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• W2000571185 abstract "Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidine-rich glycoprotein (HRG), an abundant (∼100 μg/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes. Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidine-rich glycoprotein (HRG), an abundant (∼100 μg/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes. Cell death is vital for the morphological shaping of tissues during development and for the sculpting of functionally appropriate cellular repertoires as well as for protecting an individual from viral infections and pathogenic microorganisms (1Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (934) Google Scholar, 2Cecconi F. Alvarez-Bolado G. Meyer B.I. Roth K.A. Gruss P. Cell. 1998; 94: 727-737Abstract Full Text Full Text PDF PubMed Scopus (820) Google Scholar). Selective cell death continues to play a role in the homeostasis of mature tissues, such as the deletion of immune cells in the attenuation of an immune response (3Webb S. Morris C. Sprent J. Cell. 1990; 63: 1249-1256Abstract Full Text PDF PubMed Scopus (851) Google Scholar) and the elimination of cells that have become functionally inappropriate, including virally infected and transformed cells (4Kagi D. Seiler P. Pavlovic J. Ledermann B. Burki K. Zinkernagel R.M. Hengartner H. Eur. J. Immunol. 1995; 25: 3256-3262Crossref PubMed Scopus (254) Google Scholar). Apoptosis and necrosis represent two different forms of cell death and are characterized by distinct morphologies. Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis, whereas the role and extent of phagocytosis in the clearance of necrotic cells is not well documented. It is thought, however, that by engulfing necrotic and apoptotic cells phagocytes of the innate immune system not only provide a first line of defense against microbial pathogens but also dispose of self-antigens that are released from dying cells (5Franc N.C. White K. Ezekowitz R.A. Curr. Opin. Immunol. 1999; 11: 47-52Crossref PubMed Scopus (103) Google Scholar). Apoptosis is characterized by an orderly sequence of internal events, including chromatin condensation that precedes the loss of cellular integrity (6Harvey K.J. Lukovic D. Ucker D.S. J. Cell Biol. 2000; 148: 59-72Crossref PubMed Scopus (83) Google Scholar). Apoptotic cells also display phosphatidylserine and altered membrane carbohydrates on their surface. Multiple ligands and receptors have been implicated in the recognition and uptake of apoptotic cells by phagocytes prior to membrane lysis, thus preventing release of potentially toxic and immunogenic intracellular substances into tissues. In addition, the binding and/or uptake of apoptotic cells inhibits proinflammatory cytokine production (7Huynh M.L. Fadok V.A. Henson P.M. J. Clin. Investig. 2002; 109: 41-50Crossref PubMed Scopus (1029) Google Scholar). When there are disturbances in either apoptosis or the phagocytosis of apoptotic cells, antibodies against subsequently exposed nucleosomes may be formed, leading to the development of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In contrast to apoptosis, necrotic cell death has usually been defined as a disordered mode of cell death, occurring either in cases of severe and acute injuries such as sudden shortage of nutrients and abrupt anoxia or in extreme injuries such as exposure to heat, detergents, strong bases, and irradiation (8Fishelson Z. Attali G. Mevorach D. Mol. Immunol. 2001; 38: 207-219Crossref PubMed Scopus (170) Google Scholar, 9Proskuryakov S.Y. Konoplyannikov A.G. Gabai V.L. Exp. Cell Res. 2003; 283: 1-16Crossref PubMed Scopus (557) Google Scholar). More recently, the existence of a necrotic-like cell death pathway regulated by an intrinsic death program distinct from that of apoptosis has also become apparent (9Proskuryakov S.Y. Konoplyannikov A.G. Gabai V.L. Exp. Cell Res. 2003; 283: 1-16Crossref PubMed Scopus (557) Google Scholar, 10Kitanaka C. Kuchino Y. Cell Death Differ. 1999; 6: 508-515Crossref PubMed Scopus (352) Google Scholar). Necrotic cell death is characterized by the rapid and disorganized swelling and rupture of a cell (11Shacter E. Williams J.A. Hinson R.M. Senturker S. Lee Y.J. Blood. 2000; 96: 307-313Crossref PubMed Google Scholar). As with apoptotic cells, necrotic cells are usually rapidly cleared from the circulation by phagocytic cells. A large number of soluble extracellular proteins have been described (12Savill J. Dransfield I. Gregory C. Haslett C. Nat. Rev. Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1326) Google Scholar) that bind to apoptotic cells and facilitate their uptake by macrophages. In contrast, few proteins have been identified that can specifically opsonize necrotic cells. Histidine-rich glycoprotein (HRG) 3The abbreviations used are: HRGhistidine-rich glycoproteinOVAovalbuminN1N2amino-terminal domain of histidine-rich glycoproteinCHOChinese-hamster ovaryGAGglycosaminoglycanFCSfetal calf serumePBSphosphate-buffered salineBSAbovine serum albuminmAbmonoclonal antibodySNARFcarboxy-seminaphthorhodafluorCFSEcarboxy-fluorescein diacetate succinimidyl ester. (13Jones A.L. Hulett M.D. Parish C.R. Immunol. Cell Biol. 2005; 83: 106-118Crossref PubMed Scopus (255) Google Scholar) is a 75-kDa plasma glycoprotein (∼100 μg/ml) that binds to numerous ligands, including heparan sulfate on the cell surface and in extracellular matrices (14Heimburger N. Haupt H. Kranz T. Baudner S. Hoppe-Seyler's Z. Physiol. Chem. 1972; 353: 1133-1140Crossref PubMed Scopus (120) Google Scholar, 15Lijnen H.R. van Hoef B. Collen D. Thromb. Haemostasis. 1983; 50: 560-562Crossref PubMed Scopus (29) Google Scholar, 16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), plasminogen (17Kluft C. Los P. Thromb. Haemostasis. 1988; 60: 411-414Crossref PubMed Scopus (10) Google Scholar, 18Leung L.L. J. Clin. Investig. 1986; 77: 1305-1311Crossref PubMed Scopus (119) Google Scholar, 19Jones A.L. Hulett M.D. Altin J.G. Hogg P. Parish C.R. J. Biol. Chem. 2004; 279: 38267-38276Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 20Borza D.B. Morgan W.T. J. Biol. Chem. 1997; 272: 5718-5726Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), thrombospondin (21Walz D.A. Bacon-Baguley T. Kendra-Franczak S. DePoli P. Semin. Thromb. Hemostasis. 1987; 13: 317-325Crossref PubMed Scopus (8) Google Scholar, 22Silverstein R.L. Leung L.L. Harpel P.C. Nachman R.L. J. Clin. Investig. 1985; 75: 2065-2073Crossref PubMed Scopus (81) Google Scholar, 23Simantov R. Febbraio M. Crombie R. Asch A.S. Nachman R.L. Silverstein R.L. J. Clin. Investig. 2001; 107: 45-52Crossref PubMed Scopus (92) Google Scholar), tropomyosin (24Donate F. Juarez J.C. Guan X. Shipulina N.V. Plunkett M.L. Tel-Tsur Z. Shaw D.E. Morgan W.T. Mazar A.P. Cancer Res. 2004; 64: 5812-5817Crossref PubMed Scopus (55) Google Scholar, 25Guan X. Juarez J.C. Qi X. Shipulina N.V. Shaw D.E. Morgan W.T. McCrae K.R. Mazar A.P. Donate F. Thromb. Haemostasis. 2004; 92: 403-412Crossref PubMed Google Scholar), IgG (26Gorgani N.N. Parish C.R. Easterbrook Smith S.B. Altin J.G. Biochemistry. 1997; 36: 6653-6662Crossref PubMed Scopus (72) Google Scholar, 27Gorgani N.N. Parish C.R. Altin J.G. J. Biol. Chem. 1999; 274: 29633-29640Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), C1q (26Gorgani N.N. Parish C.R. Easterbrook Smith S.B. Altin J.G. Biochemistry. 1997; 36: 6653-6662Crossref PubMed Scopus (72) Google Scholar), and Fc receptor (28Gorgani N.N. Altin J.G. Parish C.R. Int. Immunol. 1999; 11: 1275-1282Crossref PubMed Scopus (29) Google Scholar, 29Gorgani N.N. Smith B.A. Kono D.H. Theofilopoulos A.N. J. Immunol. 2002; 169: 4745-4751Crossref PubMed Scopus (47) Google Scholar). HRG is a multidomain molecule consisting of an amino-terminal domain composed of two cystatin-like modules (N1N2), a central histidine-rich region, and a carboxyl-terminal domain (30Koide T. Foster D. Yoshitake S. Davie E.W. Biochemistry. 1986; 25: 2220-2225Crossref PubMed Scopus (112) Google Scholar, 31Hulett M.D. Parish C.R. Immunol. Cell Biol. 2000; 78: 280-287Crossref PubMed Scopus (35) Google Scholar). Such a molecular structure allows the molecule to act as an adaptor protein that cross-links different ligands in solution or on cell surfaces (13Jones A.L. Hulett M.D. Parish C.R. Immunol. Cell Biol. 2005; 83: 106-118Crossref PubMed Scopus (255) Google Scholar). Furthermore, the histidine-rich region of HRG has recently been shown to mediate anti-angiogenic effects (32Juarez J.C. Guan X. Shipulina N.V. Plunkett M.L. Parry G.C. Shaw D.E. Zhang J.C. Rabbani S.A. McCrae K.R. Mazar A.P. Morgan W.T. Donate F. Cancer Res. 2002; 62: 5344-5350PubMed Google Scholar, 33Olsson A.K. Larsson H. Dixelius J. Johansson I. Lee C. Oellig C. Bjork I. Claesson-Welsh L. Cancer Res. 2004; 64: 599-605Crossref PubMed Scopus (81) Google Scholar), with HRG also being implicated in plasminogen activation (20Borza D.B. Morgan W.T. J. Biol. Chem. 1997; 272: 5718-5726Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 34Borza D.B. Shipulina N.V. Morgan W.T. Blood Coagul. Fibrinolysis. 2004; 15: 663-672Crossref PubMed Scopus (8) Google Scholar), particularly on cell surfaces (19Jones A.L. Hulett M.D. Altin J.G. Hogg P. Parish C.R. J. Biol. Chem. 2004; 279: 38267-38276Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 34Borza D.B. Shipulina N.V. Morgan W.T. Blood Coagul. Fibrinolysis. 2004; 15: 663-672Crossref PubMed Scopus (8) Google Scholar), and in immune complex clearance (28Gorgani N.N. Altin J.G. Parish C.R. Int. Immunol. 1999; 11: 1275-1282Crossref PubMed Scopus (29) Google Scholar, 35Gorgani N.N. Altin J.G. Parish C.R. Immunology. 1999; 98: 456-463Crossref PubMed Scopus (24) Google Scholar). A recent study by Gorgani et al. (29Gorgani N.N. Smith B.A. Kono D.H. Theofilopoulos A.N. J. Immunol. 2002; 169: 4745-4751Crossref PubMed Scopus (47) Google Scholar) also suggested that HRG can potentiate the ingestion of late stage apoptotic cells by macrophages. histidine-rich glycoprotein ovalbumin amino-terminal domain of histidine-rich glycoprotein Chinese-hamster ovary glycosaminoglycan fetal calf serume phosphate-buffered saline bovine serum albumin monoclonal antibody carboxy-seminaphthorhodafluor carboxy-fluorescein diacetate succinimidyl ester. While investigating the interaction of HRG with different cell populations, it became apparent that the molecule bound strongly to dead cells; thus we undertook a detailed study of the interaction of HRG with necrotic, apoptotic, and viable cells. It was found that HRG binds avidly to necrotic cells, but not to early stage apoptotic cells, and aids the uptake of necrotic cells by a phagocytic cell line. Although HRG interacts with cell surface heparan sulfate on viable cells, additional experiments revealed that HRG binds via its N1N2 domain to a cytoplasmic ligand within necrotic cells that is not heparan sulfate. These data demonstrate, for the first time, that HRG represents a novel plasma protein that specifically facilitates the ingestion of necrotic cells by phagocytes and thus may play a key role in maintaining the efficient clearance of necrotic cells and necrotic cell debris from the circulation. Cell Lines—THP-1 and Jurkat cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS). pgsA-745 cells are a mutant form of the CHO-KI parent cell line and are unable to express any cell surface heparan sulfate due to a deficiency in xylosyltransferase. Both CHO-KI (GAG+ve) and pgsA-745 (GAG-ve) cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 21 μg/ml l-proline and 10% FCS. Mammalian cell lines were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The Spodoptera frugiperda-derived insect cell line (Sf-9) was cultured in Sf-900 II serum-free medium (Invitrogen) at 27 °C. Purification of HRG—HRG was purified from human plasma based on a previously described method (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 36Rylatt D.B. Sia D.Y. Mundy J.P. Parish C.R. Eur. J. Biochem. 1981; 119: 641-646Crossref PubMed Scopus (52) Google Scholar). Briefly, human plasma was passed through a phosphocellulose column equilibrated with 0.5 m NaCl, 10 mm sodium phosphate, 1 mm EDTA, pH 6.8. Bound HRG was eluted with 2.0 m NaCl, 10 mm sodium phosphate, 1 mm EDTA, pH 6.8. Recombinant full-length HRG (507 amino acids) (30Koide T. Foster D. Yoshitake S. Davie E.W. Biochemistry. 1986; 25: 2220-2225Crossref PubMed Scopus (112) Google Scholar) and the N1N2 amino-terminal domain (amino acids 1-112) consisting of two cystatin-like modules, were produced in insect Sf9 cells using a baculovirus expression system as previously described (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Immunofluorescence Flow Cytometry—Cell lines were analyzed for HRG binding by immunofluorescence flow cytometry. Typically, plasma-purified or recombinant HRG and recombinant N1N2 domain (100 μg/ml) were added to 5 × 105 cells in PBS/0.1% BSA ± 20 μm Zn2+ for 60 min at 4 °C and washed three times with PBS/0.1% BSA. Cell-bound HRG or N1N2 domain was detected using the HRG-specific mAb HRG-4 (AGEN, Brisbane, Australia). Cell surface heparan sulfate expression was detected by the heparan sulfate-specific mAb F58-10E4 (Seikagku Corp., Tokyo, Japan), followed by secondary detection with sheep anti-mouse Ig fluorescein isothiocyanate (Amrad Biotech, Melbourne, Australia). Cells were analyzed by immunofluorescence flow cytometry using an LSR Flow Cytometer (BD Biosciences). Flow cytometry data were analyzed using Cell Quest Pro software (BD Biosciences). Each treatment condition was typically repeated in triplicate, and each experiment was repeated two to three times. In some experiments human HRG (100 μg/ml) was co-incubated with 100 μg/ml of 12.5-kDa bovine lung heparin (Sigma). Induction and Detection of Necrotic Cells—Jurkat T-cells were induced into necrosis by exposure to hyperthermic conditions. Cells were resuspended to a cell concentration of 1 × 107 cells/ml in RPMI 1640/10% FCS and placed into a 56 °C water bath for 45 min. Necrotic cells were detected by incubating cells with the DNA intercalating dye 7-AAD (0.3 μg/ml) (Molecular Probes Inc., Eugene, OR) in PBS/0.1% BSA (pH 7.2) for 15 min at 4 °C in the dark. Cells were then analyzed by flow cytometry as described. Necrotic cells were detected by gating on 7-AAD-positive cells. Induction and Detection of Apoptotic Cells—Jurkat T-cells were resuspended to 5 × 105 cell/ml and incubated with 1 μm camptothecin (Sigma) at 37 °C in a humidified atmosphere of 5% CO2 for 6 h. Detection of apoptotic cells was achieved by Annexin-V-PE cell surface staining (BD Biosciences) and 7-AAD nuclear uptake (0.3 μg/ml) (Molecular Probes) using flow cytometry as described. Early stage apoptotic cells were defined as Annexin-V-positive, 7-AAD-negative cells. Dye Labeling—THP-1 cells were labeled with carboxy-seminaphthorhodafluor (SNARF-1) (Molecular Probes) or PKH26 (Sigma), and Jurkat cells were labeled with carboxy-fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes). For SNARF-1 labeling, THP-1 cells (5 × 107 cell/ml) diluted in RPMI 1640 were incubated with SNARF-1 (25 μm) for 15 min in a 37 °C water bath. Cells were then washed four times in RPMI 1640/10% FCS to remove unbound SNARF-1. For PKH26 labeling, THP-1 cells (≤5 × 107 cell) were washed in PBS, pelleted, and resuspended in 250 μl of PBS. PKH26 was pre-diluted in 19.5 μl of diluent C (Sigma) and immediately added to cells at a final concentration of 20 nm for 5 min. The reaction was stopped by adding 275 μl of FCS for 1 min. Cells were washed four times in RPMI 1640/10% FCS to remove unbound PKH26. For CFSE labeling, Jurkat T-cells (5 × 107 cell/ml) diluted in RPMI 1640 were incubated with CFSE (2 μm) for 15 min in a 37 °C water bath. Cells were washed four times in RPMI 1640/10% FCS to remove unbound CFSE. Some of the Jurkat T-cells were then induced into necrosis or apoptosis as described. Confocal Microscopy—Viable and necrotic CHO-KI and pgsA-745 cells were incubated with HRG as described above for immunofluorescence flow cytometry and examined by confocal microscopy for HRG localization. In the case of the phagocytosis assay, PKH26-labeled THP-1 cells were incubated with CFSE-labeled necrotic Jurkat cells for 80 min at 37 °C. Cell suspensions were mounted onto glass slides, and confocal images were visualized on a Nikon Eclipse TE 300 confocal microscope with a Nikon super high pressure mercury lamp power supply (Nikon Corp., Tokyo, Japan) and a Radiance 2000 laser scanning system (Bio-Rad). Phagocytosis Assay—Equal volumes of SNARF-1-labeled THP-1 cells (1 × 106 cell/ml) and CFSE-labeled Jurkat cells (1 × 107 cell/ml) in RPMI 1640/10% FCS were mixed and incubated (37 °C, 5% CO2, 0-80 min) with either ovalbumin (OVA) (100 μg/ml; Sigma) or different concentrations of HRG in the presence or absence of 12.5-kDa bovine lung heparin (100 μg/ml), porcine muscle tropomyosin (200 μg/ml; Sigma), a CD32 (FcγRII) mAb (clone 8.26) (37Ierino F.L. Powell M.S. McKenzie I.F. Hogarth P.M. J. Exp. Med. 1993; 178: 1617-1628Crossref PubMed Scopus (76) Google Scholar), and a CD64 (FcγRI) mAb (clone 10.1) (BD Biosciences) in a pre-warmed 96-well plate. Cells were then immediately placed on ice and analyzed by flow cytometry. Rate of phagocytosis was calculated as the percentage of SNARF+ THP-1 that were CFSE+. Proper inter- and intralaser compensations were determined using single color controls before each experimental run. Binding of HRG and OVA to Intracellular Ligands—Recombinant full-length HRG or the N1N2 domain of HRG (20 μl, 100 μg/ml) or OVA (20 μl, 100 μg/ml) diluted in PBS was incubated with fixed monolayers of HEp-2 human epithelial cells (HEp-2 slides; INOVA Diagnostics, San Diego, CA) for 30 min. The slides were extensively washed with PBS before detection of bound HRG using a HRG-specific mAb, HRG-4 (AGEN), or OVA using an OVA-specific mAb (Sigma) followed by secondary detection with sheep anti-mouse Ig fluorescein isothiocyanate (Amrad Biotech). Slides were mounted with coverslips and fluorescent mounting medium (DakoCytomation, Carpinteria, CA) before viewing immediately using an Olympus fluorescence microscope (Olympus Optical Co. Ltd, Tokyo, Japan). In some experiments HRG (100 μg/ml) was co-incubated with 12.5-kDa bovine lung heparin (100 μg/ml) (Sigma) or porcine muscle tropomyosin (200 μg/ml) (Sigma). In other experiments, slides were treated with Escherichia coli-derived RNase I (100 μg/ml; Promega Corp., Madison, WI) or bovine pancreas-derived RQ1 DNase I (100 units/ml, Promega) for 30 min at 37 °C prior to HRG binding and detection. Binding of HRG to Necrotic Cells Is Heparan Sulfate-independent and Is Mediated by Its N1N2 Domain—Our laboratory recently demonstrated that HRG binds to cell surface heparan sulfate on viable cells via its N1N2 domain and that this binding is almost entirely abolished by high concentrations of heparin (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). While investigating the binding of HRG to transfected cell lines exposed to selective antibiotics, it was noted that the late stage apoptotic cells produced by antibiotic selection exhibited enhanced binding of HRG that was not inhibited by heparin (data not shown). A previous report suggested that HRG interacts with late stage apoptotic cells (29Gorgani N.N. Smith B.A. Kono D.H. Theofilopoulos A.N. J. Immunol. 2002; 169: 4745-4751Crossref PubMed Scopus (47) Google Scholar), but we wished to further investigate the molecular basis of this phenomenon and determine whether HRG can interact with necrotic and early stage apoptotic cells. CHO cell lines that either express cell surface GAGs (CHO-KI) or lack cell surface GAGs (pgsA-745) due to a deficiency in xylosyltransferase (38Esko J.D. Stewart T.E. Taylor W.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3197-3201Crossref PubMed Scopus (485) Google Scholar, 39Esko J.D. Elgavish A. Prasthofer T. Taylor W.H. Weinke J.L. J. Biol. Chem. 1986; 261: 15725-15733Abstract Full Text PDF PubMed Google Scholar) were used in these initial experiments. GAG+ve and GAG-ve CHO cells were induced into necrosis by exposure of the cells to hyperthermic conditions (56 °C) for 45 min. Cell death was confirmed as >99% using the DNA intercalating dye 7-AAD with viability being assessed by flow cytometry (Fig. 1A). Both viable and necrotic GAG+ve and GAG-ve CHO cells were analyzed for their capacity to bind HRG. In agreement with previous findings (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), viable GAG+ve CHO cells bound human HRG (100 μg/ml), binding that was almost entirely abolished by the presence of 12.5-kDa bovine lung heparin (100 μg/ml) (Fig. 1, B and C). In contrast, HRG exhibited little binding to viable GAG-ve CHO cells either in the presence or absence of heparin (Fig. 1, B and C). On the other hand, HRG bound strongly to necrotic GAG+ve CHO cells at levels 2-3-fold higher than to viable GAG+ve CHO cells. Binding to necrotic GAG-ve CHO cells was at least 100-fold higher than binding to viable GAG-ve CHO cells (Fig. 1, B and C). Necrotic cell binding was only partially inhibited by heparin (∼20-30%), suggesting that the majority of HRG binding to necrotic cells is mediated through a heparan sulfate-independent ligand. We have previously shown that heparan sulfate-mediated HRG binding to viable cells is highly dependent on the presence of physiological concentrations of free Zn2+ (20 μm) (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Interestingly, HRG binding to necrotic cells is not dependent on the presence of physiological concentrations of free Zn2+ (data not shown), indicating that HRG binding to necrotic cells is Zn2+-independent. Confocal microscopy was used to visualize the binding of HRG to both viable and hyperthermia-induced necrotic CHO cells using the HRG-specific mAb HRG-4 (Fig. 1D). Viable GAG+ve CHO cells exhibited HRG binding (green) that was predominantly localized to the outer surface of all viable cells. As expected, viable GAG-ve CHO cells did not exhibit HRG binding (data not shown). Conversely, HRG binding to necrotic GAG+ve (data not shown) and GAG-ve CHO cells was localized within the cytoplasm, but not the nucleus (Fig. 1D). Necrotic cells exhibited positive nuclei staining for the DNA intercalating dye 7-AAD, whereas viable cells were negative for 7-AAD staining (Fig. 1D). Recombinant full-length and the amino-terminal (N1N2) domain of HRG were produced in insect cells using the baculovirus expression system as previously described (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Flow cytometry studies were performed to determine whether the N1N2 domain of HRG also binds to necrotic cells. In agreement with our previous studies (16Jones A.L. Hulett M.D. Parish C.R. J. Biol. Chem. 2004; 279: 30114-30122Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), the N1N2 domain bound to viable GAG+ve CHO cells, binding that was completely inhibited by 12.5-kDa bovine lung heparin (100 μg/ml) (Fig. 2, A and B). Consistent with this observation, the N1N2 domain of HRG did not bind to GAG-deficient CHO cells (Fig. 2, A and B). However, the N1N2 domain of HRG exhibited comparable binding to both necrotic GAG+ve and GAG-ve CHO cells, binding that was not blocked by heparin (Fig. 2, A and B). Again, binding was not affected by the presence of physiological concentrations of Zn2+ (data not shown). Collectively, these data indicate that the N1N2 domain of HRG interacts with necrotic cells and that the necrotic cell ligand is unrelated to heparan sulfate. HRG Binds Poorly to Early Stage Apoptotic Cells—The capacity of HRG to bind to early stage (6 h) apoptotic (pre-necrotic) cells was then investigated. Jurkat T cells were induced into early stage apoptosis (1 μm camptothecin, 6 h, 37 °C), and the cells were analyzed by flow cytometry for Annexin-V binding and uptake of the DNA intercalating dye 7-AAD. Viable cells were defined as 7-AAD- and Annexin-V-negative, early stage apoptotic cells (30% of camptothecin-treated cells) were classified as Annexin-V-positive, 7-AAD-negative, and necrotic cells (hyperthermia treatment) were identified as 7-AAD-positive, Annexin-V-negative (Fig. 3A). The ability of HRG to bind to early stage apoptotic cells, compared with viable or necrotic cells, and the effect of heparin on this binding was then assessed. In contrast to viable cells, early stage apoptotic cells exhibited very low levels of HRG binding that was marginally affected by heparin, whereas necrotic cells bound high levels of HRG that was also only slightly affected by heparin (Fig. 3, B and C). Based on reactivity with a heparan sulfate-specific mAb, early stage apoptotic cells were also shown to exhibit very low levels of cell surface heparan sulfate (data not shown). Thus the predominant cell surface ligand for HRG is lost from early stage apoptotic cells, a process that results in these cells reacting very weakly with HRG. Furthermore, Jurkat cells behave in a similar manner to GAG+ve CHO cells in that when they are necrotic they bind HRG in a heparan sulfate-independent manner. These binding data also suggest that the ligand recognized by HRG in necrotic cells is not exposed until the cell membrane has become permeable, allowing HRG access to cytoplasmic ligands within necrotic cells. Early stage apoptotic cells have intact cell membranes and thus do not appear to allow HRG access to the cytoplasmic ligands. HRG Binds Specifically to an Intracellular Cytoplasmic Ligand via Its N1N2 Domain—Based on the above data, experiments were under-taken to characterize the interaction of HRG with intracellular ligands within necrotic cells. Our confocal studies (Fig. 1D) indicated that HRG interacts with necrotic cells via a cytoplasmic ligand. To confirm this observation, we examined the binding of HRG to monolayers of fixed human HEp-2 epithelial cells. This fixed epithelial cell line is routinely used clinically to screen for anti-nuclear antibodies that are present in the sera of systemic lupus erythematosus patients. It was found that both recombinant full-length HRG and the N1N2 domain of HRG bound uniformly to the cytoplasm of the HEp-2 cells, with no binding being detected in the cell nuclei (Fig. 4A), suggesting that the HRG ligand is evenly distributed throughout the cytoplasm of cells. Treatment of the slides with RNase (100 μg/ml), DNase I (100 units/ml), and co-incubation of HRG with 12.5-kDa bovine lung heparin (100 μg/ml) or tropomyosin (200 μg/ml) did not modify the intensity or binding pattern of HRG or the N1N2 domain (data not shown), suggesting that the cytoplasmic ligand for HRG is unrelated to RNA, DNA, heparin-like molecules, or tropomyosin. As a specificity control, the binding of an irrelevant protein (OVA) to the HEp-2 cells was examined. OVA did not show any detectable binding to the fixed epithelial cells when incubated at similar concentrations to HRG (Fig. 4B), indicat" @default.
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• W2000571185 date "2005-10-01" @default.
• W2000571185 modified "2023-10-09" @default.
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