Erythrophagocytosis by Liver Macrophages (Kupffer Cells) Promotes Oxidative Stress, Inflammation, and Fibrosis in a Rabbit Model of Steatohepatitis
2007; Elsevier BV; Volume: 170; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2007.060441
ISSN1525-2191
AutoresKohji Otogawa, Kohji Kinoshita, Hideki Fujii, Masahide Sakabe, Ryoko Shiga, Kazuki Nakatani, Kazuo Ikeda, Yuji Nakajima, Yoshihiro Ikura, Makiko Ueda, Tetsuo Arakawa, Fumihiko Hato, Norifumi Kawada,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoNonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-β1, and collagen α1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis. Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-β1, and collagen α1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis. Obesity and lifestyle-related diseases have become a concern worldwide. In the United States, ∼47 million individuals are estimated to have metabolic syndrome characterized by obesity, hyperlipidemia, hyperglucosemia, and hypertension.1Ford ES Giles WH Dietz WH Prevalence of the metabolic syndrome among US adults: findings from the Third National Health and Nutrition Examination Survey.JAMA. 2002; 287: 356-359Crossref PubMed Scopus (5712) Google Scholar Nonalcoholic fatty liver disease (NAFLD) is closely related to this syndrome and has become an urgent clinical issue in the field of hepatology. Approximately 12 to 15% of the population is estimated to have NAFLD, and 3 to 4% is considered to suffer from nonalcoholic steatohepatitis (NASH). NASH is also caused by the use of some drugs, parenteral nutrition, hypothyroidism, and gastric bypass surgery.2Bacon BR Farahvash MJ Janney CG Neuschwander-Tetri BA Nonalcoholic steatohepatitis: an expanded clinical entity.Gastroenterology. 1994; 107: 1103-1109Abstract PubMed Scopus (1019) Google Scholar, 3Pinto HC Baptista A Camilo ME Valente A Saragoca A de Moura MC Nonalcoholic steatohepatitis. 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NASH progresses to liver fibrosis and cirrhosis that lead to life-threatening liver failure and the development of hepatocellular carcinoma.2Bacon BR Farahvash MJ Janney CG Neuschwander-Tetri BA Nonalcoholic steatohepatitis: an expanded clinical entity.Gastroenterology. 1994; 107: 1103-1109Abstract PubMed Scopus (1019) Google Scholar, 9Ludwig J Viggiano TR McGill DB Oh BJ Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.Mayo Clin Proc. 1980; 55: 434-438PubMed Google Scholar, 10Haque M Sanyal AJ The metabolic abnormalities associated with non-alcoholic fatty liver disease.Best Pract Res Clin Gastroenterol. 2002; 16: 709-731Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 11Matteoni CA Younossi ZM Gramlich T Boparai N Liu YC McCullough AJ Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity.Gastroenterology. 1999; 116: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (2750) Google Scholar The histological features of NASH such as macrovesicular steatosis, nuclear glycogenation, lobular and portal inflammation, and iron deposition resemble those of alcoholic liver damage.2Bacon BR Farahvash MJ Janney CG Neuschwander-Tetri BA Nonalcoholic steatohepatitis: an expanded clinical entity.Gastroenterology. 1994; 107: 1103-1109Abstract PubMed Scopus (1019) Google Scholar, 3Pinto HC Baptista A Camilo ME Valente A Saragoca A de Moura MC Nonalcoholic steatohepatitis. Clinicopathological comparison with alcoholic hepatitis in ambulatory and hospitalized patients.Dig Dis Sci. 1996; 41: 172-179Crossref PubMed Scopus (208) Google Scholar, 4Lee RG Nonalcoholic steatohepatitis: tightening the morphological screws on a hepatic rambler.Hepatology. 1995; 21: 1742-1743PubMed Google Scholar, 9Ludwig J Viggiano TR McGill DB Oh BJ Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease.Mayo Clin Proc. 1980; 55: 434-438PubMed Google Scholar, 10Haque M Sanyal AJ The metabolic abnormalities associated with non-alcoholic fatty liver disease.Best Pract Res Clin Gastroenterol. 2002; 16: 709-731Abstract Full Text PDF PubMed Scopus (120) Google Scholar, 12Neuschwander-Tetri BA Bacon BR Nonalcoholic steatohepatitis.Med Clin North Am. 1996; 80: 1147-1166Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 13Brunt EM Janney CG Di Bisceglie AM Neuschwander-Tetri BA Bacon BR Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.Am J Gastroenterol. 1999; 94: 2467-2474Crossref PubMed Scopus (3074) Google Scholar Peroxidation of accumulated lipids may play an important role in the pathogenesis of NASH because it is considered to trigger hepatocellular injury, hepatic fibrogenesis, and the carcinogenesis of hepatocytes.14Sheth SG Gordon FD Chopra S Non alcoholic steatohepatitis.Ann Intern Med. 1997; 126: 137-145Crossref PubMed Scopus (557) Google Scholar, 15Ludwig J McGill DB Lindor KD Nonalcoholic steatohepatitis.J Gastroenterol Hepatol. 1997; 12: 398-403Crossref PubMed Scopus (179) Google Scholar, 16Sanyal AJ Campbell-Sargent C Mirshahi F Rizzo WB Contos MJ Sterling RK Luketic VA Shiffman ML Clore JN Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities.Gastroenterology. 2001; 120: 1183-1192Abstract Full Text Full Text PDF PubMed Scopus (1716) Google ScholarMost studies of NASH that use animals use rodents with genetic alterations (eg, ob/ob mice, fa/fa rats, or phosphatase and tensin homologue deleted on chromosome 10 knockout mice) or those fed a methionine- and choline-deficient diet.17Kurlawalla-Martinez C Stiles B Wang Y Devaskar SU Kahn BB Wu H Insulin hypersensitivity and resistance to streptozotocin-induced diabetes in mice lacking PTEN in adipose tissue.Mol Cell Biol. 2005; 25: 2498-2510Crossref PubMed Scopus (156) Google Scholar, 18Koteish A Mae Diehl A Animal models of steatohepatitis.Best Pract Res Clin Gastroenterol. 2002; 16: 679-690Abstract Full Text PDF PubMed Scopus (150) Google Scholar, 19Nanji AA Animal models of nonalcoholic fatty liver disease and steatohepatitis.Clin Liver Dis. 2004; 8: 559-574Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 20Sahai A Malladi P Pan X Paul R Melin-Aldana H Green RM Whitington PF Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin.Am J Physiol. 2004; 287: G1035-G1043Google Scholar, 21Carmiel-Haggai M Cederbaum AI Nieto N A high-fat diet leads to the progression of non-alcoholic fatty liver disease in obese rats.FASEB J. 2005; 19: 136-138Crossref PubMed Scopus (262) Google Scholar However, these models do not always reflect the clinical and histological features of human NASH. There have been no reports on genetic alterations among patients with NASH, and NASH is closely related to overnutrition and obesity but not to a deficiency of amino acids such as methionine and choline. Therefore, a proper animal model is urgently needed to clarify the molecular pathogenesis of this disease.In 1983, Buja et al22Buja LM Kita T Goldstein JL Watanabe Y Brown MS Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia.Arteriosclerosis. 1983; 3: 87-101Crossref PubMed Google Scholar established an animal model of human familial hypercholesterolemia with developing atherosclerosis using Watanabe heritable hyperlipidemic rabbits that were fed rabbit chow supplemented with 2% cholesterol and 10% corn oil for 2 weeks. They also reported that New Zealand White rabbits fed a diet high in cholesterol and fat for 2 weeks showed early intimal lipid accumulation in the aorta and prominent lipid accumulation in hepatocytes and macrophages of the liver.22Buja LM Kita T Goldstein JL Watanabe Y Brown MS Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia.Arteriosclerosis. 1983; 3: 87-101Crossref PubMed Google Scholar These animal models have since been used for the study of hypertension, hyperlipidemia, arteriosclerosis, and hyperglucosemia, that is, metabolic syndrome. In the course of re-exploring the models, we discovered that these rabbits exhibit steatohepatitis with fibrosis and thus could be a new model of NASH. Here, the mechanism by which iron accumulates in the liver and its role in the pathogenesis of NASH will be discussed.Materials and MethodsMaterialsMonoclonal antibody against smooth muscle α-actin (SM α-actin), Dulbecco's modified Eagle's medium, and fetal bovine serum were obtained from Sigma Chemical Co. (St. Louis, MO). Hamster antibodies against milk fat globule-EGF-factor 8 (MFG-E8) were purchased from Medical & Biological Laboratories (Nagoya, Japan). Monoclonal antibody against glycophorin-A and peroxidase-conjugated secondary antibodies against mouse and rabbit immunoglobulins were from DAKO (Glostrup, Denmark). Alexa Fluor 488 goat anti-mouse IgG2a antibodies and Alexa Fluor 488 goat anti-rabbit IgG antibodies (Molecular Probes, Eugene, OR) were also used as secondary antibodies. Armenian hamster IgG was purchased from BioLegend (San Diego, CA). Enhanced chemiluminescence detection reagent was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Immobilon P membranes were from Millipore Corp. (Bedford, MA). Cell culture inserts were from Falcon (Beckon Dickinson, Franklin Lakes, NJ). Na251CrO4 in sterile saline (37 MBq/ml) was obtained from Daiichi Radioisotope Laboratories Ltd. (Tokyo, Japan). All other reagents were purchased from Sigma Chemical Co. or Wako Pure Chemical Co.Animals and Induction of Liver SteatosisPathogen-free male Japanese White rabbits weighing 3.0 to 3.5 kg were obtained from SLC (Shizuoka, Japan). The rabbits were housed at a constant temperature and supplied with a high-fat diet (HFD) consisting of a standard diet (SD), CR3 (CLEA Japan Inc., Tokyo, Japan), supplemented with 20% corn oil and 1.25% (w/w) cholesterol, ad libitum for 8 weeks. Control rabbits were fed SD. The amount of iron, carbohydrate, amino acids, and other minerals was equivalent between the diets. In some rabbits, a phlebotomy of 35 to 40 ml was performed from the auricular artery weekly during the period. After 8 weeks, the rabbits were anesthetized with an intramuscular injection of ketamine/xylazine (40/5 mg/kg; Sankyo Yell Yakuhin Co. Ltd., Tokyo, Japan/Bayer Corp., Shawnee Mission, KS) and intravenous administration of 1 to 1.5 ml of sodium pentobarbital (20 to 25 mg/kg; Abbott Laboratories, Abbott Park, IL) and then laparotomized. The experiments were humanely conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of Osaka City University School of Medicine.Laboratory Data for Blood SamplesAspartate aminotransferase (AST), alanine aminotransaminase (ALT), total amount of cholesterol (T-cho), triglyceride (TG), lipid peroxide (LPO), complete blood count (CBC), and hydroxyproline (HP) in the serum were measured at Special Reference Laboratories (Osaka, Japan). The blood sugar (BS) concentration was determined by an enzymatic method using glucose oxidase and peroxidase (Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan).Determination of the Serum Insulin LevelThe immunoreactive insulin (IRI) level in serum was assayed using a rat insulin enzyme-linked immunosorbent assay kit (Shibayagi Co. Ltd., Gunma, Japan) and rabbit insulin standard solution (Shibayagi Co. Ltd.).Assay of Hydroxyproline and LPO Levels in the LiverWet liver samples (100 mg) were subjected to acid hydrolysis to determine the amount of hydroxyproline as previously described.23Okuno M Akita K Moriwaki H Kawada N Ikeda K Kaneda K Suzuki Y Kojima S Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-beta.Gastroenterology. 2001; 120: 1784-1800Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar The hepatic level of LPO was determined using a Bioxytech LPO-586 assay kit (Oxis Health Products Inc., Portland, OR) according to the manufacturer's directions. The data were expressed as the amount of hydroxyproline (mg) or LPO (μmol)/wet liver weight (g).Terminal dUTP Nick-End Labeling (TUNEL) StainingFor the detection of apoptosis, paraffin-embedded sections were stained with the TUNEL technique using an In Situ Apoptosis Detection kit (Takara Shuzo Co. Ltd., Ohtsu, Japan) according to the manufacturer's directions. For the semiquantitative analysis, the number of TUNEL-positive cells was counted in five randomly selected fields from each slide at a magnification of ×400.Histochemical and Immunohistochemical Analyses of Rabbit LiverImmunohistochemistry was performed according to methods described elsewhere.22Buja LM Kita T Goldstein JL Watanabe Y Brown MS Cellular pathology of progressive atherosclerosis in the WHHL rabbit. An animal model of familial hypercholesterolemia.Arteriosclerosis. 1983; 3: 87-101Crossref PubMed Google Scholar, 23Okuno M Akita K Moriwaki H Kawada N Ikeda K Kaneda K Suzuki Y Kojima S Prevention of rat hepatic fibrosis by the protease inhibitor, camostat mesilate, via reduced generation of active TGF-beta.Gastroenterology. 2001; 120: 1784-1800Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Rabbits were anesthetized, and the portal vein was cannulated with an 18-gauge Teflon catheter. The liver of each animal was perfused with 100 ml of phosphate-buffered saline (PBS) to remove the blood and then fixed with 4% paraformaldehyde or Bouin's fluid. Each sample was embedded in paraffin or frozen according to methods described elsewhere.24Nishihara E Nagayama Y Inoue S Hiroi H Muramatsu M Yamashita S Koji T Ontogenetic changes in the expression of estrogen receptor alpha and beta in rat pituitary gland detected by immunohistochemistry.Endocrinology. 2000; 141: 615-620Crossref PubMed Scopus (95) Google Scholar, 25Nakatani K Seki S Kawada N Kitada T Yamada T Sakaguchi H Kadoya H Ikeda K Kaneda K Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis.Virchows Arch. 2002; 441: 466-474Crossref PubMed Scopus (50) Google Scholar Paraformaldehyde-fixed specimens were cut into 5-μm-thick sections and stained for 1 hour with 0.1% (w/v) Sirius Red (Direct Red 80; Aldrich, Milwaukee, WI), hematoxylin and eosin (H&E), and Azan-Mallory.26Armendáriz-Borunda J Rojkind M A simple quantitative method for collagen typing in tissue samples: its application to human liver with schistosomiasis.Coll Relat Res. 1984; 4: 35-47Crossref PubMed Scopus (38) Google Scholar Berlin blue, Nile blue, Sudan black, and silver stainings were performed at Kyodo Byori Inc. (Kobe, Japan). Immunostaining of 4-hydroxy-2-nonenal (4-HNE) was performed at Biopathology Institute Co. Ltd. (Oita, Japan). The specimens were subjected to a morphometric analysis of fibrotic areas or fat accumulation using Mac SCOPE version 2.5 (Mitani Corp., Shizuoka, Japan). Immunostaining for SM α-actin and hemoglobin-α was performed using frozen specimens by methods described previously.27Kobayashi S Seki S Kawada N Morikawa H Nakatani K Uyama N Ikeda K Nakajima Y Arakawa T Kaneda K Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells.Cell Tissue Res. 2003; 311: 353-364PubMed Google Scholar After being treated with 5% bovine serum albumin/PBS, the specimens were incubated overnight with primary antibodies in 5% bovine serum albumin/PBS. They were then incubated with both 20 μg/ml Alexa Fluor 488 goat anti-mouse IgG2a antibodies and 20 μg/ml Alexa Fluor 594 goat anti-mouse IgG1 antibodies for 2 hours. Specimens were counterstained for 4,6-diaminodino-2-phenylindole (DAPI) in the nucleus. The sections were observed under an LSM510 confocal laser-scanning microscope (Carl Zeiss, Jena, Germany).Electron MicroscopyElectron microscopy was performed on sections of rabbit liver according to methods described elsewhere.27Kobayashi S Seki S Kawada N Morikawa H Nakatani K Uyama N Ikeda K Nakajima Y Arakawa T Kaneda K Apoptosis of T cells in the hepatic fibrotic tissue of the rat: a possible inducing role of hepatic myofibroblast-like cells.Cell Tissue Res. 2003; 311: 353-364PubMed Google Scholar The liver was perfusion-fixed with 1.5% glutaraldehyde in 0.0062 mol/L cacodylate buffer, pH 7.4, plus 1% sucrose. The fixed livers were cut into small pieces. They were postfixed in 1% OsO4 in 0.1 mol/L phosphate buffer, pH 7.4, dehydrated, and embedded in Polybed. Semithin sections were stained with toluidine blue. Thin sections were stained with saturated uranyl acetate and lead citrate and observed under a light microscope and a JEM-1200EX electron microscope (JEOL, Tokyo, Japan) at 100 kV.Preparation of Nuclear ExtractsFor the separation of cytoplasmic and nuclear extracts, NE-PER nuclear and cytoplasmic extraction reagents (Pierce Biotechnology Inc., Rockford, IL) were used according to the manufacturer's directions.ImmunoblottingProtein samples (10 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto Immobilon P membranes. After blocking, the membranes were treated with primary antibodies and then with peroxidase-conjugated secondary antibodies. Immunoreactive bands were visualized using the enhanced chemiluminescence system and documented with a LAS 1000 (Fuji Photo Film, Kanagawa, Japan). The density of bands was analyzed using a Bio-Rad GS-700 densitometer (Bio-Rad, Hercules, CA).Quantitative Real-Time Polymerase Chain ReactionTotal RNA was extracted from liver tissues using Isogen (Nippon Gene Co. Ltd., Tokyo, Japan).28Kristensen DB Kawada N Imamura K Miyamoto Y Tateno C Seki S Kuroki T Yoshizato K Proteome analysis of rat hepatic stellate cells.Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (204) Google Scholar The expression of mRNA was measured using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA), One-Step SYBR RT-PCR Kit (Perfect Real Time; Takara Bio Inc., Ohtsu, Japan), and Applied Biosystems Prism 7700 (Applied Biosystems) according to a previously reported procedure.29Uyama N Shimahara Y Okuyama H Kawada N Kamo S Ikeda K Yamaoka Y Carbenoxolone inhibits DNA synthesis and collagen gene expression in rat hepatic stellate cells in culture.J Hepatol. 2003; 39: 749-755Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Primers and oligonucleotide probes were designed by using Primer Express (Applied Biosystems) and are listed in Table 1. The primers for interleukin (IL)-8 were generated according to a previous report.30Palacios I Lopez-Armada MJ Hernandez P Sanchez-Pernaute O Gutierrez S Miguelez R Martinez J Egido J Herrero-Beaumont G Tenidap decreases IL-8 and monocyte chemotactic peptide-1 (MCP-1) mRNA expression in the synovial tissue of rabbits with antigen arthritis and in cultured synovial cells.Clin Exp Immunol. 1998; 111: 588-596Crossref PubMed Scopus (25) Google Scholar The primers and dual-labeled probe of MFG-E8 were designed according to the cloned rabbit MFG-E8 cDNA sequence (Supplementary Figure 1 at http://ajp.amjpathol.org). Individual gene expression was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As a standard reaction, cDNA corresponding to 1.6, 8, 40, and 200 ng of total RNA was examined and used as a reference value. The conditions for the TaqMan One-Step RT-PCR Master Mix reagents were as follows: 30 minutes at 48°C (stage 1, reverse transcription), 10 minutes at 95°C (stage 2, reverse transcription inactivation and Ampli Taq Gold activation), and then 40 cycles of amplification for 15 seconds at 95°C and 1 minute at 60°C (stage 3, PCR). The conditions for the One-Step SYBR RT-PCR kit (Perfect Real Time) were as follows: an initial step of 15 minutes at 42°C, 2 minutes at 95°C, and then 40 amplification cycles of denaturation at 94°C for 15 seconds, and annealing and extension at 60°C for 1 minute.Table 1Primer Pairs and Probes Used for RT-PCRPrimer nameSequenceReferenceNoteCollagen α1(I)AY633663TaqMan Forward5′-ACTGGATTGACCCCAACCAA-3′ Reverse5′-TTGCCCCAGTGTCCATGTC-3′ Probe5′-CTGCAACCTGGATGCCATCAAGGTC-3′GAPDHAB231852TaqMan Forward5′-GCCAAAAGGGTCATCATCTCA-3′ Reverse5′-GTGGTTCACGCCCATCACA-3′ Probe5′-CCTCCGCCGATGCCCCCA-3′Heme oxygenase-1AY421756TaqMan Forward5′-GGAGAACGCCGAGTTCATGA-3′ Reverse5′-GGCCATCACCAGCTTAAAACC-3′ Probe5′-AACTTTCAGAAGGGCCAGGTGACTGCC-3′IL-1βM26295TaqMan Forward5′-TCCAGACGAGGGCATCCA-3′ Reverse5′-CTGCCGGAAGCTCTTGTTG-3′ Probe5′-CTGCGCATCTCCTGCCAACCCT-3′IL-8Ref. 30Palacios I Lopez-Armada MJ Hernandez P Sanchez-Pernaute O Gutierrez S Miguelez R Martinez J Egido J Herrero-Beaumont G Tenidap decreases IL-8 and monocyte chemotactic peptide-1 (MCP-1) mRNA expression in the synovial tissue of rabbits with antigen arthritis and in cultured synovial cells.Clin Exp Immunol. 1998; 111: 588-596Crossref PubMed Scopus (25) Google ScholarSYBR Green Forward5′-AACCTTCCTGCTGTCTCTGA-3′ Reverse5′-TCTGCACCCACTTTTTCCTTG-3′MCP-1M57440TaqMan Forward5′-CTCTCACCCTCCAGCATGAAG-3′ Reverse5′-AGCTGAAGGCGACTGCTATGA-3′ Probe5′-TCTCTGCAACGCTTCTGTGCCTGC-3′MMP9D26514TaqMan Forward5′-GCTCCGGTGGATCAGATGTT-3′ Reverse5′-AAGCGGTCCTGGCAGAAGT-3′ Probe5′-CACACGACGTCTTCCAGTACCGAGAG-3′TGF-β1AF000133TaqMan Forward5′-AAGGGCTACCACGCCAACTT-3′ Reverse5′-CGGGTTGTGCTGGTTGTACA-3′ Probe5′-TGCCTGGGACCCTGCCCCTAC-3′TIMP2AF069713TaqMan Forward5′-TCACGCTCTGTGACTTCATCGT-3′ Reverse5′-TGTGGTTCAGGCTCTTCTTCTG-3′ Probe5′-CCCTGGGACTCCCTGAGCAGCA-3′TNF-αM12845SYBR Green Forward5′-TGAAGCTCACGGACAACCA-3′ Reverse5′-TGTGAGTGAGGAGCACGTAGGA-3′NF-κBAY753407TaqMan Forward5′-CACAGCGCTTCTCAGGAGTTC-3′ Reverse5′-CCGCCGGGCCACAT-3′ Probe5′-AGCTATGATGGAAGGTTCGTGGC-3′MFG-E8TaqMan Forward5′-TGGGCGCAAGTTCCAGTT-3′ Reverse5′-GTTACCGGCAAACACCTTGTC-3′ Probe5′-ATCCAGGAAGCGGACGGGTCTGG-3′MMP, matrix metalloproteinase. Open table in a new tab Subcloning of MFG-E8 cDNARabbit MFG-E8 cDNA was cloned by homologous PCR using a primer pair, 5′-TGGATCCAGGTGAACCT-3′ (forward) and 5′-TAACAGCCCAGCAGCTC-3′ (reverse), designed from two common nucleotide sequences of human and mouse MFG-E8, which have already been cloned and registered in the NCBI database (accession no. NM005928 and NM008594, respectively). The 880-bp PCR product was purified and ligated into pGEM-T Easy (Promega, Madison, WI). Automatic DNA sequencing was performed to confirm the proper sequence of the inserted DNA using ABI Prism 310 (Supplementary Figure 1 at http://ajp.amjpathol.org).Osmotic Fragility Test for ErythrocytesOsmotic fragility was assessed in freshly isolated erythrocytes collected from the auricular vein. Blood was washed three times with PBS to remove serum. Packed erythrocytes (0.1 ml) were incubated in 1 ml of 7 g/L NaCl for 15 minutes and then centrifuged at 1000 × g for 5 minutes. Absorption of the supernatant was read at 540 nm. Data are presented as adjusted percentages of the maximum optical density obtained. The value for hemolysis was calculated, assuming a linear response between the minimum and maximum optical density readings.Flow Cytometric Binding Assay of Fluorescent Phospholipid VesiclesThe externalization of phosphatidylserine (PS) on the outer leaflet of the erythrocyte membrane was detected by flow cytometry using an Annexin V-Biotin apoptosis detection kit (Medical & Biological Laboratories Co. Ltd.). Erythrocytes were separated from the serum, and then incubated with serum from the same rabbit for 24 hours. They were washed, mixed with Annexin V-Biotin reagent in a binding buffer containing Ca2+, and incubated with streptavidin-phycoerythrin (PE) as described previously.31Martin SJ Reutelingsperger CP McGahon AJ Rader JA van Schie RC LaFace DM Green DR Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.J Exp Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2551) Google Scholar Flow cytometry was performed with FACS Calibur (BD Biosciences, San Jose, CA) as reported.32Lanier LL Le AM Phillips JH Warner NL Babcock GF Subpopulations of human natural killer cells defined by expression of the Leu-7 (HNK-1) and Leu-11 (NK-15) antigens.J Immunol. 1983; 131: 1789-1796PubMed Google Scholar, 33Reichert RA Weissman IL Butchr EC Phenotypic analysis of thymocytes that express homing receptors for peripheral lymph nodes.J Immunol. 1986; 136: 3521-3528PubMed Google Scholar Samples were analyzed on a flow cytometer with forward and side scatter gates set to include cells but exclude cellular debris and phospholipid vesicles. Fluoresceins were excited at 488 nm and detected at 575 ± 13 nm for PE. The amount of fluorescent vesicles bound per cell was then calculated from the mean fluorescence intensity of the gated cell population. Specific binding was calculated by subtracting nonspecific binding from total binding.Assay of PhagocytosisIn Vitro StudyBlood from C57BL/6 mice (12 weeks old, male) was collected and washed three times with PBS. Erythrocytes were incubated with saline containing Na251CrO4 (0.1 mCi/ml packed cells) for 30 minutes at 37°C.34Harrison Jr, JH Jollow DJ Role of aniline metabolites in aniline-induced hemolytic anemia.J Pharmacol Exp Ther. 1986; 238: 1045-1054PubMed Google Scholar The 51Cr-labeled erythrocytes were washed, resuspended (hematocrit, 5%), and incubated with 3 mmol/L tert-butyl hydroperoxide (t-BHP; Sigma-Aldrich Co., St. Louis, MO) in PBS at 37°C.35Mandal D Moitra PK Saha S Basu J Caspase 3 regulates phosphatidylserine externalization and phagocytosis of oxidatively stressed erythrocytes.FEBS Lett. 2002; 513: 184-188Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar The cells were washed and used for in vitro phagocytosis assay conducted with a mouse macrophage cell line, RAW 264.7, expressing integrin αvβ3.36Gao C Guo H Wei J Kuo PC Osteopontin inhibits expression of cytochrome c oxidase in RAW 264.7 murine macrophages.Biochem Biophys Res Commun. 2003; 309: 120-125Crossref PubMed Scopus (18) Google Scholar The t-BHP-treated erythrocytes (1 × 106) were added to RAW 264.7 cells (1 × 105) on glass microscope slides (Nalge Nunc International, Naperville, IL) in the presence or absence of anti-M
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