Propionibacterium acnes-induced hepatic ... - Wiley Online Library

Journal of Pathology J Pathol 2005; 206: 486–492 Published online 19 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1793

Original Paper

Propionibacterium acnes-induced hepatic granuloma formation is impaired in mice lacking tetraspanin CD9 Hiroyuki Yamane,1 Isao Tachibana,1 * Yoshito Takeda,1 Yoshiyuki Saito,1 Yoshio Tamura,1 Ping He,1 Mayumi Suzuki,1 Yoshihito Shima,1 Tsutomu Yoneda,1 Shigenori Hoshino,1 Koji Inoue,1 Takashi Kijima,1 Mitsuhiro Yoshida,1 Toru Kumagai,1 Tadashi Osaki,1 Yoshinobu Eishi2 and Ichiro Kawase1 1 Department 2 Department

of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan of Human Pathology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan

*Correspondence to: Isao Tachibana, Department of Molecular Medicine, Osaka University Graduate School of Medicine, 2-2 Yamada-Oka, Suita, Osaka 565-0871, Japan. E-mail: [email protected]

Received: 25 January 2005 Revised: 9 March 2005 Accepted: 20 March 2005

Abstract The granuloma is a host defence response to persistent pathogenic irritants. In the process of granuloma formation, the activation, migration, and fusion of macrophages occur locally, but the mechanisms involved remain elusive. Tetraspanins regulate cell migration and fusion by organizing functional molecular complexes in membrane microdomains. Here we investigated the role of tetraspanin CD9 in hepatic granuloma formation. Immunostaining of the liver of untreated wild-type mice showed that CD9 was expressed by vascular endothelial cells and perivenular hepatocytes. When intrahepatic granulomas were induced by intravenous injection of Propionibacterium acnes, hepatocyte CD9 was extensively upregulated, while inflammatory cells constituting granulomas were mostly negative for CD9. Compared with wild-type littermates, CD9-knockout mice showed dissemination of Propionibacterium acnes and reduced number and size of granulomas after the injection. Moreover, production of granuloma-inducing cytokines, TNF-α and IFN-γ , was delayed and chemotactic activity for macrophages was suppressed in the liver of mutant mice. These results suggest that CD9 is one of the proteins that promotes granuloma formation in the liver. Copyright  2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: adhesion knockout mice

molecules;

Introduction Granulomas are compact focal collections of inflammatory cells that are observed in infections, vasculitis, immunological aberrations, and hypersensitivity pneumonitis. They are considered to function as a host defence response by preventing the diffusion of intracellular pathogens. The macrophage is a major cell component of granulomas and, during the process of granuloma formation, macrophages are activated, recruited to inflammatory sites, and transformed to epithelioid cells. A proportion of macrophages fuse to form multinucleated giant cells (MGC), a hallmark of the granuloma [1,2]. Several cytokines, chemokines, and adhesion molecules are thought to play pivotal roles in granuloma formation through humoral and cell–cell interaction mechanisms [3–8]. However, the mechanisms involved still remain poorly understood. The tetraspanin proteins comprise at least 32 members in mammals and include CD9, CD37, CD53, CD63, CD81, CD82, and CD151. They are characterized by a structure that spans the plasma membrane four times. Tetraspanins form complexes with each other and with other membrane proteins including

inflammation;

monocytes/macrophages;

transgenic/

integrins, major histocompatibility complex antigens, signalling molecules, and membrane-anchored growth factors. By facilitating the formation of multimolecular functional complexes in raft-like membrane microdomains, tetraspanins regulate cell migration, invasion, and fusion events [9]. However, precise mechanisms by which tetraspanins modulate these events remain elusive. Genetic studies have revealed their essential roles in fertilization [10,11], neuromuscular synapse formation [12], the immune response [13–15], brain development [16], and retinal maintenance [17]. On the other hand, there have been few studies on the role of tetraspanins in in vivo pathological conditions including inflammation and granuloma formation, although these are caused by inflammatory cell migration and fusion. In the present study, the involvement of CD9 in Propionibacterium acnes (P acnes)-induced hepatic granuloma formation was investigated. Intravenous injection of this bacterium into mice causes acute and chronic inflammation of the parenchyma and portal triads with mononuclear cell infiltration in the liver and leads to a self-limiting granulomatous hepatitis [18]. Using this model, we show data suggesting that CD9

Copyright  2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

P acnes-induced granuloma formation in CD9-knockout mice

is upregulated in hepatocytes and promotes granuloma formation.

Materials and methods Mice The CD9-knockout (KO) mice were provided by Dr E Mekada (Osaka University, Osaka, Japan) [11]. These mice were backcrossed more than five generations into the C57BL/6J background. The genotyping of all breeding pairs was confirmed by PCR analysis. The mice were maintained in a barrier facility, and all animal procedures were performed in accordance with the Osaka University (Osaka, Japan) guidelines on Animal Care. Seven- to 12-week-old CD9-KO mice and their wild-type (WT) littermates matched for age and sex were used in all experiments.

Induction of intrahepatic granulomas P acnes (ATCC 11 828; American Type Culture Collection) was a kind gift from Dr H Okamura (Hyogo College of Medicine, Hyogo, Japan). Mice were injected intravenously with 1 mg of heat-killed and sonicated P acnes. After 3–28 days, mice were sacrificed and the livers were removed. Liver specimens were fixed in 10% neutral buffered formalin, dehydrated, and embedded in paraffin wax. Sections (5 µm thick) were stained with haematoxylin and eosin and analysed by light microscopy. Granulomas were defined as being composed of at least 10 white blood cells. At least 20 granulomas were randomly selected from multiple sections, their areas were quantified using the image analysis software, MacScope (Mitani Corp, Fukui, Japan), and the average size was calculated. The number of granulomas per 1 cm2 section was also determined. Reproducibility of these quantitative assessments was confirmed by multiple blind evaluations by two of us.

Immunoblotting Livers were homogenized and lysed in lysis buffer containing 1% Brij99, 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM MgCl2 , 2 mM phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Samples containing equal protein amounts were electrophoresed on SDS-PAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were probed with rat antimouse CD9 monoclonal antibody (KMC8) (BD Biosciences, San Diego, CA, USA). For quantification, blots were analysed on a FluorChem (Alpha Innotech, San Leandro, CA, USA) using AlphaEase software.

Immunohistochemistry Liver sections (5 µm thick) were fixed in acetone and immunostained with F4/80 (IgG2b, Cl : A3-1;

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Serotec, Oxford, UK) or KMC8 (IgG2a) followed by peroxidase-labelled anti-rat IgG. Sections were also immunostained with mouse monoclonal antibody (mAb) to P acnes recognizing lipoteichoic acid of the plasmalemma [19], followed by MOM biotinylated anti-mouse IgG reagent (Vector Laboratories, Burlingame, CA, USA) and peroxidase-labelled streptavidin. After visualization with 3,3
-diaminobenzidine (DAB) substrate solution, the sections were counterstained with haematoxylin.

Cell culture and treatment Human hepatocellular carcinoma cell line, HepG2, and mouse macrophage cell line, RAW264.7, were seeded onto a six-well plate at a density of 2 × 106 cells/well and incubated in DMEM for 24 h. After washing, medium was replaced with DMEM containing 1.0–30 ng/ml of recombinant human and murine TNF-α (PeproTech, London, UK), respectively, and cells were further incubated for 24 h. Expression of tetraspanins was then evaluated by RT-PCR. In another experiment, HepG2 cells were treated with 10 ng/ml TNF-α for 24–96 h, and the expression of CD9 was analysed by RT-PCR.

RT-PCR analysis Total RNA was extracted from cultured HepG2 cells, RAW264.7 cells, and mouse liver homogenates with Isogen (Nippon Gene, Tokyo, Japan) and reversetranscribed. The thermal cycling parameters were 25 cycles (35 cycles for CD9) of 40 s at 94 ◦ C, 40 s at 60 ◦ C, and 90 s at 72 ◦ C for human CD9, CD63, CD82, and β-actin, 25 cycles of 1 min at 94 ◦ C, 1 min at 55 ◦ C, and 1 min at 72 ◦ C for mouse CD9, and 30 cycles of 50 s at 94 ◦ C, 50 s at 60 ◦ C, and 1 min at 72 ◦ C for mouse TNF-α, IFN-γ , IL1-β, and βactin. The sequences of upstream and downstream oligonucleotide primers were previously described [20–22]. We confirmed that these parameters yielded amplification of template DNAs within a linear range.

Migration assay RAW264.7 cells (1.5 × 105 ) were resuspended in 100 µl of DMEM containing 0.1% BSA and loaded into the upper chamber of tissue culture-treated Transwell membranes (pore size, 5 µm; Costar, Cambridge, MA). After incubation for 30 min, the chambers were soaked into lower chambers that were filled with 600 µl DMEM/0.1% BSA containing 0, 1.5, and 15 µg/ml liver protein, which was prepared after removal of the cell debris by centrifugation of homogenized liver samples. After incubation for 3 h, cells remaining on the upper surface of the membrane were wiped off with cotton swabs, and cells migrating to the lower surface were visualized with Diff-Quick stain and counted in 12 independent high-power fields at ×400 magnification. J Pathol 2005; 206: 486–492

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Results Expression of CD9 in the liver of P acnes-challenged mice Injection of P acnes into the mice via tail vein causes acute inflammation in the liver and, at a later stage, induces granulomatous lesions mostly composed of macrophages as a result of chronic inflammation. Foci of granulomas usually appear as early as day 3, progressively extend to a maximum at day 7, and are sustained until day 14 [18]. We first injected heatkilled P acnes intravenously into C57BL/6J mice and examined the expression of CD9 protein in the liver. As shown in Figure 1 (upper panel), CD9 was present in the liver of untreated mice and, after P acnes administration, was upregulated and reached a peak between day 3 and 7. We repeated these experiments twice and confirmed the increase of CD9 in the liver (Figure 1, lower panel). Meanwhile, CD9 expression was unchanged in the lung, where no granulomas appeared throughout the course of experiments. We also stained histological sections of the liver using a macrophage marker, F4/80, and anti-mouse CD9 mAb, KMC8. Compared with untreated mice (Figure 2 A, E), many granulomas mostly consisting of F4/80 (+) inflammatory cells were observed in the perivenular region and hepatic lobules at day 7 after P acnes injection (Figure 2B, F). CD9 was strongly expressed in biliary and vascular endothelial cells and moderately expressed by hepatocytes in the perivenular region in untreated mice (Figure 2C, G). Remarkably, priming with P acnes caused the elevation of CD9 expression by hepatocytes in a

Figure 2. Upregulation of hepatocyte CD9 in P acneschallenged mice. Immunohistological liver sections before (A, C, E, G, I) and 7 days after (B, D, F, H, and J) P acnes administration are shown at low (A–D), medium (E–H), and high (I, J) magnification. The sections were stained with F4/80 (A, B, E, F) or KMC8 (C, D, G, H, I, J). Note that CD9 staining in hepatocytes increased extensively after P acnes treatment (D, H, J). Arrowheads indicate granulomas; b, bile duct. Sections shown are from one representative of three similar experiments. Bars: 50 µm for A–H, 10 µm for I and J

Figure 1. Upregulation of CD9 in the liver of P acnes-challenged mice. C57BL/6J mice were injected intravenously with 1 mg of heat-killed P acnes. After the number of days indicated, lysates containing an equal amount of proteins from the liver were electrophoresed on SDS-PAGE, transferred to membranes, and blotted with anti-mouse CD9 mAb, KMC8. The blots were quantified by densitometry (upper panel). This experiment was repeated twice and lysates from the liver and lung were analysed in parallel for CD9 expression (lower panel) J Pathol 2005; 206: 486–492

more extensive area. Whereas KMC8 staining of endothelial cells appeared not to be changed, that of hepatocytes became more intense (Figure 2D, H). It was upregulated especially at the cell periphery, as shown at higher magnification (Figure 2I, J). On the other hand, the inflammatory cells recruited into granulomas were negative or weakly positive for CD9 (Figure 2H); this was in contrast to strong staining for F4/80 (Figure 2F).

Stimulation with TNF-α increases the expression of CD9 by HepG2 cells TNF-α is one of the central mediators that recruit macrophages and induce granuloma formation [4,23].

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Figure 3. Stimulation with TNF-α increases the expression of CD9 in HepG2 but not in RAW264.7 cells. (A) Total RNA was extracted from HepG2 cells cultured for 1 day in the presence of the indicated concentrations of TNF-α and analysed by RT-PCR for CD9, CD63, and CD82. β-Actin amplification was used as the internal control. (B) HepG2 cells were stimulated with 10 ng/ml of TNF-α for the times indicated, and then CD9 expression was analysed. (C) RAW264.7 cells were stimulated with the indicated concentrations of TNF-α for 1 day and CD9 expression was analysed. An additional experiment gave similar results

Since this proinflammatory cytokine was reported to be upregulated in the P acnes-primed liver [24], we examined if it alters the expression of CD9 in hepatocytes in vitro. As shown in RT-PCR analysis, stimulation of a hepatocellular carcinoma cell line, HepG2, with TNF-α dose-dependently promoted the transcription of CD9, which reached a plateau at a concentration of 3 ng/ml (Figure 3A). Time course analysis showed that it was fully induced within 48 h (Figure 3B). By contrast, the expression of another tetraspanin CD63 was constant, and that of CD82 was rather decreased after the treatment (Figure 3A). TNF-α failed to alter the level of CD9 transcription in a macrophage line, RAW264.7, even at a concentration of 30 ng/ml (Figure 3C).

Impaired granuloma formation and dissemination of P acnes in CD9-KO mice Upregulation of hepatocyte CD9 after stimulation with P acnes in vivo and with TNF-α in vitro suggests its putative role in liver inflammation and granuloma formation. To investigate if CD9 is required for the development of granulomas, we injected P acnes into CD9-KO mice. F4/80 staining showed no difference in the number of resident Kupffer cells between untreated wild-type and mutant mice (data not shown). However, the number of granulomas after P acnes administration was reduced by 80% in CD9-KO mice when compared with wild-type littermates (Figure 4Aa, b, B). Their average size was also 50% smaller in the mutant mice (Figure 4Ac, d, B); this was largely due to

Figure 4. Reduced granuloma formation in the liver of CD9-KO mice. (A) P acnes was administered intravenously to wild-type (a, c, e) and CD9-KO (b, d, f) mice. Histological liver sections at day 7 were stained with haematoxylin and eosin (a–d) and F4/80 (e, f). Sections shown are from one representative of four mice in each group. Bars: 250 µm for a and b, 50 µm for c–f. B, The number and size of granulomas in the liver from P acnes-treated wild-type and CD9-KO mice were quantified using the image analysis software MacScope (upper panels). The serum level of alanine aminotransferase was also determined (lower panel). Each bar represents the mean ± SE (n = 4). ∗∗ p < 0.01; NS, not significant in Student’s t-test

the decrease of macrophages in each granuloma as shown by F4/80 staining (Figure 4Ae, f). In addition, although not statistically significant, the serum level of alanine aminotransferase of the mutant mice was lower than that of control mice. This could reflect milder inflammation in the liver of CD9-KO mice (Figure 4B). We next examined the intrahepatic distribution of P acnes by staining with anti-P acnes mAb [19]. This bacterium was well confined to granulomas, probably after phagocytosis by macrophages, in both wild-type and CD9-KO mice, although granuloma size was smaller in the latter (Figure 5Aa, b, B). Remarkably, in non-granulomatous areas, P acnes was markedly disseminated in the mutant mice (Figure 5Ac, d, B). J Pathol 2005; 206: 486–492

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Figure 5. Dissemination of P acnes in the liver of CD9-KO mice. (A) Histological liver sections from wild-type (a and c) and CD9-KO (b, d) mice at day 7 after P acnes injection were stained with anti-P acnes mAb. P acnes was stained in brown in granulomatous areas (encircled in a and b) and non-granulomatous areas (c, d). Sections shown are from one representative of four mice in each group. Bar, 50 µm. (B) The number of P acnes was determined in granulomatous and non-granulomatous areas. Each bar represents the mean ± SE (n = 4). ∗∗ p < 0.01; NS, not significant

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Figure 6. Expression of TNF-α and IFN-γ is delayed in the liver of CD9-KO mice. (A) Time course analysis of the number and size of intrahepatic granulomas from wild-type (open square) and CD9-KO (closed square) mice after P acnes administration. Each data point represents the mean ± SE (n = 3). ∗∗ p < 0.01. (B) Total RNA was extracted from liver homogenates from wild-type and CD9-KO mice at the indicated days after P acnes administration. Expression of TNF-α, IFN-γ , and IL−1β was analysed by RT-PCR. β-Actin amplification was used as the internal control. Data shown are from one representative of three similar experiments

Production of TNF-α and IFN-γ is delayed and macrophage-chemotactic activity is reduced in CD9-KO mice We further performed detailed time course analysis of hepatic granuloma formation in these mice. Reflecting the variability in in vivo experiments, numerous granulomas were formed in this experiment (Figure 6A). Consistent with the previous report [18], the number and size of granulomas reached a peak between 7 and 14 days after P acnes administration in wildtype mice. CD9-KO mice displayed similar kinetics of granuloma appearance and, at day 14, the number of granulomas was comparable to that of wild-type mice. However, the increase in number and size of granulomas was decelerated, showing the largest difference at day 7. In terms of granuloma size, fully enlarged granulomas had not been formed in the mutant mice. To explore mechanisms underlying defective granuloma formation, the expression of IFN-γ and TNF-α, the two essential cytokines for granuloma formation, was studied by RT-PCR. The time course of the production of these cytokines was consistent with that of granuloma formation. Their production reached a peak between day 7 and 14 in both wild-type and CD9-KO mice, and delayed expression was again observed in the mutant mice (Figure 6B). When compared with J Pathol 2005; 206: 486–492

Figure 7. Chemotactic activity for macrophages is reduced in the liver of CD9-KO mice. RAW264.7 cells were applied to the upper chamber of Transwells, and the indicated concentrations of liver proteins from wild-type (open bars) and CD9-KO (closed bars) mice at day 0 (D0) or at day 7 (D7) of P acnes administration were added into the lower chambers. Cells migrating to the lower surface of the membrane were counted after Diff-Quick stain. Each bar represents the mean ± SE number of cells per mm2 in triplicate cultures. Data shown are from one representative of three similar experiments. ∗ p < 0.05; ∗∗ p < 0.01; NS, not significant

wild-type mice by densitometry, IFN-γ in the mutant mice was reduced by 31% and 24% at day 3 and day 7, respectively. Likewise, TNF-α was decreased by 25% and 26% at day 3 and day 7, respectively. Interestingly, downregulation of IFN-γ and TNF-α was

P acnes-induced granuloma formation in CD9-knockout mice

also decelerated in the mutant as shown by amplified bands at day 21. In contrast to these cytokines, no difference was observed in the transcription of IL-1β. Similar results were obtained by ELISA using liver homogenates (data not shown). Migration of macrophages to inflammatory sites is an essential event for the formation of granulomas. In fact, TNF-α induces chemotactic activity that recruits macrophages into granulomas [4,23]. Thus, we further performed RAW264.7 cell migration assays to examine whether there was any decrease in macrophage-chemoattractant activity in CD9-KO mice (Figure 7). Before P acnes administration (day 0) there was no difference in the chemotactic activity of liver homogenates between wild-type and CD9-KO mice. However, at day 7 after the P acnes injection, wild-type homogenate contained increased chemotactic activity when compared with day 0, whereas such an increase was not detected in CD9-KO mice and the number of migrating RAW264.7 cells was significantly lower than wild-type.

Multinucleation of intrahepatic macrophages is not different between wild-type and CD9-KO mice We additionally evaluated MGC formation using histological liver sections from the time course experiment. In both wild-type and CD9-KO mice, intrahepatic MGCs increased and their numbers reached a peak (∼50/cm2 liver section) at 14 days after P acnes administration. However, we failed to detect significant differences in the number and size of MGCs throughout the course of experiment (data not shown).

Discussion Using KO mice, inflammatory cytokines and their receptors, chemokines and their receptors, adhesion molecules, and extracellular matrix proteins have been shown to be required for the formation of granulomas [3–8]. The present study has added the tetraspanin CD9 to the list of molecules that elaborate granulomas in the liver. In particular, hepatocyte CD9 appears to be most likely the key player because the treatment of P acnes extensively upregulated its expression. The experiments using cell lines were in line with this. TNF-α, which plays a pivotal role in Th1-mediated granuloma formation, promoted the transcription of CD9 in HepG2 hepatocytes but failed to affect CD9 in RAW264.7 macrophages. Moreover, the finding that the deletion of CD9 resulted in defective granuloma formation suggests that CD9-positive cells are required for well-organized granulomas. There is also a possibility that CD9 on vascular endothelial cells, which were invariably positive in wild-type mice, may be involved. For example, the deletion of endothelial CD9 may cause impaired transmigration of macrophages, lymphocytes, or dendritic cells into the liver [25]. It may also be possible that the loss of CD9 causes certain defects in systemic immune response in mice. A

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few in vitro experiments have proposed a role for CD9 in T-cell activation [26,27]. However, although four (CD37, CD81, CD151, and Tssc6) of five tetraspanindeficient mice generated have altered immune cell function [13,15,28,29], defective function has not yet been reported in immune cells from CD9-KO mice. Thus, we prefer the hypothesis that CD9 protein on liver parenchymal cells, especially on hepatocytes, functions to induce mature granulomatous foci after priming with P acnes. Granulomas are considered to function as a host defence response by preventing the diffusion of pathogens [1]. Dissemination of P acnes in nongranulomatous areas probably reflects the defective granuloma formation in CD9-KO mice. In granulomatous areas, however, the number of P acnes per unit area was not different between wild-type and CD9KO mice, suggesting that phagocytosis of P acnes by macrophages is not compromised. Rather, the step of recruiting macrophages into granulomas after phagocytosis could be impaired. The finding that P acnesprimed liver of CD9-KO mice contained a lower level of macrophage-chemotactic activity supports this hypothesis. It was previously shown that deletion of TNF-α results in s delay in chemokine induction, defective granuloma formation, and dissemination of mycobacteria [4,23]. IFN-γ is an inducer of TNF-α production and was also shown to be required for macrophage infiltration and granuloma formation in P acnes-primed liver [3]. Notably, the expression of these cytokines was delayed in CD9-KO liver, most likely correlating with failure to elevate macrophagechemotactic activity, defective granuloma formation, and dissemination of P acnes. Previous studies showed that TNF-α is immunohistochemically detectable in hepatocytes in the early phase after P acnes injection and proposed that the hepatocyte is one of the major producers of this cytokine, thus suggesting an important role for hepatocytes in this granuloma model [18,24]. Although further study will be required to clarify detailed mechanisms, our data raise the hypothesis that TNF-α induces hepatocyte CD9 expression, and overexpressed hepatocyte CD9 functions to increase the production of TNF-α and IFN-γ and thereby amplifies macrophage-chemotactic activities that are required for the formation of hepatic granulomas. However, the 25–30% decrease in TNF-α and IFN-γ production may not fully account for the markedly suppressed RAW264.7-chemotactic activity. Thus, other mechanisms that prevent macrophage migration may be present in CD9-KO mice. We previously reported that CD9-KO mice are prone to elicit the fusion of macrophages in the lung after intratracheal administration of P acnes and that CD9/CD81-double KO mice spontaneously develop MGCs in the lung and multinucleated osteoclasts in the bone [30]. Unexpectedly, in the liver, such enhanced macrophage fusion was not observed in P acnes-treated CD9-KO mice as described in the J Pathol 2005; 206: 486–492

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current study and in na¨ıve CD9/CD81-double KO mice (unpublished data). Thus, macrophage fusion in vivo appears to be affected by tissue conditions. In conclusion, although detailed mechanisms remain to be elucidated, this paper has demonstrated a novel function of the tetraspanin CD9 in the pathophysiology of granuloma formation. When challenged with P acnes, CD9 is upregulated in hepatocytes and promotes the formation of well-organized granulomas to prevent its dissemination in the liver.

Acknowledgements We thank Dr E Mekada for providing CD9-KO mice, Dr H Okamura for providing P acnes, M Kobayashi (Osaka University) for assistance in mouse genotyping, Drs T Takehara and T Kanto (Osaka University Graduate School of Medicine) for helpful comments on liver histology, and Y Habe for secretarial assistance. This work was supported by the Osaka Foundation for Promotion of Clinical Immunology and the Ministry of Education, Science, and Culture, Japan.

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