15-deoxy-Δ12,14-prostaglandin J2 reduces recruitment of bone marrow–derived monocyte/macrophages in chronic liver injury in mice

Authors

  • Zhen Han,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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    • These authors contributed equally to this work.

  • Tieping Zhu,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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    • These authors contributed equally to this work.

  • Xihong Liu,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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    • These authors contributed equally to this work.

  • Changyong Li,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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  • Shi Yue,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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  • Xin Liu,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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  • Le Yang,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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  • Lin Yang,

    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
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  • Liying Li

    Corresponding author
    1. Department of Cell Biology, Municipal Laboratory for Liver Protection and Regulation of Regeneration, Capital Medical University, Beijing, China
    • No.10 Xitoutiao, You An Men, Beijing 100069 P.R. China===

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    • fax: +861083950101


  • Potential conflict of interest: Nothing to report.

Abstract

15-Deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2), a natural peroxisome proliferator-activated receptor gamma (PPAR-γ) ligand, has been implicated as a new antiinflammatory compound with possible clinical applications. Based on this concept, this study was designed to evaluate the effects of 15d-PGJ2 on bone marrow–derived monocyte/macrophage (BMM) migration, phagocytosis, and cytokine expression after liver injury using mouse models induced by cholestasis or carbon tetrachloride. Mice were lethally irradiated and received bone marrow transplants from enhanced green fluorescent protein transgenic mice. Our results showed that recruitment of BMM was significantly increased during chronic liver injury, and that 15d-PGJ2 administration reduced BMM, but not neutrophil, dendritic, or T cell migration toward the damaged liver, involving reactive oxygen species generation and independently of PPAR-γ. Moreover, 15d-PGJ2 inhibited the phagocytic activity of BMM and down-regulated inflammatory cytokine expression in vivo and in vitro. Accordingly, hepatic inflammation and fibrosis were strikingly ameliorated after 15d-PGJ2 administration. Conclusion: Our findings strongly suggest the antiinflammation and antifibrogenic potential of 15d-PGJ2 in chronic liver diseases. (HEPATOLOGY 2012;56:350–360)

Macrophages are a key cellular component of the innate immune system. Kupffer cells (KCs), the resident macrophages of the liver, constitute 80%-90% of the tissue macrophages present in the body. They are found in greatest number in the periportal area and constitute the first macrophage population of the body to come in contact with bacteria, bacterial endotoxins, and microbial debris derived from the gastrointestinal tract and transported to the liver by way of the portal vein. Upon activation, KCs release various products, including multiple inflammatory cytokines and chemokines, nitric oxide, and reactive oxygen species (ROS). These factors regulate the phenotype of the KCs that produce them, as well as the phenotypes of neighboring cells, such as hepatocytes, stellate cells, endothelial cells, and other immune cells that traffic through the liver.1 Therefore, KCs are intimately involved in the hepatic response to various hepatic injuries.

In general, tissue macrophages are replenished from bone marrow (BM)-derived monocyte/macrophages (BMM). Upon inflammatory signals, monocyte/macrophages are rapidly recruited to the liver, and these cells have similar functional profiles to KCs.2 Indeed, KCs are derived from circulating monocytes that arise from BM and, once localized in the liver, dwell within the lumen of the liver sinusoids, predominantly in the periportal area. There is now considerable interest in the effects of BM-derived cells on liver injury and repair.3, 4 Previous studies have shown that the sphingosine 1-phosphate system is involved in homing of BM cells to the injured liver after liver injury.5, 6 Recently, Seki et al.7 demonstrated that CC-chemokine receptors (CCR1 and CCR2) expressed in the BM cells enhanced macrophage recruitment in early phases of liver injury. Importantly, multiple lines of evidence indicate that following liver injury, a significant number of BMM migrate and accumulate at the sites of inflammation, and consequently play an important role in liver inflammation, regeneration, remodeling of extracellular matrix, and fibrogenesis.8, 9

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural peroxisome proliferator-activated receptor gamma (PPAR-γ) ligand, has garnered much interest because of its ability to modulate the inflammatory response.10 In particular, several reports have demonstrated that 15d-PGJ2 inhibits cell migration in vitro and in vivo.11-14 Based on these findings, we hypothesized that 15d-PGJ2 reduces BMM migration to the lesion and reveals the antiinflammatory functions in chronic liver injury. In this study, we first investigated the effect of 15d-PGJ2 on BMM recruitment in mouse models of bile duct ligation (BDL)- or carbon tetrachloride (CCl4)-induced liver injury. In addition, we determined the effects of 15d-PGJ2 on migration, phagocytic activity, and inflammatory cytokines generation in BMM in vitro, and further analyzed the mode of action of 15d-PGJ2. To assess the therapeutic potential of 15d-PGJ2 in hepatic inflammation and fibrosis, we also examined the inflammation/necrosis area, production of inflammatory cytokines, and deposition of collagen in chronic liver injury. We report that 15d-PGJ2 significantly inhibits BMM migration, phagocytosis, and cytokine expression, three actions relevant to the process of hepatic inflammation and fibrogenesis. Accordingly, 15d-PGJ2 administration attenuates hepatic inflammation and fibrosis. Our data point to a potential antiinflammatory and antifibrotic activity of 15d-PGJ2 during BDL- or CCl4-induced liver injury.

Abbreviations

15d-PGJ2, 15-deoxy-Δ12,14-prostaglandin J2; BM, bone marrow; BMM, BM-derived monocyte/macrophages; BDL, bile duct ligation; EGFP, enhanced green fluorescent protein; KC, Kupffer cell; NAC, N-acetylcysteine; PPAR-γ, peroxisome proliferator-activated receptor gamma; ROS, reactive oxygen species; α-SMA, smooth muscle α-actin.

Materials and Methods

Materials.

Dulbecco's modified Eagle's medium (DMEM) was from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) was from Biochrom (Berlin, Germany). Polymerase chain reaction (PCR) reagents were from Applied Biosystems (Foster City, CA). 15d-PGJ2 was from Cayman Chemical (Ann Arbor, MI). Troglitazone and ciglitazone were from Biomol (Tebu, France). GW9662, N-acetylcysteine (NAC), latex beads, collagenase type IV, Histopaque-1077, and other common reagents were from Sigma (St. Louis, MO).

BM Isolation and BMM Preparation.

Wildtype or enhanced green fluorescent protein (EGFP)-transgenic ICR mice (3 weeks old) were sacrificed by cervical dislocation at the time of BM harvest. BM cells were extracted from the tibias and femurs by flushing with culture medium using a 25G needle. The cells were then passed through a 70-mm nylon mesh and were washed three times with phosphate-buffered saline (PBS) containing 2% FBS. All animal work was performed under the ethical guidelines of the Ethics Committee of Capital Medical University.

BM cells were grown in culture dishes for 7 days in the presence of L929-conditioned medium. The identification of BMM was assessed using immunocytochemistry analysis of F4/80 expression. The purity of BMM was >95%.

BM Transplantation.

BM transplantation was performed as described.6 Briefly, ICR mice aged 6 weeks received lethal irradiation (8 Grays) and immediately received transplantation by a tail vein injection of 1.5 × 107 whole BM cells obtained from 3-week-old EGFP transgenic mice. Four weeks later mice were subjected to the BDL- or CCl4-induced liver fibrosis as follows. After 1, 3, 7, 14, or 28 days, mice were sacrificed and liver tissue was harvested.

Mouse Models of Chronic Liver Fibrosis.

BDL or sham operations and CCl4 administration were performed as described.5, 6

Immunofluorescence Staining.

Liver samples were fixed in 4% paraformaldehyde and embedded in Tissue Tek OCT compound (Electron Microscopy Sciences, Japan). Six micrometers of frozen section were used for immunofluorescence. They were blocked with 3% bovine serum albumin, then incubated with antiF4/80 rat monoclonal antibody (1:100, Santa Cruz Biotechnology, CA) or antiCD3e hamster antibody (1:50, BD Biosciences, Franklin Lakes, NJ) and Rhodamine-AffiniPure goat antirat immunoglobulin G (IgG) (1:100,) or Cy3-AffiniPure goat antihamster IgG (1:100, both Jackson Immunoresearch, West Grove, PA) as secondary antibodies, respectively. The sections were covered with Vectashield mounting medium containing DAPI and observed under confocal microscope (LSM510, Carl Zeiss MicroImaging, Germany). The proportion of BMM (number of both F4/80+ and EGFP+ cells/number of F4/80+ cells), T cells (number of both CD3e+ and EGFP+ cells/number of CD3e+ cells), and non BM-recruited macrophages (resident macrophage, number of F4/80+ cells, subtract number of both F4/80+ and EGFP+ cells/field) was measured with Image-Pro Plus.

Flow Cytometric Analysis.

Nonparenchymal cells of mouse liver were isolated as described by Baeck et al.15 with modification. Briefly, livers were perfused with 20 mL of PBS, minced with scissors, and digested for 24 minutes with collagenase type IV at 37°C with gentle shaking. Digested extracts were pressed through 70-mm cell strainers to achieve single cell suspensions. The cell suspension was subjected to density gradient (Histopaque-1077) centrifugation at 2,000 rpm for 20 minutes. The cells were collected from the interface after centrifugation, washed twice with PBS, resuspended in PBS at 1.5 × 106 cells/100 μL. Subsequently, antibodies: APC-F4/80 (eBioscience, San Diego, CA), APC-CD11c, PE-Ly-6G (both BD Biosciences), and their isotype-matched negative control antibodies were added to the cell suspension. After 15 minutes of incubation in the dark, the cells were washed with PBS and subjected to flow cytometric analysis (FACS). FACS was performed on a FACSAria and analyzed with FACSDiva4.1 (BD Biosciences).

Cell Migration Assay.

BMM, monocytes (as BMM preparation method in the absence of L929-conditioned medium), or bone marrow mesenchymal stem cells (BMSCs)6 migration were determined in Boyden chambers as described.16 Briefly, cells were serum-starved for 24 hours and then exposed to 15d-PGJ2, troglitazone, ciglitazone, or vehicle (negative control) for 1 hour. Then 4 × 104 cells were seeded to the upper chamber. Cell migration was allowed to proceed for 4 hours at 37°C in 5% CO2. Cells migrated to the lower surface of the filter were stained and quantified by cell counting.

Phagocytic Activity of BMM.

Phagocytic activity of BMM was analyzed as described17 with minor modification. Briefly, serum-starved BMM were exposed to various concentrations of 15d-PGJ2 for 1 hour with or without pretreatment of GW9662 (antagonist of PPAR-γ) or NAC (inhibitor of ROS). The latex beads were added (10%, 3 μm size) and incubated for an additional 4 hours. The cells were fixed in 4% paraformaldehyde and noninternalized beads were removed by copious washes with PBS. The cells were then scraped and beads were counted using a hemacytometer.

Assay of Cell Proliferation.

Cell proliferation was assessed with the Cell Counting Kit-8 (CCK-8, Dojin, Tokyo, Japan). Briefly, serum-starved BMM were exposed to various concentrations of 15d-PGJ2 for 24 or 48 hours. Then cells were incubated with CCK-8 solution for 1 hour at 37°C and the absorbance at 450 nm was measured with a spectrophotometer.

Real-Time Reverse Transcription (RT)-PCR.

Extraction of total RNA from mice BMM or liver frozen specimen and real-time RT-PCR were performed as described.18 Primers were as follows: mouse tumor necrosis factor alpha (TNF-α): sense, 5′-GGCAGGTT CTGTCCCTTTCA-3′; antisense, 5′-CTGTGCTCAT GGTGTCTTTTCTG-3′. MCP-1: sense, 5′-TCT GGGCCTGCTGTTCACA-3′; antisense, 5′-GGATCA TCTTGCTGGTGAATGA-3′. Transforming growth factor beta (TGF-β)1: sense, 5′-TGCGCTTGCAGAG ATTAAAA-3′; antisense, 5′-CTGCCGTACAACTCCA GTGA-3′. Interleukin (IL)-6: sense, 5′-CTCTGGGAA ATCGTGGAAATG-3′; antisense, 5′-AAGTGCATCA TCGTTGTTCATACA-3′. F4/80: sense, 5′-AGCACA TCCAGCCAAAGCA-3′; antisense, 5′-CCATCTCCC ATCCTCCACAT-3′. 18S rRNA: sense, 5′-GTAACC CGTTGAACCCCATT-3′; antisense, 5′-CCATCCAA TCGGTAGTAGCG-3′. Probes (Applied Biosystems) used for real-time RT-PCR were as follows: procollagen α1(I): Mm00801666_g1; procollagen α1(III): Mm00802331_m1; smooth muscle α-actin (α-SMA): Mm00725412_s1.

Quantitative Analysis of Liver Fibrosis and Necrosis.

The fibrotic or necrotic area were assessed as described.5

Hydroxyproline Content Assay.

Hydroxyproline content of the liver was measured as described.5

Statistical Analysis.

The results are expressed as mean ± standard error of the mean (SEM). Statistical significance was determined by Student's t test or analysis of variance (ANOVA). P < 0.05 was considered significant.

Results

Cholestasis Increases the Recruitment of BMM to the Injured Liver.

To examine the role of BM-derived cells in cholestasis-induced liver injury, we reconstituted BM in the irradiated mice by transplantation of the genetic EGFP-labeled BM cells. Liver injury was induced by BDL and double positive for F4/80 and EGFP macrophages were counted. As shown in Fig. 1A-C, significant numbers of EGFP-positive cells (BM origin) in the injured areas were found to be positive for F4/80, a representative marker of mouse monocyte/macrophage.19 There were 35% BMM in sham-treated liver, indicating KCs turnover from BM origin (Fig. 1D). The proportion of BMM increased markedly from 37% to 80% after BDL for 1, 3, 7, or 14 (n = 6 per group) days (Fig. 1D). These findings revealed that recruitment of BMM was significantly increased during cholestasis-induced liver injury.

Figure 1.

Cholestasis increases the recruitment of BMM to the injured liver and 15d-PGJ2 inhibits this process. Mice were lethally irradiated and received whole BM transplants from EGFP transgenic mice. Then livers were analyzed from mice after BDL at the indicated timepoints. (A-C) Representative images of immunofluorescence analysis by confocal microscopy to track the macrophages (F4/80+, red) of BM origin (EGFP+, green) in the BDL-treated livers. DAPI was used to visualize nuclei (blue). (D) The proportion of monocyte/macrophages from BM (numbers of cells with yellow color/numbers of cells with red color) was measured with Image-Pro Plus. (E) Effect of 15d-PGJ2 (200 μg/kg body weight, twice per week, intraperitoneally) on the recruitment of BMM to the injured liver (n = 6 per group). (F) Effect of 15d-PGJ2 on the resident macrophage (non-BM recruited macrophages, number of F4/80+ cells, subtract number of both F4/80+ and EGFP+ cells/field). *P < 0.05, compared with the sham group. #P < 0.05, compared with BDL group without 15d-PGJ2.

15d-PGJ2 Reduces BMM Migration in Cholestatic Liver Injury.

15d-PGJ2 has been shown to play an important role in the regulation of inflammatory reactions involving suppressing the activation and migration of inflammatory cells.13, 20, 21 To investigate the potential role of 15d-PGJ2 in BMM migration during cholestasis-induced liver injury, we performed an EGFP-positive BM cell transplantation experiment followed by BDL-induced liver injury with or without 15d-PGJ2 administration (n = 6 per group). Treatment with 15d-PGJ2 (200 μg/kg body weight, twice per week, intraperitoneally) had no effect on the number of BMM in the sham-operated liver (Fig. 1E, second column), and 15d-PGJ2 did not induce inflammation and necrosis in the sham liver (data not shown). However, over the 2 weeks of 15d-PGJ2 administration the proportion of BMM in the damaged liver decreased markedly compared with that in the liver without 15d-PGJ2 treatment (Fig. 1E). Meanwhile, 15d-PGJ2 administration did not significantly alter the number of resident macrophages in mouse liver (Fig. 1F). These results have shown that 15d-PGJ2 reduces the population of BMM in cholestatic liver injury.

To further demonstrate the inhibition of 15d-PGJ2 on BMM migration in cholestatic liver injury, FACS analyses were performed. After 2 weeks of 15d-PGJ2 administration, the proportion of BMM (both F4/80+ and EGFP+) in the damaged liver decreased markedly compared with that in the liver without 15d-PGJ2 treatment (Fig. 2A). However, after 15d-PGJ2 administration there were no significant changes in the proportion of BM-derived neutrophils (both LY-6G+ and EGFP+, Fig. 2B), dendritic cells (both CD11c+ and EGFP+, Fig. 2C), and T cells (both CD3e+ and EGFP+, Fig. 3) in the liver compared with that 15d-PGJ2 nontreatment.

Figure 2.

Flow-cytometric analysis for the effects of 15d-PGJ2 on the recruitment of BMM, neutrophils, and dendritic cells to cholestasis-induced liver injury. Mice were lethally irradiated and received whole BM transplants from EGFP transgenic mice, followed by 2 weeks of sham or BDL operation in the absence or presence of 15d-PGJ2 administration (n = 7 per group). Then the nonparenchymal cells of the liver were analyzed for BMM, neutrophils, and dendritic cells specific markers (F4/80, LY-6G, and CD11c, respectively). Representative scatterplots are shown with the percentage of BMM (A), neutrophils (B), and dendritic cells (C) in sham or BDL mice treated with or without 15d-PGJ.2

Figure 3.

15d-PGJ2 does not change the recruitment of T cells to cholestasis-induced liver injury. Mice were lethally irradiated and received whole BM transplants from EGFP transgenic mice followed by BDL operation with or without 15d-PGJ2 treatment. (A,B) Representative images of immunofluorescence analysis by confocal microscopy to track the T cells (CD3e+, red) of BM origin (EGFP+, green) in the BDL-treated livers with or without 15d-PGJ2. DAPI was used to visualize nuclei (blue). The proportion of T cells from BM (numbers of cells with yellow color/numbers of cells with red color) was measured with Image-Pro Plus. (C) Effect of 15d-PGJ2 (200 μg/kg body weight, twice per week, intraperitoneally) on the recruitment of T cells to the injured liver (n = 6 per group). *P < 0.05 compared with the sham group. #P < 0.05 compared with the BDL group without 15d-PGJ2.

To further evaluate the effect of 15d-PGJ2 on BMM migration, a transwell migration assay was performed in vitro. As expected, treatment with 15d-PGJ2 (1-5 μM, as demonstrated by nontoxic concentrations in BMM, data not shown) caused a concentration-dependent decrease in BMM migration (Fig. 4A). Meanwhile, the effects of 15d-PGJ2 on monocytes or BMSCs migration were evaluated in the same assay. As shown in Fig. 4B,C, a high dose of 15d-PGJ2 (5 μM) significantly blocked monocytes migration, exclusively, but there was no effect on the recruitment of BMSCs. These results suggest that 15d-PGJ2 exerted more powerful inhibition on BMM other than monocytes and earlier precursors. Furthermore, 15d-PGJ2 is known to work through two basic mechanisms: PPAR-γ activation and ROS formation.22, 23 Thus, we next investigated whether suppression of BMM migration resulted from the direct activation of PPAR-γ and/or the production of ROS by 15d-PGJ2. Neither troglitazone nor ciglitazone (PPAR-γ agonists) had an effect on BMM migration (Fig. 4D). Moreover, pretreatment with GW9662 (PPAR-γ antagonist) did not affect the inhibitory effect of 15d-PGJ2 on BMM migration (Fig. 4D). Interestingly, depletion of ROS with NAC (general ROS inhibitor) abrogated the inhibitory effect of 15d-PGJ2 on cell migration (Fig. 4E). In addition, treatment with GW9662 or NAC alone had no effect on BMM migration. These findings indicated that 15d-PGJ2 reduced BMM migration through ROS formation, independently of PPAR-γ.

Figure 4.

Effect of 15d-PGJ2 on cell migration and proliferation. Serum-starved BMM (A), monocytes (B), or BMSCs (C) were allowed to migrate for 4 hours in the presence of varying concentrations of 15d-PGJ2 or its vehicle. (D) BMM were preincubated with 10 μM PPAR-γ antagonist GW9662 for 1 hour or were directly exposed to 10 μM PPAR-γ agonist troglitazone (Trog) or ciglitazone (Cig). (E) BMM were preincubated with 2.5 mM NAC (inhibitor of ROS) for 1 hour. (F) Serum-starved BMM were exposed to various concentrations of 15d-PGJ2 for 24 or 48 hours. Assay of cell proliferation was performed with the Cell Counting Kit-8. *P < 0.05 compared with control. #P < 0.05 compared with 15d-PGJ2 group without NAC.

Because 15d-PGJ2 is recognized as a potent apoptotic and growth inhibitory factor,23 we next measured the effect of 15d-PGJ2 on cell proliferation with the Cell Counting Kit-8. As shown in Fig. 4F, at concentrations ranging from 1-5 μM there were no significant changes in the number of living cells.

Taken together, these results suggest that 15d-PGJ2 inhibits cholestasis-induced migration of BMM to the injured liver, but does not affect cell apoptosis and proliferation in BMM.

15d-PGJ2 Inhibits the Phagocytic Activity of BMM In Vitro.

The resident macrophages of the liver (KCs) are shown to exhibit potent phagocytic activity following hepatic injury.24 We next examined the effect of 15d-PGJ2 on phagocytic activity of BMM. Treatment with 15d-PGJ2 markedly inhibited the phagocytic activity in a dose-dependent manner (Fig. 5A). To clarify the mechanisms by which 15d-PGJ2 modulated phagocytic activity of BMM, PPAR-γ agonists and antagonists were also used. As shown in Fig. 5B, pretreatment with GW9662 (PPAR-γ antagonist) partially lessened the decreased phagocytic activity exerted by 15d-PGJ2; moreover, troglitazone and ciglitazone (PPAR-γ agonists) also mimicked the effect of 15d-PGJ2. More interestingly, pretreatment with NAC (ROS inhibitor) partially reversed the inhibitory effect of 15d-PGJ2 (Fig. 5C). In addition, treatment with GW9662 or NAC alone had no effect on phagocytosis. These results suggest that the effect of 15d-PGJ2 on phagocytic activity of BMM is mediated through both PPAR-γ-dependent and independent mechanisms.

Figure 5.

15d-PGJ2 inhibits the phagocytic activity of BMM. Serum-starved BMM were exposed to various concentrations of 15d-PGJ2 for 1 hour with or without pretreatment of GW9662 or NAC. (A) Effect of 15d-PGJ2 on BMM phagocytosis. (B) BMM were preincubated with 10 μM GW9662 or were directly exposed to 10 μM troglitazone (Trog) or ciglitazone (Cig). (C) BMM were preincubated with 2.5 mM NAC. *P < 0.05 compared with control. #P < 0.05 compared with 15d-PGJ2 group without GW9662 or NAC.

Effect of 15d-PGJ2 on Inflammatory Cytokines Generation in BMM.

Because monocytes/macrophages regulate inflammatory and immune responses by generating multiple inflammatory cytokines and chemokines, we next investigated the potential role of 15d-PGJ2 in their production. Cultured BMM showed a significant decrease in TNF-α and monocyte chemotactic protein-1 (MCP-1) messenger RNAs (mRNAs) following treatment with 15d-PGJ2. However, there was no significant difference in TGF-β1 or IL-6 mRNAs in 15d-PGJ2-treated BMM compared to vehicle control (Fig. 6A). Moreover, we also measured the effect of troglitazone/ciglitazone and NAC on these cytokines expression. Similar to their effect on cell phagocytosis, troglitazone and ciglitazone decreased the expression of TNF-α and MCP-1, but NAC blocked the inhibitory effect of 15d-PGJ2 (Fig. 6B,C). These results suggest that 15d-PGJ2 activity interfere with, at least in part, release of the inflammatory cytokines in BMM through both PPAR-γ-dependent and ROS-dependent pathways.

Figure 6.

15d-PGJ2 alters inflammatory cytokines expression in BMM. (A) Serum-starved BMM were exposed to 5 μM 15d-PGJ2 for 0.5, 1, 2, and 6 hours. mRNA levels of TNF-α, MCP-1, TGF-β1, and IL-6 were measured by real-time RT-PCR. (B,C) BMM were preincubated with 2.5 mM NAC or were directly exposed to 10 μM troglitazone (Trog) or ciglitazone (Cig). *P < 0.05 compared with control (Ctrl). #P < 0.05 compared with 15d-PGJ2 group without NAC.

15d-PGJ2 Administration Attenuates Cholestasis-Induced Hepatic Inflammation and Fibrosis.

The intrahepatic inflammatory response following liver injury is a highly regulated process that involves the activation of resident hepatic macrophages. We thus assessed the potential effect of 15d-PGJ2 on cholestasis-induced hepatic inflammation. One or 2 weeks after BDL operation and 15d-PGJ2 administration, F4/80 mRNA was significantly decreased compared to BDL liver without 15d-PGJ2 administration (Fig. 7A), suggesting a decrease in the accumulation of monocytes/macrophages. Moreover, following liver injury, mice pretreated with 15d-PGJ2 presented lower levels of inflammatory mediators including TGF-β1, TNF-α, MCP-1, and IL-6 when compared with mice pretreated with vehicle (Fig. 7A). Importantly, there were positive correlations between F4/80 and these inflammatory cytokines mRNA expression (Fig. 7B-E), suggesting that there might be a link between the reduction of macrophage infiltration and the reduction of inflammatory cytokines.

Figure 7.

15d-PGJ2 administration decreases cholestasis-induced hepatic inflammatory cytokines, possibly due to the reduction of macrophage infiltration. (A) One week or 2 weeks after BDL operation and 15d-PGJ2 administration (n = 6 per group), mRNA levels of F4/80, TGF-β1, TNF-α, MCP-1, and IL-6 in mice livers were measured by real-time RT-PCR. The correlation between expression of TGF-β1 (B), TNF-α (C), MCP-1 (D), or IL-6 (E) and expression of F4/80 in mouse liver. *P < 0.05 compared with the sham group. #P < 0.05 compared with the BDL group without 15d-PGJ2.

Furthermore, hematoxylin and eosin (H&E)-stained sections showed that after 1 week of BDL operation, necrotic areas were significantly reduced by 15d-PGJ2 administration (Fig. 8A,B), although there was no significant difference in necrotic areas following 2 weeks of BDL (data not shown).

Figure 8.

Antiinflammatory and antifibrotic effects of 15d-PGJ2 on cholestasis-induced liver fibrosis in mice. Mice underwent sham or BDL in the absence or presence of 15d-PGJ2 administration (n = 6 per group). (A) Representative H&E-staining liver sections (original magnification, ×100) after 1 week BDL with or without 15d-PGJ2 administration. (B) Quantification of necrotic areas. (C) Representative images of Sirius Red staining (original magnification, ×100) in the BDL- or sham-operated livers. Inset: Sirius Red staining for sham-operated livers. (D) Quantitative analysis of liver fibrosis. (E) Expression of procollagen α1(I) (Col α1(I)), procollagen α1(III) (Col α1(III)), and α-SMA mRNA levels in liver, measured by real-time RT-PCR. (F) Hydroxyproline content in the liver. *P < 0.05 compared with the sham group. #P < 0.05 compared with the BDL group without 15d-PGJ2.

Next, we tested the antifibrotic effect of 15d-PGJ2 on cholestatic liver injury. Hepatic collagen deposition was also evaluated by morphometric analysis of Sirius Red staining (Fig. 8C) and quantified by digital image analysis (Fig. 8D). The data showed that collagen deposition was markedly attenuated by 15d-PGJ2 administration. mRNA expression of procollagen α1(I), procollagen α1(III), and α-SMA in liver tissue were markedly down-regulated after 15d-PGJ2 administration (Fig. 8E). In addition, after 15d-PGJ2 administration there was a significant decrease in total liver hydroxyproline content compared with that in untreated mice (Fig. 8F).

15d-PGJ2 also reduces BMM migration, inflammation, and fibrosis in CCl4-induced liver injury (see Supporting Figs. 1, 2 and Supporting Information).

Discussion

An increasing body of in vivo evidence shows that 15d-PGJ2 possesses antiinflammatory properties in a number of experimental models.13, 25, 26 However, very few studies have investigated whether 15d-PGJ2 plays an important role in the regulation of inflammatory reactions during liver injury. In the present study, we demonstrated that the endogenous ligand for PPAR-γ, 15d-PGJ2 mediates suppression of BMM recruitment toward the damaged liver in a model of chronic liver injury. In addition, 15d-PGJ2 inhibits the phagocytic activity and inflammatory cytokines generation in BMM. Furthermore, our findings have shown that hepatic inflammation and fibrosis are significantly ameliorated after 15d-PGJ2 administration.

Prostaglandins of the J series, and more specifically 15d-PGJ2, exhibit multifaceted biological properties including growth arrest, apoptosis, differentiation, and suppression of macrophage activation and inflammation.10, 27 Importantly, accumulating data have demonstrated that 15d-PGJ2 inhibits cell migration.11-14 Here, we demonstrated for the first time that a 2-week treatment with 15d-PGJ2 (200 μg/kg) significantly reduced BMM recruitment to the injured regions. Besides its in vivo inhibitory effect on BMM migration, 15d-PGJ2 also exhibited direct inhibitory effects on BMM chemotactic activity in vitro. In addition, our data have shown that there are no significant changes in the number of living cells after treatment of 15d-PGJ2 at concentrations ranging from 1-5 μm, thus excluding the effect of 15d-PGJ2 on cell apoptosis and proliferation in BMM.

There are two main mechanisms that have been described to explain the biological effects of 15d-PGJ2. First, 15d-PGJ2 has been proposed as the endogenous ligand for PPAR-γ, a ligand-inducible transcription factor. Indeed, the best-studied effects of 15d-PGJ2 relate to activation of PPAR-γ. The second mechanisms of action by which 15d-PGJ2 alters cellular signaling pathways is through modification of redox-sensitive signaling molecules, such as ROS generation.27 In this study we showed that troglitazone and ciglitazone (PPAR-γ agonists) had no effect on BMM migration. Moreover, GW9662 (PPAR-γ antagonist) did not block the inhibitory effect of 15d-PGJ2 on BMM migration. However, the antioxidant NAC substantially abrogated the inhibitory response to 15d-PGJ2. These results suggest that production of ROS serves as a second messenger of the inhibitory effect of 15d-PGJ2 on BMM migration, which is independent of PPAR-γ. Interestingly, the inhibitory effect of 15d-PGJ2 on phagocytic activity and inflammatory cytokines production in BMM is mediated through both PPAR-γ-dependent and independent mechanisms. These findings raise the possibility that different mechanisms are involved in different biological actions induced by 15d-PGJ2, which is in keeping with a recent report demonstrating that the mechanism by which 15d-PGJ2 causes focal adhesion disassembly appears to be distinct from those responsible for migration.11

Tissue-specific macrophages, represented by KCs in the liver, contribute to the systemic response to local inflammation, clearance of pathogen-derived soluble molecules and toxins from the circulation, and engulfment of apoptotic bodies. These cells are also professional phagocytes and have been widely implicated in various liver injuries.24 Emerging data suggest that recruitment of BMM following liver injury contributes to liver inflammation and fibrogenesis.8, 9 Our findings further argue for an important role of BMM in a model of chronic liver injury. Interestingly, our previous studies indicated that BM-derived cells, predominantly BM-derived mesenchymal stem cells, contributed to myofibroblast population in the injured liver and promoted fibrogenesis.6 Here we have shown that a significant proportion of F4/80+ macrophages is of BM origin following liver injury. It is possible that diverse BM cells may manifest diverse effects. These findings further support previous studies demonstrating a role of BM in liver inflammation and fibrosis development.3, 4 Moreover, macrophages have assumed an important role in matrix degradation, which is profibrogenic during progression of fibrosis but antifibrotic during fibrosis resolution.28 Therefore, for the rational development of antifibrotic therapy, it should be kept in mind that BM contains diverse cell types that may manifest both profibrotic and antifibrotic phenotypes, depending on both their differentiation potential and the phase of liver injury when they are used.

An intriguing aspect of the present results is related to the implications for antifibrotic therapy. In our model system, 15d-PGJ2 inhibited phagocytic activity of BMM, reduced BMM migration, and down-regulated expression of inflammatory cytokines in vivo and in vitro, three actions involved in hepatic inflammation and fibrogenesis. Accordingly, hepatic inflammation and fibrosis in mouse was significantly ameliorated by 15d-PGJ2 administration. The previous studies indicated that 15d-PGJ2 induced apoptosis of human hepatic myofibroblasts, the key fibrogenic cells, by way of a mechanism involving oxidative stress and unrelated to PPAR-γ activation.23 Marra et al.12 reported that treatment of cultured human hepatic stellate cells with 15d-PGJ2 inhibited cell proliferation, migration, and chemokine expression, which were relevant to liver wound healing and fibrogenesis. Our reports further demonstrate antifibrogenic properties of 15d-PGJ2 in human hepatic myofibroblasts involved a stress-inducible protein, heme oxygenase-1.29, 30 These studies raise the therapeutic potential of 15d-PGJ2 in the treatment of liver fibrosis. In addition, both natural (15d-PGJ2) and synthetic (ciglitazone, rosiglitazone, or troglitazone) PPAR-γ agonists inhibit pulmonary myofibroblast differentiation and collagen production, induce antifibrotic hepatocyte growth factor expression in mesangial cells, and reduce human cardiac myofibroblasts proliferation, consequently eliciting potential antifibrogenic effects in lung,31 renal,32 and myocardial fibrosis,33 respectively. Our current data are the first to provide direct evidence for 15d-PGJ2-mediated antifibrotic effects during chronic liver injury. Importantly, data from other groups as well as our own suggest that the antifibrotic effects of 15d-PGJ2 may result from the multiple biological actions induced by 15d-PGJ2. Further studies are required to give a full picture of antifibrotic properties of 15d-PGJ2 in various liver diseases.

In summary, as we hypothesized, 15d-PGJ2 suppresses BMM migration through ROS generation, independently of PPAR-γ, during chronic liver injury. Moreover, 15d-PGJ2 inhibits phagocytic activity and reduces inflammatory cytokines expression in BMM. Intriguingly, 15d-PGJ2 administration markedly attenuates hepatic inflammation and fibrosis. These findings strongly suggest the antifibrogenic potential of 15d-PGJ2 in the future.

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