The Lutheran Blood Group Glycoproteins, the Erythroid Receptors for Laminin, Are Adhesion Molecules*

Wassim El NemerDagger , Pierre GaneDagger , Yves ColinDagger , Viviane BonyDagger , Cécile RahuelDagger , Frédéric Galactéros§, Jean Pierre CartronDagger , and Caroline Le Van KimDagger

From Dagger  INSERM U76, Institut National de la Transfusion Sanguine, Paris 75015 and § INSERM U91, Hopital Henri Mondor, Créteil 94100, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The Lutheran (Lu)1 antigens belong to a polymorphic blood group system in which two main alleles, Lua and Lub, generate the current phenotypes Lu(a-b+), Lu(a+b-), and Lu(a+b+). A null phenotype lacking all Lu antigens, including Lua and Lub, is called Lunull or Lu(a-b-) and results from one of three different genetic backgrounds as follows: homozygosity for an extremely rare recessive allele at the LU locus; heterozygosity for a dominant inhibitor gene, unlinked to the LU locus, called In(Lu); and hemizygosity for a recessive X-linked suppressor gene, called XS2 (1). Interestingly, the In(Lu) gene has been shown to severely depress all Lu antigens and to down-regulate the phenotypic expression of several other blood group antigens, including CD44 (carrier of Indian antigens), MER2, Knops, P1, and i antigens (2).

The Lu antigens are carried by two membrane glycoproteins (gp) of 85 and 78 kDa (3, 4). The 85-kDa isoform has been cloned from human placenta and was shown to represent a new member of the immunoglobulin superfamily with five extracellular Ig-like domains (2 V-set and 3 C2-set domains from the NH2 terminus), a transmembrane domain, and a cytoplasmic tail of 60 amino acids (5). The extracellular domains and the cytoplasmic domain contain consensus motifs for the binding of integrins and Src homology 3 (SH3) domains, respectively.

The LU gene is located on chromosome 19q13.2 (5, 6). It is composed of 15 exons extending on approximately 12 kilobase pairs of DNA, and the molecular basis for the major alleles Lua and Lub was shown to result from a single A229G nucleotide substitution changing His to Arg at position 77 of the Lu gps (7, 8). Nucleotide sequence comparison indicated that the Lu gp of 85 kDa was virtually identical to the basal cell adhesion molecule (B-CAM) epithelial cancer antigen of 78 kDa cloned from the colon cancer HT29 cell line (9). The B-CAM antigen (78 kDa) exhibits the same NH2-terminal amino acid sequence as the Lu gp of 85 kDa but lacks the last 40 COOH-terminal amino acids of the cytoplasmic tail that carries the proline-rich motif for SH3 binding domains and potential phosphorylation motifs that could be involved in intracellular signaling pathways (10).

Recently, we demonstrated that the Lu gp of 85 kDa and the B-CAM gp of 78 kDa represent isoforms of the same protein encoded by two mRNA spliceoforms of the LU gene (6). We also showed that alternative splicing of intron 13 generates the two transcript spliceoforms encoding the long tail (Lu) and the short tail (B-CAM) gps, and we proposed to refer to these molecules as the Lu and Lu(v13) species, respectively, since they originate from the same gene. Moreover, we demonstrated that Chinese hamster ovary cells expressing these two recombinant gps reacted as well with anti-Lu as with anti-B-CAM antibodies (8). These findings provided the definitive proof that the Lu and B-CAM antigens are carried by the same gps present under two isoforms of 85 and 78 kDa. They also indicate that the Lu species of 78 kDa identified on red cells (see above) is identical to the Lu(v13) isoform species (5, 6). Besides their expression on red blood cells, Lu and Lu(v13) antigens are also expressed constitutively in many other tissues (5). Moreover, these antigens are overexpressed in ovarian carcinomas in vivo and up-regulated following malignant transformation in some cell types (5, 11, 12).

Because the Lu/B-CAM antigens represent new immunoglobulin superfamily members, they were suspected to have some recognition or receptor functions (5, 11). Preliminary investigations from Udani et al. (13) indicated that soluble laminin, a basement membrane-specific protein involved in cell differentiation, adhesion, migration, and proliferation (14-16), binds to red cells. These authors examined the binding of laminin in solution to intact red cells and red cell proteins in Western blot experiments. By analyzing samples from Lu-positive and Lunull donors as well as from SS patients whose red cells expressed more Lu antigens than normal red cells, they suggested that the Lu gps are a novel laminin receptor overexpressed in sickle red cells. In the present study, we demonstrated that both isoforms of the Lu gp are adhesion molecules, by analyzing the binding of transfected mouse fibroblast L929 cells or human erythroleukemic K562 cells to various immobilized extracellular matrix proteins, including laminin. We investigated how Lu antigens and laminin binding appear during erythroid differentiation in vitro, which provided further evidence of the strict correlation between Lu antigen expression and laminin binding to red cells.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Blood Samples and Antibodies-- Whole blood from healthy donors or patients homozygous for HbS (SS) was collected. The Lu phenotypes were determined by agglutination studies using the antiglobulin gel test (Diamed SA, Morat, Switzerland). Mature red blood cells (erythrocytes) were cryopreserved by standard methods. Flow cytometry and laminin binding assays were performed using the following mouse mAbs: anti-Lub LM342 (17), anti-A, B1 or B2 laminin chain (Boehringer Ingelheim, Niederlassung, Germany), and the irrelevant anti-Fy6 (iA3) (18). The antibodies used for the two-phase liquid cultures of human peripheral blood erythroid progenitors are mouse mAb anti-GPC MR4-130 (INTS, Paris, France), and fluorescein (FITC)- or phycoerythrin (PE)-conjugated anti-GPA mAbs, and isotypic controls (Immunotech, Marseille, France).

Cell Culture and Transfection-- Mouse fibroblasts L929 and human K562 cells were obtained from the American Type Culture Collection and were grown in Dulbecco's modified Eagle's medium Glutamax I and RPMI 1640 medium, respectively, supplemented with 10% fetal calf serum. To obtain stable transfectants expressing Lub or Lub(v13) isoforms, Lub or Lub(v13) cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen, Leek, The Netherlands) and transfected into the cells (107 cells/assay) using Lipofectin reagent (Life Technologies, Inc.). Stably transfected L929 and K562 cells were maintained in culture medium supplemented with 0.6 and 0.8 g/liter neomycin (G418), respectively. Lub-positive cells stained with the anti-Lub mAb LM342 were amplified by a round of selection using magnetic beads coated with anti-mouse IgG (Dynabeads-M-450, Dynal) as recommended by the manufacturer. Stably transfected clones were then isolated, and their level of expression of the Lu antigens was estimated by flow cytometry (see below).

Production of Soluble Chimeric Lu-Fc-- The recombinant DNA encoding the extracellular domain of the Lu gp with the five Ig-like motifs was obtained by polymerase chain reaction using the cDNA GC-rich kit (CLONTECH, Palo Alto, CA) with the Lub cDNA as template and 10 pM of the following primers: sense (5'-GCGCTCTAGACCACCATGGAGCCCCCGGACGCACCG-3') and antisense (5'-GCGCGGATCCACATGGCGCTTGTTCCCGTGG-3') under the following conditions: 1 min at 94 °C, 30 cycles of 30 s at 94 °C and 2 min at 68 °C, 5 min of final elongation at 68 °C. The polymerase chain reaction product of 1.7 kilobase pairs was subcloned in the pIg plus vector (pIg-Tail Expression System, Ingenius, R & D Systems) and used for transient transfection of COS-7 cells by DEAE-dextran (ProFection mammalian transfection system, Promega). The cell culture supernatant was applied to a protein A-Sepharose column, and the Lu-Fc fragment was eluted as recommended by the manufacturer.

Two-phase Liquid Cultures of Human Peripheral Blood Erythroid Progenitors-- Peripheral blood (buffy coat) was obtained from healthy adult volunteers. Mononuclear cells were separated by Ficoll-Paque density gradient centrifugation (Amersham Pharmacia Biotech, Uppsala, Sweden). The mononuclear cells were collected and cultured using a two-phase liquid culture system described originally by Fibach and Rachmilewitz (40) with some modifications.2 with some modifications. Lu antigens and laminin binding properties of cells of the erythroid lineage were monitored by flow cytometry analysis (see below).

Flow Cytometry Analysis-- Expression of Lub antigen on red cells or transfectant cell lines was measured on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using LM342 or BRIC108 mAb as described (8). Lu(a-b-) red cells, wild type L929 and K562 cells, and irrelevant mouse and human mAb were used as negative controls.

Double staining analyses for Lub and laminin were carried out as follows: normal Lu(a-b+) red cells (106 cells) were incubated 30 min at room temperature with the anti-Lub mAb LM342. The cells were washed twice with phosphate-buffered saline (PBS) and incubated for 20 min at room temperature with an anti-mouse IgG coupled to FITC (Immunotech). After two washes with PBS the cells were incubated with mouse IgG1 (Becton-Dikinson, San Jose, CA) to saturate the free sites of the previous antibody. The cells were then incubated with 1.5 µg of purified laminin 1 (Life Technologies, Inc.) for 1 h at 37 °C, washed twice with PBS, and incubated with the anti-A laminin chain (IgG2a) for 30 min. The cells were washed twice and incubated with an anti-mouse-IgG2a-PE (Becton Dickinson) for 20 min at room temperature. After a final wash with PBS the cells were suspended in 500 µl of PBS/bovine serum albumin, 0.2%, and treated for flow cytometry analysis as described (8). As negative controls, an isotypic mouse IgG1 mAb was used or laminin was omitted prior to incubation with the anti-laminin mAb.

Laminin Binding Assay-- Normal and SS red cells, erythroid progenitor cells, and stably transfected L929 and K562 expressing Lub or Lu(v13)b were analyzed for the ability to bind laminin. Cells (5 × 105/assay) were incubated for 45 min at 37 °C with different dilutions of purified laminin 1 in PBS, supplemented with 0.5% bovine serum albumin. After two washes with PBS, cells were incubated with the anti-laminin (anti-A chain) (Boehringer Mannheim) for 30 min at 4 °C. The cells were then washed twice with PBS and incubated with PE-conjugated anti-mouse IgG (Immunotech) for 20 min at 4 °C. After another washing step, 0.1 ng of TO-PRO-1 was added, and positive cells (dead cells) were excluded from analysis.

Laminin binding inhibition assay by secreted chimeric Lu-Fc fragment was performed as follows: 500-ng aliquots of laminin were incubated with different dilutions of the Lu-Fc fragment or a control Fc fragment for 1 h at 37 °C. The suspensions were then added to 5 × 105 red cells or L929 clone cells expressing Lub isoform and incubated for 45 min at room temperature. Washing and staining of the cells were performed as described above.

For biotin labeling, 100 µg of laminin were dissolved in 300 µl of a 0.2 M NaHCO3 solution containing 0.15 M NaCl before adding 6 µl of N-Hydroxysuccinimidobiotin solution (Sigma) (4 mg/ml in dimethylformamide). The reaction was allowed to stand for 1 h at room temperature with stirring. The sample was dialyzed against PBS, collected, and stored at 4 °C. For laminin binding inhibition, 5 × 105 L929 transfectant cells were incubated with increasing amounts of cold laminin for 45 min at 37 °C. The cells were washed twice with PBS and incubated with 0,1 µg of biotinylated laminin for another 45 min at 37 °C. 50 µl of PE-conjugated streptavidin (diluted to 1/50; Immunotech, Marseille) were added to the cell suspensions, and the cells were analyzed using the FACScan flow cytometer.

Adhesion Assays-- Purified laminin, fibronectin, transferrin, fibrinogen, fibrin, and vitronectin (Life Technologies, Inc.) were diluted in water and coated on a 96-well microplate (MaxiSorp F96 Nunc-Immuno Plate, Nunc A/S, Roskilde, DK) at 4 °C overnight. The wells were washed once with a 1% non-fat milk solution and preincubated with this solution for 2 h at room temperature to block the nonspecific cell adhesion. Adhesion assays were performed using wild type and transfected human K562 cells. Cells were washed three times with 30 ml of serum-free RPMI 1640 and diluted to 1.5 × 106 cells/ml, and then aliquots of 100 µl (1.5 105 cells) were added to each well. After a 1-h incubation in a CO2 incubator at 37 °C the wells were filled with PBS, and the microplate was put to float upside down for 30 min in a PBS solution before microscopic observation.

For the inhibition assays the wells coated overnight with 500 ng of laminin were incubated with 2.5, 1, or 0.5 µg of the secreted chimeric Lu-Fc fragment prior to the addition of non-fat milk.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Laminin Binding to Lu and Lu(v13) Glycoproteins Expressing Cells-- To provide direct evidence for the binding of Lu gps to laminin, L929 cells stably transfected with pcDNA Lu or pcDNA Lu(v13) were analyzed for laminin binding in flow cytometry experiments. Fig. 1A shows that purified laminin bound equally well to cells expressing Lu or Lu(v13) gps (200,000 apparent Lub sites/transfected cell versus 2,000-5,000 sites/red cell) with a plateau reached at 2 µg of laminin, whereas no binding was observed with mock-transfected L929 cells (mean fluorescence intensity 38 versus 2%). As shown in Fig. 1B, preincubation of Lu-transfected L929 cells with increasing amounts of cold laminin dose-responsively inhibited subsequent binding of biotin-labeled laminin. On the other hand, preincubation of laminin with a Lu-Fc molecule gave a dose-response curve of inhibition, whereas preincubation with a control Fc fragment (Fig. 1C), or with BRIC108 or LM342 anti-Lub mAbs (not shown) failed to inhibit laminin binding. Similar results were obtained after stable transfection and expression of the two Lu gps isoforms in erythroleukemic K562 cells which do not express endogenous Lu antigens (data not shown). The results obtained with the two Lu-transfected cell types, adherent cells and cells growing in suspension, indicated that both Lu and Lu(v13) gps could directly bind to soluble laminin.


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Fig. 1.   Binding of soluble laminin to L929 cell transfectants. A, flow cytometry assays showing laminin binding to L929 stably transfected clones expressing Lu (black-diamond ) or Lu(v13) (bullet ) gp isoforms. L929 cells transfected by the expression vector alone were used as negative control (black-square). Cells (5 × 105) were incubated with increasing concentrations of laminin (0-2,000 ng) prior to the addition of the anti-laminin chain A mAb. The results are expressed as the mean fluorescence intensity versus laminin concentration, after incubation with a phycoerythrin-labeled second antibody. B, inhibition assay by unlabeled laminin. Lu expressing cells (5 × 105) were preincubated with increasing amounts of laminin (0-50 µg) before addition of biotin-labeled laminin (0.1 µg). Results are expressed as a percent of specifically bound biotinylated laminin versus unlabeled laminin concentration. No binding of biotinylated laminin to mock transfectant was observed (not shown). C, inhibition assay by recombinant Fc fragments. Laminin aliquots (500 ng) were preincubated with increasing amounts of the recombinant Lu-Fc fragment (open circle ) or of a control Fc fragment (bullet ) before addition to L929 cells expressing the Lu isoform. The percentage of bound laminin is expressed as the relative fluorescence intensity versus Fc fragment concentration. Similar results were obtained with cells expressing the Lu(v13) isoform (not shown).

Adhesion of Lu Glycoproteins Expressing Cells to Laminin-- We next examined the ability of Lu gps expressing cells to adhere to laminin. Microwell culture dishes were coated with increasing concentrations of purified laminin or with 500 ng of fibronectin, vitronectin, transferrin, fibrinogen, and fibrin as controls. Since non-transfected L929 cells adhered nonspecifically to laminin (not shown), only K562 cells were used in these experiments. As shown in Fig. 2A, Lu- and Lu(v13)-transfected cells adhered to the laminin-coated wells after 1 h, whereas parental K562 cells did not (Fig. 2B). Neither the transfectants nor the parental cells adhered to the wells coated with fibronectin, vitronectin, transferrin, fibrinogen, and fibrin (Fig. 2C). Adhesion of transfected cells to laminin gave a dose-response curve between 100 and 500 ng of laminin, and a plateau was reached with 500 ng of laminin (Fig. 3A). Incubation of precoated laminin (500 ng), with increasing amounts (500-2,500 ng) of a recombinant chimeric Lu-Fc molecule corresponding to the entire extracellular NH2-terminal region of the Lu gp, dose-responsively inhibited subsequent adhesion of transfected cells (Fig. 3B). These data demonstrated that the long-tail as well as the short-tail Lu gps have similar adhesion activity to laminin.


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Fig. 2.   Adhesion of K562 cell transfectants to coated laminin. K562 cells were stably transfected by the pcDNA3 expression vector alone or the recombinant vector containing the cDNA encoding the Lu gp isoform. Clones were incubated for 1 h at 37 °C in microplate wells (1.5 105 cells/well) pre-coated with laminin or vitronectin (500 ng/well). The wells were washed with PBS before microscopic observation. A, adhesion to coated laminin of K562 cells stably expressing the Lu gp; B, control showing no adhesion of mock-transfected K562 to laminin; C, control showing no adhesion of Lu-transfected K562 cells to vitronectin (nor to transferrin, fibronectin, fibrin, and fibrinogen, not shown). Similar results were obtained with cells expressing the Lu(v13) isoform (not shown).


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Fig. 3.   Dose-response adhesion of K562 transfectants to laminin and inhibition assays by recombinant Fc fragments. A, adhesion of Lu-transfected K562 cells to increasing amounts of coated laminin (Lm, bullet ) and vitronectin (Vn, black-triangle). The cells were incubated with increasing amounts of laminin or vitronectin and analyzed as in Fig. 2. The percentage of adherent cells is shown as a function of substrate concentration. B, microplate wells coated with laminin (500 ng/well) were incubated with increasing amounts of the recombinant Lu-Fc (open circle ) or of control Fc (bullet ) fragments before adding 1.5 × 105 Lu-transfected K562 cells. The percentage of adherent cells is shown as a function of Fc fragment concentration. Similar results were obtained with cells expressing the Lu(v13) isoform (not shown).

Binding of Purified Laminin to Human Erythrocytes-- Binding experiments were performed by preincubating Lu(a-b+) red cells with increasing concentrations of purified laminin in solution prior to the addition of anti-A, -B1, or -B2 laminin chain mAbs. The cells were then incubated with a phycoerythrin-conjugated antibody, and laminin binding was measured by flow cytometry. As shown in Fig. 4, laminin bound to red cells in a dose-dependent fashion, and a plateau was reached with 1 µg of laminin. The mean fluorescence intensity at saturation concentration of laminin was much higher when the cells were incubated with anti-laminin A chain than with anti-B1 and -B2 laminin chains (not shown). The latter antibodies might have lower affinities for laminin or might partially displace the binding of laminin to red cells. Thus, only the mAb anti-A chain was used in further experiments.


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Fig. 4.   Laminin binding to red cells of different Lu phenotypes. The binding assays were performed using 5 × 105 red cells of different phenotypes: Lu(a-b+) (black-diamond ), Lu(a-b-) (XS2 type) (bullet ), and Lu(a-b-) (In(Lu) type) (black-triangle). Laminin binding was detected by flow cytometry with the mAb anti-A laminin chain, and the results are expressed as in Fig. 1A.

The specificity of laminin binding was examined by studying red cells from two Lunull individuals (In(Lu) and XS2 types). As shown in Fig. 4, there was a complete absence of laminin binding to Lunull red cells of both types as compared with Lu-positive erythrocytes, indicating that laminin binding was dependent on the presence of Lu antigens.

Correlation between the Level of Lu Antigens and the Degree of Laminin Binding-- Red cells from 23 patients homozygous for HbS (SS red cells) and from 14 control individuals (AA red cells) were compared for Lub antigen expression and laminin binding (all controls and SS patients were homozygous for Lub antigen).

As shown in Fig. 5A, the mean value of Lub antibody binding capacity was increased on the total red cell population from SS as compared with AA donors (1276 ± 311 versus 794 ± 203, p < 0.001). The overexpression of Lub on SS erythroid cells was even more striking on reticulocytes as detected by positive thiazole orange (TO+ subpopulation) staining (3092 ± 929 versus 1595 ± 642, p < 0.001; Fig. 5B) than on mature red cells not stained by thiazole orange (TO- subpopulation) (1030 ± 224 versus 768 ± 192, p < 0.001; Fig. 5C). As expected, the number of reticulocytes was increased in SS patients as compared with healthy donors, with a large variation in both type of blood samples (13 ± 6% versus 1.6 ± 0.6%). Thus, only the TO- red cell population, obtained from cryopreserved red cell samples, were used in further analyses to avoid individual variation due to the heterogeneity of the reticulocyte content between different fresh blood samples.


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Fig. 5.   Lub antigen density on AA and SS reticulocytes and mature red cells. The Lub antibody binding capacities of the total red cell population (TO+ and TO- populations) (A), TO+ red cell population (reticulocytes) (B), and the TO- red cell population (mature erythrocytes) (C) from 14 control donors (AA) and 23 sickle cell patients (SS) were estimated as described under "Experimental Procedures." Each point represents one sample, and the horizontal bar indicates the mean values.

The binding of soluble laminin to each TO- red cell sample was evaluated as described above. Fig. 6 shows that there is a good correlation between the level of Lu antigen expression and the capacity of red cells to bind laminin, both in control and SS samples (r = 0.91 and r = 0.79, respectively). In addition, although the mean laminin binding capacity was increased in sickle red cells as compared with normal red cells (125 ± 35 versus 95 ± 25, p < 0.01), most SS mature red cell samples exhibited laminin binding and Lub expression values similar to those obtained with control samples (data not shown). According to the observation that the level of Lub expression was more different between SS and AA reticulocytes than between SS and AA mature red cells (see Fig. 5), the increase of the laminin binding capacity was also more striking when reticulocytes were analyzed instead of mature red cells. Indeed, laminin binding values of 300 ± 15 and 170 ± 10 were obtained with reticulocytes from selected SS and AA donors (n = 3) exhibiting the average level of Lub expression (3,200 and 1,600 antigen/cell, respectively, Fig. 5B). Double staining experiments (see "Experimental Procedures") revealed that within the red cell population of an individual (AA), only cells heavily stained by the anti-Lub antibody bound detectable amounts of laminin (Fig. 7A). The anti-laminin mAb alone (Fig. 7B) or used in double staining with an irrelevant antibody (anti-Fy6) (Fig. 7C) revealed a heterogeneity of laminin binding within red cells but failed to reveal a clear double population, as observed with the anti-Lu mAb (Fig. 7D). Altogether, these experiments indicated that the level of laminin binding to normal and sickle red cells is directly correlated with the level of Lu antigen expression.


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Fig. 6.   Correlation between Lub expression and laminin binding to red cells. The binding of soluble laminin and the expression of the Lub antigen on TO- red cells from each sample studied in Fig. 5 were quantified by flow cytometry. The linear correlation coefficient (r) was calculated for the control (A) and the sickle cell samples (B) using the KaleidaGraphTM program (version 3.08 for Apple Macintosh computer).


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Fig. 7.   Double staining of red cells with anti-Lub and anti-laminin mAbs. A, double staining with the anti-Lub and anti-laminin mAbs. The binding capacities of normal Lu(a-b+) red cells (106 cells) for the anti-Lub mAb (LM342, IgG1) and the anti-laminin chain A (IgG2a) mAb after binding of purified laminin (1.5 µg) were simultaneously analyzed, as described under "Experimental Procedures." The results show the fluorescence intensity related to the Lub antigen expression (Y axis) and to the laminin binding (X axis), as measured after incubation with FITC-labeled and PE-labeled second antibodies, respectively. B, double staining with an isotypic (IgG1) negative control and the anti-laminin mAbs. C, double staining with an unrelated IgG1 (anti-Fy-6) and the anti-laminin mAbs. D, double staining as in A but, as a negative control (X axis), laminin was omitted prior to the incubation with the anti-laminin mAb.

Inhibition of Laminin Binding by a Soluble Lu-Fc Fragment-- To confirm that the laminin binding to red cells was Lu-specific, an increasing amount of the recombinant chimeric Lu-Fc molecule was preincubated with 500 ng of laminin prior to the binding assay with normal and sickle red cells. The soluble Lu-Fc molecule dose-responsively inhibited the binding of laminin to both red cell samples (Fig. 8), as also shown with transfected cells (see above). A 90-100% inhibition was obtained with 1 µg of Lu-Fc, whereas no inhibition was detected using 1 µg of a control Fc molecule. Similar results were obtained when these experiments were carried out either with total red cells, reticulocytes, or mature red cells (data not shown).


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Fig. 8.   Inhibition of soluble laminin binding to red cells by a Lu-Fc fragment. 500 ng of laminin aliquots were preincubated with increasing amounts of the recombinant Lu-Fc or of a control Fc fragment and were added to 5 × 105 control (bullet ) or SS (open circle ) red cells. Laminin binding was estimated as in Fig. 1.

Lu Antigen Expression and Laminin Binding during in Vitro Erythropoiesis-- To investigate whether receptor(s) other than Lu antigens could mediate binding to laminin during erythropoiesis, we analyzed Lu antigen expression and laminin binding properties during in vitro two-phase liquid culture of peripheral blood erythroid progenitors (see "Experimental Procedures"). Lu antigen expression was detected at day 8, in the erythropoietin-dependent phase of the culture, as previously shown.2 This stage corresponds to late erythroid differentiation since GPC and GPA antigens appeared on a significant percentage of erythroid progenitors as early as days 4 and 7 of culture, respectively (Fig. 9). The proportion of erythroid cells that expressed the Lub antigen increased until day 13 of culture and remained stable even after enucleation. Whereas 100% of erythroid cells became GPA- and GPC-positive during maturation, a plateau of 40% of Lub-positive cells was reached, in agreement with the detection of a clear double population with the Lub mAb in circulating red cells (see Fig. 7). No laminin binding to progenitor cells was observed before the appearance of Lu antigens at day 8 of culture, whereas the proportion of laminin-positive cells increased in parallel with the proportion of Lu-positive cells (Fig. 9). Only 50% of the Lu-positive cultured cells exhibited laminin binding, which could account for the observation that only the fraction of red cells highly expressing Lub bound laminin (see Fig. 7A). Thus, laminin binding to erythroid cells appears to be strikingly correlated with the expression of Lu antigens during human erythroid differentiation. This strongly suggests that Lu gps are the only receptors for laminin, not only on mature red cells but most likely also on erythroid progenitors as well.


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Fig. 9.   Lub antigen expression and laminin binding during in vitro differentiation of human peripheral blood erythroid progenitors. GPA (square ), GPC (black-triangle), and Lu (bullet ) antigen expression and laminin binding (black-diamond ) were detected by flow cytometry during in vitro differentiation of human peripheral blood erythroid progenitors in a two-phase liquid culture system. The results show the percentage of erythroid cells expressing each antigen and binding laminin in terms of culture days. Erythropoietin was added at day 6 and enucleation began at days 16-18.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell adhesion molecules belonging to the Ig superfamily show diversity in function and participate in a variety of homophilic and heterophilic cellular interactions (19). Three (V)2-(C)3 Ig superfamily molecules have been presently identified as follows: ALCAM, the human homologue of chicken SC1, that functions as an accessory protein in T-cell activation and that is an adhesion ligand for CD6 (20); MUC18 that may have a role in tumor progression (21); and Lu or B-CAM gps (5, 9).

In this report, we have provided the definitive proof that the Lu gps are receptors for laminin, since erythroid and non-erythroid cell lines acquired laminin binding capacity and adhesion properties to coated laminin once they were transfected with the Lu cDNAs. Moreover, recent investigations performed by Southcott et al. (22) indicated that the recombinant extracellular domains of the Lu gps represent high affinity laminin receptors (KD = 5.6 ± 0.7 nM), as measured in a biosensor assay. Since the Lu and Lu(v13) gps have identical adhesive properties and bind equally well to soluble laminin, it is likely that the potential SH3 binding domain and phosphorylation sites carried only by the long-tail cytoplasmic domain of the Lu isoform are not involved in inside-out signaling that could regulate laminin binding. Conversely, it has been shown that a specific isoform of the adhesion molecule platelet endothelial cell adhesion molecule-1 mediates heterophilic binding, whereas a truncated isoform lacking a phosphorylation site in the cytoplasmic domain mediates homophilic binding (23). Further investigations should focus on the characterization of the laminin binding site(s) on the Lu gps. In contrast with Udani et al. (13), however, our data suggested that the Lub epitope is not part of the laminin binding site(s).

By analyzing Lu-positive, Lunull, and SS red cells, we confirmed that there is a correlation between the level of Lu antigen expression and the level of laminin binding in solution to red cells. We also confirmed that CD44, whose expression is associated with the Lu blood group system, did not participate in laminin binding to red cells, since the absence of laminin binding was observed both with the In(Lu) type of Lu(a-b-) red cells, which exhibit a severe reduction of Lu antigens and CD44 expression, and with XS2 Lu(a-b-) red cells, which exhibit reduced expression of the Lu antigens only (1, 2).

It has been proposed that the ability of circulating red cells to bind to vascular endothelium and to exposed components like thrombospondin (through CD36 receptor), fibronectin (through VLA-4), and laminin may play an important role in the evolution of vascular pathology particularly in sickle cell disease (24-27). Hence, there is a shedding of endothelial cells (28) suggesting some vascular damage and potential access to the laminin component of the basement membrane. Our present data, together with those of Udani et al. (13), suggest that the overexpression of the Lu antigen on SS red cells may contribute to vaso-occlusion pain episodes.

It is known that the Lu antigens are very variable in strength (29, 30) and that this heterogeneity can also be detected between individual red cells within a person. Indeed, red cell staining with anti-Lu antibodies reveals two subpopulations, Lu-positive and Lu-negative, which may account for the mixed-field agglutination pattern usually seen with Lu antisera (31). In the present paper, we showed by double staining experiments that for each individual donor laminin binds only to the red cell subpopulation heavily stained by anti-Lub mAb but not to the poorly or unstained subpopulation. Accordingly, during in vitro culture of erythroblasts (see below), laminin bound to only 50% of the Lu-positive cells, which represent 40% of mature red cells. Since anti-laminin mAb revealed a continuum from unstained to strongly stained red cells but not a clear double population, it is assumed that the double population detected by the anti-Lu antibodies may account for a true quantitative heterogeneity of the Lu antigen expression on individual red cells.

The complete inhibitory effect of a soluble recombinant Lu-Fc chimeric gp on the binding of laminin to normal and SS red cells demonstrated the laminin binding specificity to Lu gps on red cells and indicated that there is no other laminin receptors on sickle cells as compared with normal red cells. This conclusion is reinforced by the observation that normal and sickle red cells, when displaying the same level of Lu antigen expression, exhibited the same degree of laminin binding.

During in vitro erythropoiesis, Lu antigens were detected during late erythroid differentiation. We found that the time course of laminin binding to erythroid progenitor cells under in vitro differentiation was strictly correlated with the time course of Lu antigen expression. Particularly, laminin binding was not observed at early stages of the culture when CD44, but not Lu gps, is expressed,2 further indicating that CD44, the hyaluronate receptor (32), is not involved in laminin binding to erythroid cells, as discussed above.

It is known that hematopoietic progenitor cells interact with extracellular matrix in the bone marrow. This is mainly mediated by VLA-4 but also probably by CD44 adhesion molecules whose expression is down-regulated during erythroid differentiation (33). The late appearance of Lu expression/laminin binding features during in vitro erythropoiesis (our present results) and the adhesion of red cells to laminin (26) suggest that the Lu antigens may play some role in terminal erythroid differentiation, perhaps in the enucleation process.

In sickle cell disease, the typical lipid bilayer is altered with loss of the normal phospholipid asymmetry (34). Such modification could expose membrane proteins like Lu gps, thereby increasing binding to exposed components of the extracellular matrix. This could participate in the enhanced adhesion of red cells to sub-endothelial components. It has been shown recently that red cells adhere to thrombospondin and laminin in dynamic flow conditions with a reinforced adhesion of sickle red cells (26). These authors suggested that sulfated glycolipids could be the receptors for these two molecules, since high molecular weight anionic polysaccharides like dextran sulfate or chondroitin sulfate A inhibited the adhesion of normal and sickle red cells. However, our data identifying the Lu gps as the unique laminin receptors in normal and sickle red cells (see above) did not support a direct role of red cell lipids in erythrocyte adhesion to laminin. The reasons for these discrepancies need to be clarified.

In conclusion, we have provided direct evidence that the Lu gps are the only laminin receptors in normal and sickle erythrocytes as well as in erythroid progenitors. We also demonstrated that the two Lu isoforms are adhesion molecules. Further investigations will be necessary to analyze the potential interactions between laminin and the Lu gps expressed in a variety of non-erythroid tissues, since laminin is involved in cell differentiation (35), cell migration (36), and cancer metastases (37) in addition to cell attachment (38, 39). In addition, since the B-CAM antigens, carried by the Lu gps (8), are overexpressed in some tumoral cells (11, 12), it is assumed that these studies might help in elucidating the potential implication of the Lu gps in malignant transformation.

    ACKNOWLEDGEMENTS

We thank P. Hermand and P. Bailly for the gift of purified control Fc protein and for helpful discussions. We are indebted to R. H. Fraser (Regional Donor Center, Glasgow, UK) and D. Blanchard (CTS Nantes, France) for supplying the LM 342 and iA3 mAbs, respectively.

    FOOTNOTES

* This investigation was supported in part by INSERM.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: INSERM U76, Institut National de la Transfusion Sanguine, 6 Rue Alexandre Cabanel, 75015 Paris, France. Tel.: 33 01 44 49 30 46; Fax: 33 01 43 06 50 19; E-mail: clvkim{at}infobiogen.fr.

1 The abbreviations used are: Lu, Lutheran; B-CAM, basal cell adhesion molecule; TO, thiazole orange; PBS, phosphate-buffered saline; mAb, monoclonal antibody; PE, phycoerythrin; FITC, fluorescein; SH3, Src homology 3; gp(s), glycoprotein(s); GPA, glycophorin A; GPC, glycophorin C.

2 V. Bony, P. Gane, C. G. Gahmberg, J. P. Cartron, and P. Bailly, manuscript in preparation.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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