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Activating Immunity in the Liver. I. Liver Dendritic Cells (but Not Hepatocytes) Are Potent Activators of IFN- Release by Liver NKT Cells¹

Zlatko Trobonjaca^*, Frank Leithäuser, Peter Möller, Reinhold Schirmbeck^* and Jörg Reimann^2,*

Departments of ^* Medical Microbiology and Immunology and Pathology, University of Ulm, Ulm, Germany

	Abstract

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A prominent subset of the hepatic innate immune system is -galactosylceramide (GalCer)-reactive, (CD4⁺ and CD4^-CD8^-) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with GalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN- release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after GalCer injection. Although liver CD11c⁺ DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN- and IL-4 release by liver NKT when pulsed with GalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following GalCer stimulation.

	Introduction

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The regulation of immune responses in the liver is not well understood. Hepatotropic virus infections demonstrate that acute (fulminant) destruction of the large majority of hepatocytes or chronic (aggressive) necroinflammatory processes (that do not clear the infection) are more frequently observed during infections of the liver than during infections of other organs. Because the cytopathic effect of most hepatotropic viruses is limited, it seems that immune cell-mediated responses drive these disease processes (reviewed in Ref. 1). This suggests that hepatic immune responses are prone to dysregulation. The liver immune system comprises a major component of the innate immune system and a minor component of the specific (adaptive) immune system. The interactions between the two compartments of the hepatic immune system are not well defined.

CD4⁺ and CD4^-CD8^- (double negative) T cells with an invariant TCR and intermediate level NK1 surface expression (in appropriate mouse strains) represent 60–80% of the murine hepatic T cell population (2, 3). This T cell subset is also prevalent in the human liver (4, 5). The development of NKT cells in the murine liver is LFA-1 dependent (6). The liver NKT cell population is expanded during hepatocyte regeneration (7). NKT cells rapidly disappear within hours from the liver of anti-CD3- or IL-12-treated mice, but are regenerated within a few days (8). Liver NKT cells seem to play a role in the intrahepatic immunity to salmonella (7, 9), malaria (10), and hepatitis B virus (HBV)³ infection (11). NKT cells also play a role in experimental mouse hepatitis models (12, 13, 14) and accumulate in the human liver chronically infected with hepatitis C virus (15). Although liver NKT cells are the major T cell subset within this large organ, many aspects of their intrahepatic activation and their regulatory effect on other (innate or specific) components of the hepatic immune system are unknown.

The glycolipid -galactosylceramide (GalCer) binds to CD1d and stimulates V14⁺ NKT cells, a specific recognition that is highly conserved through mammalian evolution (16, 17, 18, 19). Human NKT cells are activated and expand when cocultured with GalCer-pulsed dendritic cells (DC) (20, 21). In vivo activation of murine NKT cells by injection of GalCer can facilitate priming of either Th1 or Th2 immunity. It can induce liver injury (14) and suppress liver metastases of the B16 melanoma (22), suggesting Th1-biased immunity. GalCer has been reported to introduce a Th2 bias into an immune response when codelivered with a protein Ag (23) and to protect mice against an experimentally induced Th1-type colitis (24). In a tumor model, GalCer-stimulated IFN- release by NKT cells has been shown to depend on IL-12 released by the CD40/CD40 ligand (CD40L)-dependent interaction of NKT cells with DC (25). The role of intrahepatic DC in activating liver NKT cells after GalCer injection has not been elucidated. GalCer bound to CD1d is a potent stimulator of NKT cells but natural glycolipids that stimulate liver NKT cells have not yet been identified.

Because GalCer is a potent stimulus with a well-defined restriction specificity and responder cell population, it can be used as a well-defined point of entry into the innate immune system of the liver. We use this system to follow early events that can initiate, regulate, and/or support intrahepatic immune responses. We found intrahepatic DC pulsed in vitro or in vivo with GalCer to be the most potent activators of NKT cells and to be activated themselves in the process of NKT cell stimulation. Activated DC are thus a candidate immune cell type that can transmit the NKT cell-initiated signal to other liver regulator, memory, or effector cell subsets. Although liver NKT cells are eliminated early after GalCer injection, liver injury develops 2–3 days after a single i.v. injection of a low dose of GalCer into normal or B6 mice expressing a transgene-encoded hepatitis B surface Ag in the liver (HBs-tg). This indicates that NKT cells can initiate damaging immune reactions in the liver without being the ultimate effector cell population. Liver injury induced by the same dose of GalCer in HBs-tg mice is more severe than liver injury induced in normal mice (11). HBs-tg animals display a characteristic liver pathology because they overproduce abnormally large amounts of the large envelope protein of HBV in the endoplasmic reticulum of their hepatocytes and develop ground glass cells and spontaneous liver disease (26, 27, 28). These mice are extremely hypersensitive to the toxic effects of IFN- (29, 30) which makes them a sensitive model to evaluate magnitude and kinetics of IFN release in the liver in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6J mice (H-2^b; B6) and MHC class II^-/- (A^-/- knockout (KO)) B6 mice (31) were kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). C57BL/6J-TgN(Alb1HBV)44Bri-transgenic (HBs-tg) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were used at 10–16 wk of age.

Injection of GalCer and IL-12

GalCer was kindly provided by Y. Koezuka (Kirin Brewery, Pharmaceutical Research Laboratory, Gunma, Japan). GalCer (1–100 ng/mouse) dissolved in 0.5 ml of PBS was injected i.v. In some groups, IL-12 (100 ng/mouse) was injected i.p. 6 h after the GalCer injection, following an established protocol (25).

In vivo suppression of CD4 and/or CD8 T cells

CD4⁺ T cells were eliminated by two i.p. injections of 100 µg of anti-CD4 mAb YTS 191.1 (in 200 µl of PBS) as described previously (32). Similarly, CD8⁺ T cells were depleted by two injections of 100 µg of anti-CD8 mAb YTS 169.4. Flow cytometric analyses (FCM) of PBMC (MNC) populations demonstrated that >99% of the T cells expressing the respective phenotype were deleted.

Serum alanine aminotransferase (ALT) determination

Blood was collected from the tail of mice, centrifuged at 5000 x g for 10 min, and the serum was collected. Serum ALT activity was determined in blood using the Reflotron test (catalog no. 745138; Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.

Isolation of hepatocytes and liver MNC populations

Mice were anesthetized by methoxyfluran (Metofane; Janssen-Cilag,Neuss, Germany) and their abdomens were opened. A needle was inserted into the portal vein. The inferior caval vein was cut to enable blood outflow. The liver was perfused with 20 ml of liver perfusion medium (catalog no. 17701-038; Life Technologies, Rockville, MD) followed by an injection of 5 ml of liver digestion medium (catalog no. 17703-034; Life Technologies). The liver was removed and gently pressed through a mesh. The liver cell suspension was collected and parenchymal cells (pellet) were separated from the MNC by centrifugation at 50 x g for 5 min. Hepatocytes (parenchymal cells) were washed twice in complete RPMI 1640 and resuspended either in RPMI 1640 supplemented with 5% FCS (catalog no. 10270-106; Life Technologies) or in HepatoZYME-SFM medium (catalog no. 17705-021; Life Technologies). MNC populations were purified by centrifugation through a Percoll gradient. Cells were collected, washed in PBS, and resuspended in 40% Percoll (catalog no. L6145; Biochrom, Berlin, Germany) in complete RPMI 1640 medium. The cell suspension was gently overlayed onto 70% Percoll and centrifuged for 20 min at 750 x g. MNC were collected from the interface. Cells were washed twice in PBS and resuspended in medium.

Histology and TUNEL staining

Thin slides of liver tissue (<3 mm) were fixed in 4% Formalin (pH 7.0) for 24 h and embedded in paraffin. Two-micrometer-thick paraffin sections were stained with H&E. Cells undergoing apoptosis were detected in situ by labeling DNA strand breaks using TUNEL (33). Briefly, tissue sections of paraformaldehyde-fixed thymus were digested with proteinase K (300 µg/ml, catalogue no. P2308; Sigma, St. Louis, MO) for 15 min at 37°C. The labeling reaction was conducted using 10 U of TdT (catalogue no. M1871; Promega, Madison, WI) and 17 µM biotin-16-dUTP (catalogue no. 1093070; Boehringer Mannheim, Mannheim, Germany) in 50 µl of TdT buffer (0.5 M cacodylic acid, sodium salt (pH 6.8), 1 mM CoCl₂, 0.5 mM DTT, 0.05% w/v BSA, and 0.15 M NaCl). Labeled cells were detected using HRP-conjugated streptavidin diluted 1:100 (catalogue no. RPN1051; Amersham Life Science, Freiburg, Germany). Bound HRP was visualized by the substrate 3-amino-9-ethylcarbazole (0.1 mg/ml in 0.17 M sodium acetate (pH 5.2) plus 0.01% H₂O₂). Sections were counterstained in Mayer’s hematoxylin.

FCM analyses

Cells were suspended in PBS/0.3% w/v BSA supplemented with 0.1% w/v sodium azide. Nonspecific binding of Abs to Fc receptor was blocked by preincubating cells with the mAb 2.4G2 (catalog no. 01241D) directed against the FcRIII/II CD16/CD32 (1 µg mAb/10⁶ cells/100 µl). Cells were incubated with 0.5 µg/10⁶ cells of the relevant mAb for 30 min at 4°C and washed. In most experiments, cells were subsequently incubated with a second-step reagent for 10 min at 4°C. Three-color FCM analyses were performed on a FACSCalibur (BD Biosciences, Mountain View, CA). The forward narrow angle light scatter was used as an additional parameter to facilitate exclusion of dead cells and aggregated cell clumps. The following reagents and mAb were obtained from BD PharMingen (San Diego, CA): PE-conjugated anti-CD3 mAb 145-2C11 (catalog no. 01085B), FITC- and PE-conjugated anti-CD4 mAb GK1.5 (catalog nos. 09424D and 09425B), biotinylated anti-CD4 mAb RM4-5 (catalog no. 01062D), FITC-conjugated anti-CD8 mAb 53-6.7 (catalog no.01351), biotinylated anti-CD44 (Pgp-1) mAb IM7 (catalog no. 01222D), PE-conjugated anti-NK1.1 mAb PK136 (catalog no. 01295B), and biotinylated anti-CD40L mAb MR1 (catalog no. 09022D). PE-conjugated streptavidin (catalog no. 13025D) was obtained from BD PharMingen. SA-Red670 was obtained from Life Technologies (catalog no. 19543-024).

MACS purification of CD4⁺ and CD11c⁺ cells

Spleen and liver MNC were obtained from normal B6, HBs-tg B6, or MHC-II-deficient A^-/- B6 mice. CD8⁺ T cells were depleted from these spleen cells by treatment with anti-CD8 Ab and low-toxicity rabbit complement (catalog no. CL3051; Cedarlane Laboratories, Hornby, Ontario, Canada) according to the manufacturer’s instructions. CD4⁺ T cells and CD11c⁺ were enriched to >98% purity by positive selection using the MACS system (Miltenyi Biotec, Auburn, CA). Briefly, 10⁷ cells were incubated with mouse CD4 (L3T4) MicroBeads (1:10 dilution, catalog no. 130-049-201; Miltenyi Biotec) or with mouse CD11c (N418) MicroBeads (1:10 dilution, catalogue no. 130-052-001; Miltenyi Biotec) in 100 µl of MACS buffer (PBS buffer supplemented with 2 mM EDTA and 0.5% BSA) for 30 min at 4°C. Cells were washed twice in MACS buffer, resuspended in 500 µl of MACS buffer, and transferred onto a prerinsed LS separation column (catalog no. 130-042-401; Miltenyi Biotec) attached to a MidiMACS separation unit (catalog no. 130-042-302; Miltenyi Biotec). After washing three times with 3 ml of MACS buffer, columns were removed from the separation unit. To elute bound CD4⁺ or CD11c⁺ cells, 6 ml of MACS buffer was passed through the columns using a plunger supplied with the columns. We could not detect CD3⁺ T cells, NK1⁺ NK cells, or CD68⁺F4/80⁺ macrophages in the purified CD11c⁺ DC cell preparations from liver or spleen.

Cell cultures

Cells were cultured in 200-µl flat-bottom microwells in RPMI 1640 medium supplemented with 5% FCS. NKT cells (1 x 10⁵/well) were stimulated with either 2 x 10⁴ hepatocytes/well or 2 x 10⁴ DC/well. In some experiments, titrated numbers of NKT cells (ranging from 2 x 10⁴ to 2 x 10⁵/well) were cocultured with a constant number of presenting cells (1 x 10⁴/well). Supernatants were collected after 24 and 48 h of culture for cytokine determination.

Cytokine detection by ELISA

Cytokines released into culture supernatants were detected by a conventional double-sandwich ELISA. For detection and capture, the following mAbs (from BD PharMingen) were used: mAb R4-6A2 (catalog no. 18181D) and biotinylated mAb XMG1.2 (catalog no. 18112D) were used for IFN-, and mAb BVD4-1D11 (catalog no. 18031D) and biotinylated mAb BVD6-24G2 (catalog no. 18042D) were used for IL-4. Extinction was analyzed at 405/490 nm on a TECAN microplate ELISA reader (TECAN) using the EasyWin software (TECAN, Crailsheim, Germany).

	Results

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Liver NKT cells

NK1^high CD3^- NK cells, (NK1^int or NK1^low) CD3^int NKT cells, and (CD4⁺ or CD8⁺) CD3^high T cells were found in the liver of normal and A^-/- KO B6 mice (Fig. 1). In all tested strains, 60–80% of all liver CD3⁺ T cells were NKT cells. After a single i.v. injection of GalCer, >98% of the CD3^int T cell subset disappeared within 12 h postinjection. Liver NKT cells hence seem to be best defined as CD3^intTCR^int T cells. Neither the NK1^int nor the CD4⁺ phenotype reliably identified this T cell subset. In the liver NKT cell population from normal B6 mice, 70–80% of the cells were NK1^int and 20–30% were NK1^low (Fig. 1). Within the (NK1^int and NK1^low) CD3^int T cell subset, 60–70% of the cells were CD4⁺. All NKT cells were CD44^high. Livers from A^-/- KO B6 mice contained lower numbers of NK cells, similar numbers of NKT cells but lower numbers of T cells when compared with normal B6 mice (Fig. 1). Liver CD4⁺CD3^int T cells from A^-/- KO B6 mice are highly enriched in NKT cells. Most NKT cells showed low CD1d surface expression but a 3–5% subset of the CD3^int cell population was CD1d^high. As responder cells we used either nonfractionated liver MNC or fractionated liver CD4⁺ T cells from normal or A^-/- KO B6 mice.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Surface phenotype of liver NKT cells. MNC were obtained from the livers of normal or MHC-II-deficient A-/- KO B6 mice. Cells were stained with Abs and analyzed by three-color flow cytometry. The profile of the surface coexpression of CD3 and NK1.1 allowed identification of three subsets (upper panel of the figure for a normal B6 mouse): NK1.1highCD3- NK cells, NK1int or NK1lowCD3int NKT cells, and NK1lowCD3high T cells. The mean of the total cell numbers per liver and the mean numbers of cells in the three subsets (and the relative percent) (±SEM) from three analyzed mice of the two strains are shown in the table. Surface expression of CD1d, CD44, CD4, and CD8 of liver (NK1int or NK1low) CD3int NKT cells from a representative normal B6 mouse and A-/- KO B6 mice is shown in the lower half of the figure. The representative examples were selected from three individual mice analyzed per group.

GalCer-pulsed hepatocytes stimulate IL-4 release by liver NKT cells

We isolated hepatocytes from in vivo perfused livers of B6 mice. Freshly isolated hepatocytes expressed CD1d and MHC-I (K^b) molecules on the surface (Fig. 2) as described elsewhere (34, 35, 36). Culture of hepatocytes for 24 h in vitro did not modulate their surface expression of CD1d and MHC-I (data not shown). Surface expression of the costimulator molecules CD40, CD80, and CD86 on freshly isolated (Fig. 2) or in vitro-cultured hepatocytes was low or undetectable. IL-12 release was not inducible by various stimuli in hepatocytes cultured in vitro (data not shown). Surface expression of MHC-I (K^b), CD1d, CD40, CD80, CD86, and CD95 molecules by hepatocytes was not up-regulated 12 h after the injection of 10–200 ng of GalCer i.v. (Fig. 2). We tested whether liver NKT cells cocultured with GalCer-pulsed CD1d⁺ hepatocytes release IL-4 and IFN-.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Surface phenotype of hepatocytes from nontreated or GalCer-treated mice. Hepatocytes were isolated either from normal B6 mice or from B6 mice injected 12 h previously with 100 ng of GalCer i.v. Surface expression of MHC class I (Kb), CD1d, CD40, CD80, and CD86 was analyzed by flow cytometry. The representative examples shown were selected from three independent experiments.

View larger version (25K): [in this window] [in a new window]   FIGURE 3. GalCer-pulsed hepatocytes trigger IL-4 release by liver NKT cells. B6 mice were injected i.v. with either PBS or 100 ng of GalCer, and their hepatocytes were isolated 12 h postinjection. Hepatocytes from PBS-injected mice were either nonpulsed (groups 1–3) or pulsed in vitro with 10 ng/ml GalCer (groups 7–9). Hepatocytes pulsed in vivo by GalCer injection were washed and transferred into cultures (groups 4–6). Nonpulsed or in vitro or in vivo GalCer-pulsed hepatocytes were either cultured in vitro for 24 h without NKT cells (groups 1, 4, and 7) or cocultured with purified liver CD4+ NKT cells from A-/- B6 mice (groups 2, 3, 5, 6, 8, and 9). In some groups (groups 3, 6, and 9), murine IL-12 p70 (20 ng/ml) was added to the medium. Mean IFN- or IL-4 release (of triplicates) ± SEM of a representative experiment (of four independent experiments) are shown.

Liver CD4⁺ NKT cells may autopresent GalCer

NK1^int or NK1^low (CD4⁺ or CD4^-) CD3^int NKT cells express CD1d (Fig. 1). NKT cells have been shown to autopresent glycolipids in the context of CD1d (37). We isolated the liver MNC population and separated the CD4⁺ T cell subset from this MNC population. When cells from either population were cultured with 0.1–1000 pg/ml GalCer, they released IFN- and IL-4 in a dose-dependent manner (data not shown). Cytokine release was triggered 100-fold more efficiently by GalCer in nonfractionated liver MNC populations than in purified liver CD4⁺ cell populations. We cannot formally exclude that a contamination of liver CD4⁺ DC population mediated the NKT cell stimulation in the purified liver CD4⁺ cell populations (see below). Thus, liver CD4⁺ NKT cells may autopresent GalCer but a more potent stimulator cell for CD1d-restricted NKT cell activation is present in the nonfractionated liver MNC population.

Liver DC are potent activators of liver NKT cells

CD11c⁺ DC are present in the nonfractionated liver MNC population. CD11c⁺ liver DC were isolated by MACS to >98% purity. Liver DC from nontreated B6 mice showed readily detectable surface expression of CD1d but low surface expression of MHC-II molecules, CD40, CD80, and CD86 costimulator molecules (Fig. 4A). About 20% of the liver DC were either CD8⁺ or CD4⁺, indicating that lymphoid as well as myeloid CD11c⁺ DC are found in the liver. We further isolated liver DC from B6 mice injected 12 h previously with 100 ng of GalCer i.v. These liver DC showed an activated phenotype as their surface expression of I-A^b (MHC-II), CD40, CD80, and CD86 was up-regulated (Fig. 4B). Surface expression of CD4 and CD8 was down-regulated by activated liver DC (Fig. 4B).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Surface phenotype of freshly isolated liver CD11c+ DC from nontreated (A) or GalCer-injected (B) mice. Liver MNC were isolated from either untreated B6 mice or B6 mice injected i.v. 12 h previously with 100 ng of GalCer. The cells were stained with conjugated mAbs specific for CD11c, I-Ab (MHC-II), CD1d, CD40, CD80, CD86, CD4, or CD8 and analyzed by three-color FCM. The histograms show gated CD11c+ cells within the liver MNC population. Representative examples of an individual mouse per group (of three individual mice per group analyzed) are shown.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Liver CD11c+ DC are potent activators of liver NKT cells. B6 mice were injected i.v. with either PBS or 100 ng of GalCer, and their liver CD11c+ DC were isolated 12 h postinjection. Liver CD11c+ DC from PBS-injected mice were either nonpulsed (groups 1–3) or pulsed in vitro with 10 ng/ml GalCer (groups 7–9). Liver CD11c+ DC from GalCer-injected mice were washed and transferred into cultures (groups 4–6). Nonpulsed or in vitro or in vivo GalCer-pulsed liver CD11c+ DC (2 x 104 cells/well) were cultured in vitro for 24 h either without NKT cells (groups 1, 4, and 7) or cocultured with purified liver CD4+ NKT cells (105 cells/well) from A-/- B6 mice (groups 2, 3, 5, 6, 8, and 9). In some groups (groups 3, 6, and 9), murine IL-12 p70 (20 ng/ml) was added to the medium. Mean IFN- or IL-4 release (of triplicates) ± SEM of a representative experiment (of four independent experiments) are shown.

Serum levels of transaminases (e.g., ALT) are an indicator of liver cell integrity or injury. B6 mice given a single injection of GalCer(100 ng/mouse) showed a rise in serum ALT levels 24 h postinjection that returned to baseline values within 2–3 days postinjection (Fig. 6A). Injection of IL-12 (100 ng/mouse) alone did not trigger a serum ALT response (Fig. 1A). An injection of GalCer followed 6 h later by an IL-12 injection did not amplify the serum ALT response triggered by injection of GalCer alone (Fig. 6A). As reported previously (14, 25), hepatic NKT cell activation thus can elicit an immune response in the liver of normal B6 mice that results in transient hepatocyte injury.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Normal B6 mice (A) or HBs-tg B6 mice (B and C) were i.v. injected with either 100 ng of GalCer, 100 ng of IL-12, or 100 ng of GalCer followed 6 h later by 100 ng of IL-12. Serum ALT levels were determined at the indicated time points. HBs-tg B6 mice from which either CD4+ T cells, CD8+ T cells, or both were suppressed by anti-CD4 and/or anti-CD8 Ab treatment were similarly treated (C). Mean serum ALT levels of four mice/group (+SEM) are shown.

Histopathology of the liver injury in mice treated with GalCer

In contrast to untreated B6 mice with a normal liver histology (Fig. 7, A and B), hepatocytes from HBs-tg B6 mice were enlarged and had the granular, pale, eosinophilic cytoplasm characteristic for "ground glass hepatocytes" (Fig. 7C) as described previously (38). Their liver cell nuclei were moderately swollen and irregularly shaped. Binucleated liver cells were occasionally observed. Minimal infiltrations of lymphoid cells that did not transverse the limiting plate were found in the portal fields. TUNEL staining of untreated normal B6 and HBs-tg B6 mice showed only minimal apoptotic cell death (Fig. 7, B and D).

View larger version (179K): [in this window] [in a new window]   FIGURE 7. Histopathology of liver damage. A, Untreated B6 mice showed a normal histology of the liver. B, No apoptotic bodies were found in the liver of normal B6 mice (TUNEL staining). C, Untreated HBs-tg mice showed a ground glass appearance and nuclear enlargement of hepatocytes but only minimal inflammation. D, TUNEL staining revealed no apoptotic cells in the liver of nontreated HBs-tg B6 mice. E, An endothelialitis with a mixed inflammatory infiltrate, subendothelial edema, fibrin thrombi (arrows), and endothelial desquamation was observed in normal B6 mice treated with GalCer. F, In addition, multifocal centrilobular infarctions were found in normal B6 mice treated with GalCer (star, central vein; arrows, margin of coagulation necrosis). G, Some apoptotic bodies were seen in periportal and lobular regions of treated normal B6 mice by TUNEL staining (circles). H and I, HBs-tg mice treated with GalCer showed dense mononuclear and polymorphonuclear cell infiltrates of the portal fields and the lobular parenchyma. J, Diffuse parenchymal damage with ballooning of hepatocytes and nuclear hyperchormasia was prominent in treated HBs-tg B6 mice. Arrows point to hepatocytes with nuclear pyknosis and intensely eosinophilic cytoplasm indicating beginning apoptotic cell death. K, Extensive coagulation necrosis was found in treated HBs-tg B6 mice (arrows). L, Numerous apoptotic hepatocytes were detected by TUNEL staining in treated HBs-tg B6 mice. Representative examples selected from four individual (untreated or treated) mice per group examined are shown.

GalCer/IL-12-treated HBs-tg mice also showed a prominent endothelialitis. However, the periportal inflammatory cell infiltrates were more pronounced in treated HBs-tg as compared with treated B6 mice, often extending across the limiting plate into the adjacent parenchyma (Fig. 7H). Mononuclear and, to a lesser extent, polymorphonuclear leukocytes were abundant in the lobular parenchyma (Fig. 7I). Hepatocytes were diffusely damaged (Fig. 7J) and showed a ballooned appearance due to a marked cellular edema. Extensive coagulation necrosis of the liver parenchyma was obvious (Fig. 7K). Large numbers of apoptotic hepatocytes were revealed by TUNEL staining (Fig. 7L). Nuclear hyperchromasia and mitotic activity indicated an increased regeneration of liver cells. Taken together, the GalCer/IL-12-induced vascular changes in HBs-tg as well as normal B6 mice are suggestive for a primary damage of venular endothelial cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The (CD4⁺ or CD4^-CD8^-, NK1^int or NK1^low) CD3^int NKT cell subset is the only cell population known to specifically recognize GalCer presented by CD1d-bearing presenting cells. As shown in studies that tracked GalCer-reactive NKT cells using CD1d tetramers (3), neither the CD4⁺ nor the NK1⁺ surface phenotype are reliable markers of GalCer-reactive NKT cells. The assumption that the CD3^int phenotype characterizes the liver NKT cell population is supported by the finding that a single injection of a low dose of GalCer selectively eliminates >98% of all CD3^int T cells from the liver of mice as has been previously reported (8). We used as responder NKT cells either the nonfractionated liver MNC populations or the CD4⁺ fraction of the liver MNC populations from normal or MHC-II-deficient B6 mice. The liver CD4⁺ cell subset from A^-/- KO mice contained >90% CD3^int T cells that were eliminated after GalCer treatment. This is thus a highly enriched source of NKT cells.

CD1d⁺ APC found in the liver include hepatocytes, NKT cells themselves, and DC. Although liver DC express only low levels of CD1d on the surface, they were as potent as hepatocytes (that expressed higher surface CD1d levels) in stimulating NKT cells. A similar low expression level but potent Ag-presenting function of CD1d has been described for monocyte lineage cells (39). Production of IFN- by NKT cells in response to GalCer has been shown to require direct contact between NKT cells and DC through CD40-CD40L(CD154) interaction (25). DC express CD40 whereas hepatocytes do not. A major signal that triggers IL-12 release by DC is the ligation of CD40 (40). Surface expression of this costimulator molecule by DC and DC-derived IL-12 thus seem required to stimulate IFN- release by NKT. In the absence of IL-12, NKT cell activation seems to default to IL-4 (but not IFN-) release. This supports a report (41) that different GalCer-pulsed APC can modulate the polarization of the NKT cell response toward Th2 or Th1. We show in this paper that hepatocyte stimulation can result in IFN- release of NKT cells when paracrine IL-12 is provided. IL-12 plays a pivotal role in Th1-dependent mouse liver injury (42). These data support the notion that DC but not hepatocytes are the critical initial trigger for the IFN--dependent liver injury response.

IL-12 acts in synergy with IL-18 that is constitutively expressed in the liver. Although IL-18 alone favors Th2-biased specific or innate immunity, it strikingly enhances Th1 immunity in synergy with IL-12 (reviewed in Ref. 43), as shown in many systems (44, 45, 46). IL-18 and IL-12 synergistically stimulate IFN- production by T cells (47, 48, 49), B cells (50), NK cells (51), macrophages (52), and DC (53, 54). If IL-12 is released in the liver its effect is therefore expected to be strikingly amplified by IL-18. In addition, the enhanced release of IL-4 in cocultures of liver NKT cells with GalCer-pulsed DC is of interest. IL-4 (often considered the major Th2-driving cytokine), along with GM-CSF or IFN-, enhances the production of bioactive IL-12 heterodimer and selectively reduces production of the antagonistic IL-12 p40 homodimer by DC (55). This can explain why Con A-induced liver injury is IL-4 as well as IL-12 dependent (12, 13, 56). The DC-NKT cell interaction initiated by GalCer binding to CD1d on DC can thus trigger a cascade in which the production of IL-12 (by IL-4) and its bioactivity (by IL-18) are strikingly amplified. This may explain the extraordinary potency of GalCer to induce T cell-dependent liver injury initiated by the specific and restricted interaction of NKT cells with DC in the liver. The potentiating effect of exogenous IL-12 indicates that endogenous IL-12 release is limiting. This may point to an efficient negative regulation of the release of this potentially harmful cytokine in this organ.

Injection of GalCer into mice elicited a severe endothelialitis with the formation of venular fibrin thrombi and multifocal infarctions, leading to coagulation necrosis predominantly located in the subcapsular and centrilobular regions. A similar picture has been described in hyperacute (Ab-mediated) or acute (cellular) rejection of liver allotransplants (57). Endothelialitis is not pathognomonic for this condition but is an indication of intimate T cell-endothelial cell interactions universally associated with active hepatic inflammation (58). Vascular changes and necrosis were present in GalCer-treated normal and HBs-tg mice. The severe damage of the venular endothelium and the confinement of the inflammatory reaction to the immediate perivenular space point to a pathogenic stimulus that directly acts on the endothelial lining. Studies designed to elucidate the nature of this stimulus of venular endothelia damage are in progress. A striking feature in HBs-tg mice was the severe, diffuse hepatocellular damage with an abundance of apoptotic hepatocytes and dense periportal and lobular inflammatory infiltrates that frequently crossed the limiting plate.

Liver injury induced by the i.v. injection of Con A or GalCer displays similarities. Eliciting liver injury by Con A injection requires NKT cells (12). The effector phase of the response depends on IFN- (59, 60, 61) and/or (through the TNFR1 and TNFR2) TNF- (62, 63, 64, 65). CD95-dependent hepatocyte apoptosis is associated with the parenchymal lesions (62, 66). IL-6 (56, 67), phosphodiesterase inhibitors (68), or depletion of peptidergic sensory nerve fibers (69) can block or attenuate liver injury after Con A injection. Although Con A efficiently activates most CD3⁺ T cells (including NKT cells), it is surprisingly inefficient in eliciting T cell-dependent liver injury in vivo: at least 10⁴-fold more Con A than GalCer has to be injected to induce a comparable level of liver injury (500 µg of Con A vs 50 ng of GalCer/mouse). Con A binds to the surface of many cell types and triggers many activities, which makes it very difficult to identify the initiating cell type in this system. This makes the extensively investigated Con A-induced liver injury model in mice unattractive for the study of the cascade of events that lead to immune cell-mediated liver injury. In contrast, GalCer-induced liver injury has a well-defined trigger event (i.e., NKT cell activation) and operates efficiently in a low-dose range. This model is thus potentially more informative in elucidating the cascade of intrahepatic immune cell interactions that trigger liver injury.

Immature DC constantly enter the liver from the blood and are first found in the perisinusoidal space (70). Immature DC seem to induce preferentially tolerance. They need an additional early signal from the innate immune system to gain the competence to prime immunity. NKT cells in the liver are short-lived: they are specifically activated, deliver their effector function, and die within hours. The interaction between both cell types releases IFN- and IL-4 by NKT cells and IL-12 by DC. Where this interaction takes place in situ remains to be identified. Although these cytokines up-regulate cytokine release by NKT cells (IL-12 enhances IFN- release) and by DC (IL-4 enhances IL-12 p70 release), their main target seem to be other intrahepatic immune cells that promote and amplify the response. This is likely as one of the actors (NKT cells) rapidly disappears from the scene. Recruitment of other hepatic immune cells into the response may be mediated by activated DC that migrate from the perisinusoidal space to the periportal fields (70). In further studies, we will address the role of NK cells and T cells recruited into the NKT cell-triggered immune responses in the liver.

	Acknowledgments

 
The expert technical assistance of Beate Wotschke and Anja Müller is gratefully acknowledged. We are very grateful for the discussions, help, and comments of Dr. F. V. Chisari (Scripps Clinic, La Jolla, CA). We greatly appreciate the generous gift of GalCer from Dr. Y. Koezuka (Kirin Brewery, Pharmaceutical Research Laboratory).

	Footnotes

 
¹ This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Deutsche Forschungsgemeinschaft Re549/9-1) and the Bundesministerium für Forschung und Technologie (GE9907; to J.R. and R.S.), by Interdisziplinaeres Zentrum fuer Klinische Forschung (University of Ulm) Grant A7 (to F.L. and J.R.), and Grant A10 (to R.S. and P.M.).

² Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany. E-mail address: joerg.reimann{at}medizin.uni-ulm.de

³ Abbreviations used in this paper: HBV, hepatitis B virus; KO, knockout; -GalCer, -galactosylceramide; CD40L, CD40 ligand; MNC, mononuclear cell; ALT, alanine aminotransferase; DC, dendritic cell; FCM, flow cytometric analysis; int, intermediate.

Received for publication April 4, 2001. Accepted for publication May 22, 2001.

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