Role of Phosphatidylserine Exposure and Sugar Chain Desialylation at the Surface of Influenza Virus-infected Cells in Efficient Phagocytosis by Macrophages
2002; Elsevier BV; Volume: 277; Issue: 20 Linguagem: Inglês
10.1074/jbc.m201074200
ISSN1083-351X
AutoresYuichi Watanabe, Akiko Shiratsuchi, Kazufumi Shimizu, Takenori Takizawa, Yoshinobu Nakanishi,
Tópico(s)Diabetes and associated disorders
ResumoHeLa cells infected with influenza A virus undergo typical caspase-dependent apoptosis and are efficiently phagocytosed by mouse peritoneal macrophages in a manner mediated by the membrane phospholipid phosphatidylserine, which is translocated to the surface of virus-infected cells during apoptosis. However, the extent of phagocytosis is not always parallel with the level of phosphatidylserine externalization. Here we examined the involvement of influenza virus neuraminidase (NA) in efficient phagocytosis of virus-infected cells. HeLa cells infected with an influenza virus strain expressing temperature-sensitive NA underwent apoptosis and produced viral proteins, including the defective NA, at a non-permissive temperature to almost the same extent as cells infected with the wild-type virus. The cells were, however, phagocytosed by macrophages with reduced efficiency. In addition, phagocytosis of cells infected with the wild-type virus was severely inhibited when the cells had been maintained in the presence of the NA inhibitor zanamivir. On the other hand, the binding of sialic acid-recognizing lectins to the cell surface declined after infection with the wild-type virus. The decrease in the extent of lectin binding was greatly attenuated when cells were infected with the mutant virus or when wild-type virus-infected cells were maintained in the presence of zanamivir. These results indicate that sugar chains are desialylated by NA at the surface of virus-infected cells. We conclude that the presence of both phosphatidylserine and asialoglycomoieties on the cell surface is required for efficient phagocytosis of influenza virus-infected cells by macrophages. HeLa cells infected with influenza A virus undergo typical caspase-dependent apoptosis and are efficiently phagocytosed by mouse peritoneal macrophages in a manner mediated by the membrane phospholipid phosphatidylserine, which is translocated to the surface of virus-infected cells during apoptosis. However, the extent of phagocytosis is not always parallel with the level of phosphatidylserine externalization. Here we examined the involvement of influenza virus neuraminidase (NA) in efficient phagocytosis of virus-infected cells. HeLa cells infected with an influenza virus strain expressing temperature-sensitive NA underwent apoptosis and produced viral proteins, including the defective NA, at a non-permissive temperature to almost the same extent as cells infected with the wild-type virus. The cells were, however, phagocytosed by macrophages with reduced efficiency. In addition, phagocytosis of cells infected with the wild-type virus was severely inhibited when the cells had been maintained in the presence of the NA inhibitor zanamivir. On the other hand, the binding of sialic acid-recognizing lectins to the cell surface declined after infection with the wild-type virus. The decrease in the extent of lectin binding was greatly attenuated when cells were infected with the mutant virus or when wild-type virus-infected cells were maintained in the presence of zanamivir. These results indicate that sugar chains are desialylated by NA at the surface of virus-infected cells. We conclude that the presence of both phosphatidylserine and asialoglycomoieties on the cell surface is required for efficient phagocytosis of influenza virus-infected cells by macrophages. Cells undergoing apoptosis are selectively and rapidly eliminated from the organism by phagocytosis (1Ellis R.E. Yuan J. Horvitz H.R. Annu. Rev. 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Bull. 1997; 53: 491-508Crossref PubMed Scopus (282) Google Scholar); the membrane phospholipid phosphatidylserine (PS) 1The abbreviations used are: PSphosphatidylserinePCphosphatidylcholineFITCfluorescein isothiocyanateHAinfluenza virus hemagglutininNAinfluenza virus neuraminidaseNPinfluenza virus nucleoproteinPBSphosphate-buffered salinetstemperature-sensitive 1The abbreviations used are: PSphosphatidylserinePCphosphatidylcholineFITCfluorescein isothiocyanateHAinfluenza virus hemagglutininNAinfluenza virus neuraminidaseNPinfluenza virus nucleoproteinPBSphosphate-buffered salinetstemperature-sensitive is the best characterized phagocytosis marker (6Fadok V.A. Bratton D.L. Frasch S.C. Warner M.L. Henson P.M. Cell Death Differ. 1998; 5: 551-562Crossref PubMed Scopus (617) Google Scholar, 7Chimini G. Cell Death Differ. 2001; 8: 545-548Crossref PubMed Scopus (16) Google Scholar, 8Fadok V.A. Xue D. Henson P. 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Although the externalization of PS seems to occur in many types of apoptotic cells, the presence of other phagocytosis markers that may cooperate with PS in phagocyte recognition of apoptotic cells has been proposed (12Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (127) Google Scholar, 13Schlegel R.A. Krahling S. Callahan M.K. Williamson P. Cell Death Differ. 1999; 6: 583-592Crossref PubMed Scopus (67) Google Scholar, 14Fujii C. Shiratsuchi A. Manaka J. Yonehara S. Nakanishi Y. Cell Death Differ. 2001; 8: 1113-1122Crossref PubMed Scopus (21) Google Scholar, 15Hoffmann P.R. deCathelineau A.M. Ogden C.A. Leverrier Y. Bratton D.L. Daleke D.L. Ridley A.J. Fadok V.A. Henson P.M. J. Cell Biol. 2001; 155: 649-659Crossref PubMed Scopus (45) Google Scholar). phosphatidylserine phosphatidylcholine fluorescein isothiocyanate influenza virus hemagglutinin influenza virus neuraminidase influenza virus nucleoprotein phosphate-buffered saline temperature-sensitive phosphatidylserine phosphatidylcholine fluorescein isothiocyanate influenza virus hemagglutinin influenza virus neuraminidase influenza virus nucleoprotein phosphate-buffered saline temperature-sensitive Many viruses induce apoptosis in host cells, but the physiological consequences of this phenomenon are not fully understood (16Teodoro J.G. Branton P.E. J. Virol. 1997; 71: 1739-1746Crossref PubMed Google Scholar, 17Granville D.J. Carthy C.M. Yang D. Hunt D.W.C. McManus B.M. Cell Death Differ. 1998; 5: 653-659Crossref PubMed Scopus (32) Google Scholar). Cells infected with influenza A virus undergo typical apoptosis that has been considered to be caused by Fas ligand and Fas, the expression of which is simultaneously stimulated upon viral infection (18Takizawa T. Matsukawa S. Higuchi Y. Nakamura S. Nakanishi Y. Fukuda R. J. Gen. Virol. 1993; 74: 2347-2355Crossref PubMed Scopus (295) Google Scholar, 19Takizawa T. Fukuda R. Miyawaki T. Ohashi K. Nakanishi Y. Virology. 1995; 209: 288-296Crossref PubMed Scopus (104) Google Scholar, 20Wada N. Matsumura M. Ohba Y. Kobayashi N. Takizawa T. Nakanishi Y. J. Biol. Chem. 1995; 270: 18007-18012Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Fujimoto I. Takizawa T. Ohba Y. Nakanishi Y. Cell Death Differ. 1998; 5: 426-431Crossref PubMed Scopus (67) Google Scholar). The involvement of viral genome-encoded proteins such as neuraminidase (NA) (22Morris S.J. Price G.E. Barnett J.M. Hiscox S.A. Smith H. Sweet C. J. Gen. Virol. 1999; 80: 137-146Crossref PubMed Scopus (107) Google Scholar), nonstructural protein 1 (23Schultz-Cherry S. Dybdahl-Sissoko N. Neumann G. Kawaoka Y. Hinshaw V.S. J. Virol. 2001; 75: 7875-7881Crossref PubMed Scopus (150) Google Scholar, 24Zhirnov O.P. Konakova T.E. Wolff T. Klenk H.-D. J. Virol. 2002; 76: 1617-1625Crossref PubMed Scopus (191) Google Scholar), and the newly discovered protein PB1-F2 (25Chen W. Calvo P.A. Malide D. Gibbs J. Schubert U. Bacik I. Basta S. O'Neill R. Schickli J. Palese P. Henklein P. Bennink J.R. Yewdell J.W. Nat. Med. 2001; 7: 1306-1312Crossref PubMed Scopus (810) Google Scholar) in the regulation of apoptosis in influenza virus-infected cells has also been reported, although the mode of action of nonstructural protein 1 is controversial. Influenza virus-infected HeLa cells become susceptible to apoptosis-dependent phagocytosis by mouse peritoneal macrophages, and the phagocytic clearance of virus-infected cells appears to inhibit the expansion of viral infection (26Fujimoto I. Pan J. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 3399-3403Crossref PubMed Scopus (108) Google Scholar). It can thus reasonably be presumed that apoptosis of influenza virus-infected cells leads to elimination of the virus from the organism. We previously showed that macrophages recognized PS that was expressed at the surface of influenza virus-infected cells during apoptosis, but the extent of phagocytosis continued to increase even after the level of cell surface PS reached a maximum (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar). This indicates that PS is not solely responsible for the recognition of influenza virus-infected cells by macrophages. We therefore decided to search for other surface change(s) needed for efficient phagocytosis of virus-infected cells. Influenza virus-infected cells express viral envelope proteins on their surface, and these proteins might participate in the recognition and phagocytosis of the cells by macrophages. We first examined the involvement of NA, one of the envelope proteins, because NA inhibitors and a mutant influenza virus strain with defective NA were available. The strains of influenza A virus used in this study were derivatives of influenza A/Udorn/72 (H3N2) virus, clones SP626 (wild type) and ICRC282 (a temperature-sensitive (ts) mutant). Clone ICRC282, which produces ts NA (28Shimizu K. Mullinix M.G. Murphy B.R. Chanock R.M. Bishop D.H. Compans R.W. The Replication of Negative Strand Viruses. Elsevier Science Publishers B. V., Amsterdam1981: 369-378Google Scholar, 29Shimizu K. Aihara K. Miyamoto T. Compans R.W. Bishop D.H. Segmented Negative Strand Viruses. Academic Press, New York1984: 387-394Crossref Google Scholar), was obtained by mutagenizing the wild-type virus with the acridine-based compound ICR 191, as described previously (30Shimizu K. Mullinix M.G. Chanock R.M. Murphy B.R. Virology. 1982; 117: 38-44Crossref PubMed Scopus (17) Google Scholar, 31Shimizu K. Mullinix M.G. Chanock R.M. Murphy B.R. Virology. 1982; 117: 45-61Crossref PubMed Scopus (20) Google Scholar). For virus adsorption, HeLa S3 cells maintained in Eagle's minimal essential medium containing 10% fetal bovine serum at 37 °C in 5% CO2, 95% air were incubated with phosphate-buffered saline (PBS) containing either the wild-type or the ts mutant virus at a multiplicity of infection of one at room temperature for 30 min, as described previously (20Wada N. Matsumura M. Ohba Y. Kobayashi N. Takizawa T. Nakanishi Y. J. Biol. Chem. 1995; 270: 18007-18012Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 21Fujimoto I. Takizawa T. Ohba Y. Nakanishi Y. Cell Death Differ. 1998; 5: 426-431Crossref PubMed Scopus (67) Google Scholar). For control “mock” infection, cells were similarly treated with PBS alone. Those cells were then cultured at 40 °C, a non-permissive temperature for the ts NA, in 5% CO2, 95% air and used for further analyses. In some experiments, cells infected with the wild-type influenza virus were cultured at 37 °C in 5% CO2, 95% air in the presence of 10 μm zanamivir (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) (a gift from GlaxoSmithKline), a specific inhibitor of NA (32von Itzstein M., Wu, W.-Y. Kok G.B. Pegg M.S. Dyason J.C. Jin B. Phan T.V. Smythe M.L. White H.F. Oliver S.W. Colman P.M. Varghese J.N. Ryan D.M. Woods J.M. Bethell R.C. Hotham V.J. Cameron J.M. Penn C.R. Nature. 1993; 363: 418-423Crossref PubMed Scopus (1734) Google Scholar), and then further analyzed. All culturing was done in the absence of trypsin to avoid secondary infection by the replicated virus. The growth of influenza virus in HeLa cells was monitored by examining the production of viral proteins by immunohistochemistry or Western blotting. For immunohistochemistry, virus-infected cells were maintained in poly-d-Lys-coated culture containers, fixed, and permeabilized as described previously (26Fujimoto I. Pan J. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 3399-3403Crossref PubMed Scopus (108) Google Scholar). The cells were treated with an anti-influenza virus antiserum, which recognizes nucleoprotein (NP), matrix protein-1, hemagglutinin (HA), and NA (33Shimizu K. Mukaigawa J. Oguro M. Ono Y. Nakajima K. Kida H. Vaccine. 1985; 3: 207-210Crossref PubMed Scopus (12) Google Scholar), and then with a fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulin G antibody (Vector, Burlingame, CA). The cells were examined by fluorescence and phase-contrast microscopy (BX50 microscope; Olympus, Tokyo, Japan). For Western blotting, virus-infected cells were lysed with a buffer containing 62.5 mm Tris-HCl (pH 6.8), 2.5% SDS, and 2.5% 2-mercaptoethanol, and the lysates were separated by electrophoresis on a 15% SDS-polyacrylamide gel. The proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilon P; Millipore, Bedford, MA). The membrane was blocked with 5% dry skim milk, incubated with the anti-influenza antiserum in a buffer consisting of 20 mm Tris-HCl (pH 7.5), 0.15 m NaCl, 0.5% Tween 20, and 5% dry skim milk, washed, reacted with an alkaline phosphatase-conjugated anti-rabbit immunoglobulin G antibody (Bio-Rad), and subjected to a chemiluminescence reaction using the Immun-Star system (Bio-Rad). The signals were assessed using Fluor-S MAX (Bio-Rad). The integrity of the plasma membrane and the occurrence of chromatin condensation were determined by examination under a microscope after staining cells with trypan blue and Hoechst 33342, respectively (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar, 34Shiratsuchi A. Osada S. Kanazawa S. Nakanishi Y. Biochem. Biophys. Res. Commun. 1998; 246: 549-555Crossref PubMed Scopus (77) Google Scholar). The number of cells negative for trypan blue staining or having condensed chromatin was determined and expressed (in percentage) relative to the total number of cells. Translocation of PS from the cytoplasmic to the exoplasmic leaflet of the plasma membrane was assessed by flow cytometry using annexin V, which specifically binds to PS, as described previously (35Koopman G. Reutelingsperger C.P.M. Kuijten G.A.M. Keehnen R.M.J. Pals S.T. van Oers M.H.J. Blood. 1994; 84: 1415-1420Crossref PubMed Google Scholar, 36Martin S.J. Reutelingsperger C.P.M. Green D.R. Cotton Y.G. Martin S.J. Techniques in Apoptosis: A User's Guide. Portland Press, London1996: 107-119Google Scholar). In brief, cells were treated with FITC- or 5-carboxyfluorescein-labeled annexin V and propidium iodide, a membrane-impermeable fluorochrome, and analyzed in a flow cytometer (EPICS-XL; Coulter, Hialeah, FL). The cells that were less intensely stained with propidium iodide and thus impermeable to annexin V were gated and analyzed for the amount of bound annexin V. The activity of NA was determined using a colorimetric assay with fetuin as the substrate (37Aminoff D. Biochem. J. 1961; 81: 384-392Crossref PubMed Scopus (1441) Google Scholar, 38Shimizu K. Shimizu Y.K. Kohama T. Ishida N. Virology. 1974; 62: 90-101Crossref PubMed Scopus (62) Google Scholar). In brief, virus- or mock-infected HeLa cells (2 × 106) were suspended in 40 mm acetate buffer (pH 5.6) containing 4 mg/ml fetuin (Sigma) and incubated at 37 °C for 1 h. Sialic acids released from fetuin were brought to the coloring reaction, and their amounts were determined by measuring the A549. Either the wild-type or the NA mutant influenza virus was adsorbed to 1 × 106HeLa cells at a multiplicity of infection of 10 as described above, and the infected cells were maintained at 40 °C for 6 h. They were then incubated with 3H-labeled glucosamine (d-[1,6-3H]glucosamine hydrochloride, 49 Ci/mmol; PerkinElmer Life Sciences) (40 μCi/ml) in serum-free medium at 40 °C for 30 min. The cells were harvested using a cell scraper, suspended in the buffer used to lyse cells for Western blotting, heated at 50 °C for 10 min, lysed by passage through a 26-gauge needle 15 times, and electrophoresed on a 10% SDS-polyacrylamide gel. The gel was fixed with a solution of 20% methanol and 5% acetic acid, treated with ENHANCE (PerkinElmer Life Sciences), dried, and autoradiographed. Macrophages were isolated from the peritoneal cavity of thioglycolate-treated BDF1 mice (females, 8–12 weeks old) and maintained in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum at 37 °C until use, as described previously (34Shiratsuchi A. Osada S. Kanazawa S. Nakanishi Y. Biochem. Biophys. Res. Commun. 1998; 246: 549-555Crossref PubMed Scopus (77) Google Scholar, 39Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). The phagocytosis assay was performed essentially as described (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar, 40Shiratsuchi A. Umeda M. Ohba Y. Nakanishi Y. J. Biol. Chem. 1997; 272: 2354-2358Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Briefly, HeLa cells infected with influenza virus were labeled with biotin (NHS-LC-Biotin; Pierce), mixed with macrophages (at a ratio of three target cells to one macrophage), and incubated at 37 °C for 2 h. The cell mixture was then treated with trypsin (0.5 μg/ml) to remove HeLa cells free from or lightly attached to macrophages. The remaining cells were fixed, permeabilized, and supplemented with FITC-conjugated avidin (fluorescein-avidin D; Vector Laboratories). The number of macrophages containing engulfed cells was determined by fluorescence and phase-contrast microscopy and expressed relative to the total number of macrophages; this ratio (in percentage) was termed the phagocytic index. The means ± S. D. of a typical example from at least three independent experiments are presented. Statistical analysis was performed by the Student's t test. Phospholipids (Avanti Polar Lipids, Alabaster, AL) were dried as films, suspended in PBS, and sonicated (40Shiratsuchi A. Umeda M. Ohba Y. Nakanishi Y. J. Biol. Chem. 1997; 272: 2354-2358Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Liposomes were formed using either phosphatidylcholine only (PC liposomes) or a combination of phosphatidylcholine and PS at a molar ratio of 7:3 (PS liposomes). Either the wild-type or the NA mutant influenza virus was adsorbed to 2 × 106 HeLa cells at a multiplicity of infection of 1 as described above, and the infected cells were maintained in serum-free medium at 40 °C for 16 h. To examine the effects of zanamivir, the entire process of virus adsorption (wild type) and culturing (at 37 °C for 20 h) of the virus-infected cells was done in the presence of the drug (10 μm). The cells were suspended in PBS containing FITC-labeled lectin (Honen, Tokyo, Japan) (20–30 μg/ml) and propidium iodide and incubated at room temperature for 30 min. The samples were then washed with PBS and analyzed by flow cytometry. The cells less intensely stained with propidium iodide were gated and analyzed for the amount of bound FITC-lectins. The lectins used in this study were wheat germ agglutinin, which recognizes (GlcNAc)2, (GlcNAc)3, and Neu5Ac; Maackia amurensis lectin, which recognizes Neu5Acα2–3Galβ1–4GlcNAc; and Sambucus sieboldiana lectin, which recognizes Neu5Acα2–3Gal and Neu5Acα2–6GalNAc. HeLa cells that had been infected with either the wild-type or the ts mutant influenza virus were cultured at a non-permissive temperature (40 °C) for various lengths of time and assayed for NA activity (Fig. 1 A). The NA activity in cells infected with the wild-type virus continued to increase during the culture reaching a maximum level at 24 h. In contrast, cells infected with the mutant virus showed only the basal level of activity throughout the culture period, as did mock-infected cells (data not shown). However, we found no significant difference in the NA activity between cells infected with the wild-type and the mutant viruses when they were cultured at 34 °C, a permissive temperature (data not shown). All these results agreed well with our previous observations (28Shimizu K. Mullinix M.G. Murphy B.R. Chanock R.M. Bishop D.H. Compans R.W. The Replication of Negative Strand Viruses. Elsevier Science Publishers B. V., Amsterdam1981: 369-378Google Scholar, 29Shimizu K. Aihara K. Miyamoto T. Compans R.W. Bishop D.H. Segmented Negative Strand Viruses. Academic Press, New York1984: 387-394Crossref Google Scholar). We then used immunohistochemistry and Western blotting to examine the growth of the mutant virus by detecting viral proteins. When cells cultured at 40 °C for 16 h after virus adsorption were examined immunohistochemically with the anti-influenza virus antiserum (Fig. 1 B), the proportions of positive cells were 0, 45 ± 6.4, and 86 ± 4.2% with mock-, wild-type virus-, and mutant virus-infected cells, respectively. The proportion of positive cells was always larger with the mutant virus than with the wild-type virus. The signals were almost exclusively localized in the nucleus, whereas viral proteins seemed to be located in the cytoplasm when culturing was carried out at 34 °C. Virus-infected cells cultured at 34 °C showed nuclear signals at earlier times (data not shown). This indicated that culturing virus-infected cells at 40 °C delays the virus propagation compared with that in cells cultured at 34 °C. When lysates prepared from cells infected with the two virus strains were subjected to Western blotting, the overall pattern and intensity of the signals, which were absent in the lysates of mock-infected cells, were not significantly different between the two types of lysate (Fig. 1 C). All the above results indicate that the mutant virus with the ts NA grows normally in infected HeLa cells at a non-permissive temperature. Unfortunately, we were unable to identify the signal derived from NA, probably because its expression level was much lower than that of closely migrating NP. We thus radiolabeled glycoproteins to detect the glycoprotein NA, distinguishing it from unglycosylated NP. HeLa cells infected with the wild-type or the mutant influenza virus were incubated with3H-labeled glucosamine, lysed, and subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography (Fig. 1 D). Upon infection with influenza virus, the synthesis of cellular proteins is repressed, and proteins encoded by the viral genome are predominantly expressed. Two distinct signals with slower mobility were detected in each lysate, and these signals were considered to correspond to viral glycoproteins HA and NA based on their estimated molecular masses. The intensity of the signal derived from NA was almost equal in the lysates prepared from cells infected with either the wild-type or the mutant virus. This indicates that the ts NA is produced and glycosylated in HeLa cells as efficiently as the wild-type NA even at a non-permissive temperature and thus suggests that the defective NA is present on the surface of mutant virus-infected cells. We next examined the occurrence of apoptosis in cells that had been infected with the mutant virus and maintained at a non-permissive temperature (Fig. 2). Cells infected with either the wild-type or the NA mutant virus showed similar extents of loss of plasma membrane integrity (panel A) and condensation of chromatin (panel B). On the other hand, the level of PS externalization in the mutant virus-infected cells was slightly higher than in cells infected with the wild-type virus (panel C). These results indicate that the mutant and the wild-type influenza viruses propagate and induce apoptosis almost equally in HeLa cells at 40 °C. We then conducted phagocytosis assays with HeLa cells that had been infected with either the wild-type or the mutant influenza virus and cultured at a non-permissive temperature for 16 h (panels A and B in Fig. 3). The two cell populations used as targets of macrophages differed in the activity of NA but were at almost the same stage of apoptosis in terms of plasma membrane integrity, chromatin condensation, and PS externalization (see Figs.1 A and 2). HeLa cells became susceptible to phagocytosis by mouse peritoneal macrophages when infected with the wild-type virus, as reported previously (26Fujimoto I. Pan J. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 3399-3403Crossref PubMed Scopus (108) Google Scholar). However, cells infected with the mutant virus were phagocytosed with reduced efficiency compared with cells infected with the wild-type virus. This indicates that virus-infected cells with reduced levels of NA activity were less efficiently phagocytosed by macrophages than those with wild-type NA. We then compared the PS dependence of the phagocytosis reactions using cells infected with the two virus strains. The phagocytosis of both cell populations was severely and specifically inhibited by the addition of PS-containing liposomes (panel C in Fig. 3), indicating that influenza virus-infected cells are phagocytosed by macrophages in a PS-mediated manner irrespective of the activity of NA. The above results indicate that the activity of NA in influenza virus-infected cells is needed for the efficient phagocytosis of those cells by macrophages. To further confirm this, we examined the effect of zanamivir, a sialic acid derivative that specifically binds to and inhibits NA (32von Itzstein M., Wu, W.-Y. Kok G.B. Pegg M.S. Dyason J.C. Jin B. Phan T.V. Smythe M.L. White H.F. Oliver S.W. Colman P.M. Varghese J.N. Ryan D.M. Woods J.M. Bethell R.C. Hotham V.J. Cameron J.M. Penn C.R. Nature. 1993; 363: 418-423Crossref PubMed Scopus (1734) Google Scholar). HeLa cells were infected with the wild-type influenza virus and cultured in the presence or absence of zanamivir at a concentration that inhibits the NA activity (32von Itzstein M., Wu, W.-Y. Kok G.B. Pegg M.S. Dyason J.C. Jin B. Phan T.V. Smythe M.L. White H.F. Oliver S.W. Colman P.M. Varghese J.N. Ryan D.M. Woods J.M. Bethell R.C. Hotham V.J. Cameron J.M. Penn C.R. Nature. 1993; 363: 418-423Crossref PubMed Scopus (1734) Google Scholar). Treatment with the inhibitor did not alter the growth of virus, as indicated by the following observations. The proportions of cells positive for immunofluorescence with the anti-influenza virus antiserum were 100 and 99 ± 1.4% for cells treated and not treated with zanamivir, respectively (Fig. 4 A). We found no difference in the production of viral proteins in the two cell populations by Western blotting (Fig. 4 B). In addition, both cell populations appeared to be similarly apoptotic; there was no significant difference in the extent of plasma membrane integrity or chromatin condensation (data not shown). However, PS externalization seemed to be enhanced in the presence of zanamivir (Fig. 4 C). This, together with the result shown in Fig. 2 C, suggested that a defect in the NA activity leads to stimulation of PS externalization in influenza virus-infected cells. These results do not agree with those reported by other investigators who showed that apoptosis was inhibited by zanamivir (22Morris S.J. Price G.E. Barnett J.M. Hiscox S.A. Smith H. Sweet C. J. Gen. Virol. 1999; 80: 137-146Crossref PubMed Scopus (107) Google Scholar). The reasons for this discrepancy are not clear at present but could be due to the difference in the host cell strains used in the two studies; influenza virus propagates more actively in Madin-Darby canine kidney cells used by Morris et al. (22Morris S.J. Price G.E. Barnett J.M. Hiscox S.A. Smith H. Sweet C. J. Gen. Virol. 1999; 80: 137-146Crossref PubMed Scopus (107) Google Scholar) than in HeLa cells. When the cells were subjected to the phagocytosis assay, macrophages engulfed zanamivir-treated cells to a lesser extent than untreated cells (Fig. 5). The inhibitory effect was not observed when the drug was present only during the phagocytosis reaction (data not shown). Treatment of cells with another sialic acid derivative, 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Sigma), a weaker NA inhibitor than zanamivir (32von Itzstein M., Wu, W.-Y. Kok G.B. Pegg M.S. Dyason J.C. Jin B. Phan T.V. Smythe M.L. White H.F. Oliver S.W. Colman P.M. Varghese J.N. Ryan D.M. Woods J.M. Bethell R.C. Hotham V.J. Cameron J.M. Penn C.R. Nature. 1993; 363: 418-423Crossref PubMed Scopus (1734) Google Scholar), caused a similar effect but only at a higher concentration (20 mm) (data not shown). These results indicate that NA needs to be active for influenza virus-infected cells to be efficiently phagocytosed by macrophages. All of the above results made us anticipate that NA modifies the structure of sugar chains at the surface of influenza virus-infected cells and that the resulting sugar moieties participate in the efficient phagocytosis of infected cells by macrophages. To verify this possibility, changes in the amount of sialic acid on the surface of HeLa cells before and after influenza virus infection were analyzed. For this purpose, the binding of sialic acid-recognizing lectins to the surface of HeLa cells was assessed by flow cytometry (Fig. 6). The results clearly showed that the binding of three different lectins that recognize (though not exclusively) sialic acid was reduced upon infection with the wild-type virus (panel A). The decrease in the efficiency of lectin binding was almost completely cancelled when cells were infected with the mutant virus (panel A) or significantly weakened when cells were infected with the wild-type virus and cultured in the presence of zanamivir (panel B). These results indicate that the amount of cell surface sialic acid decreases upon infection with influenza virus in a manner dependent on the activity of NA. We then determined a time course of this event. Cells infected with the wild-type virus were collected at various time points and analyzed for the binding of wheat germ agglutinin in comparison with that of annexin V (panel C). The decrease in the efficiency of lectin binding was first detectable as early as 3 h after the culturing started, and the level of lectin binding reached a plateau by 6 h. Essentially the same results were obtained using the other two lectins (data not shown). This change occurred much earlier than the externalization of PS, which became obvious at 9 h being maximized by 16 h. Because phagocytosis of virus-infected cells by macrophages reaches significant levels at 9 h (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar), surface desialylation by itself seems insufficient for phagocytosis induction. We finally examined the effect of exogenously added sugars on phagocytosis. Phagocytosis assays with cells infected with the wild-type virus for 20 h were conducted in the presence of Neu5Ac (0.01∼10 mm), GlcNAc (20 or 50 mm), Gal (40 mm), or Man (40 mm), but they neither stimulated nor inhibited the reaction (data not shown). This suggests that macrophages recognize not monosaccharides but more complex sugar moieties that are exposed at the surface of influenza virus-infected cells as a result of desialylation. Macrophages specifically recognize “phagocytosis markers” present on the surface of apoptotic cells using corresponding receptors (4Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1273) Google Scholar, 5Savill J. Brit. Med. Bull. 1997; 53: 491-508Crossref PubMed Scopus (282) Google Scholar). Although a number of molecules have been proposed as candidate phagocytosis markers, it is unclear whether each marker functions independently or whether more than two molecules are simultaneously recognized by macrophages. PS, the best characterized phagocytosis marker, seems to be capable of inducing phagocytosis by itself in macrophages, because its surface expression independent of apoptosis makes cells susceptible to phagocytosis (34Shiratsuchi A. Osada S. Kanazawa S. Nakanishi Y. Biochem. Biophys. Res. Commun. 1998; 246: 549-555Crossref PubMed Scopus (77) Google Scholar, 41Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). On the other hand, it is also possible that PS cooperates with other phagocytosis markers to make apoptotic cells more effectively recognized by macrophages (12Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (127) Google Scholar, 13Schlegel R.A. Krahling S. Callahan M.K. Williamson P. Cell Death Differ. 1999; 6: 583-592Crossref PubMed Scopus (67) Google Scholar, 14Fujii C. Shiratsuchi A. Manaka J. Yonehara S. Nakanishi Y. Cell Death Differ. 2001; 8: 1113-1122Crossref PubMed Scopus (21) Google Scholar). Fadok, Henson, and co-workers recently showed that ligation between candidate phagocytosis markers other than PS and corresponding receptors led to the binding of macrophages to apoptotic cells, but engulfment did not take place unless the PS receptor co-existed on the surface of macrophages (15Hoffmann P.R. deCathelineau A.M. Ogden C.A. Leverrier Y. Bratton D.L. Daleke D.L. Ridley A.J. Fadok V.A. Henson P.M. J. Cell Biol. 2001; 155: 649-659Crossref PubMed Scopus (45) Google Scholar). They have proposed the “tether and tickle” mechanism for heterophagic clearance of apoptotic cells; that is, two distinct receptors, which are responsible for the binding of phagocytic cells to apoptotic cells and for transmission of the signal to induce engulfment, are required (44Henson P.M. Bratton D.L. Fadok V.A. Curr. Biol. 2001; 11: R795-R805Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). In the present study, we showed that influenza virus-infected cells need to have both externalized PS and functional NA to be maximally recognized and phagocytosed by macrophages. PS most likely serves as a phagocytosis-inducing ligand that is probably recognized by a signaling receptor, i.e. the macrophage PS receptor (42Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekewitz R.A.B. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1254) Google Scholar). As for the role of NA, it is likely that sialic acids located at the ends of sugar chains are cleaved off by NA, and the resulting asialoglycomoieties then act as tethering ligands. In fact, changes of the glycan structure by desialylation occur during apoptosis, and cells undergoing apoptosis are bound by the asialoglycoprotein receptor present on the surface of Kupffer cells (phagocytes in the liver) (45Dini L. Autuori F. Lentini A. Oliverio S. Piacentini M. FEBS Lett. 1992; 296: 174-178Crossref PubMed Scopus (161) Google Scholar, 46Falasca L. Bergamini A. Serafino A. Balabaud C. Dini L. Exp. Cell Res. 1996; 224: 152-162Crossref PubMed Scopus (70) Google Scholar). Our previous study showed that macrophage phagocytosis of influenza virus-infected cells was almost completely inhibited by the addition of PS liposomes at all times during the culture period (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar). In addition, we found in the present study that PS liposomes inhibited the phagocytosis of cells infected with the wild-type and the NA mutant virus to very similar extents and that loss of cell surface sialic acids by itself did not seem to induce phagocytosis. These results collectively suggest that the surface change(s) caused by NA act in cooperation with PS for efficient recognition and engulfment of virus-infected cells by macrophages. The extent of phagocytosis by macrophages continues to increase even after desialylation and PS externalization at the surface of influenza virus-infected cells are maximized (27Shiratsuchi A. Kaido M. Takizawa T. Nakanishi Y. J. Virol. 2000; 74: 9240-9244Crossref PubMed Scopus (53) Google Scholar). Other molecules should thus participate in macrophage recognition of virus-infected cells. Scott et al. (47Scott C.B. Ratcliffe D.R. Cramer E.B. J. Immunol. 1996; 157: 351-359PubMed Google Scholar) showed that a monoclonal antibody, which recognizes another viral envelope protein, HA, inhibits the binding of human monocytes to Madin-Darby canine kidney cells infected with influenza A/WSN (H1N1) virus. A group of molecules bound by the collagenous C-type lectin called collectin are also likely candidates; phagocytosis of apoptotic neutrophils by alveolar macrophages was specifically stimulated by the lung-specific collectins, surfactant proteins A and D (48Schagat T.L. Wofford J.A. Wright J.R. J. Immunol. 2001; 166: 2727-2733Crossref PubMed Scopus (176) Google Scholar). It can thus reasonably be speculated that many kinds of molecules cooperate in the recognition of influenza virus-infected cells by macrophages so that those cells are effectively removed by phagocytosis before the infection spreads in the body. Influenza virus-infected cells are also phagocytosed by dendritic cells in an apoptosis-dependent manner, and this phagocytosis appears to lead to antigen presentation and activation of CD8-positive T lymphocytes (43Albert M.L. Pearce S.F.A. Francisco L.M. Sauter B. Roy P. Silverstein R.L. Bhardwaj N. J. Exp. Med. 1998; 188: 1359-1368Crossref PubMed Scopus (1062) Google Scholar, 49Albert M.L. Sauter B. Bhardwaj N. Nature. 1998; 392: 86-89Crossref PubMed Scopus (2032) Google Scholar). We previously showed that phagocytosis of influenza virus-infected cells by macrophages results in the inhibition of virus growth (21Fujimoto I. Takizawa T. Ohba Y. Nakanishi Y. Cell Death Differ. 1998; 5: 426-431Crossref PubMed Scopus (67) Google Scholar). It is therefore likely that apoptosis-dependent phagocytosis of influenza virus-infected cells may protect the organism from viral invasion in two different ways. Further experiments are needed to examine whether such self-defense mechanisms against viral diseases really function in vivo. We thank GlaxoSmithKline for the gift of zanamivir, E. Nobusawa for suggestions on the NA assay, Y. Suzuki for suggestions on the use of NA inhibitors, and C. Fujii for help with macrophage preparation and flow cytometry.
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