Carbohydrate-mediated Phagocytic Recognition of Early Apoptotic Cells Undergoing Transient Capping of CD43 Glycoprotein
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m310805200
ISSN1083-351X
AutoresShigetoshi Eda, Masahiro Yamanaka, Masatoshi Beppu,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoA novel mechanism of phagocytic recognition of apoptotic cells was found and characterized. Jurkat cells incubated with appropriate concentrations of etoposide or anti-Fas antibody transiently became susceptible to binding and phagocytosis by THP-1 cell-derived macrophages at 2 h. The bound Jurkat cells showed no chromatin condensation, but the binding was prevented by a caspase inhibitor, indicating that they were recognized at an early stage of apoptosis. The ligands recognized on the apoptotic cells were sialylpolylactosaminyl sugar chains because 1) the binding was inhibited by an oligosaccharide preparation of erythrocyte membrane, and its inhibitory activity was destroyed by polylactosaminoglycan-specific endo-β-galactosidase or neuraminidase; 2) Jurkat cells pretreated with endo-β-galactosidase or neuraminidase failed to be recognized; and 3) treatment of the apoptotic cells with polylactosaminoglycan-binding Datura stramonium agglutinin prevented recognition. The sialylpolylactosaminyl chains involved were most likely those of a major sialoglycoprotein CD43 because anti-CD43 antibody inhibited recognition. CD43 on apoptotic Jurkat cells was found to form a cap at 2 h, and the cap disappeared at 4 h. This transient capping of CD43 coincided with the transient increase in the susceptibility of the cells to macrophage recognition, suggesting that CD43 capping is responsible for generation of the carbohydrate ligands for recognition. Furthermore, microscopic observation suggested that the apoptotic cells were recognized at the CD43 cap. Taken together, we conclude that apoptotic Jurkat cells transiently undergo CD43 capping at an early stage of apoptosis and are recognized by macrophages through the cluster of sialylpolylactosaminyl chains of the capped CD43. A novel mechanism of phagocytic recognition of apoptotic cells was found and characterized. Jurkat cells incubated with appropriate concentrations of etoposide or anti-Fas antibody transiently became susceptible to binding and phagocytosis by THP-1 cell-derived macrophages at 2 h. The bound Jurkat cells showed no chromatin condensation, but the binding was prevented by a caspase inhibitor, indicating that they were recognized at an early stage of apoptosis. The ligands recognized on the apoptotic cells were sialylpolylactosaminyl sugar chains because 1) the binding was inhibited by an oligosaccharide preparation of erythrocyte membrane, and its inhibitory activity was destroyed by polylactosaminoglycan-specific endo-β-galactosidase or neuraminidase; 2) Jurkat cells pretreated with endo-β-galactosidase or neuraminidase failed to be recognized; and 3) treatment of the apoptotic cells with polylactosaminoglycan-binding Datura stramonium agglutinin prevented recognition. The sialylpolylactosaminyl chains involved were most likely those of a major sialoglycoprotein CD43 because anti-CD43 antibody inhibited recognition. CD43 on apoptotic Jurkat cells was found to form a cap at 2 h, and the cap disappeared at 4 h. This transient capping of CD43 coincided with the transient increase in the susceptibility of the cells to macrophage recognition, suggesting that CD43 capping is responsible for generation of the carbohydrate ligands for recognition. Furthermore, microscopic observation suggested that the apoptotic cells were recognized at the CD43 cap. Taken together, we conclude that apoptotic Jurkat cells transiently undergo CD43 capping at an early stage of apoptosis and are recognized by macrophages through the cluster of sialylpolylactosaminyl chains of the capped CD43. Cells dying by apoptosis are swiftly ingested by phagocytes before they rupture and release injurious and immunogenic contents into the surrounding tissue, and thus clearance of apoptotic cells by phagocytes is important in maintaining tissue homeostasis (1Savill J. Fadok V. Henson P. Haslett C. Immunol. Today. 1993; 14: 131-136Abstract Full Text PDF PubMed Scopus (983) Google Scholar, 2Platt N. da Silva R.P. Gordon S. Trends Cell Biol. 1998; 8: 365-372Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 3Savill J. Fadok V. Nature. 2000; 407: 784-788Crossref PubMed Scopus (1271) Google Scholar, 4Fadok V.A. Bratton D.L. Henson P.M. J. Clin. Invest. 2001; 108: 957-962Crossref PubMed Scopus (402) Google Scholar). Moreover, phagocytosis of apoptotic cells by macrophages has been suggested to induce anti-inflammatory functions of macrophages by suppressing pro-inflammatory cytokine production and by stimulating anti-inflammatory cytokine release (3Savill J. Fadok V. 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These observations have been reasonably explained by the hypothesis that membrane glycoproteins aggregate to form clusters upon cell oxidation, and the resultant clusters of their extracellular polylactosaminoglycans provide multivalent high affinity ligands for macrophage receptors and for anti-band 3 antibody (“glycoprotein clustering hypothesis”) (27Beppu M. Eda S. Fujimaki M. Hishiyama E. Kikugawa K. Biol. Pharm. Bull. 1996; 19: 188-194Crossref PubMed Scopus (21) Google Scholar, 28Beppu M. Yokoyama N. Motohashi M. Kikugawa K. Biol. Pharm. Bull. 2001; 24: 19-26Crossref PubMed Scopus (9) Google Scholar, 29Beppu M. Ando K. Saeki M. Yokoyama N. Kikugawa K. Arch. Biochem. Biophys. 2000; 384: 368-374Crossref PubMed Scopus (11) Google Scholar, 30Beppu M. Mizukami A. Ando K. Kikugawa K. J. Biol. Chem. 1992; 267: 14691-14696Abstract Full Text PDF PubMed Google Scholar, 31Beppu M. Mizukami A. Nagoya M. Kikugawa K. J. Biol. 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Materials—Etoposide, cycloheximide, bisbenzimide (Hoechst 33258), ethidium bromide, and trypsin (E.C. 3.4.21.4, porcine pancreas) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin (BSA), RNase A, proteinase K, and phorbol myristate acetate were obtained from Sigma. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk) was the product of the Peptide Institute (Osaka, Japan). Rhodamine-labeled concanavalin A and Datura stramonium agglutinin (DSA) were purchased from Vector Laboratories (Burlingame, CA). Endo-β-galactosidase (E.C.3.2.1.103, Escherichia freundii), α-galactosidase A (melibiase) (E.C.3.2.1.22, Mortierella vinacea), and endo-β-N-acetylglucosaminidase H (E.C.3.2.1.96, Streptomyces griseus) were obtained from Seikagaku Fine Chemicals (Tokyo, Japan). Neuraminidase (E.C.3.2.1.18, Vibrio cholelae) was purchased from Behringwerke AG (Marburg, Germany). Anti-Fas antibody (clone CH-11) was purchased from Medical & Biological Laboratories (Nagoya, Japan). Anti-CD43 mouse monoclonal antibody (clone DF-T1) and control mouse IgG1 were obtained from DAKO (Glostrup, Denmark). Anti-CD2 and anti-CD3 mouse monoclonal antibodies were obtained from the Nichirei Corporation (Tokyo, Japan). Alexa Fluor-488 goat anti-mouse IgG (H+L) conjugate was the product of Molecular Probe (Eugene, OR). Oligosaccharides of human erythrocyte membrane glycoprotein, mainly those from band 3 glycoprotein, were prepared by hydrazinolysis of defatted human erythrocyte ghosts as described before (27Beppu M. Eda S. Fujimaki M. Hishiyama E. Kikugawa K. Biol. Pharm. Bull. 1996; 19: 188-194Crossref PubMed Scopus (21) Google Scholar). Induction and Measurement of Apoptosis—Apoptosis of Jurkat cells (Riken Cell Bank, Tsukuba, Japan) was induced by incubation of the cells (4.0 × 106 cells/ml) in RPMI 1640 medium containing 5% fetal bovine serum with appropriate concentrations of etoposide, anti-Fas antibody (clone CH-11), or cycloheximide at 37 °C in 5% CO2 atmosphere for various hours. Apoptosis of the treated cells was assessed by chromatin condensation and by DNA fragmentation. For measurement of chromatin condensation, etoposide-treated Jurkat cells were fixed with 1% glutaraldehyde in Ca2+,Mg2+-free Dulbecco's phosphate-buffered saline (DPBS(-)) at room temperature for 30 min followed by staining with 0.1 mm bisbenzimide (Hoechst 33258) in DPBS(-) at room temperature overnight. Morphology of the nuclei of Jurkat cells was observed under a fluorescence microscope. For measurement of DNA fragmentation, etoposide-treated Jurkat cells were washed twice with DPBS(-). The cell pellets (4 × 106 cells) were resuspended in 100 μl of DPBS(-), and then 10 μl of 10 mg/ml RNase, 10 μl of 10 mg/ml proteinase K, and 20 μl of 10% sodium dodecyl sulfate were sequentially added to this solution. After incubation at 37 °C for 30 min, 300 μl of NaI solution (26 mm Tris/HCl, pH 8.0, 6 m NaI, 13 mm EDTA, 0.5% sodium N-lauroylsarcosinate, 10 mg/ml glycogen) was added to the solution. The solution was incubated at 37 °C for 30 min and then chilled on ice for 20 min. DNA was precipitated by addition of 500 μl of isopropanol and followed by centrifugation at 15,000 rpm for 15 min at 4 °C. The DNA pellet was sequentially washed with 50 and 100% isopropanol and then with diethylether. The air-dried DNA pellet was dissolved in 100 μl of TE buffer (10 mm Tris/HCl, pH 7.4, 5 mm EDTA) and electrophoresed on 2% agarose gels at 50 V for 2 h, and DNA bands were visualized by staining with ethidium bromide. Binding and Phagocytosis Assays—THP-1 cells (Japanese Cancer Research Resources Bank, Osaka, Japan) in RPMI 1640 with 5% fetal bovine serum were plated in 24-well plates at 1 × 105 cells/well in which round coverslips (15-mm diameter) were placed and cultured with 50 nm phorbol myristate acetate at 37 °C in 5% CO2 atmosphere for 4-5 days. The monolayers of THP-1 cells differentiated into macrophages on coverslips were washed in DPBS(-) before use. Jurkat cells (4 × 106 cells/ml) treated with apoptosis-inducing agents at appropriate concentrations for various hours at 37 °C in RPMI 1640 with 5% fetal bovine serum were washed three times with DPBS(-), resuspended in RPMI 1640 medium bufferized with 20 mm HEPES, pH 7.2 (RPMI 1640-HEPES) at 4 × 106 cells/ml, and coincubated with macrophage monolayers on coverslips at 37 °C for 2 h with gentle shaking. In the case of time course experiments, the beginning of the Jurkat cell incubation with an apoptosis-inducing agent was staggered for each time group so that all of the binding assays may be done at one time. Unbound cells were removed by gentle washing, and then bound Jurkat cells and macrophages were fixed with 1.25% glutaraldehyde and stained with Mayer's hematoxylin solution. The number of bound Jurkat cells and macrophages was counted under a light microscope (×400 magnification). The data are expressed as the number of bound Jurkat cells/100 macrophages as counting more than 300 macrophages. For phagocytosis assay, etoposide-treated Jurkat cells were labeled with fluorescein isothiocyanate (4 μg/ml) at 37 °C for 10 min. The labeled Jurkat cells were coincubated with macrophage monolayers similarly to the binding assay. After coincubation, unbound and lightly attached Jurkat cells were removed by washing and by treatment with 0.5 mg/ml trypsin at room temperature for 2 min. Then the cell surface facing the medium was stained with rhodamine-labeled concanavalin A (20 μg/ml) at 0 °C for 10 min, and Jurkat cells taken up by macrophages were identified under a confocal laser scanning fluorescence microscope (μ-Radiance; Bio-Rad). Measurement of Cell Surface CD43—Jurkat cells or Jurkat cell-bound macrophage monolayers were treated with 10 μg/ml anti-CD43 mouse monoclonal antibody (clone DF-T1) in RPMI 1640-HEPES with 0.2% BSA at 0 °C for 30 min and washed several times with DPBS(-) at 0 °C. Bound antibody was detected by treatment of the cells with 10 μg/ml Alexa Fluor-488 goat anti-mouse IgG (H+L) conjugate in RPMI 1640-HEPES with 0.2% BSA at 0 °C for 30 min and washing several times with DPBS(-) at 0 °C. The cells were resuspended in Hanks' balanced salt solution at 0 °C and immediately subjected to microscopic observation by a fluorescence microscope (Axiovert 200M; Carl Zeiss) or a confocal laser scanning fluorescence microscope and flow cytometry analysis by a flow cytometer (FACSCalibur; Becton Dickinson) using CELLQUEST software. Three-dimensional analysis of confocal images was performed using a software LaserSharp. Throughout the immunofluorescence staining process, the cell suspensions were kept at 0 °C to prevent antibody-induced antigen redistribution that may occur at higher temperature. Phagocytic Recognition of Early Apoptotic Jurkat Cells—First, we tried to determine conditions for induction of apoptosis that are suitable for macrophage recognition assay, using etoposide as an apoptosis-inducing agent and Jurkat cells as target cells. Human monocytic THP-1 cells differentiated by phorbol myristate acetate into adherent cells were used as macrophages. Fig. 1A demonstrates the relationship between concentrations of etoposide or the time of etoposide treatment and binding of the etoposide-treated Jurkat cells to macrophages. Jurkat cells became susceptible to binding to macrophages when treated with the proper concentrations of etoposide for proper periods. Maximal binding was observed when treated with 10 μm of etoposide for 1 or 2 h (Fig. 1A, left and right panels, filled circles). Treatment with etoposide at higher concentrations or for longer periods resulted in decreased binding. Thus, we hereafter used Jurkat cells treated with etoposide at 10 μm for 2 h, unless otherwise indicated. The extent of apoptosis was assessed by chromatin condensation staining with Hoechst 33258. The percentage of chromatin-condensed cells in Jurkat cells treated with various concentrations of etoposide for 2 h (Fig. 1A, left panel, filled squares) and those treated with 10 μm etoposide for various hours (Fig. 1A, right panel, filled squares) increased with dose of etoposide (left panel) and with time of treatment (right panel), respectively. The percentage of the chromatin-condensed cells at each point increased slightly after an additional 2 h of incubation without etoposide (filled triangles), the same conditions under which the Jurkat cell-macrophage binding assay was performed. DNA fragmentation was not significant for the Jurkat cells treated with 10 μm etoposide for 2 h and was slightly detectable after an additional 2 h of incubation without etoposide (data not shown). Thus, during etoposide treatment and the following binding assay without etoposide, only small proportions of Jurkat cells underwent nuclear changes. The Jurkat cell specimens of the binding assay were then examined for chromatin condensation by staining with Hoechst 33258. None of the Jurkat cells binding to the macrophages showed chromatin condensation (data not shown), indicating that etoposide-treated Jurkat cells were recognized by macrophages at an early stage of apoptosis before chromatin condensation occurred. To exclude the possibility that recognition occurred independently on the apoptotic process, the effect of the caspase inhibitor Z-VAD-fmk (35Slee E.A. Zhu H. Chow S.C. Nicholson MacFarlane, D.W. Cohen G.M. Biochem. J. 1996; 315: 21-24Crossref PubMed Scopus (400) Google Scholar) on recognition was tested. As shown in Fig. 1B, recognition by macrophages and chromatin condensation of etoposide-treated Jurkat cells were prevented by Z-VAD-fmk to the level of etoposide-untreated cells, indicating that the observed susceptibility of the etoposide-treated Jurkat cells to macrophage recognition was dependent on the apoptotic process. We examined whether the apoptotic cell recognition observed herein resulted in phagocytosis. After the binding assay procedure using fluorescein isothiocyanate-labeled apoptotic Jurkat cells and the following removal of lightly attached Jurkat cells by trypsin treatment, the cell surface was labeled with rhodamine-labeled concanavalin A and then observed under a confocal laser scanning fluorescence microscope. As shown in Fig. 1C, Jurkat cells remaining on macrophage surface were observed (left panel, arrowheads), suggesting that these cells began being phagocytosed. Jurkat cells being inside the macrophages were also observed (left and right panels, arrows), which obviously indicated that phagocytosis was taking place. Then the values of binding and phagocytosis (the mean number of Jurkat cells binding and phagocytosed per 100 macrophages ± S.D., respectively) were determined for the same assay. When the binding was 29.7 ± 5.1, the phagocytosis was 7.6 ± 2.5 for the etoposide-treated Jurkat cells, whereas the binding and the phagocytosis for control cells were 3.8 ± 0.8 and 2.6 ± 1.0, respectively. The data suggest that approximately 20-25% of the apoptotic cells bound to macrophages were taken up during the 2-h binding assay. Phagocytosis of etoposide-treated cells increased up to 11.0 ± 1.7 when the binding assay was prolonged to 4 h. Involvement of Carbohydrate Chains of CD43 in Recognition—We next examined whether recognition of the early apoptotic Jurkat cells by macrophages occurred through carbohydrate chains on Jurkat cells, as was the case with recognition of oxidized Jurkat cells (29Beppu M. Ando K. Saeki M. Yokoyama N. Kikugawa K. Arch. Biochem. Biophys. 2000; 384: 368-374Crossref PubMed Scopus (11) Google Scholar). Recognition was inhibited when oligosaccharides prepared from human erythrocyte membrane were present during the binding assay (Fig. 2A). The inhibitory activity of the oligosaccharides was destroyed when treated with endo-β-galactosidase, an enzyme that specifically cleaves poly-N-acetyllactosaminyl structure (i.e. Galβ1-4GlcNAcβ1-3 repeats) at the β-galactosidic bond (36Fukuda M.N. Fukuda M. Hakomori S. J. Biol. Chem. 1979; 254: 5458-5465Abstract Full Text PDF PubMed Google Scholar) or with neuraminidase that removes sialic acid residues from the nonreducing termini of carbohydrate chains (Fig. 2A). As shown in Fig. 2B, when Jurkat cells had been pretreated with endo-β-galactosidase or neuraminidase prior to induction of apoptosis, recognition did not occur. These data suggest that sialylated polylactosaminoglycans are recognition sites on apoptotic cells, although the possibility that polylactosaminoglycans and other sialylated chains independently contributed to recognition cannot be ruled out. In support of these results, DSA, a lectin known to preferentially bind to polylactosamine-type carbohydrate chains (37Yamashita K. Totani K. Ohkura T. Takasaki S. Goldstein I.J. Kobata A. J. Biol. 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A. 1989; 86: 2819-2823Crossref PubMed Scopus (118) Google Scholar) that shows multiple functions such as cell adhesion, anti-adhesion, signal transduction, and cytoskeletal interaction (40Ostberg J.R. Barth R.K. Frelinger J.G. Immunol. Today. 1998; 19: 546-550Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 41Fukuda M. Cancer Res. 1996; 56: 2237-2244PubMed Google Scholar), possesses sialylated polylactosaminoglycans (42Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar). To see whether sialyl residues or/and polylactosaminyl chains of CD43 on apoptotic Jurkat cells are recognized by macrophages, the effect of anti-CD43 monoclonal antibody (clone DF-T1), an antibody directed against a sialic acid-dependent unidentified epitope of CD43 (43de Smet W. Walter H. van Hove L. Immunology. 1993; 79: 46-54PubMed Google Scholar), on the recognition of apoptotic cells was tested. Recognition was effectively blocked by the anti-CD43 antibody, whereas irrelevant control IgG1 had no effect (Fig. 2D), suggesting the involvement of carbohydrate chains of CD43 in recognition. The antibody bound to endo-β-galactosidase-treated Jurkat cells as equally well as to untreated cells, although it did not bind to neuraminidase-treated cells, as analyzed by flow cytometry (data not shown). This suggests that the carbohydrate ligands of CD43 involved in the macrophage recognition are distinct from the epitope for the anti-CD43 monoclonal antibody. Because sialylpolylactosaminyl sugar chains of CD43 were shown to be O-glycans (42Maemura K. Fukuda M. J. Biol. Chem. 1992; 267: 24379-24386Abstract Full Text PDF PubMed Google Scholar), the effect of pretreatment of Jurkat cells with endo-β-N-acetylglucosaminidase H, an e
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