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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The healthy adult human liver expresses low levels of major histocompatibility complex class II (MHC II) and undetectable levels of immune costimulatory molecules. However, high levels of MHC II, CD40, and B7 family molecules are expressed in the activated Kupffer cells and hepatocytes of patients with viral hepatitis. The precise role of these molecules in viral clearance and immune-mediated liver injury is not well understood. We hypothesized that parenchymal CD40 expression enhances T cell recruitment and effector functions, which may facilitate viral clearance and alleviate liver injury. To test this hypothesis, we generated novel liver-specific, conditional CD40 transgenic mice, and we challenged them intravenously with a recombinant replication-deficient adenovirus carrying Cre recombinase (AdCre). Wild-type mice infected with AdCre developed a relatively mild course of viral hepatitis and recovered spontaneously. CD40 expression in the livers of transgenic animals, however, resulted in CD80 and CD86 expression. The dysregulation of population dynamics and effector functions of intrahepatic lymphocytes (IHLs) resulted in severe lymphocytic infiltration, apoptosis, necroinflammation, and serum alanine aminotransferase elevations in a dose-dependent fashion. To our surprise, an early expansion and subsequent contraction of IHLs (especially CD8+ and natural killer cells), accompanied by increased granzyme B and interferon-γ production, did not lead to faster viral clearance in CD40 transgenic mice. Conclusion: Our results demonstrate that hepatic CD40 expression does not accelerate adenoviral clearance but rather exacerbates liver injury. This study unveils a previously unknown deleterious effect of hepatic CD40 on adenovirus-induced liver inflammation. (Hepatology 2011;)

Adenoviruses are responsible for approximately 5% of all upper respiratory infections and for considerable numbers of cases of gastroenteritis in the developing world and among immunosuppressed individuals globally. In addition to their role as important pathogens, recombinant adenoviruses, especially adenovirus serotype 5 (Ad5), are among the preferred vectors for gene therapy and experimental vaccines for human immunodeficiency virus. More than 250 clinical trials of Ad5 were conducted from 1993 to 2007 (http://www. wiley.com//legacy/wileychi/genmed/clinical). This virus targets the liver, airways, and lymphocytes preferentially. However, it can also induce strong T helper, cytotoxic T lymphocyte (CTL), and B cell responses against the viral vector and the transgene in the presence of CD40/CD40 ligand (CD40L) and B7/CD28 costimulatory signals.1 The failure to constrain these responses can lead to necroinflammatory hepatitis, treatment failure, and even patient death.2 Disruption of the costimulatory pathways and immune responses, on the other hand, can enhance adenovirus-mediated gene transfer into the liver.3 The involvement of costimulatory pathways in T cell–mediated hepatitis is not peculiar to adenoviruses. In patients with hepatitis C virus infections, high levels of major histocompatibility complex class I (MHC I), MHC II, CD40, and B7 family costimulatory molecules are strongly expressed on activated Kupffer cells and hepatocytes in the liver, and these levels have been closely correlated with intrahepatic inflammation, necrosis, and elevations of serum alanine aminotransferase (ALT) levels.4-8 Despite these apparent associations, however, the precise role of parenchymal B7 superfamily molecules in viral clearance and liver inflammation is not entirely clear, partly because of severe restrictions on human studies and a general lack of suitable small-animal models.

The goal of this study was to examine the role of parenchymal CD40 in the course of adenovirus-induced hepatitis. We previously showed that CD86 expression in hepatitis C virus transgenic animals resulted in T cell activation and accumulation in the liver, which led to pronounced hepatic inflammation.9 On the basis of these observations, we speculate that parenchymal CD40 expression is critical in regulating B7 molecule expression and hepatic inflammation, and we also question whether the host may benefit from the hepatic expression of costimulatory molecules (e.g., faster viral clearance in vivo). To address these possibilities, we generated novel liver-specific, conditional CD40 transgenic mice. Upon the injection of these animals with a replication-deficient adenovirus carrying Cre recombinase (AdCre), the transgene underwent DNA recombination, and this resulted in CD40 expression. We report for the first time that CD40 expression on hepatocytes mediated a transient surge of intrahepatic lymphocytes (IHLs) accompanied by augmented granzyme B and interferon-γ (IFN-γ) production. The activated intrahepatic T and natural killer (NK) cells did not promote faster viral clearance but instead resulted in more severe liver inflammation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Generation of Liver-Specific, Conditional CD40 Transgenic Mice.

A 1.5-kb, locus of X-over P1 (loxP)–flanked DNA fragment was polymerase chain reaction (PCR)–amplified from a pAlbSVPA-HCV-S–derived construct containing loxP.9 Through the insertion of murine CD40 (mCD40) cDNA (a gift from Dr. E. Clark of the University of Washington10) into the plasmid pLIVE (Mirus Bio LLC, Madison, WI) at the SacI/XhoI sites, a CD40-expressing plasmid (pLIVE-mCD40) was produced. A conditional CD40-expressing plasmid (pLIVE-loxP-mCD40) was constructed through the insertion of the loxP fragment into pLIVE-mCD40 at the AscI/SacI sites. Recombination was induced by an infection with an adenovirus encoding Cre recombinase11 and was detected by PCR amplification with the following primer pair: forward primer 5′-ggaaccaatgaaatgcgagg-3′ (P5) and reverse primer 5′-gcacagccgaggcaaagacacc-3′ (P6). Transgenic mice were produced through the microinjection of a 4.0-kb BglII/SbfI fragment containing the mouse CD40 expression cassette into pronuclei of fertilized eggs of C57BL/6J×C3H mice. Transgene-positive (Tg+) founders were identified by PCR amplification with primers P5 and P6. The cycling conditions were as follows: 94°C for 45 seconds, 58°C for 60 seconds, and 72°C for 120 seconds for 30 cycles. Experiments were performed with two lineages of mice with similar levels of CD40 expression. Mice were maintained under specific pathogen-free conditions and were housed in a conventional animal facility at the University of Texas Medical Branch.

Animal Experiments.

We used age- and sex-matched CD40 transgenic mice with a C57BL/6J×C3H background and their littermate controls. In most experiments, the animals were injected intravenously with 2 × 109 pfu of AdCre in 200 μL of phosphate-buffered saline (PBS). Negative control mice were injected with PBS. On postinfection days 7 and 14, the mice were either suffocated with carbon dioxide without perfusion or anesthetized by an intraperitoneal injection of 50 mg/kg sodium pentobarbital before perfusion. For nonperfused mice, blood was withdrawn by heart puncture, and serum was obtained. Serum ALT levels were measured in the clinical chemistry laboratory at the University of Texas Medical Branch. At the same time, mouse liver and spleen tissues were also collected for further analyses. The liver histology, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays, immunostaining, and quantitative PCR assays are described in the supporting information.

Isolation of Hepatocytes and IHLs.

Hepatocytes from wild-type and transgenic mice were isolated as described by Klaunig et al.12 Briefly, each mouse liver was first perfused with Hank's balanced salt solution without calcium and magnesium, and this was followed by Hank's buffer with calcium and magnesium plus collagenase D (Roche Applied Science, Indianapolis, IN). Isolated hepatocytes were suspended in L-15 medium. For IHL isolation, liver tissues were removed and pressed through a 200-gauge stainless steel mesh. The liver cell suspension was collected and suspended in Roswell Park Memorial Institute 1640 medium (HyClone, Logan, UT). Liver mononuclear cells were purified by density gradient centrifugation in Lympholyte-M (Burlington, NC). The total numbers of IHLs per liver were calculated. The relative percentages of CD4+, CD8+, NK, and natural killer T (NKT) cells were measured by fluorescence-activated cell sorting (FACS), and the absolute numbers of these lymphocyte subpopulations per liver were calculated according to their percentages and the total IHL numbers in individual livers.

Flow Cytometry Analysis.

The following specific monoclonal antibodies and their corresponding isotype controls were purchased from BD Pharmingen (San Diego, CA) and eBiosciences (San Diego, CA): phycoerythrin (PE)-conjugated anti-CD40 (3.23) and rat immunoglobulin G2a (IgG2a); fluorescein isothiocyanate–conjugated anti–IFN-γ (XMG1.2), CD49b (DX5), and rat IgG1 and IgM; PE-conjugated anti–granzyme B (16G6) and rat IgG2b; allophycocyanin (APC)–conjugated anti-CD4 (GK1.5) and rat IgG2b; PE–cyanine 7 anti-CD8 (53-6.7) and rat IgG2a; and APC-Alexa750–conjugated anti-CD3 (17A2) and rat IgG1. All cell staining procedures were performed on ice. Briefly, cells were blocked with 2% rat/mouse serum and 1 μg/mL Fc gamma receptor blocker (CD16/32), stained for specific surface molecules, fixed/permeabilized with a Cytofix/Cytoperm kit (BD Biosciences, Franklin Lakes, NJ), and then stained for intracellular molecules. To detect intracellular cytokines, 1 μL/mL GolgiPlug (BD Biosciences) was added for the last 4 hours of cultivation. To detect granzyme B, we performed intracellular staining of freshly isolated IHLs. Annexin V Apoptosis Detection Kit I (BD Biosciences) was used for T lymphocyte apoptosis analysis. Data were acquired with the FACSCanto system (BD Biosciences) and were analyzed with FlowJo 8.5 software (TreeStar, Ashland, OR).

Western Blot Assay.

Proteins were extracted from frozen liver tissues by homogenization with a syringe plunger on ice in a lysis buffer [50 mM tris(hydroxymethyl)aminomethane (pH 8.0), 150 mM sodium chloride, 1% Nonidet P40, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma)]. After centrifugation at 20,000g and 4°C for 15 minutes, the supernatant was collected so that the protein concentration could be measured with a protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts (70 μg) of the proteins were loaded onto 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories). Membranes were incubated with goat anti-mouse CD40 (clone T-20, Santa Cruz, CA) or anti–β-actin (clone AC-15, Sigma), and this was followed by incubation with horseradish peroxidase–conjugated secondary antibodies for 1 hour. Blots were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Statistical Analysis.

An analysis of variance (ANOVA) was performed. For group-to-group comparisons, the unpaired Student t test was employed. A P value less than 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Generation of Liver-Specific, Conditional CD40 Transgenic Mice.

We generated conditional CD40 transgenic mice that expressed CD40 molecules on the surfaces of hepatocytes only after induction. The CD40 gene was regulated by a chimeric mouse liver promoter, and the two elements were separated by a loxP-flanked DNA spacer, which could be deleted by Cre-mediated recombination (Fig. 1A). Transgenic founders were identified by both PCR (Fig. 1B) and slot blot analyses (data not shown). PCR analysis of the F2 generation from lineage 21 demonstrated a 2.0-kb amplicon that was indicative of the unrecombined transgene, whereas Cre-mediated recombination generated a 0.6-kb DNA fragment (Fig. 1B). After AdCre transduction, abundant amounts of CD40 messenger RNA (mRNA) were evident in the livers of Tg+ mice but not transgene-negative (Tg) mice (Fig. 1C). Transgenic mice began to express CD40 in the liver as early as day 3 after AdCre induction, and they maintained high levels of transgene expression during the 2 weeks (Fig. 1D and Supporting Fig. 1); this was similar to our previous observations.9 Nearly all hepatocytes in the transgenic mice expressed CD40 molecules on their surfaces according to flow cytometry (Fig. 1E). The transgenic mice were healthy and had normal histological findings for the liver, spleen, lungs, and kidneys (Supporting Fig. 3C and data not shown) as well as normal liver function (average ALT level = 50.4 ± 6.6 U/L).

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Figure 1. Transgene construct and characterization of liver-specific, conditional CD40 transgenic mice. (A) A liver-specific, conditional CD40 transgene construct and its induction via Cre-mediated recombination. PCR primers P5 and P6 amplified a 2.0-kb fragment and a 0.6-kb fragment for unrecombined and recombined transgenes, respectively. Note the three stop codons in different reading frames in the spacer flanked by loxP; Poly A, polyadenylation signal. (B) PCR analysis of CD40 transgene recombination in vivo. Tg and Tg+ mice from a lineage were intravenously injected with AdCre or PBS. Seven days later, mouse liver genomic DNA was extracted and used for PCR analysis. (C) Quantitative RT-PCR analysis of liver CD40 mRNA. Total liver RNA was extracted from AdCre-infected mice 7 days after infection. (D) Western blot analysis of CD40 transgene expression. Liver lysates, as described previously, were subjected to electrophoresis, and the blot was probed with an antibody specific to the C-terminus of mCD40 (see the Materials and Methods section). The positive control (lane 1) in this experiment was the liver tissue of a double-transgenic mouse (CD40×Alb-Cre), which constitutively expressed CD40. Animals were sacrificed on day 7 (lanes 3 and 5) after the AdCre injection. The results are representative of two repeated experiments. (E) Flow cytometry analysis of the surface CD40 expression on hepatocytes. Mice were intravenously injected with AdCre or PBS, and 5 days later, hepatocytes were obtained from perfused and collagenase-digested livers.

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CD40 Expression Exacerbating Liver Inflammation in Viral Hepatitis.

To examine the role of CD40 in viral hepatitis, we challenged CD40 transgenic mice intravenously with 2 × 109 pfu of AdCre (Tg+ AdCre). Two additional groups of wild-type littermates were included as controls, and they were treated similarly with PBS (Tg PBS) or AdCre (Tg AdCre). No pathological changes appeared in the PBS-treated wild-type mice according to the liver histology and the serum ALT levels (Figs. 2 and 3A). AdCre injection in wild-type mice caused liver inflammation characterized by hepatocytes with megaloblastic changes and single-cell necrosis. Sporadic apoptosis and mitosis were observed in the liver on postinfection day 7 (arrows in Fig. 2A). Compared to the control animals, the transgenic mice expressed high levels of CD40 in the liver after AdCre injection on postinjection days 7 and 14 and suffered from severe liver injury on day 7 (Supporting Fig. 1 and Fig. 2). The hepatic inflammation in these Tg+ AdCre mice was characterized by prominent portal and lobular lymphocytic infiltration, which included CD4+, CD8+, CD45R+, CD11b+, and NK cells and granulocytes (Supporting Fig. 2 and data not shown). Bridging necrosis, which was accompanied by many apoptotic bodies, was found in all three adjacent zones on day 7 (arrows in Fig. 2A). On day 14, there were fewer aggregates of lymphocytes in the lobules, and no obvious hepatocyte necrosis or apoptotic bodies were observed (data not shown).

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Figure 2. Parenchymal CD40 expression exacerbates liver injury. Mice were divided into three groups: Tg PBS (tissue from a PBS-injected nontransgenic mouse), Tg AdCre (tissue from an AdCre-injected nontransgenic mouse), and Tg+ AdCre (tissue from an AdCre-injected transgenic mouse). The animals were intravenously injected with 2 × 109 pfu of AdCre. (A) Liver tissues were obtained from transgenic and nontransgenic mice on day 7 after the AdCre injection, and the tissue sections were stained with hematoxylin and eosin. The arrows indicate megaloblastic changes and mitosis in nontransgenic mice and apoptotic bodies in transgenic animals. The TUNEL assay and the immunohistochemical analysis are shown in Supporting Fig. 2A,B. (B) Scores for liver injury. Liver injury was scored with respect to portal and intralobular inflammation, degeneration, and necrosis (see the supporting information). The extent of the pathology was scored from 0 (no pathology) to 3 (severe pathology). The liver tissues were obtained on postinfection days 7 and 14. The plots summarize the results of two repeated experiments and show the mean scores of three animals in the Tg PBS group, three to four mice in the Tg AdCre group, and four to six mice in the Tg+ AdCre group. A significant difference was detected by ANOVA on days 7 and 14. The two-tailed t test was used for additional group-to-group comparison. The results are indicated by asterisks (*P < 0.05 and **P < 0.01). Abbreviation: NS, not significant.

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Figure 3. Exacerbated liver injury is not associated with faster viral clearance. (A) ALT levels of the related mice in Fig. 2. The results are representative of two experiments. (B) Clearance of intrahepatic AdCre from infected mice. The AdCre genome in the livers of the infected nontransgenic and transgenic mice was quantitated by real-time PCR analysis. Each dot represents an individual mouse, and the data were pooled from three independent experiments. The results are indicated by asterisks (*P < 0.05, **P < 0.01, and ***P < 0.001). Abbreviation: NS, not significant.

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To quantify the histopathological changes, three individuals scored the results in a double-blinded fashion, and the scores were then subjected to ANOVA. Our findings demonstrated that viral infection was associated with higher histopathological scores in both transgenic and wild-type animals on postinfection day 7 (P < 0.05; Fig. 2B). Furthermore, CD40 expression resulted in exacerbated liver injuries in the transgenic mice versus the infected nontransgenic mice on day 7 (2.0 ± 0.8 versus 0.9 ± 0.2, P < 0.05). Despite persistent CD40 expression in the liver (Supporting Fig. 1), the histopathological scores of the transgenic mice subsided considerably on day 14. Although the average score of the transgenic group remained slightly higher than that of the infected nontransgenic mice, the difference between the two groups of animals became statistically insignificant (1.4 versus 1.0, P > 0.05).

Additional experiments demonstrated that the aforementioned pathological changes were not due to an inherent, unrelated property of the CD40 transgenic mice (see the supporting information). When transgenic mice were intravenously injected with 0, 0.5 × 109, 1.5 × 109, 2 × 109, or 3 × 109 pfu of AdCre, they displayed a dose-dependent CD40-mediated effect on liver inflammation (Supporting Fig. 3). Finally, the wild-type adenovirus and its replication-defective counterpart (AdCre) elicited similar types of viral hepatitis in CD40 transgenic animals (Supporting Fig. 4).

Increased Apoptosis and Hepatic Injury Are Not Associated With Faster Viral Clearance.

Apoptosis has long been considered to be a natural mechanism of cell removal without pathogenic consequences for the tissue; however, excessive apoptosis can cause tissue injury and is emerging as an important feature of liver injury.13 Using hematoxylin and eosin staining and TUNEL assays, we found no apoptosis and low levels of apoptosis in PBS-injected wild-type mice and AdCre-injected wild-type mice, respectively, on postinfection day 7. Many more apoptotic bodies and TUNEL-positive cells were found in the transgenic mice on day 7 (Fig. 2A and Supporting Fig. 2A). The significant morphological differences in the initial liver injury between the transgenic and wild-type mice were further confirmed by the measurement of liver injury on day 7, which resulted in average serum ALT levels of 1256 U/L for CD40 transgenic mice and 263 U/L for wild-type animals (Fig. 3A). By using quantitative PCR analysis, we found no significant difference in the viral copy numbers between the CD40 transgenic and wild-type groups on day 7 (P > 0.05; Fig. 3B). Although the viral copy numbers in both groups decreased steadily from day 7 to day 14 (P < 0.01), no statistical difference was found between the two groups on day 14 (P > 0.05). These results demonstrate that increased lymphocyte infiltration and hepatic inflammation are not associated with enhanced viral clearance in the liver.

Effect of Parenchymal CD40 Expression on Lymphocyte Recruitment and Effector Functions.

To test how parenchymal CD40 expression exacerbates liver injury in viral hepatitis, we examined population dynamics and effector functions of IHLs in all three groups of mice. As expected, the total numbers of IHLs in the AdCre-infected mice, regardless of their transgenic status or the point in time, were significantly higher than those in the PBS group (Fig. 4A). The effect of parenchymal CD40 expression on lymphocyte accumulation in the liver was most evident on day 7 because the average number of IHLs rose significantly higher in transgenic animals versus wild-type animals (29.3 versus 18.2 × 105, P < 0.01). Although the increased IHL numbers were sustained in the wild-type mice into the second week (18.5 × 105), the IHL numbers in the transgenic animals declined nearly 3-fold to 10.1 × 105, which was significantly lower than the value for the nontransgenic animals (P < 0.01).

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Figure 4. Apoptosis results in the shrinkage of the IHL population in CD40 transgenic mice. Mice were intravenously injected with 2 × 109 pfu of AdCre and were sacrificed on days 7 and 14. (A) IHLs on postinfection days 7 and 14. (B) Apoptosis in IHLs induced by an AdCre infection on postinfection day 7 was analyzed with 7-AAD labeling and annexin V binding on CD8+ and CD8 T cells; the results are shown as mean percentages of cells per liver (with standard errors). The data were analyzed for statistical significance by the t test (*P < 0.05, **P < 0.01, and ***P < 0.001). Density plots are shown in Supporting Fig. 6. Abbreviation: NS, not significant

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By using flow cytometry, we found that the adenoviral infection resulted in increases in the percentages of intrahepatic CD8+ cells in both groups of mice on day 7 (57.9% and 62.0%; Table 1); these levels were higher than the level of the PBS group (21.4%, P < 0.001). This CTL expansion was more vigorous in the CD40 transgenic mice versus their wild-type counterparts (18.2 versus 10.5 × 105) and contributed to their more expanded IHL populations (Fig. 4A). Although both AdCre-infected groups maintained high percentages of CD8+ T cells in the liver on day 14 (76.3% and 77.5%), the transgenic mice had far lower numbers of CD8+ cells than the wild-type animals because of their greatly diminished IHL pools on day 14 (7.8 versus 14.1 × 105). In comparison with the wild-type animals, more intrahepatic CD8+ cells in the CD40 transgenic mice entered the apoptosis process [annexin V–positive and 7-aminoactinomycin D (7-AAD)–negative] as early as day 7 (Fig. 4B and Supporting Fig. 6). This accelerated rate of apoptosis occurred only among CD8+ effector cells in the transgenic mice and not in CD8 cells (presumably CD4+, B, and NK cells). Indeed, the percentages of intrahepatic CD4+ T cells in the CD40 transgenic mice were comparable to those in the wild-type animals, although their numbers were slightly higher and later became lower than those of the nontransgenic animals (Table 1).

Table 1. IHL Populations in Mice
 Tg PBSTg AdCreTg+ AdCre
  • The mean absolute numbers (×105) are shown in parentheses. The percentages of CD4+ T, CD8+ T, and NKT cells are gated on CD3+ events. The percentages of NK cells are gated on CD3 events. For data not marked with asterisks or daggers, the P value is >0.05.

  • *

    P < 0.05 versus Tg PBS mice.

  • P < 0.01 versus Tg PBS mice.

  • P < 0.001 versus Tg PBS mice.

Day 7 [% (n)]   
 NKT cells25.4 ± 2.9 (0.9)7.8 ± 0.9 (1.4)7.5 ± 0.8 (2.2)
 NK cells22.4 ± 2.6 (0.8)22.4 ± 2.2 (4.1)21.5 ± 1.6 (6.3)
 CD4+ T cells51.9 ± 5.5 (1.8)32.3 ± 3.3* (5.9)28.6 ± 2.6 (8.4)
 CD8+ T cells21.4 ± 3.2 (0.7)57.9 ± 4.3 (10.5)62.0 ± 2.7 (18.2)
Day 14 [% (n)]   
 NKT cells23.9 ± 0.9 (0.8)3.7± 1.4 (0.7)3.5 ± 1.3 (0.4)
 NK cells19.2 ± 3.4 (0.6)17.4 ± 2.5 (3.2)20.0 ± 2.7 (2.0)
 CD4+ T cells48.5 ± 4.5 (1.6)17.3 ± 1.5 (3.2)16.6 ± 1.5 (1.7)
 CD8+ T cells24.0 ± 5.6 (0.8)76.3 ± 2.8 (14.1)77.5 ± 2.3 (7.8)

NK cells are some of the early effector cells that respond to an adenovirus infection.14, 15 In the wild-type mice infected with AdCre, the intrahepatic NK cell population remained relatively stable between postinfection days 7 and 14 in terms of both the percentages (22.2% and 17.4%, respectively) and the absolute numbers (4.1 and 3.2 × 105, respectively; Table 1). Despite the similarity of their percentages (21.5% and 20.0%), the average number of intrahepatic NK cells in the CD40 transgenic mice decreased considerably because of the much contracted IHL pools from days 7 to 14 (from 6.3 to 2.0 × 105). Like other viral infections,16 the AdCre infection resulted in significant declines in the NKT percentages in comparison with the percentages for PBS-injected animals on postinfection days 7 and 14 (Table 1).

To test whether hepatic CD40 expression modulated phenotypical changes, cytokine production, and cytotoxicity in IHLs,14 we measured CD40L levels and intracellular IFN-γ in CD8+ and CD4+ T cells ex vivo (Supporting Fig. 5 and Fig. 5). The presence of CD40 on hepatocytes did not change the expression of CD40L in CD4+ T cells in the liver (Supporting Fig. 5). After the viral infection, higher percentages of CD8+ and CD4+ T cells in the transgenic mice expressed IFN-γ in comparison with those in the uninfected control animals on day 7 (P < 0.05 and P < 0.01, respectively; Fig. 5 and Supporting Fig. 8A). Nearly one half of the CD4+ cells from both transgenic and wild-type mice expressed IFN-γ on postinfection day 14, and there were no differences between these two groups of cells (Fig. 5). Granzyme B resides in cytotoxic granules and is a key effector molecule of CD8+ CTLs and NK cells.15 To assess the effect of CD40 on granzyme B–mediated target cell destruction, we measured granzyme B–expressing CD8+ T cells and NK cells. Similar percentages of CD8+ cells in the transgenic and control mice expressed granzyme B on postinfection day 7 (Fig. 6 and Supporting Fig. 8B). After a phase of increased apoptosis and population contraction (Fig. 4 and Table 1), however, a small percentage of CTLs (11.3%) produced greater amounts of this lytic molecule, as measured by the mean fluorescence intensity (MFI), in the transgenic mice on day 14 (P < 0.05; Fig. 6 and Supporting Fig. 8B). After a similar course of population changes (Fig. 4A, and Table 1), higher percentages of intrahepatic NK cells in the transgenic mice secreted granzyme B on day 14 (P < 0.05; Fig. 6 and Supporting Fig. 8B). Overall, these results suggest that parenchymal CD40 expression can perturb the population dynamics of CTL and NK cells in the liver and alter their effector functions in adenoviral infections.

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Figure 5. CD40 expression on hepatocytes increases IFN-γ production by intrahepatic CD4+ and CD8+ T cells. Lymphocytes were obtained from perfused livers of nontransgenic and transgenic mice 7 or 14 days after the PBS or AdCre injection. After 4 hours of stimulation with phorbol 12-myristate 13-acetate and ionomycin in the presence of GolgiPlug, IFN-γ production was assessed by intracellular staining and FACS analysis. Representative data from a mouse from each group with CD8+ and CD4+ T cells are shown in Supporting Fig. 8A. Shown here is a comparison of the percentages of IFN-γ–producing CD4+ and CD8+ T cells in the three groups. Each group contained four to six mice, and the Tg PBS group was pooled from animals on days 7 and 14. Asterisks indicate statistically significant differences against the Tg PBS group (*P < 0.05 and **P < 0.01), and pound signs indicate statistically significant differences between the Tg+ and Tg groups by the t test (##P < 0.01). For data not marked with asterisks or pound signs, the P value is >0.05.

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Figure 6. CD40 expression on hepatocytes increases granzyme B production by intrahepatic CD8+ T and NK cells. Lymphocytes were obtained from perfused livers of nontransgenic and transgenic mice 7 or 14 days after the injection with PBS or AdCre. Granzyme B production was immediately assessed by intracellular staining and FACS analysis. Representative data from a mouse in each group with CD8+ T cells and NK cells are shown in Supporting Fig. 8B. Shown here is a comparison of the percentages (upper panels) and MFIs (lower panels) for granzyme B expression by CD8+ T and NK cells in the groups of mice. Each group contained four to six mice, and the Tg PBS group was pooled from animals on days 7 and 14. Asterisks indicate statistically significant differences against the Tg PBS group (*P < 0.05 and **P < 0.01), and pound signs indicate statistically significant differences between the Tg+ and Tg groups by the t test (#P < 0.05). For data not marked with asterisks or pound signs, the P value is >0.05.

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Kupffer cells are the resident macrophages in the liver and are closely situated next to their neighboring hepatocytes. To test whether CD40 expression on hepatocytes affects Kupffer cells, we measured CD40L levels on Kupffer cells by flow cytometry ex vivo (Supporting Fig. 7). On day 3, Kupffer cells expressed higher levels of surface CD40L than they did at the later stage (day 12) in both groups. Furthermore, Kupffer cells from the CD40+ transgenic mice had higher levels of CD40L than those from the control animals on day 3. These data suggest that CD40 expressed on hepatocytes can activate Kupffer cells in the early stage of an adenovirus infection. The full implication of this interaction, however, requires further investigation.

B7 Family Molecules in CD40 Transgenic Mice.

Hepatic CD86 expression is associated with increased T cell activation and retention, which contribute to hepatitis in mice.9 In an attempt to test whether parenchymal CD40 expression affects the regulation of B7 family members in the liver, we used quantitative reverse-transcription polymerase chain reaction (RT-PCR) and flow cytometry analyses to examine CD80 and CD86 molecules in transgenic mice 7 days after AdCre injection. The CD40 transgenic mice displayed 1.63- and 1.82-fold increases in CD80 and CD86 mRNA, respectively, over their wild-type littermates (Fig. 7A,B), although the differences were not statistically significant. Furthermore, purified hepatocytes from transgenic mice expressed detectable surface expression of CD80 and CD86 (Fig. 7C-E). The effect of parenchymal CD40 expression was not limited to these two molecules in the B7 superfamily17; in transgenic mice, the relative copy numbers of programmed death ligand 1 (PD-L1; B7-H1) and B7-H4 mRNA were 2.71- (P < 0.01) and 1.84-fold (P > 0.05), respectively, versus those in the nontransgenic mice (Supporting Fig. 9). Blocking the programmed death 1 (PD-1)/PD-L1 pathway with an anti–PD-L1 antibody further enhanced the proliferation (but not IFN-γ expression) of intrahepatic CD8+ T cells (Supporting Fig. 10). In agreement with several previous reports,18 the mRNA levels of several adhesion molecules (especially E-selectin) also appeared to be up-regulated in the CD40 transgenic mice (Supporting Fig. 9). These data suggest the possible involvement of B7 family members and adhesion molecules in the pathogenesis of adenovirus-induced hepatitis.

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Figure 7. CD40 expression on hepatocytes up-regulates downstream costimulatory molecules. (A,B) Total liver RNA was extracted from PBS-injected mice and AdCre-injected mice (2 × 109 pfu) 7 days after infection. Quantitative RT-PCR assays were carried out for (A) CD80 and (B) CD86. The fold increase was calculated by normalization to 18S RNA with respect to the quantity of CD80 or CD86 in the Tg PBS group (P = 0.06 and P = 0.07 by ANOVA, respectively). Each group contained two to four animals, and all samples were assayed in triplicate. (C-E) Hepatocytes were purified from PBS-injected mice and AdCre-injected mice (2 × 109 pfu) 5 days after infection. Hepatocytes were stained for the surface expression of CD80 and CD86, and data were acquired with flow cytometry. Each group contained two to four animals. (C) A representative histogram of CD80 and CD86 in a Tg+ AdCre mouse is shown. (D,E) Average MFIs for CD80 and CD86, respectively, are shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

CD40 is a member of the tumor necrosis factor receptor superfamily and is expressed on the surfaces of professional APCs as well as vascular endothelial cells and parenchymal cells during inflammation.4-7 The binding of CD40 by CD40L induces the up-regulation of MHC and B7 family members on professional APCs and leads to a broad range of immune and inflammatory responses.7, 19, 20 CD40 engagement on vascular endothelial cells induces cell proliferation and expression of adhesion molecules (e.g., E-selectin, vascular cell adhesion molecule 1, and intercellular cell adhesion molecule 1)19, 21 and results in microvasculature changes in patients with inflammatory bowel disease.22 On primary human hepatocytes, crosslinking CD40 with a monoclonal antibody leads to a rise in nuclear factor kappa B and activator protein 1 activity and apoptosis.19 To date, the role of CD40 in the liver parenchyma of patients with virus- and immune-mediated hepatitis is not entirely clear, and this remains one of the obstacles to gene therapy and orthotopic liver transplantation.2, 23, 24

The liver is a functionally unique organ in which hepatic sinusoids allow circulating lymphocytes to make direct contact with underlying hepatocytes through perforated fenestrations of liver sinusoidal endothelial cells.25 These interactions have been revealed by electron microscopy,26 and ample evidence supports the contention that hepatocytes can act as APCs to direct T cell activation.27-29 We previously reported that hepatic CD86 expression led to hepatitis through T cell activation and accumulation, and we speculated that CD40 expression is essential to signaling B7 molecule expression and downstream effects in the liver.9 In this study, we generated transgenic mice that conditionally expressed CD40 on their hepatocytes. Parenchymal CD40 expression upon AdCre infection resulted in the increased expression of CD80 and CD86 molecules, which led to an early expansion and subsequent contraction of CD8+ T cells in the liver (Table 1). Intrahepatic NK and CD4+ cells in CD40 transgenic mice followed a similar course of population changes, though to a lesser degree, and produced greater amounts of granzyme B and IFN-γ, respectively (Table 1 and Figs. 5 and 6). These data reveal that activation of the parenchymal CD40 and B7 signaling pathway disrupts IHL regulation and leads to necroinflammation and severe liver injury. Previous reports have indicated roles for NK cells and CD8+ CTLs in different stages of adenovirus infections.14, 15, 30 Dysregulation of IHLs can also play a role in other acute and chronic inflammatory liver diseases.4-8, 31

CD8+ CTLs and NK cells are capable of migrating to the liver to produce IFN-γ or degranulating; this leads to viral clearance.14, 15, 32 In this study, despite vigorous CD8+ T and NK cell responses (Figs. 5 and 6), CD40 transgenic mice did not show enhanced viral clearance in vivo. In a study designed to dissect the effector functions of virus-specific CTLs, the primary CTL clones were reported to produce IFN-γ (cytokine production) or degranulate (cytotoxicity); this depended on the antigen concentration.33 Cytotoxicity can be triggered at antigenic peptide concentrations that are 10- to 100-fold less than those required for IFN-γ production.33 Indeed, most hepatitis B virus and hepatitis C virus infections have been found to be purged from the liver by a cytokine-mediated, noncytolytic mechanism rather than direct target destruction.34 Adenovirus-induced hepatotoxicity has been linked to granzyme B–producing and perforin-producing NK cells and CTLs.15, 35 More interestingly, a recent study has suggested that CD4+ T cells can mediate tissue inflammation and liver injury via CD154 in the presence of CD40.36 Whether this notion is applicable to this and other viral and immune-mediated forms of hepatitis requires further investigation.

Patients with chronic necroinflammatory liver disease had increased percentages of PD-1+ IHLs, and their hepatocytes expressed its ligands, PD-L1 and B7-DC.8 However, the PD-1/PD-L1 pathway did not seem to affect acute viral hepatitis in our model (Supporting Fig. 10). In mice, disruption of the costimulatory molecule PD-L1 resulted in impaired CD8+ T cell contraction and thus led to accelerated hepatocyte damage and hepatitis.37 In costimulatory signaling pathways, CD40 is located upstream of CD80 and CD86; however, whether it interacts with other molecules, including PD-L1, B7-H4, and E-selectin, remains unclear.17

In summary, we generated a novel transgenic mouse model that allows parenchymal CD40 expression after an adenovirus infection in the liver. Our results suggest that hepatocyte CD40 expression and the activation of its downstream signaling events alter the effector functions of IHLs and exacerbate the liver injury. These data highlight a previously unknown deleterious effect of CD40 engagement and signaling in vivo. These CD40 transgenic mice also provide a valuable model for investigating the relevance of CD40 as the second hit in the oxidative stress and altered homeostasis of lymphocytes in alcoholic liver disease and alcoholic steatohepatitis.20, 38

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Maki Wakamiya (University of Texas Medical Branch Transgenic Core) and Yixiao Sun for their technical assistance, Tian Wang and Yingzi Cong for their critical comments, and Mardelle Susman for her assistance with the preparation of this article.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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