Original Paper International Journal of Cell Cloning 9:233-238 (1991)
Carbohydrate Composition of Human Megakaryocyte Membranes in Culture: Identification Using Binding of Seven Lectins Jean-Francois Abgrall, Isabelle Renard, Christian Berthou, Luc Sensebe, Genevikve Guern, Claudie Autrand, Guy LeGall, Jean Briere Department of Hematology, Augustin Morvan Hospital, Brest University, School of Medicine, Brest, France
Key Words. Human megakaryocytopoiesis
Lectins
Carbohydrates
Abstract. Seven tetramethylrhodamineB isothiocyanate-(TRITC) labeled lectins: lens culinaris (LCH), ulex europeus-1 (UEA-l), lycopersicon esculentum (LEA), wheat germ agglutinin (WGA), dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA) were applied on cultured human megakaryocytes (Megs) detected by immunofluorescence. All stages of Megs (from lymphocyte-like Megs to mature Megs) and platelets were labeled by LCH, LEA, UEA-1 and WGA. ECA binds to platelets but only to some Megs. DBA did not bind to platelets but did bind to some Megs, irrespective of stage. SBA binds to all stages of Megs, but did not bind to platelets. These results indicate the presence of mannose, glucose (LCH), sialic acid (WGA), and glucosamine (UEA-1, LEA, WGA) on the surface of all cells of the Meg lineage, a variable presence of galactosamine (DBA, SBA, ECA), and a discrepancy in the presence of some galactosamine compounds between platelets and Megs (DBA, SBA).
Introduction The carbohydrate composition of human megakaryocyte (Meg) membranes has never been extensively studied. In guinea pigs, 19 K . Schick [l] has shown that sialic acid is present much more on mature Megs than on immature Megs as indicated by binding of wheat germ agglutinin on isolated Megs. Carbohydrates are thought to play an important role in platelet activation [2], in cell regulation [3], and in regulation of erythropoiesis [4]. Lectin binding was also recently used to characterize myeloid progenitor cells: wheat germ agglutinin-positive cells [ 5 ] , and soybean agglutinin-negativecells [6] being recognized as hemopoietic progenitors. The aim of the present study was to identify carbohydrates present on ~
Correspondence: Dr. J.F. Abgrall, Service d'H&matologie,H6pital A. M o m , 29285 Brest Cedex, France. Received October 31, 1990; provisionally accepted January 7, 1991; accepted for publication February 14, 1991. 0737-1454191/$2.0010 oAlphaMed Press
Lectins Binding on Cultured Megakaryocytes
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the surface of human Megs in culture by comparison with platelets, using binding of lectins.
Materials and Methods Assay for Meg Progenitors
Human bone marrow cells were obtained from three hematologically normal individuals during surgery, after consent. Bone marrow specimens were collected on 1 ml alpha modified minimal essential medium (a-MEM; Eurobio, Paris, France) containing 0.1 ml heparin 20 Ulml (Roche, Neuilly, France). Bone marrow was diluted (1:l) in a-MEM and centrifuged at 100 g for 10 min at 20°C in order to prevent contamination by platelets. Mononuclear cells were separated on lymphocyte separating medium (LSM, 1.077 density; Eurobio) and washed twice in IY-MEM.After adherence on plastic for 24 h, nonadherent mononuclear cells (NAMC) were collected and suspended in (u-MEMcontaining0.15 mg/ml glutamine (Sigma Chemical Co., St. Louis, MO), 2 mglml asparagine (Sigma), 15 pglml streptomycin and 15 U/ml penicillin (GIBCO, Paisley, Scotland, UK). Cells were plated in duplicate, and incubated for 12 days with 5% C 0 2 in a humidified atmosphere at 37°C. Meg progenitor cells were cultured using an improved plasma clot technique, previously described [7]. Briefly, 2 X los NAMC were plated in 1.05 ml culture medium containing 10%human normal AB serum, 5 % leukocyte-conditioned medium stimulated by phytohemagglutinin HA 15 (PHA-LCM; Wellcome, Paris, France), 10%citrated bovine plasma, and 1% BSA. After 12 days of incubation, cultures were dehydrated and stored at 4°C until needed. Platelets Platelets from normal individuals were collected on citrate, then platelet-rich plasma was obtained and immediately used for binding of lectins. Lectins Seven tetramethylrhodamine B isothiocyanate- (TRITC) labeled lectins (Sigma Chemical Co., St. Louis, MO) were used: a) one lectin specific for mannose and glucose, lens culinaris (LCH); b) three lectins specific for N-acetylglucosamine, ulex europeus (UEA-l), lycopersicon esculentum (LEA) and wheat germ agglutinin (WGA), which also binds to sialic acid; c) three lectins specific for N-acetylgalactosamine, dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA), which is specific for Each lectin was reconstituted in phosphateP-D-galactose (I-4)-D-N-acetylglucosamine. buffered saline (PBS; BioMerieux, Marcy, France), pH 7.2, to obtain a protein concentration of 1 mglml . The best staining of Megs was obtained with a concentrationof 0.1 mglml of each lectin. Lectins were applied directly in the culture dishes and incubated for 30 min at 37°C. The culture dishes were then washed three times in PBS, and Megs were stained by a monoclonal antibody specific for the glycoprotein IIb/IIIa complex (HuP1-ml; AMD, Artarmon, Australia) at a dilution of 1:40, incubated 30 min at 37°C. After three washes in PBS, a goat anti-mouse fluorescent antibody (G+M) (Tmrnunotech, France) was incubated 30 min at 37°C. Thus, a double labeling of Megs was obtained. Negative controls were done using PBS instead of lectin in the first step of the procedure. The double labeling was observed under fluorescent microscopy (200x, 5 0 0 ~or 1OOOX) and photographed (Fig. 1). Platelets (3 x lo6)were labeled by lectin using the same procedure without fixation, then centrifuged in cytospin (Shandon, Runcorn, UK) before staining by HuP1-ml. Three experiments were done for Megs labeling with DBA, two experiments for Megs labeling with SBA, LCH, ECA, WGA, UEA-1 and LEA. Four experiments were done
Abgrall et al.
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Fig. 1. Photomicrographsof Megs and platelets labeled by fluorescent monoclonal antibody anti-glycoprotein IIb/IEa complex (A, C, E) and by tetramethylrhodamineB isothiosyanate-labeled lectin (B, D,F). A and B) A large plylobdated Meg and a Meg with a round nucleus (500X). C) A lymphocyte-like Meg (1OOOX) with a D) positively lectin-labeled granulomonocyte colony. E and F) Aggregates of platelets (1OOOX).
Lectins Binding on Cultured Megakaryocytes
236
for platelets labeling with UEA-1, WGA, DBA and ECA;two experiments with LEA and SBA; and no experiment with LCH.
Results Meg Labeling All Megs present in culture were labeled by five lectins: SBA, LCH, LEA, UEA-1 and WGA. An example of labeling is shown in Figure 1. All stages of Meg maturation were labeled by lectins, from lymphocyte-like Megs to large mature Megs (Fig. 1). With LEA, small Megs were more weakly stained than large Megs. DBA and ECA bind to some Megs, but other Megs were negative, irrespective of the stage of maturation. Some lymphocyte-like Megs were positively labeled with DBA and ECA while others were negative. Platelet Labeling All lectins bind to platelets except DBA and SBA. Platelets were stimulated by lectins as indicated by the presence of aggregates (Fig. 1). With DBA and SBA, platelets were neither labeled nor aggregated. The results are summarized in Table I. These experiments demonstrated that the following carbohydrates are present on the surface of all Megs (from lymphocyte-like Megs to large mature Megs) and platelets: D-mannose (LCH), N-acetyl-P-D-acetylglucosamine (WGA, UEA-1, LEA), a-L-fucose (UEA-l), and sialic acid (WGA). Galactose and N-acetylgalactosamine were not observed at the surface of all Megs. SBA labeled all stages of Megs, but not platelets. DBA labeled a few Megs of all stages of maturation, but did not label platelets, and ECA labeled a few Megs of all stages and platelets. Binding of LCH was not tested on platelets.
Discussion No data are presently available on the carbohydratecomposition of the membrane of human Megs. Using the binding of seven lectins on Megs in culture, we can conclude that several carbohydrates are present on the surface of all Megs identified by fluorescent monoclonal antibody anti-IIb/IIIa. These are D-mannose, a-L-fucose, sialic acid and N-acetylglucosamine. Galactose and N-acetylgalactosamine are heterogeneously distributed on the Meg surface. All seven lectins bind on the surface of lymphocyte-like Megs. LEA, which binds to N-acetylglucosamine, gave a weak labeling of lymphocyte-likeMegs and small Megs, and a strong labeling of mature Megs, indicating a difference in the amount of this carbohydrate according to maturation. There was no labeling of platelets by DBA and SBA. However, there was strong labeling of some mature Megs by the same lectins, which may indicate disappear-
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Table I. Binding of lectins on the surface of Megs and platelets
Lectin
Lymphocyte -like Megs
Megs with round nucleus
Polybbulated Megs
LCH LEA WGA UEA-1 DBA SBA
+++ + +++ +++ + +++ +++
+++ + +++ +++ + +++ +++
+++ +++ +++ +++ + +++ +++
ECA
Platelets ND
+++ +++ +++ -
+++
~
+++ = strong positive labeling of all Megs of the same stage + = weak labeling - = platelets not labeled
ND = lectin not tested
ance of N-acetyl-D-galactose when platelets are formed. The positive labeling of platelets by ECA can be explained by the fact that ECA recognizes a peculiar form of carbohydratethat contains galactose: P-D-galactose (14)-D-N-acetylglucosamine. The carbohydrates recognized by these seven lectins are probably not constitutive of the glycoprotein IIb/IIIa complex because: 1) in the procedures used, the antibody was applied after binding of lectin; 2) the size of the seven lectins is large (MW 35,000- 110,000) and could interfere with the binding of antibody if lectin was bound to the glycoprotein IIb/IIIa complex. From the present study showing binding of WGA on lymphocyte-like Megs and recent data from the literature of the binding of WGA on early hemopoietic progenitors [ 5 ] , we can hypothesize that carbohydrates recognized by this lectin are present both on the surface of pluripotent progenitors and on all cells of Meg lineage, including platelets. On the contrary, carbohydrates recognized by SBA are not present on early hemopoietic progenitors [ 6 ] ,but are observed on the surface of cells committed in the Meg lineage. The role of carbohydrates in human megakaryocytopoiesis is currently under study in our laboratory.
Acknowledgment We thank f! McLuughlin for technical assistance in translation.
References 1 Schick PK, Filmer Jr WG. Sialic acid in mature megakaryocytes: detection by wheat germ agglutinin. Blood 1985;65:ll20-1126. 2 Ganguly P, Fossett NG. The role of sialic acid in the activation of platelets by wheat germ agglutinin. Blood 1984;63:181-187.
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3 Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem 1989;264:13369-13372. 4 LaRussa VF, Sieber F, Sensenbrenner LL, Sharkis SJ. Effects of neuraminidase on the regulation of erythropoiesis. Blood 1984;63:784-788. 5 Hara H, Mori K, Misawa M, et al. Wheat germ agglutinin (WGM) or My10 (CD34), which is better marker of human hemopoietic progenitors? Exp Hemato1 1990;18:643a. 6 Simmons PJ, Bartelmez S, Torok-Storb B. Human long-term culture (LTC)repopulating cells are CD34+/SBA-/RH123. Exp Hematol 1990;18:7a. 7 Han ZC, Briere J, Abgrall JF, Sensebe L, Parent D, Guern G. Characteristics of megakaryoqte colony formation in normal individuals and in primary thrombocythemia: studies using an optimal cloning system. Exp Hematol 1989;17:46-52.
Carbohydrate Composition of Human Megakaryocyte Membranes in Culture: Identification Using Binding of Seven Lectins Jean-Francois Abgrall, Isabelle Renard, Christian Berthou, Luc Sensebe, Genevikve Guern, Claudie Autrand, Guy LeGall, Jean Briere Department of Hematology, Augustin Morvan Hospital, Brest University, School of Medicine, Brest, France
Key Words. Human megakaryocytopoiesis
Lectins
Carbohydrates
Abstract. Seven tetramethylrhodamineB isothiocyanate-(TRITC) labeled lectins: lens culinaris (LCH), ulex europeus-1 (UEA-l), lycopersicon esculentum (LEA), wheat germ agglutinin (WGA), dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA) were applied on cultured human megakaryocytes (Megs) detected by immunofluorescence. All stages of Megs (from lymphocyte-like Megs to mature Megs) and platelets were labeled by LCH, LEA, UEA-1 and WGA. ECA binds to platelets but only to some Megs. DBA did not bind to platelets but did bind to some Megs, irrespective of stage. SBA binds to all stages of Megs, but did not bind to platelets. These results indicate the presence of mannose, glucose (LCH), sialic acid (WGA), and glucosamine (UEA-1, LEA, WGA) on the surface of all cells of the Meg lineage, a variable presence of galactosamine (DBA, SBA, ECA), and a discrepancy in the presence of some galactosamine compounds between platelets and Megs (DBA, SBA).
Introduction The carbohydrate composition of human megakaryocyte (Meg) membranes has never been extensively studied. In guinea pigs, 19 K . Schick [l] has shown that sialic acid is present much more on mature Megs than on immature Megs as indicated by binding of wheat germ agglutinin on isolated Megs. Carbohydrates are thought to play an important role in platelet activation [2], in cell regulation [3], and in regulation of erythropoiesis [4]. Lectin binding was also recently used to characterize myeloid progenitor cells: wheat germ agglutinin-positive cells [ 5 ] , and soybean agglutinin-negativecells [6] being recognized as hemopoietic progenitors. The aim of the present study was to identify carbohydrates present on ~
Correspondence: Dr. J.F. Abgrall, Service d'H&matologie,H6pital A. M o m , 29285 Brest Cedex, France. Received October 31, 1990; provisionally accepted January 7, 1991; accepted for publication February 14, 1991. 0737-1454191/$2.0010 oAlphaMed Press
Lectins Binding on Cultured Megakaryocytes
234
the surface of human Megs in culture by comparison with platelets, using binding of lectins.
Materials and Methods Assay for Meg Progenitors
Human bone marrow cells were obtained from three hematologically normal individuals during surgery, after consent. Bone marrow specimens were collected on 1 ml alpha modified minimal essential medium (a-MEM; Eurobio, Paris, France) containing 0.1 ml heparin 20 Ulml (Roche, Neuilly, France). Bone marrow was diluted (1:l) in a-MEM and centrifuged at 100 g for 10 min at 20°C in order to prevent contamination by platelets. Mononuclear cells were separated on lymphocyte separating medium (LSM, 1.077 density; Eurobio) and washed twice in IY-MEM.After adherence on plastic for 24 h, nonadherent mononuclear cells (NAMC) were collected and suspended in (u-MEMcontaining0.15 mg/ml glutamine (Sigma Chemical Co., St. Louis, MO), 2 mglml asparagine (Sigma), 15 pglml streptomycin and 15 U/ml penicillin (GIBCO, Paisley, Scotland, UK). Cells were plated in duplicate, and incubated for 12 days with 5% C 0 2 in a humidified atmosphere at 37°C. Meg progenitor cells were cultured using an improved plasma clot technique, previously described [7]. Briefly, 2 X los NAMC were plated in 1.05 ml culture medium containing 10%human normal AB serum, 5 % leukocyte-conditioned medium stimulated by phytohemagglutinin HA 15 (PHA-LCM; Wellcome, Paris, France), 10%citrated bovine plasma, and 1% BSA. After 12 days of incubation, cultures were dehydrated and stored at 4°C until needed. Platelets Platelets from normal individuals were collected on citrate, then platelet-rich plasma was obtained and immediately used for binding of lectins. Lectins Seven tetramethylrhodamine B isothiocyanate- (TRITC) labeled lectins (Sigma Chemical Co., St. Louis, MO) were used: a) one lectin specific for mannose and glucose, lens culinaris (LCH); b) three lectins specific for N-acetylglucosamine, ulex europeus (UEA-l), lycopersicon esculentum (LEA) and wheat germ agglutinin (WGA), which also binds to sialic acid; c) three lectins specific for N-acetylgalactosamine, dolichos biflorus (DBA), soybean agglutinin (SBA) and erythrina cristagalli (ECA), which is specific for Each lectin was reconstituted in phosphateP-D-galactose (I-4)-D-N-acetylglucosamine. buffered saline (PBS; BioMerieux, Marcy, France), pH 7.2, to obtain a protein concentration of 1 mglml . The best staining of Megs was obtained with a concentrationof 0.1 mglml of each lectin. Lectins were applied directly in the culture dishes and incubated for 30 min at 37°C. The culture dishes were then washed three times in PBS, and Megs were stained by a monoclonal antibody specific for the glycoprotein IIb/IIIa complex (HuP1-ml; AMD, Artarmon, Australia) at a dilution of 1:40, incubated 30 min at 37°C. After three washes in PBS, a goat anti-mouse fluorescent antibody (G+M) (Tmrnunotech, France) was incubated 30 min at 37°C. Thus, a double labeling of Megs was obtained. Negative controls were done using PBS instead of lectin in the first step of the procedure. The double labeling was observed under fluorescent microscopy (200x, 5 0 0 ~or 1OOOX) and photographed (Fig. 1). Platelets (3 x lo6)were labeled by lectin using the same procedure without fixation, then centrifuged in cytospin (Shandon, Runcorn, UK) before staining by HuP1-ml. Three experiments were done for Megs labeling with DBA, two experiments for Megs labeling with SBA, LCH, ECA, WGA, UEA-1 and LEA. Four experiments were done
Abgrall et al.
235
Fig. 1. Photomicrographsof Megs and platelets labeled by fluorescent monoclonal antibody anti-glycoprotein IIb/IEa complex (A, C, E) and by tetramethylrhodamineB isothiosyanate-labeled lectin (B, D,F). A and B) A large plylobdated Meg and a Meg with a round nucleus (500X). C) A lymphocyte-like Meg (1OOOX) with a D) positively lectin-labeled granulomonocyte colony. E and F) Aggregates of platelets (1OOOX).
Lectins Binding on Cultured Megakaryocytes
236
for platelets labeling with UEA-1, WGA, DBA and ECA;two experiments with LEA and SBA; and no experiment with LCH.
Results Meg Labeling All Megs present in culture were labeled by five lectins: SBA, LCH, LEA, UEA-1 and WGA. An example of labeling is shown in Figure 1. All stages of Meg maturation were labeled by lectins, from lymphocyte-like Megs to large mature Megs (Fig. 1). With LEA, small Megs were more weakly stained than large Megs. DBA and ECA bind to some Megs, but other Megs were negative, irrespective of the stage of maturation. Some lymphocyte-like Megs were positively labeled with DBA and ECA while others were negative. Platelet Labeling All lectins bind to platelets except DBA and SBA. Platelets were stimulated by lectins as indicated by the presence of aggregates (Fig. 1). With DBA and SBA, platelets were neither labeled nor aggregated. The results are summarized in Table I. These experiments demonstrated that the following carbohydrates are present on the surface of all Megs (from lymphocyte-like Megs to large mature Megs) and platelets: D-mannose (LCH), N-acetyl-P-D-acetylglucosamine (WGA, UEA-1, LEA), a-L-fucose (UEA-l), and sialic acid (WGA). Galactose and N-acetylgalactosamine were not observed at the surface of all Megs. SBA labeled all stages of Megs, but not platelets. DBA labeled a few Megs of all stages of maturation, but did not label platelets, and ECA labeled a few Megs of all stages and platelets. Binding of LCH was not tested on platelets.
Discussion No data are presently available on the carbohydratecomposition of the membrane of human Megs. Using the binding of seven lectins on Megs in culture, we can conclude that several carbohydrates are present on the surface of all Megs identified by fluorescent monoclonal antibody anti-IIb/IIIa. These are D-mannose, a-L-fucose, sialic acid and N-acetylglucosamine. Galactose and N-acetylgalactosamine are heterogeneously distributed on the Meg surface. All seven lectins bind on the surface of lymphocyte-like Megs. LEA, which binds to N-acetylglucosamine, gave a weak labeling of lymphocyte-likeMegs and small Megs, and a strong labeling of mature Megs, indicating a difference in the amount of this carbohydrate according to maturation. There was no labeling of platelets by DBA and SBA. However, there was strong labeling of some mature Megs by the same lectins, which may indicate disappear-
Abgrall et al.
237
Table I. Binding of lectins on the surface of Megs and platelets
Lectin
Lymphocyte -like Megs
Megs with round nucleus
Polybbulated Megs
LCH LEA WGA UEA-1 DBA SBA
+++ + +++ +++ + +++ +++
+++ + +++ +++ + +++ +++
+++ +++ +++ +++ + +++ +++
ECA
Platelets ND
+++ +++ +++ -
+++
~
+++ = strong positive labeling of all Megs of the same stage + = weak labeling - = platelets not labeled
ND = lectin not tested
ance of N-acetyl-D-galactose when platelets are formed. The positive labeling of platelets by ECA can be explained by the fact that ECA recognizes a peculiar form of carbohydratethat contains galactose: P-D-galactose (14)-D-N-acetylglucosamine. The carbohydrates recognized by these seven lectins are probably not constitutive of the glycoprotein IIb/IIIa complex because: 1) in the procedures used, the antibody was applied after binding of lectin; 2) the size of the seven lectins is large (MW 35,000- 110,000) and could interfere with the binding of antibody if lectin was bound to the glycoprotein IIb/IIIa complex. From the present study showing binding of WGA on lymphocyte-like Megs and recent data from the literature of the binding of WGA on early hemopoietic progenitors [ 5 ] , we can hypothesize that carbohydrates recognized by this lectin are present both on the surface of pluripotent progenitors and on all cells of Meg lineage, including platelets. On the contrary, carbohydrates recognized by SBA are not present on early hemopoietic progenitors [ 6 ] ,but are observed on the surface of cells committed in the Meg lineage. The role of carbohydrates in human megakaryocytopoiesis is currently under study in our laboratory.
Acknowledgment We thank f! McLuughlin for technical assistance in translation.
References 1 Schick PK, Filmer Jr WG. Sialic acid in mature megakaryocytes: detection by wheat germ agglutinin. Blood 1985;65:ll20-1126. 2 Ganguly P, Fossett NG. The role of sialic acid in the activation of platelets by wheat germ agglutinin. Blood 1984;63:181-187.
Lectins Binding on Cultured Megakaryocytes
238
3 Ruoslahti E. Proteoglycans in cell regulation. J Biol Chem 1989;264:13369-13372. 4 LaRussa VF, Sieber F, Sensenbrenner LL, Sharkis SJ. Effects of neuraminidase on the regulation of erythropoiesis. Blood 1984;63:784-788. 5 Hara H, Mori K, Misawa M, et al. Wheat germ agglutinin (WGM) or My10 (CD34), which is better marker of human hemopoietic progenitors? Exp Hemato1 1990;18:643a. 6 Simmons PJ, Bartelmez S, Torok-Storb B. Human long-term culture (LTC)repopulating cells are CD34+/SBA-/RH123. Exp Hematol 1990;18:7a. 7 Han ZC, Briere J, Abgrall JF, Sensebe L, Parent D, Guern G. Characteristics of megakaryoqte colony formation in normal individuals and in primary thrombocythemia: studies using an optimal cloning system. Exp Hematol 1989;17:46-52.
