The Lutheran Blood Group Glycoproteins, the Erythroid Receptors
for Laminin, Are Adhesion Molecules*
Wassim El
Nemer
,
Pierre
Gane
,
Yves
Colin
,
Viviane
Bony
,
Cécile
Rahuel
,
Frédéric
Galactéros§,
Jean Pierre
Cartron
, and
Caroline Le
Van Kim
¶
From
INSERM U76, Institut National de la Transfusion
Sanguine, Paris 75015 and § INSERM U91, Hopital Henri
Mondor, Créteil 94100, France
 |
ABSTRACT |
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 |
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 |
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 |
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 ( ) or
Lu(v13) ( ) gp isoforms. L929 cells transfected by the expression
vector alone were used as negative control ( ). 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 ( ) or of a control Fc fragment ( )
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, ) and vitronectin
(Vn, ). 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 ( ) or of
control Fc ( ) 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).
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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+) ( ),
Lu(a b ) (XS2 type) ( ), and Lu(a b ) (In(Lu) type)
( ). Laminin binding was detected by flow cytometry with the mAb
anti-A laminin chain, and the results are expressed as in Fig.
1A.
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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.
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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.
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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
( ) or SS ( ) red cells. Laminin binding was estimated as in Fig.
1.
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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 ( ), GPC ( ), and
Lu ( ) antigen expression and laminin binding ( ) 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 |
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 |
-
Daniels, G.
(1995)
Human Blood Groups, pp. 356-384, Blackwell Scientific, Oxford, UK
-
Telen, M. J.
(1995)
in
Blood Cell Biochemistry (Cartron, J. P., and Rouger, P., eds), Vol. 6, pp. 281-297, Plenum Publishing Corp., New York
-
Parsons, S. F.,
Mallison, G.,
Judson, P. A.,
Anstee, D. J.,
Tanner, M. J. A.,
and Daniels, G. L.
(1987)
Transfusion (Phila.)
27,
61-63[Medline]
[Order article via Infotrieve]
-
Daniels, G.,
and Khalid, G.
(1989)
Vox Sang
57,
137-141[Medline]
[Order article via Infotrieve]
-
Parsons, S. F.,
Mallison, G.,
Holmes, C. H.,
Houlihan, J. M.,
Simpson, K. L.,
Mawby, W. J.,
Spurr, N. K.,
Warne, D.,
Barclay, A. N.,
and Anstee, D. J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5490-5500
-
Rahuel, C.,
Le Van Kim, C.,
Mattei, M. G.,
Cartron, J. P.,
and Colin, Y.
(1996)
Blood
88,
1865-1872[Abstract/Free Full Text]
-
Parsons, S. F.,
Mallinson, G.,
Daniels, G. L.,
Green, C. A.,
Smythe, J. S.,
and Anstee, D. J.
(1997)
Blood
89,
4219-4225[Abstract/Free Full Text]
-
El Nemer, W.,
Rahuel, C.,
Colin, Y.,
Gane, P.,
Cartron, J. P.,
and Le Van Kim, C.
(1997)
Blood
89,
4608-4616[Abstract/Free Full Text]
-
Campbell, I. G.,
Foulkes, W. D.,
Senger, G.,
Trowsdale, J.,
Garin-Chesa, P.,
and Rettig, W. J.
(1994)
Cancer Res.
54,
5761-5765[Abstract]
-
Yu, H.,
Chen, J. K.,
Feng, S.,
Dalgarno, D. C.,
Brauer, A. W.,
and Schreiber, S. L.
(1994)
Cell
76,
933-945[Medline]
[Order article via Infotrieve]
-
Chesa-Garin, P.,
Sanz-Moncasi, M.-P.,
Campbell, I. G.,
and Rettig, W. J.
(1994)
Int. J. Oncol.
5,
1261-1266
-
Rettig, W. J.,
Garin-Chesa, P.,
Bersford, H. R.,
Oettgen, H.,
Melamed, M. R.,
and Old, L.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
3110-3114[Abstract]
-
Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl 1, 6 (abstr.)
-
Engel, J.
(1992)
Biochemistry
31,
10643-10651[Medline]
[Order article via Infotrieve]
-
Tryggvarson, K.
(1993)
Curr. Opin. Cell Biol.
5,
877-882[Medline]
[Order article via Infotrieve]
-
Yurchenko, P. D.,
and O'Rear.
(1993)
in
Molecular and Cellular Aspects of Basement Membranes (Rohrbach, D. H., and Timpl, R., eds), pp. 121-146, Academic Press, San Diego
-
Inglis, G., Fraser, R. H., and Mitchell, R. (1993) Transfus.
Med. 3, (suppl.) 94 (abstr.)
-
Riwom, S.,
Janvier, D.,
Navenot, J. M.,
Benbunan, M.,
Muller, J. Y.,
and Blanchard, D.
(1994)
Vox Sang.
66,
61-67[Medline]
[Order article via Infotrieve]
-
Carlos, T. M.,
and Harlan, J. M.
(1994)
Blood
84,
2068-2101[Abstract/Free Full Text]
-
Bowen, M. A.,
Patel, D. D.,
Li, X.,
Modrell, B.,
Malacko, A. R.,
Wang, W. C.,
Marquardt, H.,
Neubauer, M.,
Pesando, J. M.,
and Francke, U.
(1995)
J. Exp. Med.
181,
2213-2220[Abstract]
-
Lehman, J. M.,
Riethmuller, G.,
and Johnson, J. P.
(1989)
Proc. Natl. Acad. Sci. U. S. A.
86,
9891-9895[Abstract]
-
Southcott, M. J. G., Parsons, S. F., Anstee, D. J.,
and Tanner, M. J. (1997) Blood 90, Suppl. 2, 175 (abstr.)
-
De Lisser, H. M.,
Chilkotowsky, J.,
Yan, H. C.,
Daise, M. L.,
Buck, C. A.,
and Albelda, S. M.
(1994)
J. Cell Biol.
124,
195-203[Abstract]
-
Hebbel, R. P.
(1991)
Blood
77,
214-237[Medline]
[Order article via Infotrieve]
-
Bunn, H. F.
(1992)
N. Engl. J. Med.
337,
762-769[Free Full Text]
-
Hillery, C. A.,
Ming, C. D.,
Montgomery, R. R.,
and Scott, J. P.
(1996)
Blood
87,
4879-4886[Abstract/Free Full Text]
-
Wick, T. W.,
and Eckman, J. R.
(1996)
Curr. Opin. Hematol.
3,
118-124[Medline]
[Order article via Infotrieve]
-
Solovey, A.,
Lin, Y.,
Browne, P.,
Choong, S.,
Wayner, E.,
and Hebbel, R. P.
(1997)
N. Engl. J. Med.
337,
1584-1590[Abstract/Free Full Text]
-
Race, R. R.,
and Sanger, R.
(1975)
Blood Groups in Man, 6th Ed., pp. 261-282, Blackwell Scientific, Oxford
-
Greenwalt, T. J.,
Sasaki, T. T.,
and Steane, E. A.
(1967)
Transfusion (Phila.)
7,
189-200[Medline]
[Order article via Infotrieve]
-
Cutbush, M.,
and Chanarin, I.
(1956)
Nature
178,
855-856
-
Aruffo, A.,
Stamenkovic, I.,
Melnick, M.,
Underhill, C. B.,
and Seed, B.
(1990)
Cell
61,
1303-1313[Medline]
[Order article via Infotrieve]
-
Hanspal, M.
(1997)
Curr. Opin. Hematol.
4,
142-147[Medline]
[Order article via Infotrieve]
-
Franck, P. F. H.,
Bevers, E. M.,
Lubin, B. H.,
Comfurius, P.,
Chiu, D. T.-Y.,
Op den Kamp, J. A. F.,
Zwaal, R. F. A.,
van Deenen, L. L. M.,
and Roelofsen, B.
(1985)
J. Clin. Invest.
75,
183-190[Medline]
[Order article via Infotrieve]
-
Manthorpe, M.,
Engvall, E.,
Ruoslahti, E.,
Longo, F.,
Davis, G.,
and Varon, S. J.
(1983)
Cell Biol.
97,
1882-18906
-
McCarthy, J. B.,
Palm, S. L.,
and Furcht, L. J.
(1983)
Cell Biol.
97,
772-777
-
Terranova, V. P.,
Liotta, L. A.,
Rousso, R. G.,
and Martin, G. R.
(1982)
Cancer Res.
42,
2265-2269[Abstract]
-
Terranova, V. P.,
Rao, C. N.,
Takbic, T.,
Margulies, I. M. K.,
and Liotta, L. A.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
444-448[Abstract]
-
Johansson, S.,
Kjellen, L.,
Hook, M.,
and Timple, R. J.
(1981)
Cell Biol.
90,
260-264
-
Fibach, E.,
and Rachmielwitz, E. A.
(1990)
Am. J. Hematol
35,
151-155[Medline]
[Order article via Infotrieve]
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.