not approved for children. Further controlled trials are needed.

Complete hepatitis B virus genome analysis in HBsAg positive mothers and their infants with fulminant hepatitis B.

BACKGROUND: After perinatal transmission of hepatitis B virus, infants of anti-HBe positive HBsAg carrier mothers may develop fulminant hepatitis B. Previously it has been suggested, that fulminant hepatitis B in adults was associated with specific mutations in the HBV-genome. The aim of this study was to investigate, whether specific viral variants are associated with fulminant hepatitis B in young infants. METHODS: The complete HBV-genomes of five mothers and their infants with fulminant hepatitis were isolated from the sera, amplified and directly sequenced. RESULTS: Between 6 and 43 base pair exchanges between the HBV genomes of the infants and their mothers were identified. The mutations spread over the entire virus genome. Nucleotide exchanges in the basic core promotor and precore region were identified in all cases. A heterogeneous virus population was detected in four mothers. CONCLUSIONS: Many new mutations were proved to emerge during fulminant hepatitis B in infants, who had been perinatally infected. HBeAg negative variants were the predominant population in all children, whereas these mutants could only be detected as subpopulations in four mothers. The data suggest that the selection of a specific HBeAg negative viral strain may be associated with the development of fulminant hepatitis B in children.