CTLA-4Ig suppresses liver injury by inhibiting acquired immune responses in a mouse model of fulminant hepatitis

Authors


  • Potential conflict of interest: Nothing to report.

Abstract

Expression of costimulatory molecules is significantly upregulated in various organs in an animal model of severe hepatitis induced by injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). In the present study, we examined whether blockade of costimulatory signals by CTLA-4Ig can suppress the liver injury in this model. We injected an adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) into mice 7 days before, on the same day, or 3 days after P. acnes priming. The virus was found to infect the liver preferentially, and CTLA-4Ig was detected in the serum as early as 2 days after viral injection. After injection of LPS, liver injury and survival rates were examined. Most of the mice not injected with AdCTLA-4Ig died within 12 hours after injection of LPS. In contrast, all the AdCTLA-4Ig–injected mice survived when the virus was injected 7 days before or on the same day as P. acnes priming. Importantly, hemorrhagic liver injury and serum alanine aminotransferase levels were significantly reduced after LPS injection even when AdCTLA-4Ig was injected 3 days after P. acnes priming. Immunological analyses showed that CTLA-4Ig inhibited the activation and expansion of P. acnes–specific CD4+ T cells in the hepatic lymph nodes, leading to a reduction in the recruitment of the cells to the liver. The total amounts of interferon-γ, interleukin-12, and various chemokines in the liver were then decreased, resulting in inhibition of the secondary recruitment of not only T cells but also macrophages. In conclusion, CTLA-4Ig could be useful for treatment of severe liver injury. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;42:915–924.)

Antigen presentation to naïve T cells by dendritic cells (DCs) is essential for the activation of antigen-specific T cells, and two signals are known to be necessary for activation.1 The first signal is from the interaction between the T-cell receptor and antigen peptide-human leukocyte antigen complex, and the second, a costimulatory signal, is from the binding of CD80 or CD86 on DCs and CD28 on T cells. Once activated, T cells express cytotoxic T-lymphocyte antigen-4 (CTLA-4) on their surface, and receive a negative signal through binding with CD80 or CD86 on DCs.2 CTLA-4 thus acts as a negative feedback molecule in the immune response.3 CTLA-4Ig is a fusion protein, consisting of the extracellular domain of mouse CTLA-4 and a human immunoglobulin (Ig) Fc fragment. CTLA-4Ig binds to CD80 or CD86 on DCs and induces anergy of antigen-specific T cells through interference with binding between costimulatory molecules on DCs and CD28 on T cells.4, 5

Mice with severe liver injury induced by injection of Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) is one of the most commonly used animal models of fulminant hepatitis. In this model, all the animals die of liver injury, and there are two different phases in the immune response—a priming phase in which the injection of P. acnes generates granulomas, and an eliciting phase in which LPS activates granuloma-forming cells, leading to massive liver injury. Yoneyama et al. recently reported that P. acnes–induced activation and recruitment of DCs in the liver is an initial event and is a prerequisite for liver injury in this model.6, 7 Furthermore, activated DCs have been shown to move to the hepatic regional lymph nodes (hepatic LNs) and activate P. acnes–specific CD4+ T cells. Those are then recruited to the liver; the result of which is an accumulation of more T cells, macrophages, and DCs producing various chemokines.8, 9 All of these cells are thought to be involved in the formation of granulomas, which may be essential for the hepatocyte damage after injection of LPS.10 Based on this report, costimulatory signals were suggested to act as a second signal for the activation of P. acnes–specific CD4+ T cells in the hepatic LNs.

In the present study, we injected an adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) into mice with severe liver injury induced by P. acnes/LPS to inhibit the activation of P. acnes–specific T cells by blocking costimulatory signals.

Abbreviations:

DC, dendritic cell, CTLA-4, cytotoxic T-lymphocyte antigen-4; Ig, immunoglobulin; LPS, lipopolysaccharide; LN, lymph node; AdCTLA-4Ig, adenovirus encoding CTLA-4Ig; AdLacZ, adenovirus encoding LacZ; mAb; monoclonal antibody; Ab, antibody; RT-PCR, reverse transcription-polymerase chain reaction; pfu, plaque-forming units; ALT, alanine aminotransferase; MNC, mononuclear cell; TNF; tumor necrosis factor; IFN, interferon; MACS, magnetic cell sorting; IDO, indoleamine 2,3-dioxygenase; mRNA, messenger RNA; IL, interleukin.

Materials and Methods

Mice.

Seven- to eight-week-old female C57BL/6 (H-2Kb) mice were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were approved by The Toyama Medical and Pharmaceutical University's Committee on Animal Welfare.

Construction of Recombinant Adenoviruses.

An adenovirus encoding LacZ (AdLacZ) was purchased from Riken BRC DNA Bank (Tsukuba, Japan). A murine AdCTLA-4Ig was constructed by the COS-TPC method as described elsewhere.11 A plasmid and a cosmid cassette containing murine CTLA-4Ig and the CAG promoter12 were kindly provided by Dr. H. Hamada (Sapporo Medical University, Hokkaido, Japan) and Dr. H. Yagita (Juntendo University, Tokyo, Japan). Isolated viral clones were propagated by the standard procedure and purified by two rounds of centrifugation in a cesium chloride density gradient.

Detection of CTLA-4Ig in the Serum and Expression of CTLA-4Ig Gene in Various Tissues.

Production of CTLA-4Ig was analyzed in the cell lysate of AdCTLA-4Ig–infected HeLa cells, and in the mouse serum 2 and 7 days after injection of AdCTLA-4Ig by Western blotting with a hamster anti-mouse CTLA-4 monoclonal antibody (mAb) and horseradish peroxidase– conjugated rabbit anti-hamster IgG as a second antibody (Ab). The blots were developed with ECL Western blotting detection reagents.

CTLA-4Ig gene expression in the liver, spleen, hepatic LNs, lung, and kidney was examined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) by detecting genes encoding the Fc portion of human Ig using the primers shown in Table 1.

Table 1. PCR Primer Sequence
PrimerSense 5′-3′Antisense 5′-3′
  1. Abbreviations: IP-10, CXCL10/IFN-inducible protein-10; MCP-1, CCL2/JE/monocyte chemoattractant protein-1; MIP-1α, CCL3/macrophage inflammatory protein-1α; RANTES, CCL5/regulated upon activation, normal T-cell expressed and secreted.

β-actin5′-GTG GGC CGC TCT AGG CAC CAA-3′5′-CTC TTT GAT GTC ACG CAC GAT TTC-3′15
Fc portion of human Ig5′-ATG CCA AGA CAA AGC CGC-3′5′-GTT TTC TCG ATG GGG GCT-3′
IDO5′-GTA CAT CAC CAT GGC GTA TG-3′5′-GCT TTC GTC AAG TCT TCA TTG-3′14
IFN-γ5′-TGC ATC TTG GCT TTG CAG CTC TTC CTC ATG GC-3′5′-CG GTT CAA ACT CCA GTT GTT GGG TGT CCA GGT-3′15
IL-25′-TGT GTT GTA AGC AGG AG-3′5′-CAG AAA GTC CAC CAC AGT TG-3′16
IL-45′-CCA GCT AGT TGT CAT CCT GCT CTT CTT TCT CG-3′5′-CG TGG TAC TTA CTC AGG TTC AGG TGT AGT GAC-3′15
IL-125′-CTG GTG TCT CCA CTC ATG GCC ATG TGG GAG-3′5′-CAG TGT GAC CTG GTT TCC CTG ATA CTC TTC-3′15
IP-105′-CCC CAT CAG CAC CAT GAA C-3′5′-GCT TCA CTC CAG TTA AGG A-3′17
MCP-15′-ACC ACC ATG CAG GTC CCT GT-3′5′-GAG TCA CAC TAG TTC ACT GT-3′17
MIP-1α5′-AAC ATC ATG AAG GTC TCC AC-3′5′-CCA AGA CTC TCA GGC ATT CA-3′17
RANTES5′-GGT ACC ATG AAG ATC TCT GCA-3′5′-AAA CCC TCT ATC CTA GCT CAT-3′17

Treatment of Severe Liver Injury Induced by P. acnes Priming and LPS Injection With a Recombinant Adenovirus Encoding CTLA-4Ig.

To induce severe liver injury in C57BL/6 mice, 1 mg of heat-killed P. acnes (American Type Culture Collection [ATCC], Manassas, VA) was first injected intravenously. Seven days later, 1 μg LPS (Escherichia coli 055:B4, Sigma-Aldrich, St. Louis, MO) was injected intravenously. For treatment of the mice, viral solutions containing 1 × 109 plaque-forming units (pfu) were injected into the mice via the tail vein 7 days before, on the same day, or 3 days after P. acnes priming.

Histological and Immunohistochemical Examinations.

Liver tissues fixed in 10% formalin-buffered solution (pH 7.4) were stained with hematoxylin and eosin solution. For immunohistochemical analysis, thin-sectioned tissues fixed in periodate-lysine-paraformaldehyde solution were incubated with a hamster mAb against mouse CD80 (BD PharMingen, San Diego, CA), or a rat mAb against mouse CD86 (BD PharMingen) at 4°C overnight. The slides were then incubated with peroxidase-conjugated second antibodies: goat mAb against hamster IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit mAb against rat IgG (DakoCytomation, Glostrup, Denmark). The slides were finally incubated in a solution containing 0.2 mg/mL diaminobenzidine to develop the reaction products.

Measurement of Serum Alanine Aminotransferase.

Serum alanine aminotransferase (ALT) levels were measured with a Reflotron GPT (Roche Diagnostics, Basel, Switzerland).

Analysis of the Immunological Mechanism of the Effect of Treatment With CTLA-4Ig.

Although AdCTLA-4Ig was injected at three different time points to treat the mice with severe liver injury, the following immunological analyses were performed only in mice pretreated with the recombinant adenovirus 7 days before P. acnes priming to avoid the influence of viral infection itself.

Preparation of Mononuclear Cells From the Liver and Hepatic LNs.

Mononuclear cells (MNCs) were prepared by gentle mechanical disruption of the tissues, followed by filtration and density gradient centrifugation using Ficoll-Conray.

Enzyme-Linked Immunosorbent Assay (ELISA).

The concentrations of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the sera of mice or the supernatants of MNCs isolated from the liver were measured with ELISA kits (eBioscience, San Diego, CA) according to the manufacturer's protocol.

Flow Cytometry.

Suspensions of MNCs from the liver and hepatic LNs were incubated first with anti-CD16/CD32 Ab (BD PharMingen) to block the Fc receptor, and then with fluorescein isothiocyanate-conjugated anti–major histocompatibility complex class II (I-Ab), anti-CD3, and anti-CD4 (all from Immunotech, Marseille, France), phycoerythrin-conjugated anti-CD11c, anti-CD14 (BD Pharmingen), anti-CD8, and anti-CD44, (eBioscience), or biotin-conjugated anti-CD11b (BD PharMingen) and anti-TLR4 (eBioscience). For biotin-conjugated Abs, incubation with allophycocyanin-conjugated streptavidin (Biomeda, Foster City, CA) was then performed. Isotype controls were used for determination of negative cells. The stained cells were analyzed on a FACScalibur (Becton Dickinson, San Jose, CA).

P. acnes–Specific Lymphocyte Proliferation Assay.

Suspensions of MNCs (1 × 105 cells/well) of hepatic LNs from the mice 7 days after P. acnes priming were cultured in triplicate in 96-well flat-bottomed plates (Becton Dickinson Labware, Franklin, NJ) with (10 μg/mL or 100 μg/mL) or without heat-killed P. acnes in 200 μL of RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, and 2 × 10−2 mmol/L mercaptoethanol at 37°C under an atmosphere of 5% CO2/95% air. The cells were incubated for 72 hours, with 1 μCi of 3H-thymidine per well present for the last 12 hours. The cells were harvested on filter paper, and radioactivity was counted in a beta counter (Beckman, Fullerton, CA).

P. acnes–Specific IFN-γ Production.

Suspensions of MNCs (5 × 105 cells) from the livers isolated from mice 7 days after P. acnes priming were cultured in 500 μL of RPMI 1640 medium supplemented with 10% fetal bovine serum in 24-well plates (Corning, Corning, NY), and P. acnes was added at a concentration of 100 μg/mL. Supernatants were harvested after 24 hours of culture, and the concentrations of IFN-γ were measured by ELISA.

Separation of CD11c+, CD11b+, CD4+, and CD8+ Cells From MNCs.

Suspensions of MNCs from mice 7 days after P. acnes priming were first incubated with anti-CD16/CD32 Ab to block the Fc receptor. CD11c+ fractions were then isolated by magnetic cell sorting (MACS) after incubation with 100 μL of anti-CD11c–conjugated MACS microbeads (Miltenyi Biotec, Bergisch, Germany) using MACS separation columns (Miltenyi Biotec) according to the manufacturer's protocol. CD11b+, CD4+ and CD8+ fractions were then separated from the negative cell fraction by MACS with a rat anti-mouse CD11b IgG antibody (eBioscience), a rat anti-mouse CD4 IgG antibody (eBioscience) and a rat anti-mouse CD8 IgG antibody (eBioscience), respectively, followed by incubation with Dynabeads M-450 sheep anti-rat IgG (Dynal Biotech, Oslo, Norway)

Functional Changes in CD11c+ Dendritic Cells by Treatment With CTLA-4Ig.

As binding of CTLA-4Ig to CD80 or CD86 was shown to suppress the functions of DCs by inducing indoleamine 2,3-dioxygenase (IDO),13 CD11c+ cells isolated from the liver or LNs were examined for their allo T-cell stimulatory activity and for expression of messenger RNA (mRNA) encoding IDO by RT-PCR using the primers shown in Table 1.14 For analysis of allo T-cell stimulatory activity, isolated CD11c+ cells (1 × 104 cells) were cocultured with T cells isolated from spleens of BALB/c mice (2 × 105 cells) in 96-well flat-bottomed wells at 37°C, 5% CO2 for 5 days, with addition of 1 μCi of 3H-thymidine to each well 18 hours before harvesting. The radioactivity of each well was counted in a beta counter as described in the previous paragraph.

RT-PCR for the Analysis of Messenger RNA Expression for Cytokines and Chemokines.

RNA was isolated from total liver and LN tissues using an RNeasy Mini kit (QIAGEN, Valencia, CA), and isolated from intrahepatic CD11c+, CD11b+, CD4+, and CD8+ cells using an ISOGEN RNA isolation kit (Nippon Gene, Toyama, Japan). The first-strand complementary DNA was reverse transcribed from 0.8 μg total RNA using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA) for subsequent RT-PCR. RT-PCR was performed for various cytokines and chemokines using primers shown in Table 1. The complementary DNA was amplified using AmpliTaq Gold DNA Polymerase (Applied Biosystems, les Ulis, France). The samples with Taq-Polymerase were initially incubated for 10 min at 95°C to activate the AmpliTaq Gold and to denature the DNA. Amplification was then performed for 30 cycles of denaturation at 94°C for 0.5 minutes, annealing at 55°C for 0.5 minutes, and extension at 72°C for 0.5 minutes. The PCR products were electrophoresed on 2% agarose gels and visualized by staining with ethidium bromide.

TNF-α and Nitric Oxide Production by Intrahepatic and Splenic CD11b+ Cells Induced by LPS.

Intrahepatic CD11b+ cells (1 × 105 cells/well) isolated from mice 7 days after P. acnes priming were cultured in 96-well plates in RPMI 1640 medium supplemented with 10% fetal bovine serum, and LPS was added at a concentration of 10 ng/mL. Supernatants were harvested after 24 hours of culture for measurement of TNF-α and nitric oxide concentrations using an ELISA kit (eBioscience) and Griess Reagent System (Promega, Madison, WI), respectively.

Statistical Analysis.

Statistical analysis was performed using the Mann-Whitney U test, and the level of statistical significance was set at P value less than .05. Differences in survival rates were analyzed by the Kaplan-Meier method, and P values were evaluated by the Mantel log-rank test.

Results

Expression of Costimulatory Molecules in the Liver and Hepatic LNs.

To investigate the possible involvement of costimulatory molecules in the immunopathogenesis of acute liver injury induced by P. acnes/LPS, we performed immunohistochemical analyses of the expression of CD80 and CD86 in the liver and hepatic LNs, 7 days after injection of P. acnes. As shown in Supplementary Fig. 1 (Available at http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html), MNCs strongly expressed CD80 or CD86 after P. acnes priming in these organs, suggesting that the costimulatory molecules play a role in the immunopathogenesis.

Detection of CTLA-4Ig in the Serum and CTLA-4Ig Gene Expression in Various Organs.

CTLA-4Ig (55 kDa) was identified in the mouse serum as early as 2 days after injection with AdCTLA-4Ig and in the cell lysate of AdCTLA-4Ig–infected HeLa cells by Western blotting (Fig. 1A). Semi-quantitative RT-PCR analysis for detection of mRNA encoding the Fc portion of human Ig showed that AdCTLA-4Ig infected the liver preferentially, although all tissues examined were found to be infected by the virus (Fig. 1B).

Figure 1.

(A) Expression of CTLA-4Ig in the mouse serum 2 and 7 days after treatment with adenovirus encoding CTLA-4Ig (AdCTLA-4Ig) and in the cell lysate of AdCTLA-4Ig–infected HeLa cells determined by Western blotting analysis. (B) Detection of human immunoglobulin (Ig) Fc mRNA expression by semi-quantitative RT-PCR in various tissues 7 days after AdCTLA-4Ig injection. mCTLA-4Ig, murine cytotoxic T-lymphocyte antigen-4Ig.

Mortality and Severity of Liver Injury After LPS Injection.

Over 70% of P. acnes–primed untreated mice and of those treated with AdLacZ (n = 8) died within 12 hours in response to subsequent LPS injection (Fig. 2A-C). In contrast, all the AdCTLA-4Ig–treated mice (n = 8) survived for 24 hours when the recombinant virus was injected 7 days before or on the same day as P. acnes priming (Fig. 2A-B). More importantly, survival rates of mice were improved even when the recombinant adenovirus was injected 3 days after P. acnes priming (Fig. 2C). At 6 hours after subsequent LPS challenge, multilobular hemorrhagic injury was observed in the livers of untreated P. acnes–primed mice or those treated with AdLacZ, and these mice showed marked elevation of serum ALT levels (Figs. 2A-C, 3A-B). In contrast, treatment with AdCTLA-4Ig at three different time points caused marked reductions in both hemorrhagic liver injury and serum ALT level (Figs. 2A-C, 3F,J,N) as compared with untreated or AdLacZ-injected mice (Fig. 3D,H,L). The formation of granulomas in the liver observed in mice 7 days after P. acnes injection were markedly reduced in AdCTLA-4Ig–treated mice (Fig. 3E,I,M), but not in AdLacZ-injected mice (Fig. 3C,G,K), when the virus was injected at any of the three time points. These results suggest that the reductions in mortality and liver injury after injection of LPS are dependent on the status of the priming phase.

Figure 2.

Cumulative survival rates and serum ALT levels after LPS injection in P. acnes–primed mice with or without AdLacZ or AdCTLA-4Ig treatment. (A) Cumulative survival rates and serum ALT levels after LPS injection in P. acnes–primed mice with or without AdLacZ or AdCTLA-4Ig treatment. Either virus was injected 7 days before (A), on the same day (B), or 3 days after (C) P. acnes priming. The data represent the means ± 1 SD from at least 6 mice. *P < .05; **P < .01. ALT, alanine aminotransferase; LPS, lipopolysaccharide; AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ.

Figure 3.

Histopathological analysis of liver specimens obtained from mice 7 days after P. acnes priming (before LPS injection) (A,C,E,G,I,K,M) and 6 hours after LPS injection (B,D,F,H,J,L,N). Samples of liver tissue were obtained from untreated P. acnes–primed mice (A-B); AdLacZ-treated + P. acnes–primed mice: (C-D) 7 days before P. acnes priming, (G-H) on the same day as P. acnes priming, (K-L) 3 days after P. acnes priming; and AdCTLA-4Ig–treated + P. acnes–primed mice: (E-F) 7 days before P. acnes priming, (I-J) on the same day as P. acnes priming, (M-N) 3 days after P. acnes priming. The specimens were fixed in 10% formalin-buffered solution (pH 7.4) and stained with hematoxylin and eosin (original magnification, ×100). LPS, lipopolysaccharide; AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ.

Changes in the Numbers and the Compositions of Mononuclear Cells in the Liver and Hepatic LNs at 7 Days After P. acnes Injection.

We examined the cell numbers in the liver and hepatic LNs 7 days after P. acnes priming. Total numbers of intrahepatic MNCs in whole livers from normal mice were increased by more than 100-fold after administration of P. acnes (Supplementary Fig. 2A). However, the numbers were decreased to below one tenth by pretreatment with AdCTLA-4Ig (Supplementary Fig. 2A). Similarly, the numbers of hepatic LN cells were increased by priming, and significantly decreased by AdCTLA-4Ig (Supplementary Fig. 2A). Flow cytometric analysis for MNCs in the liver and hepatic LNs showed that CD4+ cells and CD11b+ cells (macrophages) showed an especially marked decrease in number in animals injected with AdCTLA-4Ig (Supplementary Fig. 2B).

Activation Status of CD4+ T Cells and Macrophages on Pretreatment With AdCTLA-4Ig.

The expression of CD44 (an activation/memory marker) on CD4+ T cells and CD11b+ cells was examined to analyze the activation status of the cells. The population of CD44bright CD4+ T cells was increased markedly in the liver 7 days after priming with P. acnes, and most cells became CD44bright (Fig. 4). However, the hepatic LNs contained both CD44dim and CD44bright CD4+ T cells in almost equal proportions, indicating that hepatic LNs consisted of more naive T cells than the liver even after antigenic stimulation. Importantly, the percentages of CD44bright CD4+ T cells in the hepatic LNs were significantly decreased by pretreatment with AdCTLA-4Ig (Fig. 4). In contrast, there was only a mild decrease in the expression level of CD44 on intrahepatic CD4+ T cells by AdCTLA-4Ig pretreatment (Fig. 4). While, AdCTLA-4Ig pretreatment did not alter the expression of CD44 on CD11b+ cells, not only in the liver, but also in the hepatic LNs (Fig. 4).

Figure 4.

Changes in the expression levels of CD44 on CD4+ T cells and macrophages in the liver and hepatic lymph nodes (LNs) on P. acnes priming with or without AdLacZ or AdCTLA-4Ig pretreatment analyzed by flow cytometry. AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ.

T-Cell Proliferation and IFN-γ Secretion in Response to P. acnes.

We analyzed the changes in P. acnes–specific proliferation of MNCs evoked by AdCTLA-4Ig pretreatment. Although P. acnes–specific proliferation of MNCs was observed in the hepatic LNs, it was significantly inhibited by pretreatment with AdCTLA-4Ig (Fig. 5A), and the level of interleukin (IL)-2 mRNA expression was significantly decreased by AdCTLA-4Ig pretreatment in the hepatic LNs from P. acnes–primed mice (Fig. 5B), indicating that hepatic LN cells became anergic to P. acnes by pretreatment with AdCTLA-4Ig. We next examined IFN-γ production by MNCs isolated from the liver 7 days after P. acnes priming. The concentrations of IFN-γ in the medium after 24-hour stimulation with P. acnes showed that MNCs from the liver produced significant amounts of IFN-γ. However, IFN-γ production was completely abrogated by AdCTLA-4Ig pretreatment (Fig. 5C), which is consistent with the serum concentration of IFN-γ (Fig. 5D). We then analyzed the changes in the expression of IFN-γ mRNA in each population of intrahepatic MNCs after separating CD4+, CD8+, CD11c+, and CD11b+ cells by magnetic cell sorting (Fig. 5E) Although the expression of IFN-γ mRNA was shown to be reduced in all cell populations by AdCTLA-4Ig pretreatment, the reduction was most remarkable in CD4+ T cells (Fig. 5E).

Figure 5.

P. acnes–specific immune responses of the mononuclear cells isolated from the liver and hepatic lymph nodes in unprimed and P. acnes–primed mice. (A) Proliferation of mononuclear cells in response to P. acnes. Mononuclear cells of hepatic lymph nodes isolated from P. acnes–primed, AdLacZ-pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice were re-stimulated with P. acnes for 72 hours, and their proliferation was analyzed by 3H-thymidine incorporation. The results represent the means ± 1 SD from three mice. (B) Expression of IL-2 mRNA in the hepatic lymph nodes 7 days after P. acnes priming with or without AdLacZ or AdCTLA-4Ig pretreatment analyzed by semi-quantitative RT-PCR. (C) P. acnes–specific IFN-γ production of intrahepatic mononuclear cells isolated from unprimed, P. acnes–primed, AdLacZ-pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice. Isolated mononuclear cells were re-stimulated with P. acnes for 24 hours, and IFN-γ concentrations in the medium were measured by ELISA. The data are the means ± 1 SD from 4 mice. (D) The concentrations of IFN-γ in the serum from unprimed, P. acnes–primed, AdLacZ-pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice were measured by ELISA. The data are the means ± 1 SD from 4 mice. *P < .05; **P < .01. (E) Expression of mRNA for IFN-γ in intrahepatic mononuclear cells (CD11c+, CD11b+, CD4+, and CD8+ cells) separated 7 days after P. acnes priming with or without AdCTLA-4Ig pretreatment analyzed by semi-quantitative RT-PCR. AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ; IL, interleukin; IFN, interferon; CPM, counts per minute.

Allo T-Cell Stimulatory Activity and Expression of Messenger RNA Encoding IDO of Dendritic Cells.

We examined allo T-cell stimulatory activity and mRNA expression of IDO by RT-PCR in CD11c+ DCs isolated from hepatic LNs (Fig. 6A) and the liver (Fig. 6B). The data indicated that only DCs from hepatic LNs, but not from the liver, showed significant suppression of allo T-cell stimulatory activity and a high level of IDO mRNA expression only in CTLA-4Ig–treated mice.

Figure 6.

Allo T-cell stimulatory activity (upper panel) and the expression of IDO mRNA analyzed by RT-PCR in CD11c+ dendritic cells (lower panel) isolated from hepatic lymph nodes (LNs) (A) and the liver (B) of P. acnes-primed, AdLacZ–pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice are shown. The results in the upper panel represent the means ± 1 SD from 3 mice. CPM, counts per minute; AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ; IDO, indoleamine 2,3-dioxygenase.

Expression of mRNA for Cytokines and Chemokines in the Liver Analyzed by RT-PCR.

To analyze the mechanism of the decrease in number of not only CD4+ T cells but also CD11b+ cells in the liver by AdCTLA-4Ig injection, we examined the expression of mRNA for various cytokines and chemokines in the liver by RT-PCR (Fig. 7). The levels of expression of IFN-γ, IL-12, IP-10 (CXCL10/IFN-inducible protein-10), MCP-1 (CCL2/JE/monocyte chemoattractant protein-1), MIP-1α (CCL3/macrophage inflammatory protein-1) and RANTES (CCL5/regulated upon activation, normal T-cell expressed and secreted) were upregulated 7 days after P. acnes priming. However, the expression of mRNA for IFN-γ, IL-12, MCP-1, MIP-1α and RANTES was significantly decreased by AdCTLA-4Ig pretreatment (Fig. 7).

Figure 7.

Expression of mRNA for cytokines and chemokines. Expression of mRNA for cytokines and chemokines in the liver 7 days after P. acnes priming with or without AdLacZ or AdCTLA-4Ig pretreatment analyzed by semi-quantitative RT-PCR. The expression level of β-actin mRNA was used as an internal control. AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ; IFN, interferon; IL, interleukin; IP-10, CXCL10/IFN-inducible protein-10; MCP-1, CCL2/JE/monocyte chemoattractant protein-1; MIP-1α, CCL3/macrophage inflammatory protein-1; RANTES, CCL5/regulated upon activation, normal T-cell expressed and secreted.

Serum Concentrations of IFN-γ and TNF-α After LPS Injection.

The concentration of IFN-γ peaked at 6 hours after injection (Supplementary Fig. 3A), while that of TNF-α peaked at 1 hour (Supplementary Fig. 3B) after LPS injection, and in both cases the level was markedly decreased by AdCTLA-4Ig pretreatment.

Expression of LPS Receptors and Production of TNF-α and Nitric Oxide in CD11b+ Cells Stimulated With LPS.

To analyze the contribution of the LPS receptor to susceptibility to LPS, we analyzed the expression of LPS receptors, such as CD14 and TLR4, on CD11b+ cells by flow cytometry. As shown in Fig. 8A, the expression of neither molecule was changed by P. acnes either alone or in combination with AdCTLA-4Ig. Next, to examine whether CTLA-4Ig directly affects the activation status of CD11b+ cells, we analyzed the production of TNF-α and nitric oxide by intrahepatic CD11b+ cells isolated from P. acnes–primed mice with or without AdCTLA-4Ig pretreatment after stimulation by LPS. Importantly, the concentrations of TNF-α and nitric oxide were not decreased by pretreatment with AdCTLA-4Ig (Fig. 8B-C), suggesting that AdCTLA-4Ig did not directly affect the activation of CD11b+ cells.

Figure 8.

Susceptibility to LPS in intrahepatic CD11b+ cells isolated from P. acnes–primed mice with or without AdLacZ or AdCTLA-4Ig pretreatment. (A) Flow cytometric analysis of CD14 and TLR4 on intrahepatic CD11b+ cells isolated 7 days after P. acnes priming with or without AdLacZ or AdCTLA-4Ig pretreatment. (B) TNF-α production induced by lipopolysaccharide in intrahepatic CD11b+ cells isolated from unprimed, P. acnes–primed, AdLacZ-pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice. The data are the means ± 1 SD from three mice. (C) Nitric oxide production induced by LPS in intrahepatic CD11b+ cells isolated from unprimed, P. acnes–primed, AdLacZ-pretreated + P. acnes–primed, and AdCTLA-4Ig–pretreated + P. acnes–primed mice. The results represent the means ± 1 SD from three mice. AdCTLA-4Ig, adenovirus encoding cytotoxic T-lymphocyte antigen-4Ig; AdLacZ, adenovirus encoding LacZ; TNF, tumor necrosis factor.

Discussion

The present study indicated that the levels of expression of costimulatory molecules were markedly enhanced in the liver and secondary lymphoid organs after injection of P. acnes, suggesting that costimulatory signals might be important in the immunopathogenesis of the liver injury in this P.acnes/LPS model. Based on these observations, we postulated that blockade of these signals might suppress the development of the disease. Therefore, we injected these liver injury model mice intravenously with a CTLA-4Ig–producing recombinant adenovirus. Our results clearly showed that administration of CTLA-4Ig significantly suppressed the severity of the liver disease even when the virus was administered after P. acnes priming. This dramatic effect was primarily due to inhibition of the activation of CD4+ T cells in the hepatic LNs, which was thought to be mediated by both blockade of costimulatory signals delivered by antigen-presenting cells to T cells and the suppression of DC functions through induction of IDO. Inhibition of activation of CD4+ T cells in AdCTLA-4Ig–pretreated mice was supported by a marked decrease in the number of CD44bright CD4+ T cells (activated/memory T cells) and suppression of the proliferative response to P. acnes in MNCs isolated from their hepatic LNs

In contrast to CD4+ T cells in the hepatic LNs, the expression level of CD44 on intrahepatic CD4+ T cells showed only a mild decrease. Although the reason for the difference is unknown, it is possible that small numbers of T cells activated (CD44bright) in LNs were recruited to the liver, and the expression of CD44 on these cells does not depend on the local costimulatory signals once upregulated.

We injected AdCTLA-4Ig 7 days before, on the same day, or 3 days after P. acnes priming. The virus injection rescued all the mice when injected 7 days before or on the same day as P. acnes priming with significant improvements in serum ALT levels and liver histology. Importantly, hemorrhagic liver injury and serum ALT levels after LPS injection were significantly improved even when the virus was injected 3 days after P. acnes priming, although the survival rates of the mice were only improved until 12 hours after LPS injection. It is possible that injury of tissue other than the liver, e.g., the lungs, may be a cause of death in these mice.18 Our unpublished data showed remarkable expression of cleaved caspase-3 in lymphocytes located near the interface hepatitis, suggesting that liver-infiltrating T cells die continuously, and are replaced by newly activated and recruited T cells. Therefore, it seems possible that prompt production of CTLA-4Ig after injection of AdCTLA-4Ig inhibited the generation of newly activated T cells, leading to the suppression of granuloma formation. These data would be important in considering their clinical application. In the clinical setting, although fulminant viral hepatitis in humans is also thought to be initiated by antigen-specific T cell activation in secondary lymphoid organs, immune responses are already underway when the diagnosis is made. The results of the present study suggested that inhibition of the activation of CD4+ T cells in hepatic LNs may lead to cessation of ongoing immune responses and inhibit severe hepatitis in humans. As the systemic administration of adenovirus has been reported to be associated with serious complications in humans,19 systemic administration of CTLA-4Ig protein may be an alternative method of treatment of severe liver injury in humans. However, preferential adenovirus infection of the liver suggested that producing a recombinant protein mainly in the liver may be advantageous, because it leads to efficient delivery of the protein to regional LNs, and thus results in effective suppression of liver inflammation. Careful comparisons of the effects of AdCTLA-4Ig with those of systemic administration of CTLA-4Ig protein or the development of an adenovirus vector with little immunogenicity will be necessary for application of our strategy to humans.

As costimulatory molecules are also markedly expressed in the inflamed liver, signals through these molecules in the liver may have some important roles in local immune responses.20 The results of the present study suggested that the ability to produce IFN-γ in response to P. acnes was markedly diminished by AdCTLA-4Ig injection, indicating that costimulatory signals in the liver, which is not a lymphoid organ, might be necessary for maintaining the production of antigen-specific cytokines, especially Th-1 type cytokines.

IFN-γ induces leukocyte trafficking by upregulating the secretion of chemokines in many kinds of cells,21 and increases susceptibility to LPS.22 Therefore, the decrease in the total amount of IFN-γ produced in the liver caused a decrease in the local amounts of various chemokines, such as RANTES, MCP-1, and MIP-1α.23–26 The decreases in the local production of IFN-γ and chemokines in the liver led to inhibition of the secondary recruitment of T cells as well as granuloma-forming cells, such as macrophages, and resulted in the poor response of these cells to LPS.27 Although CD14 and TLR4 are known to be receptors for LPS, their levels of expression on CD11b+ cells were not altered by P. acnes or P. acnes and CTLA-4Ig, indicating that changes in the expression of LPS receptors were not involved in the susceptibility to LPS in this model.

Importantly, the present study clearly showed that CTLA-4Ig inhibited the immunopathogenesis of the disease by inhibiting the recruitment of mainly macrophages without affecting the activation status of the cells, as estimated by the production of TNF-α or nitric oxide. The inhibition of antigen-specific T-cell activation is sufficient to abate the effector phase of the acquired immune response that is mediated by innate immune cells. The effector phase often exacerbates tissue injuries in a variety of diseases,28 thus its abatement merits significant and novel implications.

In conclusion, the results of the present study indicate an important role of costimulation in the immunopathogenesis of fulminant hepatitis in a mouse model. Administration of AdCTLA-4Ig could be useful for clinical treatment of the disease.

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