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Dimeric Galectin-1 Induces Surface Exposure of Phosphatidylserine and Phagocytic Recognition of Leukocytes without Inducing Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m306624200
ISSN1083-351X
AutoresMarcelo Dias‐Baruffi, Hui Zhu, Moonjae Cho, Sougata Karmakar, Rodger P. McEver, Richard D. Cummings,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoWe report that human galectin-1 (dGal-1), a small dimeric β-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface β-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis. We report that human galectin-1 (dGal-1), a small dimeric β-galactoside-binding protein, induces phosphatidylserine (PS) exposure, measured by Annexin V staining, on human promyelocytic HL-60 cells, T leukemic MOLT-4 cells, and fMet-Leu-Phe-activated, but not resting, human neutrophils. This effect of dGal-1 on HL-60 and MOLT-4 cells is enhanced by pretreatment of the cells with neuraminidase, but treatment of resting neutrophils with neuraminidase does not enhance their sensitivity to dGal-1. Although the induction of staining with Annexin V is often associated with apoptosis, the dGal-1-treated HL-60 cells, MOLT-4 cells, and activated neutrophils do not undergo apoptosis, and there is no detectable DNA fragmentation. HL-60 and MOLT-4 cells treated with dGal-1 continue to grow normally. By contrast, camptothecin-treated HL-60 cells, etoposide-treated MOLT-4 cells, and anti-Fas-treated neutrophils exhibit extensive DNA fragmentation and/or cell death. Lactose inhibits the dGal-1-induced effects, indicating that dGal-1-induced signaling requires binding to cell surface β-galactosides. The dimeric form of Gal-1 is required for signaling, because a monomeric mutant form of Gal-1, termed mGal-1, binds to cells but does not cause these effects. Importantly, dGal-1, but not mGal-1, treatment of HL-60 cells and activated human neutrophils significantly promotes their phagocytosis by activated mouse macrophages. These dGal-1-induced effects are distinguishable from apoptosis, but like apoptotic agents, prepare cells for phagocytic removal. Such effects of dGal-1 may contribute to leukocyte homeostasis. It is believed that the turnover of neutrophils and other leukocytes in tissues involves programmed cell death (apoptosis) and then phagocytosis by tissue macrophages (1Homburg C.H. Roos D. Curr. Opin. Hematol. 1996; 3: 94-99Crossref PubMed Scopus (44) Google Scholar, 2Savill J. J. Leukoc. Biol. 1997; 61: 375-380Crossref PubMed Scopus (562) Google Scholar, 3Fadok V.A. Bratton D.L. Henson P.M. J. Clin. Invest. 2001; 108: 957-962Crossref PubMed Scopus (402) Google Scholar, 4Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. 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Although Fas and Fas ligand (FasL) 1The abbreviations used are: FasL, Fas ligand; dGal-1, dimeric human galectin-1; mGal-1, mutated, monomeric human galectin-1; MAL, M. amurensis leukoagglutinin; HBSS, Hanks' balanced salt solution; HSA, human serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein-isothiocyanate; PI, propidium iodide; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; fMet-Leu-Phe, N-formyl-Met-Leu-Phe chemotactic peptide; 2-ME, 2-mercaptoethanol.1The abbreviations used are: FasL, Fas ligand; dGal-1, dimeric human galectin-1; mGal-1, mutated, monomeric human galectin-1; MAL, M. amurensis leukoagglutinin; HBSS, Hanks' balanced salt solution; HSA, human serum albumin; PBS, phosphate-buffered saline; FITC, fluorescein-isothiocyanate; PI, propidium iodide; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; fMet-Leu-Phe, N-formyl-Met-Leu-Phe chemotactic peptide; 2-ME, 2-mercaptoethanol. induce apoptosis of mature, circulating neutrophils in vitro (5Iwai K. 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Brown K.K. Brain J.D. Accurso F.J. Henson P.M. J. Clin. Invest. 2002; 109: 661-670Crossref PubMed Google Scholar, 16Shi J. Gilbert G.E. Kokubo Y. Ohashi T. Blood. 2001; 98: 1226-1230Crossref PubMed Scopus (127) Google Scholar). These results suggest that factors not yet defined may regulate leukocyte turnover in tissues. Such observations led us to explore whether the basement membrane and extracellular matrix might harbor other proteins capable of binding to leukocytes and inducing their apoptosis or phagocytic recognition. A candidate protein is the β-galactoside-binding protein termed galectin-1 (dGal-1), which binds to most leukocytes. dGal-1 is a widely expressed dimeric protein (subunit ∼14.6 kDa), which is a member of the galectin family of lectins (17Cooper D.N. Barondes S.H. Glycobiology. 1999; 9: 979-984Crossref PubMed Scopus (282) Google Scholar, 18Varki A. Cummings R.D. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. 1st ed. Cold Spring Harbor Laboratory Press, Inc., Boston1999Google Scholar, 19Perillo N.L. Marcus M.E. Baum L.G. J. Mol. Med. 1998; 76: 402-412Crossref PubMed Scopus (580) Google Scholar). It is secreted by many cell types, including human endothelial cells (20Lotan R. Belloni P.N. Tressler R.J. Lotan D. Xu X.C. Nicolson G.L. Glycoconj. J. 1994; 11: 462-468Crossref PubMed Scopus (125) Google Scholar, 21Baum L.G. Seilhamer J.J. Pang M. Levine W.B. Beynon D. Berliner J.A. Glycoconj. J. 1995; 12: 63-68Crossref PubMed Scopus (117) Google Scholar), and is found in the basement membrane and extracellular matrices around capillary walls (22Wasano K. Hirakawa Y. Yamamoto T. Cell Tissue Res. 1990; 259: 43-49Crossref PubMed Scopus (41) Google Scholar, 23Allen H.J. Sucato D. Gottstine S. Kisailus E. Nava H. Petrelli N. Castillo N. Wilson D. Tumour Biol. 1991; 12: 52-60Crossref PubMed Scopus (33) Google Scholar). dGal-1 has been reported to have various biological activities, including effects on neurite out-growth (24Outenreath R.L. Jones A.L. J. Neurocytol. 1992; 21: 788-795Crossref PubMed Scopus (27) Google Scholar, 25Puche A.C. Poirier F. Hair M. Bartlett P.F. Key B. Dev. Biol. 1996; 179: 274-287Crossref PubMed Scopus (180) Google Scholar), growth inhibition of non-neural cells (26Wells V. Mallucci L. Cell. 1991; 64: 91-97Abstract Full Text PDF PubMed Scopus (228) Google Scholar, 27Adams L. Scott G.K. Weinberg C.S. Biochim. Biophys. Acta. 1996; 1312: 137-144Crossref PubMed Scopus (138) Google Scholar, 28Kopitz J. von Reitzenstein C. Andre S. Kaltner H. Uhl J. Ehemann V. Cantz M. Gabius H.J. J. Biol. Chem. 2001; 276: 35917-35923Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar), cell growth stimulation (29Pitts M.J. Yang D.C. Biochem. J. 1981; 195: 435-439Crossref PubMed Scopus (32) Google Scholar, 30Sanford G.L. Harris-Hooker S. FASEB J. 1990; 4: 2912-2918Crossref PubMed Scopus (104) Google Scholar), and apoptosis of immature thymocytes (31Perillo N.L. Uittenbogaart C.H. Nguyen J.T. Baum L.G. J. Exp. Med. 1997; 185: 1851-1858Crossref PubMed Scopus (266) Google Scholar, 32Galvan M. Tsuboi S. Fukuda M. Baum L.G. J. Biol. Chem. 2000; 275: 16730-16737Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and to activate human T cells and T cell lines (33Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (940) Google Scholar, 34Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811PubMed Google Scholar). To explore the biological activity of dGal-1 toward leukocytes, we prepared a recombinant form of dimeric human dGal-1 and a mutated, monomeric form of galectin-1 (mGal-1). We explored the interactions of these lectins with HL-60 cells, MOLT-4 cells, and both resting and activated human neutrophils. Our results show that dGal-1, but not mGal-1, rapidly enhances surface staining with Annexin V (phosphatidylserine (PS) exposure) in desialylated HL-60 cells, desialylated MOLT-4 cells, and activated, but not resting, human neutrophils. The exposure of PS is often associated with apoptosis (35Martin S.J. Reutelingsperger C.P. McGahon A.J. Rader J.A. van Schie R.C. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2556) Google Scholar, 36Rimon G. Bazenet C.E. Philpott K.L. Rubin L.L. J. Neurosci. Res. 1997; 48: 563-570Crossref PubMed Scopus (112) Google Scholar, 37Homburg C.H. de Haas M. von dem Borne A.E. Verhoeven A.J. Reutelingsperger C.P. Roos D. Blood. 1995; 85: 532-540Crossref PubMed Google Scholar). However, dGal-1 does not induce apoptosis, because no DNA fragmentation is observed in dGal-1-treated cells, and HL-60 cells and MOLT-4 cells continue to grow normally. Importantly, dGal-1-treated, but not mGal-1-treated, cells are actively phagocytosed in vitro by activated mouse peritoneal macrophages. Thus, dGal-1-induced effects are distinguishable from apoptosis yet prepare cells for phagocytic recognition. These results suggest that dGal-1 could induce surface changes that play a role in leukocyte turnover in tissues independently of apoptosis. Preparation of dGal-1 and mGal-1—The cDNA for human dGal-1 was cloned by PCR from the published sequence (38Couraud P.O. Casentini-Borocz D. Bringman T.S. Griffith J. McGrogan M. Nedwin G.E. J. Biol. Chem. 1989; 264: 1310-1316Abstract Full Text PDF PubMed Google Scholar, 39Hirabayashi J. Ayaki H. Soma G. Kasai K. FEBS Lett. 1989; 250: 161-165Crossref PubMed Scopus (50) Google Scholar). The recombinant form of human dGal-1 was purified as described previously (40Cho M. Cummings R.D. J. Biol. Chem. 1995; 270: 5198-5206Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar) employing affinity chromatography on lactosyl-Sepharose, and the protein was eluted with lactose. The column was washed with 5 column volumes of PBS (0.01 m Na2HPO4, 0.01 m Na2HPO4, 0.85% NaCl, pH 7.4) containing 2-ME (14 mm), and the bound dGal-1 and mGal-1 were eluted with PBS containing 2-ME (14 mm) and lactose (0.1 m). To confirm that the purified protein quantitatively retained carbohydrate-binding activity, the protein was re-chromatographed on lactosyl-Sepharose. The elution profile of the re-purified dGal-1 revealed a single peak of protein that was quantitatively eluted with lactose (data not shown). In some studies we heat-inactivated the dGal-1 by heating the protein at a concentration of 1 mg/ml to 100 °C for 10 min. The treated sample remained in suspension, although cloudy, and was inactive, because it was unable to bind lactosyl-Sepharose. To generate a monomeric form of the protein (mGal-1), we used PCR primer-directed mutagenesis as described for the hamster-derived Gal-1 (41Cho M. Cummings R.D. Biochemistry. 1996; 35: 13081-13088Crossref PubMed Scopus (69) Google Scholar) to place a Ser residue codon at the Cys-2 position and an Asp residue at the Val-5 position. The cDNAs for dGal-1 and mGal-1 were ligated into the BamHI and HindIII sites of the plasmid PQE-50 (Qiagen). Both dGal-1 and mGal-1 were expressed at high levels in transformed Escherichia coli. dGal-1 and mGal-1 were purified from sonicated E. coli cell extracts on columns of lactosyl-Sepharose as described above. Human dGal-1 at a concentration from 2–80 μm behaves as a dimeric protein on size-exclusion chromatography, whereas mGal-1 behaves as a monomeric species under the same conditions, using experimental approaches previously described (41Cho M. Cummings R.D. Biochemistry. 1996; 35: 13081-13088Crossref PubMed Scopus (69) Google Scholar). The detailed characterization of the binding properties and kinetics of subunit association of human dGal-1 and mGal-1 will be described elsewhere. 2M. Cho, H. Zhu, C. Brower, R. P. McEver, and R. D. Cummings, submitted for publication. Cell Lines and Enzymatic Desialylation—HL-60 and MOLT-4 cells were obtained from the American Type Culture Collection and maintained at 37 °C and 5% CO2 in complete RPMI 1640 media containing 10% fetal calf serum, glutamine (2 mm), penicillin (100 milliunits/ml), and streptomycin (100 μg/ml). To desialylate the cells, HL-60 cells or MOLT-4 cells (3 × 107 cells) were treated with 100 milliunits of Arthrobacter ureafaciens neuraminidase (Sigma) in 500 μl of complete RPMI for 1 h at 37 °C. The treated cells were washed twice with RPMI before use. Isolation, Activation, and Desialylation of Neutrophils—Heparinized blood was obtained from normal donors. Neutrophils were isolated by dextran sedimentation, hypotonic lysis, and density gradient centrifugation on Histopaque-1077 (Sigma) as described previously (42Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1985; 76: 2235-2246Crossref PubMed Scopus (276) Google Scholar). Typically, the polymorphonuclear cells were >90% neutrophils by Wright-Giemsa staining and >90% CD16+ as assessed by flow cytometry. Neutrophils were activated by treatment with fMet-Leu-Phe (Sigma, 1.0 μm) for 10 min at 37 °C in 1 ml of HBSS/HSA (Ca2+-Mg2+-free Hanks' balanced salt solution with 0.2% HSA). Desialylated neutrophils were prepared by treating 1 × 107 neutrophils with A. ureafaciens neuraminidase (Sigma, 100 milliunits) in 500 μl of HBSS/HSA for 30 min at 37 °C. Cells were washed twice with HBSS/HSA before use. Flow Cytometry—Galectins and the sialic acid binding plant lectin Maackia amurensis leukoagglutinin (MAL) (Vector Laboratories) (43Wang W.C. Cummings R.D. J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar) were biotinylated by using EZ-Link™ Sulfo-NHS-Biotin (Pierce). Cells were incubated with biotinylated forms of the lectins (10 μg/ml) for 30 min on ice and washed twice with sterile PBS. The washed cells were incubated with FITC-streptavidin (1:100 dilution, Sigma), washed with sterile PBS, and analyzed by flow cytometry in a FACSCalibur equipped with a 488-nm argon laser (BD Biosciences). Cells were incubated at 37 °C in a humidified tissue culture incubator with 5% CO2 in the absence of either MAL, dGal-1, or mGal-1 in complete RPMI or HBSS/HSA. In the case of dGal-1 and mGal-1, we used control treatments in which lactose (20 mm) was included during the incubations to inhibit galectin binding. After this incubation, all cells either treated or untreated with galectins were treated with lactose (20 mm) in sterile PBS to remove galectins. The lactose-treated cells were washed at least two times in sterile PBS and then incubated in a 100-μl final volume in HEPES buffer (10 mm HEPES, 140 mm NaCl2,5mm CaCl2, pH 7.4) with a mixture of FITC-conjugated Annexin V (Roche Applied Science, 2 μlof conjugate per 100 μl of resuspended cells, as described by the manufacturer) and PI (Molecular Probes, 1 μg/ml final concentration) at room temperature for 15 min. Into this tube was pipetted 300 μl of HEPES buffer, and the sample was analyzed by flow cytometry in a FACSCalibur. As known inducers of apoptosis, we used camptothecin (Calbiochem, 0–25 μm), etoposide (Calbiochem, 20 μm), and IgM anti-Fas (Upstate Biotechnology Inc., 250 ng/ml). Resting and activated neutrophils were stained with anti-MAC 1 antibody (anti-CD11b, Caltag) and anti-L-selectin antibody (purified DREG 56) and analyzed by flow cytometry. Approximately 1 × 106 neutrophils were washed once in HBSS and resuspended in 100 μl of HBSS/HSA. The specific antibodies were added at a final concentration of 10–20 μg/ml, and the cells were incubated at 4 °C for 45 min. Then the cells were washed twice with HBSS before resuspension in 100 μlof HBSS/HSA. Goat anti-mouse F(ab)2 conjugated to FITC was then added, and the cells were incubated at 4 °C for an additional 30 min. Cells were then washed twice with HBSS before resuspending them in HBSS/HSA for flow cytometric analysis. Confocal Microscopy—At the end of incubations with galectins, cells were washed with 20 mm lactose in PBS and incubated for 20 min at 37 °C in PBS with the DNA stain Hoechst 33342 (1 μg/ml, Molecular Probes). These cells were then stained with a mixture of FITC-conjugated Annexin V and PI and analyzed by confocal microscopy on a Leica TCS NT Microscope. DNA Fragmentation Assays—To detect DNA fragmentation, two approaches were used. In the first approach, cells were subjected to the TUNEL assay. Cells were treated with or without dGal-1 (20 μm for 8 h), the topoisomerase inhibitor camptothecin (15 μm for 4 h), or anti-Fas mouse monoclonal IgG antibody (R & D Systems, 100 ng/ml for 8 h). Cells were then washed with lactose (20 mm), fixed in 1% paraformaldehyde buffered with PBS, and permeabilized with 70% ethanol on ice. The TUNEL reaction (In Situ Cell Death Detection Kit, Roche Applied Science, Indianapolis, IN) was conducted by incubating the cells for 1 h at 37 °C with 50 μl of TUNEL reaction mixture. The cells were then washed and analyzed by flow cytometry. In the second approach, we examined DNA fragmentation by gel electrophoresis. After treatments of cells with galectins or other reagents as described above, 1 × 107 cells were lysed with 1% Nonidet P-40 in EDTA (20 mm), Tris-HCl (50 mm), pH 7.5, and then microcentrifuged to remove pelleted, insoluble material. The supernatants were then treated with RNase A (5 μg/μl) for 2 h at 56 °C, followed by digestion with proteinase K (2.5 μg/μl) for 2 h at 37 °C. A half volume of ammonium acetate (10 m) was added, and DNA was precipitated with 2.5 volumes of absolute ethanol. DNA was recovered by centrifuging at 12,000 × g for 10 min and dissolved in gel loading buffer. Finally, DNA was separated by electrophoresis in a 1.6% agarose gel containing ethidium bromide (44Sorenson C.M. Barry M.A. Eastman A. J. Natl. Cancer Inst. 1990; 82: 749-755Crossref PubMed Scopus (449) Google Scholar). Determination of HL-60 and MOLT-4 Cell Growth in the Presence of dGal-1—Desialylated HL-60 cells (1.8 × 105/ml) or MOLT-4 cells (1.5 × 105 cells/ml) were incubated with or without dGal-1 (20 μm) in complete RPMI in 12-well plates (2 ml/well). Cells were collected at 24-h intervals, washed with lactose (20 mm), pelleted by centrifugation, and resuspended in PBS. Live cell counts were determined by trypan blue exclusion and counting with a hemacytometer in duplicate assays. Camptothecin (Calbiochem, 20 μm) and etoposide (Calbiochem, 20 μm) were used in control experiments to induce apoptosis. Phagocytosis Assays—The standard macrophage phagocytosis assay was utilized as described (45Fadok 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, 46Zhuang J. Ren Y. Snowden R.T. Zhu H. Gogvadze V. Savill J.S. Cohen G.M. J. Biol. Chem. 1998; 273: 15628-15632Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Briefly, peritoneal macrophages were collected from mice treated intraperitoneally with Brewer's thioglycolate (Difco, 4%), and the isolated macrophages were cultured in a 24-well plate. The macrophage monolayer was washed with PBS, before use. dGal-1- or mGal-1-treated cells, untreated cells (negative control), in vitro aged human neutrophils that had been incubated in vitro for 24 h at 37 °C in HBSS/HSA (positive control), or camptothecin-treated HL-60 cells (positive control), were added to each well containing macrophages and incubated for1hat37 °C. After incubation, each well was washed in ice-cold PBS and treated briefly with trypsin (0.25%) to remove lightly adherent cells. The macrophages were fixed with paraformaldehyde (1.8%), and phagocytosis was microscopically evaluated by counting 200 macrophages/well in each duplicate well and noting those macrophages that had visibly enlarged and contained visibly phagocytosed cells. Results were then expressed as the percentage of macrophages that had phagocytosed cells (% phagocytosis), as described by Fadok et al. (45Fadok 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) and as modified by Zhuang et al. (46Zhuang J. Ren Y. Snowden R.T. Zhu H. Gogvadze V. Savill J.S. Cohen G.M. J. Biol. Chem. 1998; 273: 15628-15632Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Binding of dGal-1 and mGal-1 to HL-60 Cells, MOLT-4 Cells, Resting Human Neutrophils, and Activated Human Neutrophils—The human recombinant galectin-1 preparation was purified by affinity chromatography on lactosyl-Sepharose, as described under "Experimental Procedures." The activity and stability of the purified protein was demonstrated by the quantitative rebinding of the material to a column of lactosyl-Sepharose (data not shown). SDS-PAGE analysis of fractions demonstrated that the protein was apparently homogeneous and migrated with a molecular mass of ∼14.9 kDa (data not shown). These results demonstrate that the protein used in the studies below was quantitatively active and pure. Similar purification and activity profiles were obtained with mGal-1, the monomeric mutant form of galectin-1 (data not shown), prepared as described under "Experimental Procedures." Human dGal-1 at a concentration from 2–80 μm behaves as a dimeric protein on size-exclusion chromatography, whereas mGal-1 behaves as a monomeric species under the same conditions, using experimental approaches previously described (41Cho M. Cummings R.D. Biochemistry. 1996; 35: 13081-13088Crossref PubMed Scopus (69) Google Scholar) (data not shown). We examined the binding of dGal-1 and mGal-1 to HL-60 cells, a human promyelocytic cell line. Both dGal-1 and mGal-1 bound to HL-60 cells, but enzymatic desialylation of the cells significantly enhanced the binding of both lectins (Fig. 1, A and B, respectively). The binding of both dGal-1 and mGal-1 was inhibitable by lactose. We confirmed the effectiveness of enzymatic desialylation by examining the binding of the sialic acid-specific lectin MAL, which recognizes NeuAcα2–3Galβ1–4Glc-NAc-R linkages (43Wang W.C. Cummings R.D. J. Biol. Chem. 1988; 263: 4576-4585Abstract Full Text PDF PubMed Google Scholar). Enzymatic desialylation of HL-60 cells (dsHL-60 cells) decreased the binding of MAL (Fig. 1C). These results demonstrate that both dGal-1 and mGal-1 bind equivalently to HL-60 cells and that enzymatic desialylation of the cells exposes more binding sites for both lectins. We then assessed the binding of dGal-1 to normal human leukocytes. For these studies we used resting neutrophils, enzymatically desialylated resting neutrophils, and fMet-Leu-Phe-activated neutrophils. dGal-1 bound to resting neutrophils, but it bound at higher levels to activated neutrophils (Fig. 1D). The binding of dGal-1 was largely inhibitable by inclusion of lactose (20 mm) (Fig. 1D). dGal-1 binding was enhanced to enzymatically desialylated resting neutrophils (Fig. 1D). Similar results were obtained using mGal-1 (data not shown). The enzymatic desialylation of resting neutrophils was effective, as shown by the higher binding of MAL to resting neutrophils than to enzymatically desialylated resting neutrophils (Fig. 1E). We also observed that activation of neutrophils without treatment of the cells with neuraminidase, also slightly reduced their binding to MAL, consistent with a possible loss or redistribution of sialic acid-containing glycoconjugates upon activation (Fig. 1E). In control studies, we confirmed the activation of neutrophils, because activation resulted in up-regulation of MAC-1 expression and down-regulation of L-selectin expression (47Kishimoto T.K. Jutila M.A. Berg E.L. Butcher E.C. Science. 1989; 245: 1238-1241Crossref PubMed Scopus (912) Google Scholar) (Fig. 1F). These results demonstrate that dGal-1 binds to resting neutrophils but binds slightly more to activated neutrophils. PS Exposure on Leukocytes Induced by Treatment with dGal-1—Based on observations by others that dGal-1 appears to induce apoptosis in some leukocytes (31Perillo N.L. Uittenbogaart C.H. Nguyen J.T. Baum L.G. J. Exp. Med. 1997; 185: 1851-1858Crossref PubMed Scopus (266) Google Scholar, 32Galvan M. Tsuboi S. Fukuda M. Baum L.G. J. Biol. Chem. 2000; 275: 16730-16737Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 33Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (940) Google Scholar, 34Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811PubMed Google Scholar), we assessed whether dGal-1 could induce apoptosis of neutrophils by assessing the staining of cells by Annexin V. Apoptosis is often accompanied by increased exposure of cell surface PS, which is recognized by Annexin V (35Martin S.J. Reutelingsperger C.P. McGahon A.J. Rader J.A. van Schie R.C. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2556) Google Scholar, 36Rimon G. Bazenet C.E. Philpott K.L. Rubin L.L. J. Neurosci. Res. 1997; 48: 563-570Crossref PubMed Scopus (112) Google Scholar, 37Homburg C.H. de Haas M. von dem Borne A.E. Verhoeven A.J. Reutelingsperger C.P. Roos D. Blood. 1995; 85: 532-540Crossref PubMed Google Scholar). dGal-1 treatment of resting human neutrophils did not significantly induce PS exposure (Fig. 2A). In addition, dGal-1 treatment of enzymatically desialylated resting neutrophils, while increasing their binding to dGal-1 (Fig. 1D), did not significantly induce surface exposure of PS (Fig. 2A). By contrast, dGal-1 treatment of fMet-Leu-Phe-activated neutrophils markedly enhanced their staining with Annexin V (Fig. 2B). Lactose inhibited the effects of dGal-1 on activated neutrophils (Fig. 2B), demonstrating that carbohydrate ligands on the cell surface are required for the dGal-1-induced staining of these cells with Annexin V (Table I and Fig. 2, A and B). dGal-1-treated neutrophils stained with Annexin V remained impermeable, as measured by the lack of staining with PI (Fig. 2B), demonstrating that changes induced by dGal-1 are not associated with membrane leakage. These results demonstrate that dGal-1 induces surface PS exposure in activated but not resting neutrophils.Table IInduction of PS exposure on human cell lines by dimeric dGal-1Treatment, Experiment 1% of cells staining with Annexin VaHL-60 cells or MOLT-4 cells (106 cells) (either untreated or desialylated by neuraminidase treatment, ds) were treated with dGal-1 (20 μm) or mGal-1 (20 μm) in the presence or absence of lactose (20 mm) for 4 h at 37 °C. The cells were then washed with lactose (20 mm) and stained with Annexin V-FITC and PI. The dashed line signifies lack of treatment. The percentage of Annexin V-positive/PI-negative cells was determined by flow cytometry. The data shown represent averages of duplicate determinations where the S.E. was <5%.Activated neutrophilsHL-60dsHL-60MOLT-4dsMOLT-4%No treatment (control)7.15.12.97.8—dGal-144.271.111.650.156.8dGal-1 plus lactose7.511.21.311.84.2mGal-1—6.4——4.3mGal-1 plus lactose—5.1——4.4a HL-60 cells or MOLT-4 cells (106 cells) (either untreated or desialylated by neuraminidase treatment, ds) were treated with dGal-1 (20 μm) or mGal-1
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