Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats
2005; Elsevier BV; Volume: 128; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2004.10.005
ISSN1528-0012
AutoresMichio Imamura, Tadashi Ogawa, Yasuyuki Sasaguri, Kazuaki Chayama, Hikaru Ueno,
Tópico(s)Liver Disease and Transplantation
ResumoBackground & Aims: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. Methods: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor β receptor (AdTβ-TR), which works as a dominant-negative receptor, bacterial β-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1–21 days. Results: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTβ-TR–, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTβ-TR–treated rats. Conclusions: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTβ-TR–treated livers indicate that transforming growth factor β is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor β is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells. Background & Aims: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. Methods: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor β receptor (AdTβ-TR), which works as a dominant-negative receptor, bacterial β-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1–21 days. Results: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTβ-TR–, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTβ-TR–treated rats. Conclusions: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTβ-TR–treated livers indicate that transforming growth factor β is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor β is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells. Inflammation is always accompanied by an infiltration by leukocytes,1Luster A.D. Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3287) Google Scholar a process that is thought to be regulated by chemotactic cytokines called chemokines.1Luster A.D. Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3287) Google Scholar, 2Rollins B.J. Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar Monocyte chemoattractant protein (MCP)-1, one of these chemokines, induces infiltration by monocytes/macrophages and lymphocytes3Baggiolini M. Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2418) Google Scholar by binding to a specific receptor, CCR2.1Luster A.D. Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3287) Google Scholar, 2Rollins B.J. Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar In animal models of liver injury4Czaja M.J. Geerts A. Xu J. Schmiedeberg P. Ju Y. Monocyte chemoattractant protein 1 (MCP-1) expression occurs in toxic rat liver injury and human liver disease.J Leukoc Biol. 1994; 55: 120-126PubMed Google Scholar, 5Yamaguchi Y. Matsumura F. Takeya M. Ichiguchi O. Kuratsu J.I. Horiuchi T. Akizuki E. Matsuda T. Okabe K. Ohshiro H. Liang J. Mori K. Yamada S. Takahashi K. Ogawa M. Monocyte chemoattractant protein-1 enhances expression of intercellular adhesion molecule-1 following ischemia-reperfusion of the liver in rats.Hepatology. 1998; 27: 727-734Crossref PubMed Scopus (67) Google Scholar and in patients with chronic hepatitis,6Marra F. DeFranco R. Grappone C. Milani S. Pastacaldi S. Pinzani M. Romanelli R.G. Laffi G. Gentilini P. Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis correlation with monocyte infiltration.Am J Pathol. 1998; 152: 423-430PubMed Google Scholar, 7Fisher N.C. Neil D.A. Williams A. Adams D.H. Serum concentrations and peripheral secretion of the beta chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1alpha in alcoholic liver disease.Gut. 1999; 45: 416-420Crossref PubMed Scopus (90) Google Scholar MCP-1 is detectable in both livers and serum. Injury-induced inflammation results in tissue remodeling or liver fibrosis. However, the actual roles performed by infiltrated monocytes/macrophages and MCP-1 in liver fibrogenesis are largely unknown. During liver fibrogenesis, hepatic stellate cells (HSC) are activated to myofibroblast-like cells expressing α-actin. These activated HSC and myofibroblasts already existing in the portal field and around central veins may play a central role in fibrogenesis,8Ramadori G. Saile B. Mesenchymal cells in the liver—one cell type or two?.Liver. 2002; 22: 283-294Crossref PubMed Scopus (100) Google Scholar after which they produce extracellular matrix through the generation of various cytokines, including transforming growth factor (TGF)-β.9Friedman S.L. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.N Engl J Med. 1993; 328: 1828-1835Crossref PubMed Scopus (0) Google Scholar For fibrogenesis, HSC are considered to be the responsible cells, and TGF-β is one of the critical factors for fibrogenesis. In fact, when we inhibited the action of TGF-β by using a dominant-negative mutated receptor for TGF-β,10Yamamoto H. Ueno H. Ooshima A. Takeshita A. Adenovirus-mediated transfer of a truncated transforming growth factor-β (TGF-β) type II receptor completely and specifically abolishes diverse signaling by TGF-β in vascular wall cells in primary culture.J Biol Chem. 1996; 271: 16253-16259Crossref PubMed Scopus (81) Google Scholar the activated HSC were markedly reduced in number, and fibrogenesis, as well as the progression of already-established fibrosis, was almost completely suppressed.11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar, 13Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats.Hum Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (139) Google Scholar This shows the essential roles played by TGF-β and HSC in fibrotic remodeling after liver injury. However, the mechanism underlying the activation of HSC is not fully understood, although TGF-β has been believed to be an activating factor.14Reeves H.L. Friedman S.L. Activation of hepatic stellate cells—a key issue in liver fibrosis.Front Biosci. 2002; 7: d808-d826Crossref PubMed Scopus (434) Google Scholar In this study, to try to answer these questions, we introduced a mutated form of MCP-1 (7ND), which is considered to inhibit the action of MCP-1 as a dominant-negative mutant,15Zhang Y. Rutledge B.J. Rollins B.J. Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis. Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.J Biol Chem. 1994; 269: 15918-15924Abstract Full Text PDF PubMed Google Scholar, 16Zhang Y. Rollins B.J. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.Mol Cell Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (145) Google Scholar into dimethylnitrosamine (DMN)-treated rats, an established model of liver fibrosis with a pathology closely resembling that of human cirrhosis.17Jenkins S.A. Grandison A. Baxter J.N. Day D.W. Taylor I. Shields R. A dimethylnitrosamine-induced model of cirrhosis and portal hypertension in the rat.J Hepatol. 1985; 1: 489-499Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 18Jezequel A.M. Mancini R. Rinaldesi M.L. Macarri G. Venturini C. Orlandi F. A morphological study of the early stages of hepatic fibrosis induced by low doses of dimethylnitrosamine in the rat.J Hepatol. 1987; 5: 174-181Abstract Full Text PDF PubMed Scopus (130) Google Scholar Some rats were given a dominant-negative TGF-β receptor to eliminate signaling by TGF-β.11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar We compared these rats in terms of (1) infiltration by monocytes/macrophages and activation of HSC, both of which occur in the acute phase after injury, and (2) fibrotic changes in the chronic phase after injury. Although inhibition of MCP-1 and blockade of TGF-β each led to a marked suppression of liver fibrogenesis, we were interested to find that some responses in the initial phase after injury were quite different between these 2 groups. Our study indicates that TGF-β is not an activating factor for HSC and suggests that infiltrated monocytes/macrophages may produce the activating factor(s). Replication-defective E1− and E3− adenoviral vectors expressing an amino-terminal deletion mutant of human MCP-1 (Ad7ND) with a FLAG epitope tag in its carboxyl-terminal (complementary DNA, a generous gift from Dr. B. Rollins, Harvard University),15Zhang Y. Rutledge B.J. Rollins B.J. Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis. Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.J Biol Chem. 1994; 269: 15918-15924Abstract Full Text PDF PubMed Google Scholar, 16Zhang Y. Rollins B.J. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.Mol Cell Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (145) Google Scholar a truncated human TGF-β type II receptor (AdTβ-TR),10Yamamoto H. Ueno H. Ooshima A. Takeshita A. Adenovirus-mediated transfer of a truncated transforming growth factor-β (TGF-β) type II receptor completely and specifically abolishes diverse signaling by TGF-β in vascular wall cells in primary culture.J Biol Chem. 1996; 271: 16253-16259Crossref PubMed Scopus (81) Google Scholar, 11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar or bacterial β-galactosidase (AdLacZ)19Ueno H. Li J.J. Tomita H. Yamamoto H. Pan Y. Kanegae Y. Saito I. Takeshita A. Quantitative analysis of repeat adenovirus-mediated gene transfer into injured canine femoral arteries.Arterioscler Thromb Vasc Biol. 1995; 15: 2246-2253Crossref PubMed Scopus (62) Google Scholar under a CA promoter comprising a cytomegalovirus enhancer and a chicken β-actin promoter20Niwa H. Yamamura K. Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4642) Google Scholar were prepared as previously described.21Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.Proc Natl Acad Sci U S A. 1996; 93: 1320-1324Crossref PubMed Scopus (789) Google Scholar COS cells were infected with either Ad7ND (multiplicity of infection [MOI] of 1, 10, and 100) or AdLacZ (MOI of 10), as previously described.10Yamamoto H. Ueno H. Ooshima A. Takeshita A. Adenovirus-mediated transfer of a truncated transforming growth factor-β (TGF-β) type II receptor completely and specifically abolishes diverse signaling by TGF-β in vascular wall cells in primary culture.J Biol Chem. 1996; 271: 16253-16259Crossref PubMed Scopus (81) Google Scholar One day after infection, the medium was replaced with serum-free medium, and cells were incubated for a further 24 hours. A mutant MCP-1 (7ND) secreted into culture media was analyzed by Western blotting by using monoclonal antibodies against either FLAG (Abcam, Cambridge, UK) or human MCP-1 (Sanbio, 5400 AM Uden, The Netherlands), as previously described.13Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats.Hum Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (139) Google Scholar 7ND and rat MCP-1 were also detectable by enzyme-linked immunosorbent assay (ELISA). Livers were homogenized in phosphate-buffered saline with 1% Triton X-100, 0.1% sodium dodecyl sulfate, and 0.5% sodium deoxycholate. The homogenates were centrifuged at 20,000g for 30 minutes. 7ND and rat MCP-1 were measured in the supernatant of liver homogenates and in sera from rats by using a human MCP-1 ELISA kit (Biosource, Camarillo, CA) and a rat kit (Biosource), respectively, according to the manufacturer’s instructions. These ELISA kits are species specific, and cross-reaction between human and rat MCP-1 is less than 5%. In fact, no human MCP-1 protein was detectable in samples from either intact or AdLacZ-infected rats (data not shown). All animals were treated under protocols approved by the institutional animal care committees, and the experiment was performed under both the institutional guidelines for animal experiments and by the Law (No. 105) and Notification (No. 6) of the Japanese government. Male Sprague–Dawley rats, 10 weeks old and weighing approximately 350 g, were given a single infusion of 0.5 mL of Ad7ND, AdTβ-TR, AdLacZ (2 × 109 plaque-forming units per milliliter), or saline via the tail vein, as previously reported.12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar By this method, virtually all cells in the liver were infected and expressed the introduced molecule.11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar Seven days later, rats were given an intraperitoneal injection of DMN (10 μg/g body weight; Wako, Osaka, Japan) either once or at the indicated times (3 consecutive daily injections or 3 consecutive daily injections and 4 days off per week for 3 weeks), as previously reported.11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar, 13Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats.Hum Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (139) Google Scholar After DMN treatment, blood was collected, and the rats were killed. Biochemical parameters were measured by using standard methods. The liver was either fixed with 4% buffered paraformaldehyde for histological examination or frozen immediately in liquid nitrogen for the extraction of hydroxyproline, the content of which was measured as described elsewhere.22Woessner J.F.J. The determination of hydroxyproline in tissue and protein samples containing small amounts of the amino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3408) Google Scholar Liver sections were stained with hematoxylin or Masson trichrome or subjected to immunohistostaining by using antibodies against either CD68 (ED-1; Serotec, Raleigh, NC) or α-actin (Dako, Tokyo, Japan). Immunoreactive materials were visualized by using a streptavidin-biotin staining kit (Histofine SAB-PO kit; Nichirei, Tokyo, Japan) and diaminobenzidine. Macrophages (CD68-positive cells) and lymphocytes were counted by a technician blinded to the treatment regimen. Four random high-power (200×) fields from each section were examined. As negative controls, immunohistostaining was performed without the first antibodies. Fragmented DNA in apoptotic cells in liver sections was stained with diaminobenzidine (dark brown) by the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique by using a commercially available kit (Roche Diagnostics, Mannheim, Germany). Then, the sections were double-stained against α-actin and visualized with the aid of 3-amino-9-ethyl carbazole liquid substrate chromogen (red; Dako). As negative controls, the TUNEL reaction mixture was used without terminal transferase. Statistical analysis was performed by 1-way analysis of variance followed by Scheffé’s test. P < .05 was considered significant. In the culture medium from Ad7ND-infected COS cells, 7ND was readily detectable in an MOI-dependent manner, as assessed by Western blotting analysis (Figure 1). Human 7ND and endogenous rat MCP-1 proteins were measured in sera (Figure 2A) and liver extracts (Figure 2B) from rats infected with Ad7ND. Seven days after gene transfer, a 3-week DMN treatment was begun. It is interesting to note that the amount of rat MCP-1 was not significantly changed by DMN treatment in either serum or liver. 7ND reached a peak on the seventh day after gene transfer and then declined gradually; however, the values were much higher than those obtained for rat MCP-1 in most time periods under DMN injury.Figure 2Amounts of human 7ND and rat MCP-1 in the sera (A) and livers (B) of DMN-injured rats. Rats were given a single infusion of Ad7ND (or saline infusion) via the tail vein. Seven days later, rats were subjected to a 3-week DMN treatment (shown as arrows). Rats were killed 5, 7 (just before the initiation of DMN treatment), 14, 21, and 28 days after Ad7ND injection. Means ± SD (n = 4) are shown.View Large Image Figure ViewerDownload (PPT) Rats were infused via the tail vein with either saline or an adenovirus expressing 1 of the following: β-galactosidase (AdLacZ), a truncated TGF-β receptor (AdTβ-TR), or a mutated MCP-1 (Ad7ND). Seven days later (when the expression of the introduced molecules had reached a submaximal level), DMN was given. One day after a single injection of DMN, we analyzed liver sections by hematoxylin staining and immunohistostaining against CD68, which is a specific marker for macrophages. Macrophages were detectable in the centrilobular area of the livers of AdLacZ-infected, AdTβ-TR–infected, or saline-injected rats: there were no differences among these 3 groups. However, macrophages were greatly reduced in Ad7ND-treated livers (Figure 3A). The numbers of CD68-positive cells (per high-power field) were 29 ± 3.5 in saline-treated livers, 27.5 ± 2.1 in AdLacZ-treated livers, 27.5 ± 11.4 in AdTβ-TR–treated livers, and only 7.1 ± 1.2 in Ad7ND-treated livers (Figure 3B). Similarly, the numbers of lymphocytes (histology not shown) were 98 ± 7.5 in saline-treated livers, 101 ± 2.5 in AdLacZ-treated livers, 93 ± 5.5 in AdTβ-TR–treated livers, and only 40 ± 3.5 per high-power field in Ad7ND-treated livers (Figure 3C). Without DMN treatment, neither macrophages nor lymphocytes (histology not shown) were increased in the livers of AdLacZ-infected, AdTβ-TR–infected, and Ad7ND-infected livers compared with intact livers (subjected to no injection of either saline or adenovirus and no DMN treatment; Figure 3B and C). Next, after a 3-day DMN treatment, we examined livers for α-actin–positive cells (a marker of activated HSC). They were readily detectable, not only in AdLacZ- or saline-treated, but also in AdTβ-TR–treated livers. In contrast, we could see none in the Ad7ND-treated livers (Figure 4). After a 3-week DMN treatment, the hydroxyproline content of livers was measured as a quantitative evaluation of fibrosis (Figure 5). The hydroxyproline contents in the livers of both AdLacZ- and saline-treated rats were approximately 3-fold higher than in intact livers, as previously observed.10Yamamoto H. Ueno H. Ooshima A. Takeshita A. Adenovirus-mediated transfer of a truncated transforming growth factor-β (TGF-β) type II receptor completely and specifically abolishes diverse signaling by TGF-β in vascular wall cells in primary culture.J Biol Chem. 1996; 271: 16253-16259Crossref PubMed Scopus (81) Google Scholar, 11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar In contrast, in the Ad7ND-treated and AdTβ-TR–treated livers, the hydroxyproline content remained close to the level seen in intact livers. After the DMN treatment, the serum levels of aspartate aminotransferase, alanine aminotransferase, and total bilirubin were all increased, and both the body and liver weights were decreased, probably because of liver dysfunction. However, these values were preserved or better maintained in the Ad7ND-treated or AdTβ-TR–treated groups (Table 1).Table 1Serum Hepatobiliary Parameters and Body and Liver WeightsVariableTotal bilirubin (mg/mL)AST (IU/mL)ALT (IU/mL)Body weight (g)Liver weight (g)Intact0.2 ± 0.168 ± 1640 ± 9350 ± 203.5 ± 0.1AdLacZ0.2 ± 0.171 ± 1139 ± 7340 ± 203.6 ± 0.1Saline + DMN0.7 ± 0.6495 ± 103245 ± 88290 ± 202.4 ± 0.4AdLacZ + DMN0.8 ± 0.7525 ± 149232 ± 97290 ± 302.3 ± 0.5Ad7ND + DMN0.3 ± 0.1aP < .05 vs. AdLacZ + DMN.134 ± 16aP < .05 vs. AdLacZ + DMN.69 ± 7aP < .05 vs. AdLacZ + DMN.350 ± 10aP < .05 vs. AdLacZ + DMN.3.4 ± 0.1aP < .05 vs. AdLacZ + DMN.AdTβ − TR + DMN0.4 ± 0.1aP < .05 vs. AdLacZ + DMN.222 ± 84aP < .05 vs. AdLacZ + DMN.69 ± 25aP < .05 vs. AdLacZ + DMN.350 ± 10aP < .05 vs. AdLacZ + DMN.3.5 ± 0.1aP < .05 vs. AdLacZ + DMN.NOTE. Rats were given a single infusion of saline, AdLacZ, Ad7ND, or AdTβ-TR via the tail vein. Seven days later, a 3-week DMN treatment was given to some rats (shown as +DMN). After a 3-week DMN treatment, blood was collected, and body and liver weights were measured. Serum total bilirubin, AST, and ALT and body and liver weights are shown as mean ± SE (n = 4). Rats never subjected to adenovirus infection or treated with DMN were also measured (shown as Intact).AST, aspartate aminotransferase; ALT, alanine aminotransferase.a P < .05 vs. AdLacZ + DMN. Open table in a new tab NOTE. Rats were given a single infusion of saline, AdLacZ, Ad7ND, or AdTβ-TR via the tail vein. Seven days later, a 3-week DMN treatment was given to some rats (shown as +DMN). After a 3-week DMN treatment, blood was collected, and body and liver weights were measured. Serum total bilirubin, AST, and ALT and body and liver weights are shown as mean ± SE (n = 4). Rats never subjected to adenovirus infection or treated with DMN were also measured (shown as Intact). AST, aspartate aminotransferase; ALT, alanine aminotransferase. After a 3-week DMN treatment, we analyzed liver histology both by Masson trichrome staining and by immunohistostaining against α-actin. In accordance with the data on hydroxyproline content (Figure 5), both Ad7ND-treated and AdTβ-TR–treated livers showed a fibrotic area that was markedly smaller than that seen in the AdLacZ- and saline-injected rats, and α-actin–positive cells were almost undetectable (Figure 6). In the AdTβ-TR–treated livers, α-actin–positive cells were readily detectable after the initial 3-day DMN treatment (Figure 4). We assumed that activated HSC disappeared through apoptosis under conditions in which the action of TGF-β was suppressed. We therefore performed TUNEL staining on the fourth day after starting DMN treatment. TUNEL-positive cells were increased in the AdTβ-TR–treated livers; however, no such apoptotic cells were observed in the AdLacZ- or saline-injected livers (Figure 7A). Immunohistostaining against α-actin confirmed that the TUNEL-positive cells in the AdTβ-TR–treated livers (Figure 7A) were indeed α-actin positive (Figure 7B). Inflammation induces infiltration by leukocytes and monocytes/macrophages into inflamed tissues.1Luster A.D. Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3287) Google Scholar Tissue remodeling or fibrosis then follows the inflammation. MCP-1, one of the CC chemokines, attracts monocytes/macrophages bearing CCR2.1Luster A.D. Chemokines—chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3287) Google Scholar, 2Rollins B.J. Chemokines.Blood. 1997; 90: 909-928Crossref PubMed Google Scholar, 3Baggiolini M. Chemokines and leukocyte traffic.Nature. 1998; 392: 565-568Crossref PubMed Scopus (2418) Google Scholar In this study, the roles of such macrophages in injury-induced liver fibrogenesis were investigated by overexpressing a mutated MCP-1 (7ND), which is reported to suppress the actions of MCP-1.15Zhang Y. Rutledge B.J. Rollins B.J. Structure/activity analysis of human monocyte chemoattractant protein-1 (MCP-1) by mutagenesis. Identification of a mutated protein that inhibits MCP-1-mediated monocyte chemotaxis.J Biol Chem. 1994; 269: 15918-15924Abstract Full Text PDF PubMed Google Scholar, 23Egashira K. Zhao Q. Kataoka C. Ohtani K. Usui M. Charo I.F. Nishida K. Inoue S. Katoh M. Ichiki T. Takeshita A. Importance of monocyte chemoattractant protein-1 pathway in neointimal hyperplasia after periarterial injury in mice and monkeys.Circ Res. 2002; 90: 1167-1172Crossref PubMed Scopus (157) Google Scholar, 24Inoue S. Egashira K. Ni W. Kitamoto S. Usui M. Otani K. Ishibashi M. Hiasa K. Nishida K. Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice.Circulation. 2002; 106: 2700-2706Crossref PubMed Scopus (238) Google Scholar, 25Ni W. Kitamoto S. Ishibashi M. Usui M. Inoue S. Hiasa K. Zhao Q. Nishida K. Takeshita A. Egashira K. Monocyte chemoattractant protein-1 is an essential inflammatory mediator in angiotensin II-induced progression of established atherosclerosis in hypercholesterolemic mice.Arterioscler Thromb Vasc Biol. 2004; 24: 534-539Crossref PubMed Scopus (97) Google Scholar In the Ad7ND-treated rats, DMN-induced infiltration by macrophages and lymphocytes into injured livers was markedly suppressed (Figure 3), the activation of HSC was eliminated (Figure 4), and liver fibrogenesis was greatly prevented (Figure 5, Figure 6). The cellular infiltration and activation of HSC observed immediately after infliction of the injury were similar between the AdTβ-TR–treated livers and the controls (saline-infused or AdLacZ-infected rats; Figure 3, Figure 4). Our study shows that infiltrated macrophages are critical for HSC activation and subsequent fibrogenesis and, importantly, that TGF-β is not an activating factor for HSC. It is suggested that the infiltrated macrophages may themselves secrete an activating factor or factors for HSC. We have previously shown that anti–TGF-β intervention inhibits liver fibrogenesis11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 13Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats.Hum Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (139) Google Scholar and its progression.12Nakamura T. Sakata R. Ueno T. Sata M. Ueno H. Inhibition of transforming growth factor β prevents progression of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats.Hepatology. 2000; 32: 247-255Crossref PubMed Scopus (254) Google Scholar In this study, we found that suppression of infiltration by macrophages and lymphocytes through overexpression of 7ND led to a powerful suppression of liver fibrogenesis to a similar degree as blockade of TGF-β but that the underlying mechanisms seem to be different. Activation of HSC in the initial stage immediately after injury was already eliminated in the Ad7ND-treated livers (Figure 4). Probably because HSC activation was inhibited, the subsequent progress toward fibrosis was suppressed in the Ad7ND-treated livers, thus supporting the idea that activation of HSC is the initial and critical event that leads to liver fibrosis. It has been considered for a long time that TGF-β is the HSC-activating factor (or at least one of the activating factors).14Reeves H.L. Friedman S.L. Activation of hepatic stellate cells—a key issue in liver fibrosis.Front Biosci. 2002; 7: d808-d826Crossref PubMed Scopus (434) Google Scholar However, our study clearly shows for the first time that TGF-β is not the HSC-activating factor, because a substantial number of activated HSC were present in the AdTβ-TR–treated livers (Figure 4); indeed, the numbers of activated HSC were the same among saline-treated, AdLacZ-treated, and AdTβ-TR–treated livers. We confirmed previously that virtually all liver cells are infected with an adenovirus when one is administered to rats with intact livers,11Qi Z. Atsuchi N. Ooshima A. Takeshita A. Ueno H. Blockade of TGF-β signaling prevents liver fibrosis and dysfunction in the rat.Proc Natl Acad Sci U S A. 1999; 96: 2345-2349Crossref PubMed Scopus (298) Google Scholar, 13Ueno H. Sakamoto T. Nakamura T. Qi Z. Astuchi N. Takeshita A. Shimizu K. Ohashi H. A soluble transforming growth factor β receptor expressed in muscle prevents liver fibrogenesis and dysfunction in rats.Hum Gene Ther. 2000; 11: 33-42Crossref PubMed Scopus (139) Google Scholar so the possibility can be excluded that all of these activated HSC were uninfected with AdTβ-TR. Although substantial numbers of activated HSC were seen after a 3-day DMN treatment, most disappeared during the next 2 weeks of DMN treatment (Figure 6). The activated HSC are probably eliminated through apoptosis under conditions in which TGF-β signaling is inhibited. Indeed, we showed that in the AdTβ-TR–treated livers, but not in the AdLacZ- or saline-injected ones, activated HSC were in apoptosis (Figure 7). Saile et al26Saile B. Knittel T. Matthes N. Schott P. Ramadori G. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair.Am J Pathol. 1997; 151: 1265-1272PubMed Google Scholar reported that HSC undergo CD95-mediated spontaneous apoptosis when they are activated, and TGF-β inhibits CD95-agonistic antibody-induced apoptosis of activated HSC in culture.27Saile B. Matthes N. Knittel T. Ramadori G. Transforming growth factor beta and tumor necrosis facto alpha inhibit both apoptosis and proliferation of activated rat hepatic stellate cells.Hepatology. 1999; 30: 196-202Crossref PubMed Scopus (159) Google Scholar On the basis of these reported findings and our present study, it is likely that TGF-β is required for the activated HSC to survive. Consequently, fibrogenesis was markedly inhibited in the AdTβ-TR–treated livers despite activation of HSC in the initial stage after injury. To judge from our findings, anti–TGF-β intervention ought to be superior to anti–MCP-1 therapy for treating liver cirrhosis patients, most of whom already have some degree of fibrosis or injury. This issue is now under further investigation in our laboratory. Marra et al28Marra F. Romanelli R.G. Giannini C. Failli P. Pastacaldi S. Arrighi M.C. Pinzani M. Laffi G. Montalto P. Gentilini P. Monocyte chemotactic protein-1 as a chemoattractant for human hepatic stellate cells.Hepatology. 1999; 29: 140-148Crossref PubMed Scopus (230) Google Scholar reported that MCP-1 enhances the migration of HSC in culture. Moreover, it has been reported that HSC themselves produce MCP-129Xu Y. Rojkind M. Czaja M.J. Regulation of monocyte chemoattractant protein 1 by cytokines and oxygen free radicals in rat hepatic fat-storing cells.Gastroenterology. 1996; 110: 1870-1877Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 30Marra F. Valente A.J. Pinzani M. Abboud H.E. Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines.J Clin Invest. 1993; 92: 1674-1680Crossref PubMed Scopus (187) Google Scholar and that TGF-β induces the secretion of MCP-1.29Xu Y. Rojkind M. Czaja M.J. Regulation of monocyte chemoattractant protein 1 by cytokines and oxygen free radicals in rat hepatic fat-storing cells.Gastroenterology. 1996; 110: 1870-1877Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Together with our present study, it is likely that MCP-1 stimulates liver fibrogenesis by 2 mechanisms: (1) MCP-1 induces macrophage infiltration, and macrophages secrete an activating factor(s) for HSC; (2) MCP-1 acts directly on activated HSC to modulate their function. In favor of this notion, it has been reported that in cultured skin fibroblasts, MCP-1 increases the gene expressions of α1 (I) procollagen and TGF-β31Gharaee-Kermani M. Denholm E.M. Phan S.H. Costimulation of fibroblast collagen and transforming growth factor beta1 gene expression by monocyte chemoattractant protein-1 via specific receptors.J Biol Chem. 1996; 271: 17779-17784Crossref PubMed Scopus (409) Google Scholar and of matrix metalloproteinase 1 and 2 (and of their inhibitor, tissue inhibitor of metalloproteinase 1)32Yamamoto T. Eckes B. Mauch C. Hartmann K. Krieg T. Monocyte chemoattractant protein-1 enhances gene expression and synthesis of matrix metalloproteinase-1 in human fibroblasts by an autocrine IL-1 alpha loop.J Immunol. 2000; 164: 6174-6179PubMed Google Scholar and that MCP-1 stimulates the proliferation of cultured vascular smooth muscle cells.33Selzman C.H. Miller S.A. Zimmerman M.A. Gamboni-Robertson F. Harken A.H. Banerjee A. Monocyte chemotactic protein-1 directly induces human vascular smooth muscle proliferation.Am J Physiol Heart Circ Physiol. 2002; 283: H1455-H1461Crossref PubMed Scopus (76) Google Scholar Collectively, these observations suggest that MCP-1 secreted from macrophages and from HSC themselves may facilitate the production of TGF-β, as well as of matrix metalloproteinases and their inhibitors, thereby enhancing inflammation and tissue remodeling (fibrogenesis). We expressed 7ND in livers expecting that it would inhibit MCP-1 as a dominant-negative mutant.16Zhang Y. Rollins B.J. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.Mol Cell Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (145) Google Scholar It has been reported that a 75:1 molar ratio of 7ND/wild-type MCP-1 is needed for a 50% inhibition of monocyte chemotaxis in vitro.16Zhang Y. Rollins B.J. A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.Mol Cell Biol. 1995; 15: 4851-4855Crossref PubMed Scopus (145) Google Scholar In our setting, the amount of 7ND was substantially higher than that of rat MCP-1 in both sera and livers for at least 2–2.5 weeks under DMN treatment (Figure 2). We have not yet determined how much 7ND is required to inhibit the actions of MCP-1 in vivo, specifically in the case of the DMN-injured rat liver. Thus, it is not certain whether the actions of MCP-1 were indeed inhibited in our experiments or whether the observed inhibition of macrophage infiltration was indeed achieved via a suppression of MCP-1 by 7ND. We would like to add that numerous reports (13, to our knowledge) have been published in which the same 7ND construct as that used in this study was introduced (either by direct injection or by electroporation with an expression plasmid) into various animal models, and suppression of macrophage infiltration and some biological effects were seen in every one of these reports. In 3 of these 13 studies, both 7ND and endogenous MCP-1 proteins in serum were measured, and the values obtained (7ND/MCP-1) were 220/71 pg/mL,23Egashira K. Zhao Q. Kataoka C. Ohtani K. Usui M. Charo I.F. Nishida K. Inoue S. Katoh M. Ichiki T. Takeshita A. Importance of monocyte chemoattractant protein-1 pathway in neointimal hyperplasia after periarterial injury in mice and monkeys.Circ Res. 2002; 90: 1167-1172Crossref PubMed Scopus (157) Google Scholar 226/85 pg/mL,24Inoue S. Egashira K. Ni W. Kitamoto S. Usui M. Otani K. Ishibashi M. Hiasa K. Nishida K. Takeshita A. Anti-monocyte chemoattractant protein-1 gene therapy limits progression and destabilization of established atherosclerosis in apolipoprotein E-knockout mice.Circulation. 2002; 106: 2700-2706Crossref PubMed Scopus (238) Google Scholar and 124/92 pg/mL25Ni W. Kitamoto S. Ishibashi M. Usui M. Inoue S. Hiasa K. Zhao Q. Nishida K. Takeshita A. Egashira K. Monocyte chemoattractant protein-1 is an essential inflammatory mediator in angiotensin II-induced progression of established atherosclerosis in hypercholesterolemic mice.Arterioscler Thromb Vasc Biol. 2004; 24: 534-539Crossref PubMed Scopus (97) Google Scholar (all in mice). We detected a peak value of 528 ± 182 pg/mL for 7ND and 62 ± 12 pg/mL for endogenous rat MCP-1. Both this peak value for 7ND and the ratio between 7ND and endogenous MCP-1 are the highest among the values reported in the literature so far. In summary, we have shown that the macrophages that infiltrate into livers immediately after an initial injury are critical both for HSC activation and for the subsequent fibrogenesis, and we also showed that TGF-β, which is required for activated HSC to survive, is not an activating factor for HSC (at least in this situation). Macrophages may themselves secrete an activating factor(s) for HSC.
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