The FASEB Journal express article 10.1096/fj.02-1157fje. Published online July 3, 2003.
Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis Esther Titos,* Joan Clària,* Anna Planagumà,* Marta López-Parra,* Neus Villamor,† Marcelina Párrizas,‡ Anna Carrió,§ Rosa Miquel,¶ Wladimiro Jiménez,‡ Vicente Arroyo,** Francisca Rivera,‡ and Joan Rodés** *DNA Unit, ‡Hormonal, †Hematopathology, ¶Pathology, and §Genetics Laboratories, and **Liver Unit, Hospital Clínic, Institut d´Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona 08036, Spain Corresponding author: Joan Clària, DNA Unit, Hospital Clínic, Villarroel 170, Barcelona 08036, Spain. E-mail:
[email protected] ABSTRACT The existence of an increased number of Kupffer cells is recognized as critical in the initiation of the inflammatory cascade leading to liver fibrosis. Because 5-lipoxygenase (5-LO) is a key regulator of cell growth and survival, in the current investigation we assessed whether inhibition of the 5-LO pathway would reduce the excessive number of Kupffer cells and attenuate inflammation and fibrosis in experimental liver disease. Kupffer cells were the only liver cell type endowed with a metabolically active 5-LO pathway (i.e., expressed mRNAs for 5-LO, 5LO-activating protein [FLAP], and leukotriene [LT] C4 synthase and generated LTB4 and cysteinyl-LTs). Both the selective 5-LO inhibitor AA861 and the FLAP inhibitor BAY-X-1005 markedly reduced the number of Kupffer cells in culture. The antiproliferative properties of AA861 and BAY-X-1005 were associated with the occurrence of condensed nuclei, fragmented DNA, and changes in DNA content and cell cycle frequency distribution consistent with an apoptotic process. In vivo, in carbon tetrachloride-treated rats, BAY-X-1005 had a significant antifibrotic effect and reduced liver damage and the hepatic content of hydroxyproline. Together, these findings indicate a novel mechanism by which inactivation of the 5-LO pathway could disrupt the sequence of events leading to liver inflammation and fibrosis. Key words: hepatic inflammation • sinusoidal liver cells • arachidonic acid metabolites
L
iver fibrosis is the accumulation of connective tissue in the liver as the result of an imbalance between production and degradation of extracellular matrix (1). Unlike cirrhosis, which is the irreversible end-stage consequence of liver disease, liver fibrosis is a reversible process. Thus, in recent years we have witnessed considerable efforts focused primarily on events that lead to the early accumulation of scarring to identify novel therapeutic targets to slow its progression. Unfortunately, to date, therapy that effectively prevents the progression of fibrosis or reverses established fibrosis remains elusive. Inflammation plays a key role in many prevalent diseases, and there is increased appreciation that liver fibrosis incorporates uncontrolled inflammation as part of its etiology (1). It is now
recognized that Kupffer cells, which represent the largest population of resident macrophages in the body, are uniquely positioned as the predominant inflammatory effector cell to initiate the inflammatory cascade leading to tissue remodeling and fibrosis (2–4). In fact, Kupffer cells actively participate in the initiation and progression of liver fibrosis by releasing many soluble factors such as cytokines, reactive oxygen species, and arachidonic acid metabolites (eicosanoids) (2–4). As a result, the constellation of inflammatory phenomena initiated by Kupffer cells leads to activation of hepatic stellate cells (HSCs) and, subsequently, to the unbalanced synthesis of collagen, proteoglycans, and hyaluronate (3). The population of Kupffer cells is self-renewing under steady-state conditions (5). However, during the onset of liver fibrosis, when inflammation is extensive, the population of liver resident macrophages increases exponentially (4–9). Indeed, during the early stages of experimental liver disease induced by administration of carbon tetrachloride (CCl4) to rats, an animal model that closely reproduces human liver disease (10), the number of Kupffer cells increases sharply (6– 9). Moreover, in this animal model, the number of Kupffer cells closely correlates with the degree of liver injury induced by the hepatotoxin (6–9). Thus, the presence of an increased population of liver macrophages and the bulk release of inflammatory mediators are critical in the development of hepatic extracellular matrix alterations. 5-Lipoxygenase (5-LO)-derived eicosanoids are key regulators of cell growth and survival (11). 5-LO inhibitors stop growth-related signals and induce programmed cell death (apoptosis) in neurons, mast cells, and mesothelial and endothelial cells, and in lung, prostate, breast, and pancreas cancer cell lines (12–19). We recently demonstrated that mRNA expression for 5-LO and the production of 5-LO products are elevated in livers from rats with CCl4-induced cirrhosis (20). On the basis of these findings, in the current investigation we tested the hypothesis that 5LO-derived products may play a role in Kupffer cell growth and survival and that inhibition of this eicosanoid-generating pathway may be anti-inflammatory and antifibrotic in vivo in CCl4treated rats. As a consequence of this investigation, we identified a novel mechanism implicating the 5-LO pathway in the pathogenesis of liver inflammation and fibrosis, which provides the basis for new approaches for prevention and treatment of these liver complications. MATERIALS AND METHODS Materials Adult male Wistar rats were from Criffa S.A. (Santa Perpètua de Mogoda, Spain). RPE-ED2 antibody was from Serotec (Raleigh, NC), and fluorescein isothiocyanate (FITC)-labeled annexin V was from Bender MedSystems (Vienna, Austria). Synthetic leukotriene (LT) D4 and 5-HETE and enzyme immunoassays (EIAs) for LTB4 and LTC4/LTD4/LTE4 (cysteinyl-LTs) were from Cayman Chemical (Ann Arbor, MI). Percoll and RNase I A (bovine pancreas) were from Pharmacia (Uppsala, Sweden). Guanidinium isothiocyanate, aprotinin, leupeptin, pepstatin A, phenylmethylsulfonyl fluoride, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide, bovine serum albumin (BSA), and high-performance liquid chromatography (HPLC) columns were purchased from Sigma Chemical (St. Louis, MO). SepPak C18 columns were from Waters (Milford, MA). Collagen was from Innogenetics (Heidelberg, Germany). Recombinant murine macrophage colony-stimulating factor (M-CSF) and TACS DNA Laddering Kit were from R&D Systems (Minneapolis, MN). The 8-well Lab-
Tek Chamber Slides were from Nalge Nunc International (Naperville, IL). Nylon mesh filters were from Becton, Dickinson Labware (Franklin Lakes, NJ). Cesium chloride and collagenase (A type) were from Roche (Basel, Switzerland). The cDNA synthesis kit was from Promega (Madison, WI). DNA amplification reagents, Dulbecco’s PBS (DPBS), Hanks’ balanced salt solution without calcium and magnesium (HBSS), RPMI 1640, and Williams’ medium E were from Invitrogen (Carlsbad, CA). FCS was from Biological Industries (Kibbutz Beit Haemek, Israel). Diff-Quik was from Dade Behring (Düdingen, Switzerland). AA861 was from Biomol (Plymouth Meeting, PA). BAY-X-1005 was kindly provided by Dr. R Müller-Peddinghaus, Pharma Research Center, Bayer AG, Wuppertal, Germany. CRL-2192 cells, a rat macrophage cell line, was from the European Collection of Cell Cultures (Salisbury, UK). Oligonucleotides were constructed by solid-phase phosphoramidite chemistry in a 394 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). Isolation and culture of rat Kupffer cells Male Wistar rats weighing 400–500 g were anesthetized with ketamine (50 mg/kg of body weight [b.w.]), and liver cells were isolated by in situ collagenase perfusion through the portal vein as described previously (20). Briefly, livers were perfused for 20 min at a flow rate of 14 ml/min at 37°C with HBSS containing 10 mmol/L HEPES (pH 7.4) and 1 mmol/L EGTA, followed by 5 min of perfusion with HBSS containing 10 mmol/L HEPES (pH 7.4), 1.3 mmol/L CaCl2, and 0.6% BSA and then 10 min with a 0.05% collagenase (A type) solution containing 10 mmol/L HEPES (pH 7.4) and 1.3 mmol/L CaCl2. The resultant digested liver was excised and minced, and the dispersed cells were passed through nylon mesh filters (100 µm). The cells were then washed twice with cold HBSS containing 10 mmol/L HEPES (pH 7.4), 1.3 mmol/L CaCl2, and 1% BSA, followed by two washes with DPBS. The pellet obtained, containing the hepatocytes, was further purified and plated on collagen-coated plastic dishes as described in Titos et al. (20). The supernatant enriched in nonparenchymal cells was centrifuged twice at 50g for 5 min at 4°C and then centrifuged again at 800g for 10 min at 4°C. The pellet obtained was resuspended in DPBS and centrifuged at 800g for 15 min through a 25%/50% Percoll gradient at 4°C. HSCs were isolated from the upper layer of the discontinuous Percoll density gradient. The interface of the gradient containing Kupffer cells and sinusoidal endothelial cells was seeded on 35-mm tissue culture plates, 48-well plates, or 8-well Lab-Tek Chamber Slides, and incubation proceeded at 37°C for 30 min. Cell monolayers that adhered to the dishes were characterized as Kupffer cells by nonspecific esterase activity staining and by immunolabeling with the monoclonal antibody RPE-ED2 (21). Kupffer cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10% FCS and were maintained in a humidified 5% CO2 incubator at 37°C. Analysis of 5-hydroxyeicosatetraenoic acid (5-HETE) by reverse-phase (RP)-HPLC Supernatants from Kupffer cells (1–2.8 × 106 cells) seeded on 48-well plates for 16 h at 37°C were collected in two volumes of MeOH and kept at –20°C until further analysis. Samples were concentrated under a stream of N2 before injection into a RP-HPLC system which consisted of a Waters integrated system controller (model 600E) equipped with a 996 Photodiode Array Detector and Millennium HPLC analysis software (Waters). 5-HETE was separated in a Tracer Supelcosil column (5 µm, 4.5 × 250 mm) and eluted at a flow rate of 1.0 ml/min with
MeOH/H2O/acetic acid (65:35:0.01; v/v/v) as phase 1 (t0 to 20 min) and a linear gradient with MeOH/acetic acid (99.9:0.01, v/v) as phase 2 (20–45 min). Analysis of LTB4 and cysteinyl-LTs by EIA Freshly isolated rat Kupffer cells (1–2.8 × 106 cells) were seeded on 48-well plates and maintained in culture for 16 h at 37°C. Cell supernatants were stored at –20°C in 2 volumes of MeOH for further analysis of LTB4 and cysteinyl-LT levels by specific EIA kits. Liver samples of about 0.2–0.4 g were individually homogenized with a Polytron in 2 ml of cold DPBS with calcium and magnesium, precipitated with 2 volumes of ice-cold MeOH, and extracted with SepPak C18 columns, before EIA analysis. The antibody against LTB4 had no significant crossreactivity with the two major nonenzymatic, biologically inactive, hydrolysis products of LTA4, namely 6-trans-LTB4 and 12-epi-6-trans-LTB4. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was obtained by the guanidinium isothiocyanate-cesium chloride method (22). Briefly, samples were homogenized in 4 M guanidinium isothiocyanate, placed on the reagent 5.7 M cesium chloride, and centrifuged at 200,000g for 20 h at 20°C. Subsequently, pellets from cell lysates were resuspended in TES solution (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, and 1% sodium dodecyl sulfate), and RNA was precipitated by the ethanol method and quantified by UV spectrophotometry (Kontron, Milan, Italy). The RT reaction was performed at 42°C for 45 min in a 20-µl sample containing 2 µg of total RNA, MgCl2 (50 mM), 1x RT buffer, deoxynucleotide mixture (10 mM), RNase inhibitor (20 U), oligo-d(T)15 primers (500 µg/ml), and avian myeloblastoma virus reverse transcriptase (15 U). PCR was performed by using oligonucleotides designed from published rat 5-LO, 5-LO-activating protein (FLAP), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) cDNA sequences (23–25). The cDNA sequence for LTC4 synthase was obtained from the GenBank database (No. AB048790). The detailed description of oligonucleotides is as follows: 5-LO: sense: 5'-TACATTTACCTCAGCCTCATTG-3' (nucleotides 152–173) and antisense: 5'-GTCCACTCCCTTTTCACTATCA-3' (nucleotides 598–619); FLAP: sense: 5'-GGGCTGATGTATCTGTTCGTG-3' (nucleotides 289–310) and antisense: 5'-GCGGGGAGATCGTCGTGGTTA-3' (nucleotides 479–499); LTC4 synthase: sense: 5'-CCGAGTTCGAGCGCGTCT-3' (nucleotides 167–184) and antisense: 5'TCTACCTGTTCGCGCGCC-3' (nucleotides 290–307); and GAPDH: sense: 5'TCCCTCAAGATTGTCAGCAA-3' (nucleotides 451–470) and antisense: 5'AGATCCACAACGGATACATT-3' (nucleotides 739–758). PCR (35 cycles) was carried out at 94°C (30 s), 58°C (1 min), and 72°C (30 s) for 5-LO and GAPDH; at 96°C (30 s), 55°C (30 s), and 72°C (45 s) for FLAP; and at 96°C (45 s), 60°C (30 s), and 72°C (1 min) for LTC4 synthase. PCR products were analyzed by electrophoresis in a 2% agarose gel and were visualized by ethidium bromide staining. The expected sizes of the PCR products were 468, 211, 141, and 309 bp for 5-LO, FLAP, LTC4 synthase, and GAPDH, respectively. Analysis of cell proliferation Rat Kupffer cells (1–2.8 × 106 cells) were seeded for up to 6 days in 200 µl of complete RPMI 1640 medium on 48-well plates at 37°C in a 5% CO2 atmosphere. Cell growth was determined by the microculture MTT assay. Briefly, at the end of the incubation period, 20 µl of MTT (5
mg/ml in RPMI 1640 without phenol red) was added to each well, and 4 h later cells were lysed by addition of 200 µl of isoamyl alcohol and shaking for 20 min. Absorbance at 570 nm was measured in a multiwell plate reader (Molecular Devices, Menlo Park, CA). In some experiments, Kupffer cells were isolated from cirrhotic rat livers and incubated with vehicle (0.1% BSA in DPBS) or M-CSF (100 and 150 ng/ml) for 5 days at 37°C. To ascertain the effect of 5-LO and FLAP inhibitors, Kupffer cells from cirrhotic rat livers were grown in the presence of vehicle (0.1% dimethyl sulfoxide [DMSO]), AA861 (5 and 10 µM), or BAY-X-1005 (30 and 40 µM) for 8 h at 37°C, and the number of cells was determined by direct counting using the Neubauer chamber. The effects of 5-LO and FLAP inhibitors on cell proliferation were also assessed in THP-1 cell cultures. THP-1 cells (50 × 103 cells) were seeded in 24-well plates and grown in serum-free RPMI 1640 medium in the absence or presence of AA861 (from 1 to 25 µM) or BAY-X-1005 (from 10 to 60 µM) for increasing periods of time (from 0 to 72 h). For these cells, we established a linear relation between the MTT values and cell number within the range of the experiments shown (r = 0.99, P
