Defective T Helper Response of Hepatocyte-Stimulated CD4 T Cells Impairs Antiviral CD8 Response and Viral Clearance
2007; Elsevier BV; Volume: 133; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2007.09.007
ISSN1528-0012
AutoresChristiane Wiegard, Petra Wolint, Christian Frenzel, Uta Cheruti, Edgar Schmitt, Annette Oxenius, Ansgar W. Lohse, Johannes Herkel,
Tópico(s)Immune Cell Function and Interaction
ResumoBackground & Aims: In hepatitis, hepatocytes gain the ability to express major histocompatibility complex (MHC) class II molecules and to present antigen to CD4 T cells. Here, we investigated whether MHC class II-expressing hepatocytes influence in vitro the differentiation of CD4 T cells and in vivo the T-cell response to and control of viral infection. Methods: Class II transactivator-transgenic hepatocytes that constitutively express MHC class II molecules were used to stimulate CD4 T cells in vitro, and the effector response type of the stimulated CD4 T cells was determined. The in vivo relevance of the obtained findings was confirmed by infecting nontransgenic or class II transactivator-transgenic mice with lymphocytic choriomeningitis virus. Results: MHC II-expressing hepatocytes induced T helper cell (Th) 2 differentiation of uncommitted CD4 T cells and abrogated the ability of previously differentiated Th1 to secrete interferon-γ, even in the presence of proinflammatory microbial signals. The suppression of Th1 responses by hepatocytes was associated with poor expression levels of Th1-promoting Delta-like Notch ligands. In vivo, MHC II expression by hepatocytes impaired the interferon-γ production by lymphocytic choriomeningitis virus-specific CD4 and CD8 T cells and prolonged viral persistence. Conclusions: By instructing infiltrating CD4 T cells to differentiate into a less inflammatory phenotype, MHC II-expressing hepatocytes seem to impair antiviral CD8 T-cell responses and viral clearance. Thus, hepatocytes may contribute to the chronicity of hepatitis virus infection. Background & Aims: In hepatitis, hepatocytes gain the ability to express major histocompatibility complex (MHC) class II molecules and to present antigen to CD4 T cells. Here, we investigated whether MHC class II-expressing hepatocytes influence in vitro the differentiation of CD4 T cells and in vivo the T-cell response to and control of viral infection. Methods: Class II transactivator-transgenic hepatocytes that constitutively express MHC class II molecules were used to stimulate CD4 T cells in vitro, and the effector response type of the stimulated CD4 T cells was determined. The in vivo relevance of the obtained findings was confirmed by infecting nontransgenic or class II transactivator-transgenic mice with lymphocytic choriomeningitis virus. Results: MHC II-expressing hepatocytes induced T helper cell (Th) 2 differentiation of uncommitted CD4 T cells and abrogated the ability of previously differentiated Th1 to secrete interferon-γ, even in the presence of proinflammatory microbial signals. The suppression of Th1 responses by hepatocytes was associated with poor expression levels of Th1-promoting Delta-like Notch ligands. In vivo, MHC II expression by hepatocytes impaired the interferon-γ production by lymphocytic choriomeningitis virus-specific CD4 and CD8 T cells and prolonged viral persistence. Conclusions: By instructing infiltrating CD4 T cells to differentiate into a less inflammatory phenotype, MHC II-expressing hepatocytes seem to impair antiviral CD8 T-cell responses and viral clearance. Thus, hepatocytes may contribute to the chronicity of hepatitis virus infection. Hepatocytes are normally separated from portal blood-borne CD4 T cells by the sinusoidal endothelial cell layer, and, under healthy conditions, hepatocytes do not express class II molecules of the major histocompatibility complex (MHC II). However, during clinical hepatitis in patients or in mice, hepatocytes have contact to inflammatory CD4 T cells and often acquire MHC II expression.1Dienes H.P. Hutteroth T. Hess G. et al.Immunoelectron microscopic observations on the inflammatory infiltrates and HLA antigens in hepatitis B and non-A, non-B.Hepatology. 1987; 7: 1317-1325Crossref PubMed Scopus (113) Google Scholar, 2Franco A. Barnaba V. Natali P. et al.Expression of class I and class II major histocompatibility complex antigens on human hepatocytes.Hepatology. 1988; 8: 449-454Crossref PubMed Scopus (171) Google Scholar, 3Toyonaga T. Hino O. Sugai S. et al.Chronic active hepatitis in transgenic mice expressing interferon-γ in the liver.Proc Natl Acad Sci U S A. 1994; 91: 614-618Crossref PubMed Scopus (200) Google Scholar Therefore, MHC II-expressing hepatocytes may interact with inflammatory CD4 T cells and influence their functional phenotype. Indeed, hepatocytes feature costimulatory molecules4Sacher T. Knolle P. Nichterlein T. et al.CpG-ODN-induced inflammation is sufficient to cause T-cell-mediated autoaggression against hepatocytes.Eur J Immunol. 2002; 32: 3628-3637Crossref PubMed Scopus (62) Google Scholar and have the ability to process and present protein antigen to CD4 T cells.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar Being the predominant liver cell population, hepatocytes may thus influence the type of hepatic immunity in clinical hepatitis. The type of an immune response is shaped by differentiation of naive or uncommitted (T helper cell [Th] 0) CD4 T cells into various effector lineages; the best characterized lineages are interferon (IFN)-γ-producing Th1 cells, which promote cellular immunity, inflammation, and viral clearance, and interleukin (IL) 4-producing Th2 cells, which favor humoral immunity.6Rengarajan J. Szabo S.J. Glimcher L.H. Transcriptional regulation of Th1/Th2 polarization.Immunol Today. 2000; 21: 479-483Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar Lineage commitment of CD4 T cells is instructed by the context in which CD4 T cells are stimulated by antigen-presenting cells with peptide antigen complexed to MHC II molecules. The major factor that drives Th1 differentiation is IL-127Moser M. Murphy K.M. Dendritic cell regulation of TH1-TH2 development.Nat Immunol. 2000; 1: 199-205Crossref PubMed Scopus (995) Google Scholar; Th2 differentiation is mainly driven by IL-4.8Murphy K.M. Reiner S.L. The lineage decisions of helper T cells.Nat Rev Immunol. 2002; 2: 933-944Crossref PubMed Scopus (1392) Google Scholar Besides these cytokines, the Notch ligand families Delta or Jagged, which are differentially expressed on antigen-presenting cells, have been shown to favor Th1 or Th2 differentiation, respectively.9Amsen D. Blander J.M. Lee G.R. et al.Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells.Cell. 2004; 117: 515-526Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar The type of hepatic immunity has been linked to the course of liver diseases; acute hepatitis virus infection and viral clearance,10Gerlach J.T. Diepolder H.M. Jung M.C. et al.Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T-cell response in acute hepatitis C.Gastroenterology. 1999; 117: 933-941Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar, 11Penna A. Del Prete G. Cavalli A. et al.Predominant T-helper 1 cytokine profile of hepatitis B virus nucleocapsid-specific T cells in acute self-limited hepatitis B.Hepatology. 1997; 25: 1022-1027Crossref PubMed Scopus (187) Google Scholar, 12Nishimura T. Ohta A. A critical role for antigen-specific Th1 cells in acute liver injury in mice.J Immunol. 1999; 162: 6503-6509PubMed Google Scholar as well as autoimmune liver disease,13Jones D.E. Palmer J.M. Burt A.D. et al.Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complex.Hepatology. 2002; 36: 679-686Crossref PubMed Scopus (45) Google Scholar seem to be associated with a preponderance of hepatic Th1 immunity. In contrast, chronic hepatitis virus infection seems to be associated with poor hepatic Th1 responses or even predominance of Th2 immunity.14Kamal S.M. Rasenack J.W. Bianchi L. et al.Acute hepatitis C without and with schistosomiasis: correlation with hepatitis C-specific CD4(+) T-cell and cytokine response.Gastroenterology. 2001; 121: 646-656Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar Thus, the contextual signals delivered by hepatic antigen-presenting cells may contribute to the pathogenesis of hepatic diseases. To explore the possibility that antigen-presenting hepatocytes may instruct CD4 T cells to differentiate into a certain immune effector type, we previously generated transgenic mice, which express the class II transactivator molecule (CIITA) under control of the C-reactive protein promoter.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar Hepatocytes from these mice constitutively express MHC II molecules and can process and present protein antigen to CD4 T cells.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar Here, we report that antigen-presenting hepatocytes, in the absence of exogenous cytokines, induced Th2 differentiation of uncommitted CD4 T cells in vitro. Previously differentiated Th1 cells, when restimulated by hepatocytes, lost their ability to secrete IFN-γ. Thus, hepatocytes may instruct infiltrating CD4 T cells to obtain a less inflammatory phenotype. Indeed, lymphocytic choriomeningitis virus (LCMV) infection of CIITA-transgenic mice resulted in impaired IFN-γ production of LCMV-specific CD4 and CD8 T cells and delayed viral elimination. These findings indicate that hepatocytes may promote chronicity of hepatitis virus infection by modulating the functional phenotype of infiltrating CD4 T cells. CIITA-transgenic mice were generated as described.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar DO11.10 BALB/c TCR transgenic mice15Murphy K.M. Heimberger A.B. Loh D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo.Science. 1990; 250: 1720-1723Crossref PubMed Scopus (1654) Google Scholar have CD4+ T cells that express a T-cell receptor, which recognizes the p323–339 chicken ovalbumin peptide complexed with the MHC class II molecule I-Ad (detected by the clonotypic monoclonal antibody KJ1.26). To allow for MHC-matched antigen-specific stimulation of T cells, antigen-presenting hepatocytes were derived from the F1 generation of CIITA-transgenic FVB mice crossed to BALB/c mice, which express H-2d molecules. Mice were bred and kept in the animal facilities at the Johannes Gutenberg-University Mainz or at the University Medical Centre Hamburg-Eppendorf and used at the age of 8 to 12 weeks; the LCMV experiments were performed at the animal facilities of the Swiss Federal Institute of Technology in Zurich. The experiments were approved by the institutional animal experimentation committee. Hepatocytes were isolated from mouse livers after perfusion with 0.05% NB8 Collagenase (Serva, Heidelberg, Germany) via the portal vein. The livers were then mechanically dissected and centrifuged (30g). To remove residual accessory cells from the hepatocyte preparation, the cells were then incubated with a mixture of monoclonal antibodies to murine CD8,16Sarmiento M. Loken M.R. Trowbridge I.S. et al.High molecular weight lymphocyte surface proteins are structurally related and are expressed on different cell populations at different times during lymphocyte maturation and differentiation.J Immunol. 1982; 128: 1676-1684PubMed Google Scholar F4/80,17Austyn J.M. Gordon S. F4/80, a monoclonal antibody directed specifically against the mouse macrophage.Eur J Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1378) Google Scholar CD13 (from Dr Veelken, Institute for Immunology, Johannes Gutenberg University, Mainz, Germany), B220,18Coffman R.L. Weissman I.L. B220: a B cell-specific member of the T200 glycoprotein family.Nature. 1981; 289: 681-683Crossref PubMed Scopus (516) Google Scholar Mac-1,19Sanchez-Madrid F. Simon P. Thompson S. et al.Mapping of antigenic and functional epitopes on the α- and β-subunits of two related mouse glycoproteins involved in cell interactions, LFA-1 and Mac-1.J Exp Med. 1983; 158: 586-602Crossref PubMed Scopus (250) Google Scholar and mouse anti-rat IgG20Lanier L.L. Gutman G.A. Lewis D.E. et al.Monoclonal antibodies against rat immunoglobulin κ chains.Hybridoma. 1982; 1: 125-131Crossref PubMed Scopus (282) Google Scholar and rabbit complement for 20 minutes at 37°C. Hepatocytes were then resuspended in Dulbecco's modified Eagle medium (Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 1% glutamine, and 1% penicillin/streptomycin and cultured in 96-well Primaria flat-bottom plates (Becton Dickinson, Heidelberg, Germany), 5 × 103 cells per well, or in collagen-R (Serva)-coated 24-well culture plates (Greiner, Solingen, Germany), 5 × 104 cells per well. Hepatocytes were cultured overnight, and experiments were performed on the next day. Isolation of dendritic cells (DC) from spleen was performed applying a modification of a previously described protocol.21Vremec D. Zorbas M. Scollay R. et al.The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.J Exp Med. 1992; 176: 47-58Crossref PubMed Scopus (547) Google Scholar Spleens were cut into small fragments and digested with collagenase A (0.5 mg/mL; Roche, Mannheim, Germany) and DNAse I (40 μg/mL; Sigma, Taufkirchen, Germany) in RPMI 1640 medium supplemented with 5% FCS for 30 minutes at 37°C. Spleen cells were then suspended and washed in phosphate-buffered saline (PBS), supplemented with 5% FCS and 5 mmol/L EDTA. Alternatively, liver nonparenchymal cells were prepared from the supernatant of the hepatocyte preparation (see above) by centrifugation over a 17% Optiprep (Sigma) gradient at 400g. DC were then isolated from spleen cells or liver nonparenchymal cells by positive selection of CD11c+ cells using magnetic cell separation (Miltenyi, Bergisch-Gladbach, Germany) according to the manufacturer's instructions. CD4 T cells were purified from the spleens of DO11.10 BALB/c mice by positive selection of cells labelled with CD4 antibody attached to FITC-labelled magnetic multisort-microbeads using the MACS system (Miltenyi). Residual non-CD4 T cells were depleted with magnetic Dynabeads (CD8, B220, and Mac1; Dynal, Hamburg, Germany). The purity of the isolated primary CD4 T cells was above 98%. Purified ovalbumin-specific DO11.10 CD4 T cells (1 × 106/mL) were activated in the presence of CIITA-transgenic hepatocytes and the specific p323–339 ovalbumin peptide (1 μg/mL). As indicated, lipopolysaccharide (LPS) (100 ng/mL; Sigma) or CpG-ODN (500 pmol/mL) was added. Seven days after primary stimulation, cells were restimulated either on freshly isolated hepatocytes or by plate-bound antibody to CD3 (145-C11, 5 μg/mL). Cells of the purified protein derivative (PPD)-specific LNC2 CD4 T cell line,22Schmitt E. Van Brandwijk R. Fischer H.G. et al.Establishment of different T-cell sublines using either interleukin 2 or interleukin 4 as growth factors.Eur J Immunol. 1990; 20: 1709-1715Crossref PubMed Scopus (58) Google Scholar 5 × 105 per well, were stimulated with 20 μg/mL PPD (kindly provided by Behringwerke, Marburg, Germany) presented by hepatocytes or splenic DC. Nonparenchymal liver cells from perfused livers of LCMV-infected mice were prepared as described,23Wiegard C. Frenzel C. Herkel J. et al.Murine liver antigen-presenting cells control the suppressive activity of CD4+CD25+ regulatory T cells.Hepatology. 2005; 42: 193-199Crossref PubMed Scopus (87) Google Scholar and the included hepatic lymphocytes were then stimulated for 6 hours with purified WE (107 plaque-forming units [pfu]/mL) or with the np118–126 peptide (RPQASGVYM; 1 μg/mL), which is an immunodominant H-2d-restricted CD8 T-cell epitope derived from the LCMV nucleoprotein. Three days after primary stimulation or 24 hours after restimulation, secretion of IFN-γ and IL-4 from triplicate cultures was assessed by specific enzyme-linked immunosorbent assay (ELISA) of culture supernatants (R&D Systems, Wiesbaden, Germany). Alternatively, intracellular cytokine staining was performed with CD4 T cells that had been stimulated twice by hepatocytes and ovalbumin peptide, followed by restimulation with plate-bound antibody to CD3, or with hepatic T cells from LCMV-infected mice. Three to 6 hours after anti-CD3 stimulation or stimulation of hepatic T cells with purified WE or np118, monensin A was added to the cells for another 4 hours of incubation. After washing, cells were fixed and permeabilized using 0.5 mL FIX/perm solution, ie, FACSLyse (Becton Dickinson) diluted to 2X concentration with water and 0.05% Tween 20 (Sigma) or with 1% paraformaldehyde and 0.5% saponin/0.5% bovine serum albumin (BSA), followed by double staining for intracellular cytokines with fluorochrome-conjugated antibodies to IFN-γ and IL-4 (Becton Dickinson). Cells were then analyzed with a FACSscan or FACScalibur (Becton Dickinson). Lymphocytes were gated according to their forward/sideward scatter profile; nonspecific binding was controlled with species- and isotype-matched fluorescent antibody. The messenger RNA (mRNA) of hepatocytes or splenic DC was isolated with Tri Reagent (Sigma), and complementary DNA (cDNA) was synthesized with a cDNA synthesis kit and oligo-dT primers according to the manufacturer's instructions (Roche). The specific cDNA of Jagged-1 and Delta-like 4 was then quantified by real-time polymerase chain reaction (PCR) (light cycler, Roche) according to the manufacturer's instructions. The β-actin housekeeping gene was used for normalization, and the Notch ligand expression levels are given in relation to the β-actin levels. The specific primers were as follows: Jagged-1 sense (5′-AGAAGTCAGAGTTCAGAGGCGTCC-3′), Jagged-1 antisense (5′-AGTAGAAGGCTGTCACCAAGCAAC-3′); Delta-like 4 sense (5′-AGGTGCCACTTCGGTTACACAG-3′), Delta-like 4 antisense (5′-CAATCACACACTCGTTCCTCTCTTC-3′); β-actin sense (5′-GAAGTCCCTCACCCTCCCAA-3′), β-actin antisense (5′-GGCATGGACGCGACCA-3′). CD4 T cells were incubated with 2 μmol/L 5-, 6-carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes, Leiden, The Netherlands) in PBS at 37°C for 10 minutes before stimulation. Proliferation was then assessed as dilution of CFSE by flow cytometry. The LCMV isolate WE was provided by Dr R. M. Zinkernagel (University Hospital, Zurich, Switzerland) and propagated on L929 fibroblast cells. Mice were infected intravenously with 106 pfu of virus. Virus titers were determined by the focus-forming assay, as described.24Battegay M. Cooper S. Althage A. et al.Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates.J Virol Methods. 1991; 33: 191-198Crossref PubMed Scopus (406) Google Scholar Data are given as mean of quadruplicate wells ± standard deviation of the mean. Each experiment is representative of at least 4 repetitions. Statistical significance of differences between data sets was tested by Student t test or the Mann–Whitney U test, as appropriate. A P value <.05 was considered significant. We have previously shown that MHC II-expressing hepatocytes can present the p323–339 ovalbumin peptide and induce the proliferation of primary CD4 T cells from DO11.10 mice.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar To learn whether hepatocytes could induce T-cell differentiation, we stimulated primary CD4 T cells from spleens of DO11.10 mice with the p323–339 ovalbumin peptide, presented by hepatocytes or, as control, by splenic DC. After 1 week, the CD4 T cells were adjusted to equal numbers and restimulated with antibody to CD3, and secretion of IFN-γ (Figure 1A, grey bars) and IL-4 (Figure 1A, black bars) was assessed. CD4 T cells prestimulated by DC secreted high amounts of IFN-γ (2463 U/mL) and low amounts of IL-4 (22 U/mL); in contrast, CD4 T cells prestimulated by hepatocytes secreted low amounts of IFN-γ (163 U/mL; P < .05) and high amounts of IL-4 (1898 U/mL; P < .05). Alternatively, primary DO11.10 CD4 T cells were repeatedly stimulated by ovalbumin peptide-presenting hepatocytes (Figure 1B). After each of 3 restimulations on hepatocytes, the amount of secreted IL-4 greatly increased, whereas the amount of secreted IFN-γ remained low. After the third restimulation by hepatocytes, the CD4 T cells also secreted low amounts of IL-10 (20 U/mL) but not transforming growth factor-β (data not shown). Thus, hepatocytes seemed to induce Th2 differentiation of primary CD4 T cells. To confirm this interpretation, we performed intracellular cytokine staining for IFN-γ and IL-4 of CD4 T cells after 2 stimulations on hepatocytes and re-stimulation with antibody to CD3 (Figure 1C). Indeed, we found that CD4 T cells stimulated by hepatocytes differentiated to Th2 cells, marked by exclusive production of IL-4 by approximately 40% of the T cells and absence of cells staining for IFN-γ. We then tested whether addition of proinflammatory microbial stimuli could restore Th1 differentiation of primary CD4 T cells stimulated by hepatocytes. Therefore, primary CD4 T cells were stimulated by hepatocytes or liver DC and restimulated by antibody to CD3, in the presence or absence of LPS or oligonucleotides with immune-stimulatory CpG sequence motifs, and the secretion of IFN-γ was determined. Both LPS and CpG oligonucleotides strongly increased IFN-γ secretion by CD4 T cells, which had been stimulated by liver DC (Figure 2, shaded bars). In contrast, even in the presence of LPS or CpG oligonucleotides, IFN-γ secretion by CD4 T cells, which had been stimulated by hepatocytes, remained poor (Figure 2, open bars). Previously, we observed that hepatocytes, which present the p323–339 ovalbumin peptide, could stimulate specific Th1 lines as well as Th2 lines; after stimulation by hepatocytes, the Th1 cells secreted IFN-γ and the Th2 cells secreted IL-4.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar Because we showed above that hepatocytes induced Th2 differentiation of uncommitted CD4 T cells, it was of interest to know whether hepatocytes could sustain Th1 responses of differentiated effector cells. To that end, Th1 differentiation of primary DO11.10 CD4 T cells was induced by stimulation on splenic DC; the Th1-committed cells were then repeatedly restimulated on hepatocytes. Figure 3 shows that, after each restimulation by hepatocytes, the Th1-committed T cells secreted less IFN-γ and more IL-4, resulting in the outgrowth of Th2 cells and gradual impairment of the Th1 response. To confirm this observation, we repeatedly restimulated established Th1 cells of the LNC2 line on hepatocytes. Indeed, we found that the differentiated LNC2 cells, 3 times restimulated by hepatocytes, secreted only negligible amounts of IFN-γ (54 U/mL; Figure 4); in contrast, LNC2 cells, when 3 times restimulated by splenic DC, secreted large amounts of IFN-γ (6704 U/mL; P < .05). Thus, differentiated Th1 cells lost their potential to secrete IFN-γ after being repeatedly stimulated by hepatocytes. The failure of hepatocytes to sustain Th1 responses was not due to insufficient stimulation of LNC2 cells because proliferation of LNC2 cells was induced by hepatocytes.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar To confirm that the observed differences in the cytokine response of stimulated T cells were not due to a different stimulatory potential of hepatocytes or DC, we assessed the T-cell activation status by staining for the activation markers CD25 (Figure 5A, left) and CD44 (Figure 5A, right) and found that CD25 and CD44 expression of the hepatocyte-stimulated T cells (Figure 5A, lower panels) was even higher than that of DC-stimulated T cells (Figure 5A, upper panels), indicating efficient stimulation. However, hepatocyte-stimulated T cells did not form the typical proliferation clusters, as do DC-stimulated T cells. Indeed, when we assessed the proliferative response to stimulation by labelling the T cells with the dye CFSE by flow cytometry (Figure 5B), we found that the hepatocyte-stimulated proliferation was retarded in comparison with the DC-stimulated proliferation by approximately 1 day. Nevertheless, we did not find a significant difference in the overall rates of proliferation or the rates of activation-induced cell death induced by DC (38%) or hepatocytes (32%), as assessed by staining with Annexin V and propidium iodide (not shown). Furthermore, we compared the relative abundance of MHC II on hepatocytes and splenic DC (Figure 5C) and found that MHC II expression by hepatocytes was similar to that of DC, suggesting that hepatocellular MHC II expression was not limiting to the T-cell response. These findings confirmed that both types of antigen-presenting cells can efficiently activate stimulated T cells but induce different response types. It has been reported that expression of Delta-like Notch ligand molecules on antigen-presenting cells, notably in the absence of IL-12, seems to favor Th1 differentiation, whereas expression of Jagged Notch ligand molecules seems to promote Th2 differentiation.9Amsen D. Blander J.M. Lee G.R. et al.Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells.Cell. 2004; 117: 515-526Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar We therefore examined whether the inability of hepatocytes to induce or sustain Th1 immunity may be correlated with the expression pattern of Notch ligands. The expression levels were determined by real-time PCR and normalized to the expression of the β-actin housekeeping gene. Figure 6A shows that the expression levels of Jagged-1 mRNA, which promotes Th2 differentiation, was almost similar in hepatocytes and splenic DC; in contrast, the levels of Delta-like 4 mRNA, which promotes Th1 differentiation, were considerably lower in hepatocytes (4.7 times β-actin mRNA) than in DC (22 times β-actin mRNA; P < .05). Of particular interest was that, even in the presence of proinflammatory microbial signals, LPS, or oligonucleotides with immune-stimulatory CpG motifs, there was no increase in the expression of Delta-like 4 mRNA in hepatocytes (1.6 and 4.2 times β-actin mRNA, respectively). In contrast, DC greatly increased Delta-like 4 mRNA expression upon stimulation with TLR4 or TLR9 ligands (126 and 212 times β-actin mRNA, respectively; Figure 6B). To learn whether our observation that hepatocytes seem to impair inflammatory Th1 responses was relevant in vivo, we infected nontransgenic or CIITA-transgenic mice with noncytopathic LCMV of the WE strain to induce immune-mediated hepatitis and determined the proportions of CD4 and CD8 T cells in the livers of nontransgenic or CIITA-transgenic mice at day 12 after infection. We found that the relative CD4 and CD8 numbers per liver were not significantly different between the nontransgenic and the CIITA-transgenic mice (Figure 7A, left and right panels). However, when we analyzed the LCMV-specific response of hepatic T cells, we found a significant impact of hepatocellular MHC II-expression on antiviral immunity. Indeed, the livers of CIITA-transgenic mice contained significantly fewer IFN-γ-producing LCMV-specific CD4 T cells than the livers of nontransgenic mice (on average, 1.7% vs 7.0%, respectively; P < .01); a representative quantification of the IFN-γ-producing CD4 T cells from 1 mouse of each group is shown in Figure 7B (upper panels). Thus, MHC II expression by hepatocytes in vivo seemed to impair the antiviral Th1 response of LCMV-infected mice. Interestingly, the impaired CD4 response to LCMV in the CIITA-transgenic mice seemed to be associated with an impaired CD8 response; note that the numbers of IFN-γ-producing CD8 T cells in the livers of CIITA-transgenic mice were significantly lower than those in nontransgenic mice (on average, 5.3% vs 16.2%, respectively; P < .02). A representative quantification of the IFN-γ-producing CD8 T cells from 1 mouse of each group is shown in Figure 7B (lower panels). Of note, the impaired IFN-γ response of LCMV-specific CD4 and CD8 T cells in the CIITA-transgenic mice seemed to be associated with impaired viral clearance because these mice still exhibited significant LCMV titers at day 12 of infection, both in the spleen and in the liver (3.0 and 2.4 × log10 pfu, respectively; Figure 7C); the nontransgenic mice, in contrast, had almost cleared LCMV with titers close to the detection limit (1.9 and 1.7 × log10 pfu; P < .05). The higher virus load of the CIITA-transgenic livers seemed to be associated with increased liver injury because we found an increase of the serum transaminases in the CIITA-transgenic mice compared with nontransgenic mice at day 7 but not day 4 or day 10 of infection (Figure 7D). However, in histologic analysis, the degree of liver inflammation at day 7 was similar in CIITA-transgenic and nontransgenic mice (Figure 7E), suggesting that the increased liver damage in CIITA-transgenic mice was virus mediated rather than immune mediated. Inflammation is a highly regulated physiologic process that facilitates tissue maintenance, wound healing, and eradication of damaged cells and pathogens; however, deregulated inflammation may contribute to the pathogenesis of various diseases.25Nathan C. Points of control in inflammation.Nature. 2002; 420: 846-852Crossref PubMed Scopus (2124) Google Scholar In the liver, exaggerated inflammatory activity seems to be involved in the pathogenesis of autoimmune liver diseases,13Jones D.E. Palmer J.M. Burt A.D. et al.Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complex.Hepatology. 2002; 36: 679-686Crossref PubMed Scopus (45) Google Scholar, 26Djilali-Saiah I. Lapierre P. 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Sato M. et al.Indispensable role for TNF-α and IFN-γ at the effector phase of liver injury mediated by Th1 cells specific to hepatitis B virus surface antigen.J Immunol. 2000; 165: 956-961Crossref PubMed Scopus (62) Google Scholar Insufficient inflammatory activity, in contrast, seems to be related to chronic hepatitis virus infection and the inability to induce viral clearance.30Chisari F.V. Ferrari C. Hepatitis B virus immunopathogenesis.Annu Rev Immunol. 1995; 13: 29-60Crossref PubMed Scopus (1498) Google Scholar, 31Cerny A. Chisari F.V. Pathogenesis of chronic hepatitis C: immunological features of hepatic injury and viral persistence.Hepatology. 1999; 30: 595-601Crossref PubMed Scopus (403) Google Scholar Regulation of hepatic inflammation, at least in part, depends on the accessory signals delivered by the hepatic antigen-presenting cells,32Crispe I.N. Hepatic T cells and liver tolerance.Nat Rev Immunol. 2003; 3: 51-62Crossref PubMed Scopus (585) Google Scholar which instruct the CD4 T-cell response type. Hepatocytes, when interacting with infiltrating CD4 T cells during hepatitis, can become antigen-presenting cells.5Herkel J. Jagemann B. Wiegard C. et al.MHC class II-expressing hepatocytes function as antigen-presenting cells and activate specific CD4 T lymphocytes.Hepatology. 2003; 37: 1079-1085Crossref PubMed Scopus (124) Google Scholar Because they are the major hepatic cell type, hepatocytes may thus be major modulators of hepatic immunity. Indeed, we report here that hepatocytes can mediate hepatic immune deviation by at least 2 mechanisms: (1) hepatocytes induced the differentiation of uncommitted CD4 T cells into Th2 cells (Figure 1), and (2) hepatocytes suppressed the effector function of differentiated Th1 cells. Th1 cells, when repeatedly stimulated by hepatocytes, lost the ability to secrete IFN-γ (Figure 3, Figure 4). Thus, antigen-presenting hepatocytes seem to instruct CD4 T cells to obtain a less inflammatory phenotype. This skewed cytokine response did not appear to result from inefficient activation by hepatocytes because the overall proliferative response to hepatocellular stimulation was not significantly altered, although the T-cell proliferation induced by repeated stimulation on hepatocytes was somewhat retarded in comparison with that induced by DC (Figure 5B). Moreover, T-cell activation markers were efficiently induced by hepatocellular stimulation (Figure 5A). Because hepatocytes up-regulate MHC II expression in inflammatory conditions, it is conceivable that the MHC II-dependent anti-inflammatory activity of hepatocytes may have an important physiologic role in the resolution of hepatic inflammation. Moreover, hepatocytes, being the most prevalent cell type in the liver, may also contribute considerably to the anti-inflammatory immune deviation in the liver that has been described as an important mechanism of hepatic immune tolerance.32Crispe I.N. Hepatic T cells and liver tolerance.Nat Rev Immunol. 2003; 3: 51-62Crossref PubMed Scopus (585) Google Scholar, 33Klugewitz K. Blumenthal-Barby F. Schrage A. et al.Immunomodulatory effects of the liver: deletion of activated CD4+ effector cells and suppression of IFN-γ-producing cells after intravenous protein immunization.J Immunol. 2002; 169: 2407-2413Crossref PubMed Scopus (92) Google Scholar Anti-inflammatory immune deviation by hepatocytes may also be responsible for the promotion of chronic infection by hepatitis virus. Indeed, we find MHC II expression by hepatocytes in vivo to be associated with impaired IFN-γ responses of antiviral CD4 and CD8 T cells and impaired clearance of LCMV infection (Figure 7). In contrast to the predominant Th2 differentiation observed in vitro (Figure 2), the differentiation of hepatocyte-primed CD4 T cells to IL-4 producers appeared to be of minor relevance in vivo; the increase in virus-specific IL-4-producing CD4 and CD8 T cells was negligible (Figure 7B). This finding indicates that, in vivo, T-cell priming occurred predominantly in the lymph nodes and not in the liver. Nevertheless, the inflammatory activity of the so primed T cells was attenuated by hepatocytes in vivo, as indicated by the reduced IFN-γ production of virus-specific T cells (Figure 7B). Indeed, the concomitant impairment of the inflammatory activity of both antiviral CD4 and CD8 T cells is remarkable because it is known that CD4 T cells are dispensable for the clearance of LCMV,34Matloubian M. Concepcion R.J. Ahmed R. CD4+ T cells are required to sustain CD8+ cytotoxic T-cell responses during chronic viral infection.J Virol. 1994; 68: 8056-8063PubMed Google Scholar which rather depends on IFN-γ-producing CD8 T cells.35Guidotti L.G. Borrow P. Brown A. et al.Noncytopathic clearance of lymphocytic choriomeningitis virus from the hepatocyte.J Exp Med. 1999; 189: 1555-1564Crossref PubMed Scopus (141) Google Scholar Thus, it appears that the poorly inflammatory CD4 T cells, which had been stimulated by hepatocytes, may actively down-regulate the capacity of CD8 T cells to secrete IFN-γ, which, in turn, seemed to impair the clearance of LCMV from spleen and liver (Figure 7). The rapidity of this functional CD8 T cell impairment by hepatocyte-stimulated CD4 T cells is noteworthy in that it was manifest already 10 days after LCMV infection. Note that the LCMV hepatitis model used here is an acute model, which is normally cleared by day 12. It remains to be seen whether and how the hepatitis B and C viruses, which are rarely cleared within such a short time, may exploit the ability of hepatocytes to down-regulate the antiviral activity of inflammatory T cells for the promotion of chronic virus persistence. Indeed, the clearance of hepatitis C virus infection also depends on antiviral IFN-γ responses, notably of CD4 T cells,36Kaplan D.E. Sugimoto K. Newton K. et al.Discordant role of CD4 T-cell response relative to neutralizing antibody and CD8 T-cell responses in acute hepatitis C.Gastroenterology. 2007; 132: 654-666Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar and CD8 T cells primed in liver gain a much less inflammatory phenotype than those primed in lymph nodes.37Bowen D.G. Zen M. Holz L. et al.The site of primary T-cell activation is a determinant of the balance between intrahepatic tolerance and immunity.J Clin Invest. 2004; 114: 701-712Crossref PubMed Scopus (284) Google Scholar Th2 immune deviation induced by hepatocytes was remarkably robust; even with the presence of potent inflammatory stimuli, such as LPS or oligonucleotides with CpG sequence motifs, the suppression of Th1 effector function by hepatocytes could not be reversed (Figure 2). In contrast, the same inflammatory stimuli greatly enhanced IFN-γ secretion by DC (Figure 2). Of note, this finding cannot be explained by insensitivity of hepatocytes to LPS or CpG oligonucleotides; hepatocytes are equipped with the signal transducing toll-like receptors 4 and 938Liu S. Gallo D.J. Green A.M. et al.Role of toll-like receptors in changes in gene expression and NF-κB activation in mouse hepatocytes stimulated with lipopolysaccharide.Infect Immun. 2002; 70: 3433-3442Crossref PubMed Scopus (226) Google Scholar and respond to LPS and CpG signals.23Wiegard C. Frenzel C. Herkel J. et al.Murine liver antigen-presenting cells control the suppressive activity of CD4+CD25+ regulatory T cells.Hepatology. 2005; 42: 193-199Crossref PubMed Scopus (87) Google Scholar Robustness of hepatic immune deviation may explain the difficulty to induce effective Th1 immunity in hepatitis virus infection. The molecular basis for the apparent inability of hepatocytes to induce and sustain Th1 responses may be their expression pattern of Notch ligands. In contrast to splenic DC, which expressed high levels of Th1-inducing Delta-like Notch ligands, hepatocytes only expressed low levels of Delta-like molecules, whereas expression of Th2-inducing Notch ligands of the Jagged family was similar in both cell types (Figure 6A). Most interesting was the observation that, in contrast to DC, microbial signals did not increase Delta-like expression in hepatocytes (Figure 6B); expression levels of Delta-like Notch ligands corresponded to IFN-γ secretion by the stimulated CD4 T cells (Figure 2). The major inducer of Th1 differentiation is IL-127Moser M. Murphy K.M. Dendritic cell regulation of TH1-TH2 development.Nat Immunol. 2000; 1: 199-205Crossref PubMed Scopus (995) Google Scholar; in the absence of IL-12, it is believed that Th1 differentiation is facilitated by Delta-like Notch-ligands.9Amsen D. Blander J.M. Lee G.R. et al.Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells.Cell. 2004; 117: 515-526Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar Interleukin-12 expression in liver cells is mostly absent or low39Frenzel C. Herkel J. Wiegard C. et al.Murine liver dendritic cells, but not Kupffer cells or sinusoidal endothelial cells, induce secretion of interferon-γ by CD4+ T cells.Hepatology. 2004; 40: 424APubMed Google Scholar; thus, the skewed Notch ligand expression pattern by hepatocytes may explain the high frequency of chronic hepatitis virus infections. The authors thank Meike Petersen and Alex Hobel for excellent technical assistance.
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