- Email: [email protected]
Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers
Hepatic macrophages promote the neutrophil-dependent resolution of fibrosis in repairing cholestatic rat livers Mark W. Harty, PhD,a Elaine F. Papa, BS,a Hannah M. Huddleston, MD,b Ezekiel Young, BS,a Samantha Nazareth, BS,a Charles A. Riley,a Grant A. Ramm, PhD,c Stephen H. Gregory, PhD,d and Thomas F. Tracy Jr, MD,e Providence, RI, Indianapolis, Ind, Brisbane, Queensland, Australia
Background. Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. Methods. Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl3) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. Results. GdCl3 treatment for either 24 or 48 hours before decompression reduced the numbers of ED2+ Kupffer cells and ED1+ inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl3 pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl3 treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. Conclusion. We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation. (Surgery 2008;143:667-78.) From the Department of Surgery, The Warren Alpert Medical School of Brown University and Hasbro Children’s Hospitals,a Providence, RI; the Department of Dermatology, Indiana University School of Medicine,b Indianapolis, Ind; The Hepatic Fibrosis Group, The Queensland Institute of Medical Research,c Brisbane, Queensland, Australia; the Department of Medicine, The Warren Alpert Medical School of Brown University, Rhode Island Hospital,d Providence, RI; and the Departments of Surgery and Pediatrics, The Warren Alpert Medical School of Brown University, Rhode Island and Hasbro Children’s Hospitals,e Providence, RI
Supported in part by NIH Grants R01 DK46831 (TFT), R01DK068097 (SHG) and RR-P20 RR17695 from the Institutional Development Award (IDeA) Program of the National Center for Research Resources (COBRE). Accepted for publication January 31, 2008. Reprint requests: Thomas F Tracy, Jr., MD, Vice Chairman, Department of Surgery, The Warren Alpert Medical School of Brown University, Pediatric Surgeon-in-Chief, Hasbro Children’s Hospital, Room 147, 593 Eddy Street, Providence, RI 02903. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2008.01.008
CHOLESTATIC LIVER DISEASE, resulting from extraand/or intrahepatic bile duct obstruction, predisposes adults and children to the development of cirrhosis. When treating patients with cholestatic liver disease, clinicians have no guarantee that medical or operative intervention will obviate ongoing injury or initiate successful repair. Over the past 20 years, numerous studies have clarified the factors that initiate injury and promote hepatic fibrosis.1,2 The mechanisms of repair, however, remain largely undefined. Extra- and/or intrahepatic bile duct obstruction induces a stereotypical pattern of liver injury SURGERY 667
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composed of bile duct epithelial cell hyperplasia, periportal fibrosis, and an inflammatory cell infiltrate3,4 that includes monocytes5 and neutrophils.6,7 Resident tissue macrophages (Kupffer cells) are thought to be key mediators of cholestatic liver injury.8-10 Activated Kupffer cells are known to stimulate hepatic stellate cells, which preferentially deposit collagen type-I, leading to a marked increase in collagen type-I relative to other matrix components.11 Neutrophils constitute a significant percentage of the cells in the inflammatory infiltrate characteristic of cholestatic liver injury.7,12 Conflicting results regarding the potential role of neutrophils in liver repair have risen from different experimental systems. Neutrophil depletion studies demonstrated reduced tissue injury in models of sepsis,13 cholestatic liver injury,12 and viral infection.14 These studies identified an exaggerated neutrophil response as an important mediator of tissue injury. In contrast, similar models of sepsis and cholestasis suggest a more complex role for neutrophils that includes a protective response from or after injury.15,16 Using a rat model of extrahepatic cholestasis that rapidly develops collagen fibrosis,17,18 we reported that increased neutrophil numbers are associated with collagen degradation after liver injury.17 We also reported altered matrix metabolism and inflammatory cell responses after Kupffer cell depletion in injured livers.19 Using the same rat model of reversible extrahepatic cholestasis, we undertook the series of experiments described herein to delineate the cellular and molecular mechanisms of liver repair. Specifically, we sought to elucidate the complex interactions of hepatic macrophages and neutrophils in fibrotic repair after cholestatic liver injury. Here, we report that the hepatic macrophage population plays a key role in resolving liver injury by promoting the accumulation of neutrophils. We suggest that neutrophils, which are known to have collagenase, gelatinase, and elastase activity, may be important in the degradation of injury induced fibrosis. MATERIAL AND METHODS Animals. Adult male rats (225--250 g, Harlan Sprague-Dawley, Indianapolis, Ind) were housed in an artificial 12-hour light--dark cycle with access to rat chow and water ad libitum according to the NIH publication Guide for the Care and Use of Laboratory Animals. Experiments were carried out in compliance with guidelines prescribed by the Institutional Animal Care and Use Committee of Rhode Island Hospital and The Warren Alpert Medical School of Brown University.
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Biliary obstruction (liver injury). Animals (n = 77) were divided into either study (n = 52) or sham-operated (n = 25) groups. The study group underwent bile duct loop suspension surgery for biliary obstruction as previously described.19,20 Briefly, animals were anesthetized with vaporized isoflurane, weighed, and prepared for surgery. A midline laparotomy was performed and the common bile duct was dissected sufficiently to allow passage of a 5-cm length of silicone vessel loop (Surg-I-Loop, Scanlan International, Saint Paul, Minn). A midsegment of this loop was premarked to 1 cm and the ends were brought through each side of the abdominal wall, 1 cm lateral to the midline at the costochondral margin. The vessel loop was stretched and sutured to the perichondrium bilaterally at the premarked points displacing the common bile duct ventrally. Bile duct obstruction was verified by visual inspection and the abdomen was closed. Sham-operated animals underwent an identical laparotomy where the common bile duct was identified, but not obstructed. Obstructed and sham-operated animals were divided into treatment groups that received intravenously via the dorsal penile vein saline (n = 37) or GdCl3 (10 mg/kg) at either 1 (n = 21) or 2 (n = 19) days before decompression (Fig 1). Biliary decompression (liver repair). After 7 days of cholestasis, rats were anesthetized, weighed, and prepared for decompression/repair. The skin of the midline incision was opened exposing the vessel loop. The anchoring sutures were cut and the vessel loop removed. The abdominal skin was closed and the animals were allowed to recover. This time point is referred to as day 0 of repair. Necropsy. Animals were necropsied after 7 days of cholestatic injury (day 0 of repair; n = 29) or 2 days after decompression (day 2 of repair; n = 23). All sham-operated groups (n = 25) were necropsied on day 0 of repair (Fig 1). Rats were anesthetized and weighed, the abdomen was opened with a U-shaped incision, and the abdominal wall was reflected superiorly. Blood (5 mL) was collected for serum bilirubin by venipuncture of the inferior vena cava. Hepatectomy was performed and livers were divided and processed as follows: frozen with dry ice in OCT Compound (Sakura, Torrance, Calif); flash frozen in liquid nitrogen; fixed in 10% neutral phosphate buffered formalin (Fisher Scientific, Fair Lawn, NJ); or fixed in formalin free zinc fixative (BD Pharmingen, San Diego, Calif). We have previously demonstrated that urine color, weight loss, and increased bilirubin levels reflect the degree of biliary obstruction achieved in this model.5,19 Here, we utilized urine
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color, weight, and bilirubin, quantified using a commercial kit (Sigma-Aldrich, St. Louis, Mo), as markers of the progression of cholestatic injury and subsequent repair. Animals that did not demonstrate obstruction or decompression were excluded from the study. Histochemistry, immunohistochemistry, and image analysis. Formalin- and zinc-fixed tissues were embedded in paraffin and sectioned at 7 mm. Histochemistry utilized picric acid Sirius red for collagen or naphthol AS-D chloracetate esterase (esterase; Sigma-Aldrich) specific for granulocytes.21 Mouse anti-rat CD68 monoclonal antibody (Serotec Inc., Raleigh, NC) specific for the majority of (ED1+) macrophages,22 mouse anti-rat CD163 monoclonal antibody (Serotec Inc.) specific for (ED2+) Kupffer cells,22,23 FITC-conjugated rabbit anti-rat neutrophil polyclonal antibody (Accurate Chemical, Westbury, NY), and mouse antirat neutrophil monoclonal antibody (RP3, a gift from F. Sendo, Yamagata University School of Medicine, Yamagata, Japan)24 were used for immunohistochemistry. Sirius red stained images were scanned (SprintScan 35 Plus, Polaroid, Cambridge, Mass) into a PowerMac G4 (Apple Computer, Inc., Cupertino, Calif) using a PathScan Enabler optical card (Meyer Instruments, Inc., Houston, Tex). NIH ImageJ imaging software (NIH, Bethesda, Md) was used to determine total tissue section area and collagen area. Results are expressed as a percentage of total section area. Liver sections stained for ED1, ED2, or esterase were digitally imaged at 3400 with a minimum of 10 fields per section. Trained laboratory personnel (blinded to the groups) quantified the resident hepatic macrophages (ED2+), recruited mononuclear phagocytes (ED1+), and neutrophils (esterase+) in each section. Data are expressed as cells per square millimeter. Tissue sections stained with RP3 antibody were labeled secondarily with Texas Red-conjugated goat anti-mouse IgM (Vector Laboratories, Burlingame, Calif). Fluorescent images of RP3 (Texas Red) and anti-neutrophil (FITC) regions of rat liver were digitally imaged at 3200 (BD IPlabs, Rockville, Md) and analyzed for distribution. Real-time RT-PCR methods and analyses. Blocks of liver tissue were snap frozen in liquid nitrogen. Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Inc., Valencia, Calif). RNA quality and quantity were determined using the RNA 6000 Nano LabChip kit on the 2600 BioAnalyzer (Agilent Technologies, Santa Clara, Calif). Five micrograms of total RNA was converted into cDNA using First Strand cDNA Synthesis kit (GE Healthcare
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Fig 1. Experimental design. Biliary obstruction surgery was performed and cholestatic injury occurred over the after 7 days. Saline or gadolinium chloride (10 mg/kg) was administered IV 1 or 2 days before bile duct decompression and liver repair. The day of decompression is defined here as day 0. Rats were humanely killed after 0 and 2 days of repair for each group.
Life Sciences, Piscataway, NJ) with random primers. The Quantitect SYBR Green real-time PCR kit (Qiagen, Inc.) was used for all quantitative PCR and run on the Stratagene MX4000 thermocycler. Primer sequences (shown in the Table) were chosen using appropriate GENBANK sequences, NCBI (www.ncbi.nih.gov), and Primer 3 software (Whitehead Institute for Biomedical Research; source code available at http://fokker.wi.mit.edu/ primer3/).25 Sequences were purchased from Integrated DNA Technologies, Inc (Coralville, Ia). Melting curves validated the utility and specificity of each primer set. Data were evaluated using the Comparative Ct Method (2( DDCt)) of relative quantification standardized to 18S rRNA and are reported as fold increases over sham controls. Because of the relative abundance of 18S rRNA, standardizing samples were diluted 1:1000. The results were equivalent to those obtained using GAPDH as an alternate reference gene (data not shown). In situ hybridization. In situ hybridization was performed on 7-mm frozen liver sections using probes (Table) designed and purchased from GeneDetect (Bradenton, Fla). Hybridization was visualized using the Dako Kit (Dako North America; Carpentaria, Calif) according to manufacturers instructions. Antisense and sense probes were run simultaneously to control for nonspecific hybridization. Statistics. Data are expressed as mean values ± standard error of the mean. Statistically significant differences were determined by ANOVA and Fisher’s PSLD (StatView 4.1+, SAS Institute Inc., Cary, NC).
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Table. Primer list for quantitative RT-PCR and probe for in situ hybridization Primer name 18S up 18S down CINC-1 up CINC-1 down MIP-2 up MIP-2 down ICAM-1 up ICAM-1 down Probe name CINC-1 anti-sense CINC-1 sense
Sequence CGA AAG CAT TTG CCA AGA AT AGT CGG CAT CGT TTA TGG TC ACC CAA ACC GAA GTC ATA GC ACT TGG GGA CAC CCT TTA GC GGG GGA GTT GGG TAC TGA CT CCT TGA AAG CCC TCT GAC TG GGA GGC CCT AAA ACT CAA GG GAG GTG GGT GAG GGG TAA AT
CTT CAG GGT CAA GGC AAG CCT CGC GAC CAT TCT TGA GTG GTG TGG CTA TGA TCA TAG CCA CAC TCA AGA ATG GTC GCG AGG CTT GCC TTG ACC CTG AAG
Fig 2. Fibrotic injury. Liver sections derived from rats on days 5, 7, 14, and 21 after biliary obstruction were stained with Sirius red and digitally imaged. Representative images from sham and 7- and 21-day injured animals illustrate staining and increased matrix (original magnification, 340). The amount of collagen is expressed as a percentage of total area. Tissue collagen was elevated compared to sham-operated controls. #P = .05; *P # .05 (ANOVA).
RESULTS Biliary obstruction, fibrosis, and the response of hepatic macrophages. During the 7-day period that followed biliary obstruction (day 0 of repair), collagen deposition in the livers of rats increased approximately 4-fold relative to sham-operated control animals. This increase represented 60%
of maximum injury that occurred after 21 days of obstruction (Fig 2). The numbers of ED1+ inflammatory macrophages and ED2+ Kupffer cells were elevated 6- and 3-fold, respectively, in the livers of bile duct-obstructed animals (Fig 3). Saline-treated animals showed a significant and rapid decline in
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Fig 3. Response of hepatic macrophages to cholestatic liver injury and repair. ED1- and ED2-stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with saline or gadolinium chloride at 24 or 48 hours before repair were digitally imaged and the positively stained cells were counted. Representative images of ED1 (A) and ED2 (C) stained liver sections from injured animals illustrate staining quality (original magnification, 3400). The numbers of ED1+ (B) and ED2+ (D) cells are increased significantly relative to the numbers found in sham-operated control animals. *P # .05 (ANOVA).
both inflammatory macrophages and Kupffer cells after biliary decompression. These macrophages were distributed throughout the parenchymal sinusoids and portal tracts during cholestatic injury and were restricted primarily to the parenchymal sinusoids during repair. Treatment with GdCl3 on day 1 before decompression diminished the injury-induced increase in ED1+ cells and totally blocked the increase in ED2+ cells otherwise observed both at the time of decompression (day 0) and after 2 days of repair. On the other hand, treatment with GdCl3 on day 2 before decompression abrogated the increase observed in ED2+, as well as ED1+, cells on day 0 and after 2 days of repair. Depletion of hepatic macrophage by treatment with GdCl3 on day 1 or 2 before biliary decompression did not alter the amount of collagen detected in the liver after 7 days of injury (day 0 of repair; Fig 4). Moreover, the decreased collagen assessed on day 2 of repair was not altered by the depletion of hepatic macrophages with GdCl3 administered on day 1 before decompression. When GdCl3 was administered 2 days before biliary decompression, however, macrophage depletion impaired liver repair and collagen levels
persisted. Taken together, these findings demonstrate the response of hepatic macrophages to cholestatic liver injury and suggest their indirect, time-dependent role in liver repair and the resolution of fibrosis. Hepatic macrophages modulate the response of neutrophils to cholestatic liver injury. Consistent with our previous observations,17 the number of esterase-positive neutrophils was significantly elevated in the livers of bile duct-obstructed animals and remained elevated after biliary decompression (Fig 5). Depletion of hepatic macrophages on day 1 before repair did not alter the neutrophil counts determined at the time of biliary decompression (day 0). In sharp contrast, neutrophil numbers were decreased significantly on day 0 in the liver of rats rendered macrophage-deficient by pretreatment with GdCl3 on day 2 before repair. Neutrophil numbers in the livers of all bile ductobstructed rats rendered macrophage deficient were diminished approximately 2-fold relative to nondeficient animals on day 2 of repair, and were not different from the number determined in saline-treated, sham-operated rats. Thus, hepatic macrophages modulate the response of neutrophils
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Fig 4. Fibrotic repair. A, Sirius red–stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with either saline or gadolinium chloride for 24 or 48 hours before bile duct decompression were digitally imaged; the amount of collagen is expressed as a percentage of total area. Biliary obstruction significantly increased the deposition of collagen in all groups determined on day 0 of repair relative to sham-operated animals. *P # .05 (ANOVA). The collagen content was significantly decreased in the saline-treated and 1 day GdCl3-treated groups after 2 days of repair (D, P # .05; Fisher’s PLSD), and significantly less than that observed in the 2 day GdCl3-treated group (P # .05; ANOVA). B, Representative images of Sirius red—stained liver sections from all groups reflect differences depicted in the histograms (original magnification, 3200).
to cholestasis; the consequences of macrophage depletion on neutrophil accumulation in cholestatic livers are most pronounced on day 2 or greater post-GdCl3 treatment. Macrophages modulate proinflammatory gene expression in cholestatic livers. Gene expression during periods of cholestatic injury and repair was evaluated by real-time RT-PCR. Consistent with the findings of other groups,7 neutrophil chemoattractant CINC-1 (CXCL1) mRNA expression increased significantly (10-fold) after cholestatic liver injury in saline-treated animals (Fig 6, A). Furthermore,
CINC-1 message expression remained elevated in the livers of saline treated rats during 2 days of repair. Hepatic CINC-1 mRNA expression was not reduced at either 0 or 2 days of repair in animals treated with GdCl3 and rendered macrophage-deficient 1 day before bile duct decompression. In sharp contrast, CINC-1 message expression at both 0 and 2 days of repair was reduced (50% relative to saline-treated controls) in rats rendered macrophage-deficient 2 days before biliary decompression. In contrast with CINC-1, neutrophil chemoattractant macrophage inflammatory protein
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Fig 5. Response of infiltrating neutrophils to cholestatic liver injury and repair. Neutrophil esterase stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with either saline or GdCl3 at 24 or 48 hours before repair were digitally imaged and the positively stained cells were counted. *Significantly more than comparably treated, sham-operated animals (P # .05). D Significantly less than saline-treated controls (P # .05).
(CXCL2) message expression did not change at any time point or with any treatment (Fig 6, B). The mechanisms of inflammatory cellular infiltration require the upregulation of both chemotactic signaling and adhesion factors, for example, intercellular adhesion molecule-1 (ICAM-1), a molecule that regulates neutrophil trafficking. ICAM-1 mRNA expression after 0 and 2 days of repair was elevated 5-fold in saline-treated, bile duct-ligated rats relative to comparably treated sham-operated controls (Fig 6, C). GdCl3 administered on day 1 before decompression reduced ICAM-1 mRNA expression (50% decrease) on day 0 of repair; however, expression was significantly elevated in this group at 2 days of repair. GdCl3 administered 2 days before repair inhibits ICAM-1 message expression at both 0 and 2 days of repair. Thus, the increased accumulation of neutrophils in the liver of saline-treated animals coincided with the elevated expression of CINC1 and ICAM-1, whereas reductions in expression of both CINC-1 and ICAM-1 after GdCl3 administered 2 days before repair coincided with reductions in neutrophil accumulation. Interestingly, CINC-1 and ICAM-1 expression after GdCl3 administration 2 days before repair was unsynchronized, which resulted in a changing neutrophil response during repair. CINC-1 message expression and localization of inflammatory neutrophils in portal tracts. CINC1 mRNA was localized to the portal tracts of injured
Fig 6. Quantitative real-time RT-PCR. CINC-1 (A), macrophage inflammatory protein (MIP-2; B), and ICAM-1 (C) mRNA expression was quantified in liver homogenates derived from sham, injured (day 0) and repairing (day 2) animals pretreated with saline or GdCl3 for 24 or 48 hours before repair. *Significantly increased relative to sham-operated controls treated comparably (P # .05; ANOVA).
and repairing livers using in situ hybridization (Fig 7). Widespread antisense hybridization was found throughout the portal tracts in sham, injured, and repairing animals; notably, hybridization was excluded from bile duct epithelial cells and cells in the parenchyma. The depletion of hepatic macrophages 2 days before biliary decomposition did not alter the distribution of CINC-1 mRNA
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Fig 7. In situ hybridization for CINC-1. Representative liver sections obtained from sham, injured (day 0) and repairing (day 2) animals treated with either saline or gadolinium chloride (48 hours before repair) were labeled with sense or anti-sense CINC1 mRNA (original magnification, 3400).
hybridization at any time point. Indeed, visualization of the hybridized product was more intense than in saline-treated controls. No CINC-1 mRNA was found in the parenchyma, and all sense hybridization controls were negative. The distribution of esterase-positive neutrophils in cholestatic livers correlated with the localization of CINC-1 mRNA; that is, the cells accumulated predominantly in the portal tracts at all time points (Fig 8, A). Interestingly, neutrophil distribution was frequently in close proximity to bile ducts. Specific labeling of rat neutrophils using rabbit antirat neutrophil (Fig 8, B) and RP3 (Fig 8, C)
confirmed the esterase histochemistry in terms of both neutrophil counts (data not shown) and distribution. DISCUSSION During cholestasis, increased concentrations of bile acids and toxins in the liver result in the activation of Kupffer and hepatic stellate cells. This is accompanied by the release of inflammatory cytokines,26-29 the infiltration of circulating monocytes and neutrophils,9 and net collagen deposition by activated myofibroblasts in the sinusoids and portal triads.30-34 If not attenuated, these
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Fig 8. Neutrophil distribution. Neutrophils in liver sections derived from sham, injured (repair day 0), and repairing (day 2) animals after saline or GdCl3 treatment were stained with naphthol AS-D chloroacetate esterase (A), FITC-conjugated rabbit anti-rat PMN polyclonal antibody (B), or mouse anti-rat neutrophil monoclonal antibody (RP3; C). (Original magnification, 3200.)
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events can lead to cirrhosis and subsequent endstage liver disease. Although the mechanisms that underlie cholestatic liver injury are becoming increasingly clearer, those that contribute to repair are less well-understood and more difficult to study. For clinical practice, surgical and other interventional decompression techniques have been developed to rapidly reverse defects in bile flow. Liver repair, however, is often complicated by progressive hepatic damage in the absence of overt cholestatic stimuli.29,32,35 For infants, children, and adults, progressive liver disease can increase clinical morbidity and mortality associated with biliary atresia, choledochal cysts, sclerosing cholangitis, and other biliary obstructive etiologies. Persistent biliary hyperplasia, fibrosis, and inflammatory responses characterize the cellular and molecular consequences associated with inadequate repair.29,32,35 Previously, we reported that the administration of GdCl3 and the depletion of hepatic macrophages throughout the course of cholestatic liver injury delayed repair after ductular decompression in our rat model.19 Persistent liver fibrosis due to the loss in macrophage activity was associated with the delayed resolution of ductal hyperplasia. By administering GdCl3 and eliminating macrophages just before decompression in the present study, we focused specifically on the macrophagedependent mechanisms that contribute to repair. Based on previous work implicating neutrophils and neutrophil collagenase in cholestatic liver repair,17 we speculated that this approach would allow us to delineate those critical interactions that occur between hepatic macrophages and immigrating neutrophils. We found that both recruited (ED1+) and resident (ED2+) hepatic macrophages were depleted and remained at or below sham levels through 2 days of repair after treatment with GdCl3 on day 1 before biliary decompression. After such treatment, the decline in neutrophil cell counts within the portal tracts of livers was not evident until day 2 of repair. However, treatment with GdCl3 on day 2 before decompression reduced the number of neutrophils, as well as the numbers of recruited and resident macrophages, in the liver at the time of repair. Thus, in the absence of hepatic macrophages, the decline in number of neutrophils recruited to the liver requires approximately 48 hours (Fig 5), a timeframe consistent with the normal survival rate of extravasated neutrophils in tissues.36,37 Morphometric analysis of collagen deposited after 7 days of cholestatic liver injury demonstrated levels that were significantly elevated relative to
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sham-operated control animals. After 2 days of repair, these levels were reduced to near those observed in shams. Rats treated with GdCl3 on day 1 before decompression (which had no effect on the number of neutrophils present in the liver on day 0 of repair) showed a comparable reduction in collagen levels assessed on day 2 of repair. In contrast, treatment with GdCl3 on day 2 before biliary decompression significantly reduced the number of neutrophils present in the liver on day 0 of repair and blocked the reduction in collagen normally seen by day 2 of repair. Taken together, these findings indicate that liver repair can occur in the absence of hepatic macrophages provided the persistence of neutrophils. It is intriguing that neutrophils, which promote tissue damage in a number of animal models including bile duct ligation and cholestatic liver injury in mice,12-14,38 can also contribute to repair. Indeed, in this regard, several studies indicate that the rat and mouse models of cholestatic liver injury differ significantly in a number of ways. In the mouse model, for example, necrotic regions develop rapidly in the liver after biliary obstruction, whereas fibrosis occurs only secondarily at a much later time.9,39 Cholestatic liver injury in the rat model, on the other hand, progresses in a manner analogous to that seen in humans; that is, proliferation of the nonparenchymal cell population and development of fibrosis that progresses in the absence of necrotic lesions.17,29 Moreover, in sharp contrast to the mouse model, neutrophil depletion fails to alter the course of tissue injury in cholestatic rats.7 Biliary obstruction increased the expression of messages for CINC-1, an important chemokine for neutrophil recruitment,40-44 and ICAM-1, required for the firm adhesion of inflammatory cells to the endothelial barrier and extravasation in conjunction with chemokine signaling.45-47 Increases in CINC-1 and ICAM-1 mRNA expression are consistent with the signaling requirement for immigration to inflammatory sites. As we reported previously,17 neutrophil counts remain elevated while ED1+ and ED2+ hepatic macrophages return to sham levels in the repairing rat liver after cholestatic injury. Additionally, we showed here that the expression both CINC-1 and ICAM-1 mRNA also remains elevated after 2 days of normal repair demonstrating a persistent chemotactic signal that continues well past operative repair and that sustains the presence of neutrophils in the liver. Loss of synchronization in the expression of CINC-1 and ICAM-1 mRNAs after GdCl3 treatment show that chemotactic signaling responds within
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the first 24 hours of treatment. This timeframe is consistent with that previously reported for GdCl3-mediated hepatic macrophage depletion, which requires 24 hours to complete and must be repeated every 48 hours to maintain in the rat.19 Taken together, these data support the conjecture that chemokine and adhesion factor expression require synchronous upregulation and their production sustained to support the continued presence of neutrophils in repairing cholestatic rat livers. Collagen deposited in the livers of cholestatic rats is principally localized to the expanding portal tracts by activated hepatic stellate cells or myofibroblasts. Desmin and a-SMA immunohistochemistry in this model has demonstrated the classic pathway, activation of hepatic stellate cells, necessary for net matrix deposition.48 Herein, we show Sirius red staining, which indicates that our gadolinium treatment does not interfere with the accumulation of matrix during injury. Therefore, differences in matrix during repair are most likely due to changes in mechanisms of matrix degradation as opposed to net deposition. In this study, infiltrating neutrophils also localize to the expanding portal tracts of the liver during cholestatic injury and normal repair. Neutrophils, however, seem to accumulate preferentially near the remodeling biliary epithelial cells. Using in situ hybridization, we localized CINC1 mRNA transcripts to the expanded nonparenchymal cells of the portal tract. Although CINC1 mRNA expression was distributed throughout the portal tract, it was predominately excluded from the bile duct epithelial cells. Our previous study indicated that neutrophils are an important mediator of cholestatic liver repair in rats.17 The data presented herein demonstrate that neither recruited nor resident hepatic macrophages are directly involved in the mechanisms of liver repair. Rather, hepatic macrophages upregulate the production of proinflammatory chemokines (eg, CINC-1) and adhesion molecules (ICAM-1) that promote neutrophil infiltration and localization at sites of tissue remodeling and fibrotic repair. Employing neutrophil-depleting antibody in vivo, experiments ongoing in our laboratory will delineate the specific role of neutrophils and the neutrophil-dependent mechanisms required for reversing fibrosis and for liver repair after cholestatic injury. REFERENCES 1. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis.
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Mechanisms and treatment strategies. N Engl J Med 1993;328:1828-35. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005; 115:209-18. Tracy TF Jr, Goerke ME, Bailey PV, Sotelo-Avila C, Weber TR. Growth-related gene expression in early cholestatic liver injury. Surgery 1993;114:532-7. Slott PA, Liu MH, Tavoloni N. Origin, pattern, and mechanism of bile duct proliferation following biliary obstruction in the rat. Gastroenterology 1990;99:466-77. Roggin KK, Kim JC, Kurkchubasche AG, Papa EF, Vezeridis AM, Tracy TF. Macrophage phenotype during cholestatic injury and repair: the persistent inflammatory response. J Pediatr Surg 2001;36:220-8. Saito JM, Maher JJ. Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant. Gastroenterology 2000;118:1157-68. Saito JM, Bostick MK, Campe CB, Xu J, Maher JJ. Infiltrating neutrophils in bile duct-ligated livers do not promote hepatic fibrosis. Hepatol Res 2003;25:180-91. Aldana PR, Goerke ME, Carr SC, Tracy TF Jr. The expression of regenerative growth factors in chronic liver injury and repair. J Surg Res 1994;57:711-7. Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ. Mechanisms of hepatotoxicity. Toxicol Sci 2002; 65:166-76. Gehring S, Dickson EM, San Martin ME, van Rooijen N, Papa EF, Harty MW, et al. Kupffer cells abrogate cholestatic liver injury in mice. Gastroenterology 2006;130: 810-22. Tsukada S, Parsons CJ, Rippe RA. Mechanisms of liver fibrosis. Clin Chim Acta 2005;364:33-60. Gujral JS, Farhood A, Bajt ML, Jaeschke H. Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice. Hepatology 2003;38:355-63. Hewett JA, Schultze AE, VanCise S, Roth RA. Neutrophil depletion protects against liver injury from bacterial endotoxin. Lab Invest 1992;66:347-61. Sitia G, Isogawa M, Kakimi K, Wieland SF, Chisari FV, Guidotti LG. Depletion of neutrophils blocks the recruitment of antigen-nonspecific cells into the liver without affecting the antiviral activity of hepatitis B virus-specific cytotoxic T lymphocytes. Proc Natl Acad Sci U S A 2002;99:13717-22. Hoesel LM, Neff TA, Neff SB, Younger JG, Olle EW, Gao H, et al. Harmful and protective roles of neutrophils in sepsis. Shock 2005;24:40-7. Arai T, Yoshikai Y, Kamiya J, Nagino M, Uesaka K, Yuasa N, et al. Bilirubin impairs bactericidal activity of neutrophils through an antioxidant mechanism in vitro. J Surg Res 2001;96:107-13. Harty MW, Huddleston HM, Papa EF, Puthawala T, Tracy AP, Ramm GA, et al. Repair after cholestatic liver injury correlates with neutrophil infiltration and matrix metalloproteinase 8 activity. Surgery 2005;138:313-20. Svegliati-Baroni G, Ridolfi F, Caradonna Z, Alvaro D, Marzioni M, Saccomanno S, et al. Regulation of ERK/JNK/ p70S6K in two rat models of liver injury and fibrosis. J Hepatol 2003;39:528-37. Roggin KK, Papa EF, Kurkchubasche AG, Tracy TF Jr. Kupffer cell inactivation delays repair in a rat model of reversible biliary obstruction. J Surg Res 2000;90:166-73. Posner MC, Burt ME, Stone MD, Han BL, Warren RS, Vydelingum NA, et al. A model of reversible obstructive jaundice in the rat. J Surg Res 1990;48:204-10.
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21. Moloney WC, McPherson K, Fliegelman L. Esterase activity in leukocytes demonstrated by the use of naphthol AS-D chloroacetate substrate. J Histochem Cytochem 1960;8:200-7. 22. Damoiseaux JG, Dopp EA, Calame W, Chao D, MacPherson GG, Dijkstra CD. Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 1994;83:140-7. 23. Dijkstra CD, Dopp EA, Joling P, Kraal G. The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology 1985; 54:589-99. 24. Sekiya S, Gotoh S, Yamashita T, Watanabe T, Saitoh S, Sendo F. Selective depletion of rat neutrophils by in vivo administration of a monoclonal antibody. J Leukoc Biol 1989; 46:96-102. 25. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 2000;132:365-86. 26. Decker K. Biologically active products of stimulated liver macrophages (Kupffer cells). Eur J Biochem 1990;192:245-61. 27. Gaillard T, Mulsch A, Busse R, Klein H, Decker K. Regulation of nitric oxide production by stimulated rat Kupffer cells. Pathobiology 1991;59:280-3. 28. Ishizaki-Koizumi S, Sonaka I, Takei Y, Ikejima K, Sato N. The glycine analogue, aminomethanesulfonic acid, inhibits LPSinduced production of TNF-alpha in isolated rat Kupffer cells and exerts hepatoprotective effects in mice. Biochem Biophys Res Commun 2004;322:514-9. 29. Tracy TF Jr, Dillon P, Fox ES, Minnick K, Vogler C. The inflammatory response in pediatric biliary disease: macrophage phenotype and distribution. J Pediatr Surg 1996;31: 121-5. 30. Schaefer B, Rivas-Estilla AM, Meraz-Cruz N, Reyes-Romero MA, Hernandez-Nazara ZH, Dominguez-Rosales JA, et al. Reciprocal modulation of matrix metalloproteinase-13 and type I collagen genes in rat hepatic stellate cells. Am J Pathol 2003;162:1771-80. 31. Benyon RC, Arthur MJ. Mechanisms of hepatic fibrosis. J Pediatr Gastroenterol Nutr 1998;27:75-85. 32. Friedman SL. Liver fibrosis—from bench to bedside. J Hepatol 2003;38(Suppl 1):S38-53. 33. Lewindon PJ, Pereira TN, Hoskins AC, Bridle KR, Williamson RM, Shepherd RW, et al. The role of hepatic stellate cells and transforming growth factor-beta(1) in cystic fibrosis liver disease. Am J Pathol 2002;160:1705-15. 34. Ramm GA, Hoskins AC, Greco SA, Pereira TN, Lewindon PJ. Signals for hepatic fibrogenesis in pediatric cholestatic
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35. 36.
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liver disease: review and hypothesis. Comp Hepatol 2004; 3(Suppl 1):S5. Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000; 46:443-6. Stabel TJ, Fedorka-Cray PJ, Gray JT. Neutrophil phagocytosis following inoculation of Salmonella choleraesuis into swine. Vet Res Commun 2002;26:103-9. Niwa M, Hara A, Kanamori Y, Kohno K, Yoshimi N, Mori H, et al. Comparison of susceptibility to apoptosis induced by rhTNF-alpha and cycloheximide between human circulating and exudated neutrophils. Life Sci 1997;61:205-15. Jaeschke H. Mechanisms of liver injury. II. Mechanisms of neutrophil-induced liver cell injury during hepatic ischemia-reperfusion and other acute inflammatory conditions. Am J Physiol Gastrointest Liver Physiol 2006;290:G1083-8. Miyoshi H, Rust C, Roberts PJ, Burgart LJ, Gores GJ. Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas. Gastroenterology 1999;117:669-77. Maher JJ. Rat hepatocytes and Kupffer cells interact to produce interleukin-8 (CINC) in the setting of ethanol. Am J Physiol 1995;269:G518-23. Maher JJ, Lozier JS, Scott MK. Rat hepatic stellate cells produce cytokine-induced neutrophil chemoattractant in culture and in vivo. Am J Physiol 1998;275:G847-53. Luster AD. Chemokines—chemotactic cytokines that mediate inflammation. N Engl J Med 1998;338:436-45. Ohkubo K, Masumoto T, Horiike N, Onji M. Induction of CINC (interleukin-8) production in rat liver by non-parenchymal cells. J Gastroenterol Hepatol 1998;13:696-702. Stefanovic L, Brenner DA, Stefanovic B. Direct hepatotoxic effect of KC chemokine in the liver without infiltration of neutrophils. Exp Biol Med (Maywood) 2005;230:573-86. Ishihara K, Yamaguchi Y, Uchino S, Furuhashi T, Yamada S, Kihara S, et al. ICAM-1 signal transduction in cells stimulated with neutrophil elastase. Dig Dis Sci 2006;51:2102-12. Diamond MS, Staunton DE, de Fougerolles AR, Stacker SA, Garcia-Aguilar J, Hibbs ML, et al. ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J Cell Biol 1990;111:3129-39. Jaeschke H, Farhood A, Bautista AP, Spolarics Z, Spitzer JJ, Smith CW. Functional inactivation of neutrophils with a Mac-1 (CD11b/CD18) monoclonal antibody protects against ischemia-reperfusion injury in rat liver. Hepatology 1993;17:915-23. Ramm GA, Carr SC, Bridle KR, Li L, Britton RS, Crawford DH, et al. Morphology of liver repair following cholestatic liver injury: resolution of ductal hyperplasia, matrix deposition and regression of myofibroblasts. Liver 2000;20:387-96.
Background. Cholestatic liver injury from extrahepatic biliary obstruction is well characterized by inflammatory and fibrogenic mechanisms. Little is known, however, about mechanisms required to reverse injury and effect liver repair. We sought to determine the cellular and molecular requirements for repair after biliary decompression, focusing on the role of hepatic macrophages in regulating inflammation and matrix resolution. Methods. Male Sprague-Dawley rats underwent bile duct obstruction for 7 days followed by ductular decompression. Rats were treated with gadolinium chloride (GdCl3) to deplete the macrophage populations 24 or 48 hours before decompression. Liver tissue obtained at the time of decompression or after 2 days of repair was processed for morphometric analysis, immunohistochemistry, quantitative RT-PCR and in situ hybridization. Results. GdCl3 treatment for either 24 or 48 hours before decompression reduced the numbers of ED2+ Kupffer cells and ED1+ inflammatory macrophages in obstructed livers; only 48 hours of pretreatment, however, reduced the neutrophil counts. Furthermore, 48-hour GdCl3 pretreatment blocked matrix degradation. Quantitative polymerase chain reaction demonstrated decreased cytokine-induced neutrophil chemoattractant-1 (CINC-1; CXCL1) and intercellular adhesion molecule-1 mRNA expression after GdCl3 treatment and the elimination of hepatic macrophages. Immunohistochemistry and in situ hybridization revealed that neutrophils and CINC-1 mRNA localize within regions of fibrotic activity during both injury and repair. Conclusion. We conclude that the macrophage population is not directly involved in fibrotic liver repair. Rather, hepatic macrophages regulate the influx of neutrophils, which may play a direct role in matrix degradation. (Surgery 2008;143:667-78.) From the Department of Surgery, The Warren Alpert Medical School of Brown University and Hasbro Children’s Hospitals,a Providence, RI; the Department of Dermatology, Indiana University School of Medicine,b Indianapolis, Ind; The Hepatic Fibrosis Group, The Queensland Institute of Medical Research,c Brisbane, Queensland, Australia; the Department of Medicine, The Warren Alpert Medical School of Brown University, Rhode Island Hospital,d Providence, RI; and the Departments of Surgery and Pediatrics, The Warren Alpert Medical School of Brown University, Rhode Island and Hasbro Children’s Hospitals,e Providence, RI
Supported in part by NIH Grants R01 DK46831 (TFT), R01DK068097 (SHG) and RR-P20 RR17695 from the Institutional Development Award (IDeA) Program of the National Center for Research Resources (COBRE). Accepted for publication January 31, 2008. Reprint requests: Thomas F Tracy, Jr., MD, Vice Chairman, Department of Surgery, The Warren Alpert Medical School of Brown University, Pediatric Surgeon-in-Chief, Hasbro Children’s Hospital, Room 147, 593 Eddy Street, Providence, RI 02903. E-mail: [email protected]. 0039-6060/$ - see front matter Ó 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.surg.2008.01.008
CHOLESTATIC LIVER DISEASE, resulting from extraand/or intrahepatic bile duct obstruction, predisposes adults and children to the development of cirrhosis. When treating patients with cholestatic liver disease, clinicians have no guarantee that medical or operative intervention will obviate ongoing injury or initiate successful repair. Over the past 20 years, numerous studies have clarified the factors that initiate injury and promote hepatic fibrosis.1,2 The mechanisms of repair, however, remain largely undefined. Extra- and/or intrahepatic bile duct obstruction induces a stereotypical pattern of liver injury SURGERY 667
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composed of bile duct epithelial cell hyperplasia, periportal fibrosis, and an inflammatory cell infiltrate3,4 that includes monocytes5 and neutrophils.6,7 Resident tissue macrophages (Kupffer cells) are thought to be key mediators of cholestatic liver injury.8-10 Activated Kupffer cells are known to stimulate hepatic stellate cells, which preferentially deposit collagen type-I, leading to a marked increase in collagen type-I relative to other matrix components.11 Neutrophils constitute a significant percentage of the cells in the inflammatory infiltrate characteristic of cholestatic liver injury.7,12 Conflicting results regarding the potential role of neutrophils in liver repair have risen from different experimental systems. Neutrophil depletion studies demonstrated reduced tissue injury in models of sepsis,13 cholestatic liver injury,12 and viral infection.14 These studies identified an exaggerated neutrophil response as an important mediator of tissue injury. In contrast, similar models of sepsis and cholestasis suggest a more complex role for neutrophils that includes a protective response from or after injury.15,16 Using a rat model of extrahepatic cholestasis that rapidly develops collagen fibrosis,17,18 we reported that increased neutrophil numbers are associated with collagen degradation after liver injury.17 We also reported altered matrix metabolism and inflammatory cell responses after Kupffer cell depletion in injured livers.19 Using the same rat model of reversible extrahepatic cholestasis, we undertook the series of experiments described herein to delineate the cellular and molecular mechanisms of liver repair. Specifically, we sought to elucidate the complex interactions of hepatic macrophages and neutrophils in fibrotic repair after cholestatic liver injury. Here, we report that the hepatic macrophage population plays a key role in resolving liver injury by promoting the accumulation of neutrophils. We suggest that neutrophils, which are known to have collagenase, gelatinase, and elastase activity, may be important in the degradation of injury induced fibrosis. MATERIAL AND METHODS Animals. Adult male rats (225--250 g, Harlan Sprague-Dawley, Indianapolis, Ind) were housed in an artificial 12-hour light--dark cycle with access to rat chow and water ad libitum according to the NIH publication Guide for the Care and Use of Laboratory Animals. Experiments were carried out in compliance with guidelines prescribed by the Institutional Animal Care and Use Committee of Rhode Island Hospital and The Warren Alpert Medical School of Brown University.
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Biliary obstruction (liver injury). Animals (n = 77) were divided into either study (n = 52) or sham-operated (n = 25) groups. The study group underwent bile duct loop suspension surgery for biliary obstruction as previously described.19,20 Briefly, animals were anesthetized with vaporized isoflurane, weighed, and prepared for surgery. A midline laparotomy was performed and the common bile duct was dissected sufficiently to allow passage of a 5-cm length of silicone vessel loop (Surg-I-Loop, Scanlan International, Saint Paul, Minn). A midsegment of this loop was premarked to 1 cm and the ends were brought through each side of the abdominal wall, 1 cm lateral to the midline at the costochondral margin. The vessel loop was stretched and sutured to the perichondrium bilaterally at the premarked points displacing the common bile duct ventrally. Bile duct obstruction was verified by visual inspection and the abdomen was closed. Sham-operated animals underwent an identical laparotomy where the common bile duct was identified, but not obstructed. Obstructed and sham-operated animals were divided into treatment groups that received intravenously via the dorsal penile vein saline (n = 37) or GdCl3 (10 mg/kg) at either 1 (n = 21) or 2 (n = 19) days before decompression (Fig 1). Biliary decompression (liver repair). After 7 days of cholestasis, rats were anesthetized, weighed, and prepared for decompression/repair. The skin of the midline incision was opened exposing the vessel loop. The anchoring sutures were cut and the vessel loop removed. The abdominal skin was closed and the animals were allowed to recover. This time point is referred to as day 0 of repair. Necropsy. Animals were necropsied after 7 days of cholestatic injury (day 0 of repair; n = 29) or 2 days after decompression (day 2 of repair; n = 23). All sham-operated groups (n = 25) were necropsied on day 0 of repair (Fig 1). Rats were anesthetized and weighed, the abdomen was opened with a U-shaped incision, and the abdominal wall was reflected superiorly. Blood (5 mL) was collected for serum bilirubin by venipuncture of the inferior vena cava. Hepatectomy was performed and livers were divided and processed as follows: frozen with dry ice in OCT Compound (Sakura, Torrance, Calif); flash frozen in liquid nitrogen; fixed in 10% neutral phosphate buffered formalin (Fisher Scientific, Fair Lawn, NJ); or fixed in formalin free zinc fixative (BD Pharmingen, San Diego, Calif). We have previously demonstrated that urine color, weight loss, and increased bilirubin levels reflect the degree of biliary obstruction achieved in this model.5,19 Here, we utilized urine
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color, weight, and bilirubin, quantified using a commercial kit (Sigma-Aldrich, St. Louis, Mo), as markers of the progression of cholestatic injury and subsequent repair. Animals that did not demonstrate obstruction or decompression were excluded from the study. Histochemistry, immunohistochemistry, and image analysis. Formalin- and zinc-fixed tissues were embedded in paraffin and sectioned at 7 mm. Histochemistry utilized picric acid Sirius red for collagen or naphthol AS-D chloracetate esterase (esterase; Sigma-Aldrich) specific for granulocytes.21 Mouse anti-rat CD68 monoclonal antibody (Serotec Inc., Raleigh, NC) specific for the majority of (ED1+) macrophages,22 mouse anti-rat CD163 monoclonal antibody (Serotec Inc.) specific for (ED2+) Kupffer cells,22,23 FITC-conjugated rabbit anti-rat neutrophil polyclonal antibody (Accurate Chemical, Westbury, NY), and mouse antirat neutrophil monoclonal antibody (RP3, a gift from F. Sendo, Yamagata University School of Medicine, Yamagata, Japan)24 were used for immunohistochemistry. Sirius red stained images were scanned (SprintScan 35 Plus, Polaroid, Cambridge, Mass) into a PowerMac G4 (Apple Computer, Inc., Cupertino, Calif) using a PathScan Enabler optical card (Meyer Instruments, Inc., Houston, Tex). NIH ImageJ imaging software (NIH, Bethesda, Md) was used to determine total tissue section area and collagen area. Results are expressed as a percentage of total section area. Liver sections stained for ED1, ED2, or esterase were digitally imaged at 3400 with a minimum of 10 fields per section. Trained laboratory personnel (blinded to the groups) quantified the resident hepatic macrophages (ED2+), recruited mononuclear phagocytes (ED1+), and neutrophils (esterase+) in each section. Data are expressed as cells per square millimeter. Tissue sections stained with RP3 antibody were labeled secondarily with Texas Red-conjugated goat anti-mouse IgM (Vector Laboratories, Burlingame, Calif). Fluorescent images of RP3 (Texas Red) and anti-neutrophil (FITC) regions of rat liver were digitally imaged at 3200 (BD IPlabs, Rockville, Md) and analyzed for distribution. Real-time RT-PCR methods and analyses. Blocks of liver tissue were snap frozen in liquid nitrogen. Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Inc., Valencia, Calif). RNA quality and quantity were determined using the RNA 6000 Nano LabChip kit on the 2600 BioAnalyzer (Agilent Technologies, Santa Clara, Calif). Five micrograms of total RNA was converted into cDNA using First Strand cDNA Synthesis kit (GE Healthcare
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Fig 1. Experimental design. Biliary obstruction surgery was performed and cholestatic injury occurred over the after 7 days. Saline or gadolinium chloride (10 mg/kg) was administered IV 1 or 2 days before bile duct decompression and liver repair. The day of decompression is defined here as day 0. Rats were humanely killed after 0 and 2 days of repair for each group.
Life Sciences, Piscataway, NJ) with random primers. The Quantitect SYBR Green real-time PCR kit (Qiagen, Inc.) was used for all quantitative PCR and run on the Stratagene MX4000 thermocycler. Primer sequences (shown in the Table) were chosen using appropriate GENBANK sequences, NCBI (www.ncbi.nih.gov), and Primer 3 software (Whitehead Institute for Biomedical Research; source code available at http://fokker.wi.mit.edu/ primer3/).25 Sequences were purchased from Integrated DNA Technologies, Inc (Coralville, Ia). Melting curves validated the utility and specificity of each primer set. Data were evaluated using the Comparative Ct Method (2( DDCt)) of relative quantification standardized to 18S rRNA and are reported as fold increases over sham controls. Because of the relative abundance of 18S rRNA, standardizing samples were diluted 1:1000. The results were equivalent to those obtained using GAPDH as an alternate reference gene (data not shown). In situ hybridization. In situ hybridization was performed on 7-mm frozen liver sections using probes (Table) designed and purchased from GeneDetect (Bradenton, Fla). Hybridization was visualized using the Dako Kit (Dako North America; Carpentaria, Calif) according to manufacturers instructions. Antisense and sense probes were run simultaneously to control for nonspecific hybridization. Statistics. Data are expressed as mean values ± standard error of the mean. Statistically significant differences were determined by ANOVA and Fisher’s PSLD (StatView 4.1+, SAS Institute Inc., Cary, NC).
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Table. Primer list for quantitative RT-PCR and probe for in situ hybridization Primer name 18S up 18S down CINC-1 up CINC-1 down MIP-2 up MIP-2 down ICAM-1 up ICAM-1 down Probe name CINC-1 anti-sense CINC-1 sense
Sequence CGA AAG CAT TTG CCA AGA AT AGT CGG CAT CGT TTA TGG TC ACC CAA ACC GAA GTC ATA GC ACT TGG GGA CAC CCT TTA GC GGG GGA GTT GGG TAC TGA CT CCT TGA AAG CCC TCT GAC TG GGA GGC CCT AAA ACT CAA GG GAG GTG GGT GAG GGG TAA AT
CTT CAG GGT CAA GGC AAG CCT CGC GAC CAT TCT TGA GTG GTG TGG CTA TGA TCA TAG CCA CAC TCA AGA ATG GTC GCG AGG CTT GCC TTG ACC CTG AAG
Fig 2. Fibrotic injury. Liver sections derived from rats on days 5, 7, 14, and 21 after biliary obstruction were stained with Sirius red and digitally imaged. Representative images from sham and 7- and 21-day injured animals illustrate staining and increased matrix (original magnification, 340). The amount of collagen is expressed as a percentage of total area. Tissue collagen was elevated compared to sham-operated controls. #P = .05; *P # .05 (ANOVA).
RESULTS Biliary obstruction, fibrosis, and the response of hepatic macrophages. During the 7-day period that followed biliary obstruction (day 0 of repair), collagen deposition in the livers of rats increased approximately 4-fold relative to sham-operated control animals. This increase represented 60%
of maximum injury that occurred after 21 days of obstruction (Fig 2). The numbers of ED1+ inflammatory macrophages and ED2+ Kupffer cells were elevated 6- and 3-fold, respectively, in the livers of bile duct-obstructed animals (Fig 3). Saline-treated animals showed a significant and rapid decline in
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Fig 3. Response of hepatic macrophages to cholestatic liver injury and repair. ED1- and ED2-stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with saline or gadolinium chloride at 24 or 48 hours before repair were digitally imaged and the positively stained cells were counted. Representative images of ED1 (A) and ED2 (C) stained liver sections from injured animals illustrate staining quality (original magnification, 3400). The numbers of ED1+ (B) and ED2+ (D) cells are increased significantly relative to the numbers found in sham-operated control animals. *P # .05 (ANOVA).
both inflammatory macrophages and Kupffer cells after biliary decompression. These macrophages were distributed throughout the parenchymal sinusoids and portal tracts during cholestatic injury and were restricted primarily to the parenchymal sinusoids during repair. Treatment with GdCl3 on day 1 before decompression diminished the injury-induced increase in ED1+ cells and totally blocked the increase in ED2+ cells otherwise observed both at the time of decompression (day 0) and after 2 days of repair. On the other hand, treatment with GdCl3 on day 2 before decompression abrogated the increase observed in ED2+, as well as ED1+, cells on day 0 and after 2 days of repair. Depletion of hepatic macrophage by treatment with GdCl3 on day 1 or 2 before biliary decompression did not alter the amount of collagen detected in the liver after 7 days of injury (day 0 of repair; Fig 4). Moreover, the decreased collagen assessed on day 2 of repair was not altered by the depletion of hepatic macrophages with GdCl3 administered on day 1 before decompression. When GdCl3 was administered 2 days before biliary decompression, however, macrophage depletion impaired liver repair and collagen levels
persisted. Taken together, these findings demonstrate the response of hepatic macrophages to cholestatic liver injury and suggest their indirect, time-dependent role in liver repair and the resolution of fibrosis. Hepatic macrophages modulate the response of neutrophils to cholestatic liver injury. Consistent with our previous observations,17 the number of esterase-positive neutrophils was significantly elevated in the livers of bile duct-obstructed animals and remained elevated after biliary decompression (Fig 5). Depletion of hepatic macrophages on day 1 before repair did not alter the neutrophil counts determined at the time of biliary decompression (day 0). In sharp contrast, neutrophil numbers were decreased significantly on day 0 in the liver of rats rendered macrophage-deficient by pretreatment with GdCl3 on day 2 before repair. Neutrophil numbers in the livers of all bile ductobstructed rats rendered macrophage deficient were diminished approximately 2-fold relative to nondeficient animals on day 2 of repair, and were not different from the number determined in saline-treated, sham-operated rats. Thus, hepatic macrophages modulate the response of neutrophils
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Fig 4. Fibrotic repair. A, Sirius red–stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with either saline or gadolinium chloride for 24 or 48 hours before bile duct decompression were digitally imaged; the amount of collagen is expressed as a percentage of total area. Biliary obstruction significantly increased the deposition of collagen in all groups determined on day 0 of repair relative to sham-operated animals. *P # .05 (ANOVA). The collagen content was significantly decreased in the saline-treated and 1 day GdCl3-treated groups after 2 days of repair (D, P # .05; Fisher’s PLSD), and significantly less than that observed in the 2 day GdCl3-treated group (P # .05; ANOVA). B, Representative images of Sirius red—stained liver sections from all groups reflect differences depicted in the histograms (original magnification, 3200).
to cholestasis; the consequences of macrophage depletion on neutrophil accumulation in cholestatic livers are most pronounced on day 2 or greater post-GdCl3 treatment. Macrophages modulate proinflammatory gene expression in cholestatic livers. Gene expression during periods of cholestatic injury and repair was evaluated by real-time RT-PCR. Consistent with the findings of other groups,7 neutrophil chemoattractant CINC-1 (CXCL1) mRNA expression increased significantly (10-fold) after cholestatic liver injury in saline-treated animals (Fig 6, A). Furthermore,
CINC-1 message expression remained elevated in the livers of saline treated rats during 2 days of repair. Hepatic CINC-1 mRNA expression was not reduced at either 0 or 2 days of repair in animals treated with GdCl3 and rendered macrophage-deficient 1 day before bile duct decompression. In sharp contrast, CINC-1 message expression at both 0 and 2 days of repair was reduced (50% relative to saline-treated controls) in rats rendered macrophage-deficient 2 days before biliary decompression. In contrast with CINC-1, neutrophil chemoattractant macrophage inflammatory protein
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Fig 5. Response of infiltrating neutrophils to cholestatic liver injury and repair. Neutrophil esterase stained liver sections derived from sham, injured (day 0), and repairing (day 2) animals treated with either saline or GdCl3 at 24 or 48 hours before repair were digitally imaged and the positively stained cells were counted. *Significantly more than comparably treated, sham-operated animals (P # .05). D Significantly less than saline-treated controls (P # .05).
(CXCL2) message expression did not change at any time point or with any treatment (Fig 6, B). The mechanisms of inflammatory cellular infiltration require the upregulation of both chemotactic signaling and adhesion factors, for example, intercellular adhesion molecule-1 (ICAM-1), a molecule that regulates neutrophil trafficking. ICAM-1 mRNA expression after 0 and 2 days of repair was elevated 5-fold in saline-treated, bile duct-ligated rats relative to comparably treated sham-operated controls (Fig 6, C). GdCl3 administered on day 1 before decompression reduced ICAM-1 mRNA expression (50% decrease) on day 0 of repair; however, expression was significantly elevated in this group at 2 days of repair. GdCl3 administered 2 days before repair inhibits ICAM-1 message expression at both 0 and 2 days of repair. Thus, the increased accumulation of neutrophils in the liver of saline-treated animals coincided with the elevated expression of CINC1 and ICAM-1, whereas reductions in expression of both CINC-1 and ICAM-1 after GdCl3 administered 2 days before repair coincided with reductions in neutrophil accumulation. Interestingly, CINC-1 and ICAM-1 expression after GdCl3 administration 2 days before repair was unsynchronized, which resulted in a changing neutrophil response during repair. CINC-1 message expression and localization of inflammatory neutrophils in portal tracts. CINC1 mRNA was localized to the portal tracts of injured
Fig 6. Quantitative real-time RT-PCR. CINC-1 (A), macrophage inflammatory protein (MIP-2; B), and ICAM-1 (C) mRNA expression was quantified in liver homogenates derived from sham, injured (day 0) and repairing (day 2) animals pretreated with saline or GdCl3 for 24 or 48 hours before repair. *Significantly increased relative to sham-operated controls treated comparably (P # .05; ANOVA).
and repairing livers using in situ hybridization (Fig 7). Widespread antisense hybridization was found throughout the portal tracts in sham, injured, and repairing animals; notably, hybridization was excluded from bile duct epithelial cells and cells in the parenchyma. The depletion of hepatic macrophages 2 days before biliary decomposition did not alter the distribution of CINC-1 mRNA
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Fig 7. In situ hybridization for CINC-1. Representative liver sections obtained from sham, injured (day 0) and repairing (day 2) animals treated with either saline or gadolinium chloride (48 hours before repair) were labeled with sense or anti-sense CINC1 mRNA (original magnification, 3400).
hybridization at any time point. Indeed, visualization of the hybridized product was more intense than in saline-treated controls. No CINC-1 mRNA was found in the parenchyma, and all sense hybridization controls were negative. The distribution of esterase-positive neutrophils in cholestatic livers correlated with the localization of CINC-1 mRNA; that is, the cells accumulated predominantly in the portal tracts at all time points (Fig 8, A). Interestingly, neutrophil distribution was frequently in close proximity to bile ducts. Specific labeling of rat neutrophils using rabbit antirat neutrophil (Fig 8, B) and RP3 (Fig 8, C)
confirmed the esterase histochemistry in terms of both neutrophil counts (data not shown) and distribution. DISCUSSION During cholestasis, increased concentrations of bile acids and toxins in the liver result in the activation of Kupffer and hepatic stellate cells. This is accompanied by the release of inflammatory cytokines,26-29 the infiltration of circulating monocytes and neutrophils,9 and net collagen deposition by activated myofibroblasts in the sinusoids and portal triads.30-34 If not attenuated, these
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Fig 8. Neutrophil distribution. Neutrophils in liver sections derived from sham, injured (repair day 0), and repairing (day 2) animals after saline or GdCl3 treatment were stained with naphthol AS-D chloroacetate esterase (A), FITC-conjugated rabbit anti-rat PMN polyclonal antibody (B), or mouse anti-rat neutrophil monoclonal antibody (RP3; C). (Original magnification, 3200.)
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events can lead to cirrhosis and subsequent endstage liver disease. Although the mechanisms that underlie cholestatic liver injury are becoming increasingly clearer, those that contribute to repair are less well-understood and more difficult to study. For clinical practice, surgical and other interventional decompression techniques have been developed to rapidly reverse defects in bile flow. Liver repair, however, is often complicated by progressive hepatic damage in the absence of overt cholestatic stimuli.29,32,35 For infants, children, and adults, progressive liver disease can increase clinical morbidity and mortality associated with biliary atresia, choledochal cysts, sclerosing cholangitis, and other biliary obstructive etiologies. Persistent biliary hyperplasia, fibrosis, and inflammatory responses characterize the cellular and molecular consequences associated with inadequate repair.29,32,35 Previously, we reported that the administration of GdCl3 and the depletion of hepatic macrophages throughout the course of cholestatic liver injury delayed repair after ductular decompression in our rat model.19 Persistent liver fibrosis due to the loss in macrophage activity was associated with the delayed resolution of ductal hyperplasia. By administering GdCl3 and eliminating macrophages just before decompression in the present study, we focused specifically on the macrophagedependent mechanisms that contribute to repair. Based on previous work implicating neutrophils and neutrophil collagenase in cholestatic liver repair,17 we speculated that this approach would allow us to delineate those critical interactions that occur between hepatic macrophages and immigrating neutrophils. We found that both recruited (ED1+) and resident (ED2+) hepatic macrophages were depleted and remained at or below sham levels through 2 days of repair after treatment with GdCl3 on day 1 before biliary decompression. After such treatment, the decline in neutrophil cell counts within the portal tracts of livers was not evident until day 2 of repair. However, treatment with GdCl3 on day 2 before decompression reduced the number of neutrophils, as well as the numbers of recruited and resident macrophages, in the liver at the time of repair. Thus, in the absence of hepatic macrophages, the decline in number of neutrophils recruited to the liver requires approximately 48 hours (Fig 5), a timeframe consistent with the normal survival rate of extravasated neutrophils in tissues.36,37 Morphometric analysis of collagen deposited after 7 days of cholestatic liver injury demonstrated levels that were significantly elevated relative to
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sham-operated control animals. After 2 days of repair, these levels were reduced to near those observed in shams. Rats treated with GdCl3 on day 1 before decompression (which had no effect on the number of neutrophils present in the liver on day 0 of repair) showed a comparable reduction in collagen levels assessed on day 2 of repair. In contrast, treatment with GdCl3 on day 2 before biliary decompression significantly reduced the number of neutrophils present in the liver on day 0 of repair and blocked the reduction in collagen normally seen by day 2 of repair. Taken together, these findings indicate that liver repair can occur in the absence of hepatic macrophages provided the persistence of neutrophils. It is intriguing that neutrophils, which promote tissue damage in a number of animal models including bile duct ligation and cholestatic liver injury in mice,12-14,38 can also contribute to repair. Indeed, in this regard, several studies indicate that the rat and mouse models of cholestatic liver injury differ significantly in a number of ways. In the mouse model, for example, necrotic regions develop rapidly in the liver after biliary obstruction, whereas fibrosis occurs only secondarily at a much later time.9,39 Cholestatic liver injury in the rat model, on the other hand, progresses in a manner analogous to that seen in humans; that is, proliferation of the nonparenchymal cell population and development of fibrosis that progresses in the absence of necrotic lesions.17,29 Moreover, in sharp contrast to the mouse model, neutrophil depletion fails to alter the course of tissue injury in cholestatic rats.7 Biliary obstruction increased the expression of messages for CINC-1, an important chemokine for neutrophil recruitment,40-44 and ICAM-1, required for the firm adhesion of inflammatory cells to the endothelial barrier and extravasation in conjunction with chemokine signaling.45-47 Increases in CINC-1 and ICAM-1 mRNA expression are consistent with the signaling requirement for immigration to inflammatory sites. As we reported previously,17 neutrophil counts remain elevated while ED1+ and ED2+ hepatic macrophages return to sham levels in the repairing rat liver after cholestatic injury. Additionally, we showed here that the expression both CINC-1 and ICAM-1 mRNA also remains elevated after 2 days of normal repair demonstrating a persistent chemotactic signal that continues well past operative repair and that sustains the presence of neutrophils in the liver. Loss of synchronization in the expression of CINC-1 and ICAM-1 mRNAs after GdCl3 treatment show that chemotactic signaling responds within
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the first 24 hours of treatment. This timeframe is consistent with that previously reported for GdCl3-mediated hepatic macrophage depletion, which requires 24 hours to complete and must be repeated every 48 hours to maintain in the rat.19 Taken together, these data support the conjecture that chemokine and adhesion factor expression require synchronous upregulation and their production sustained to support the continued presence of neutrophils in repairing cholestatic rat livers. Collagen deposited in the livers of cholestatic rats is principally localized to the expanding portal tracts by activated hepatic stellate cells or myofibroblasts. Desmin and a-SMA immunohistochemistry in this model has demonstrated the classic pathway, activation of hepatic stellate cells, necessary for net matrix deposition.48 Herein, we show Sirius red staining, which indicates that our gadolinium treatment does not interfere with the accumulation of matrix during injury. Therefore, differences in matrix during repair are most likely due to changes in mechanisms of matrix degradation as opposed to net deposition. In this study, infiltrating neutrophils also localize to the expanding portal tracts of the liver during cholestatic injury and normal repair. Neutrophils, however, seem to accumulate preferentially near the remodeling biliary epithelial cells. Using in situ hybridization, we localized CINC1 mRNA transcripts to the expanded nonparenchymal cells of the portal tract. Although CINC1 mRNA expression was distributed throughout the portal tract, it was predominately excluded from the bile duct epithelial cells. Our previous study indicated that neutrophils are an important mediator of cholestatic liver repair in rats.17 The data presented herein demonstrate that neither recruited nor resident hepatic macrophages are directly involved in the mechanisms of liver repair. Rather, hepatic macrophages upregulate the production of proinflammatory chemokines (eg, CINC-1) and adhesion molecules (ICAM-1) that promote neutrophil infiltration and localization at sites of tissue remodeling and fibrotic repair. Employing neutrophil-depleting antibody in vivo, experiments ongoing in our laboratory will delineate the specific role of neutrophils and the neutrophil-dependent mechanisms required for reversing fibrosis and for liver repair after cholestatic injury. REFERENCES 1. Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis.
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