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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-03-0796.
 Previous Article | Table of Contents | Next Article 
Blood, 1 February 2003, Vol. 101, No. 3, pp. 877-885
HEMATOPOIESIS
Laminin isoform-specific promotion of adhesion and migration
of human bone marrow progenitor cells
Yu-Chen Gu,
Jarkko Kortesmaa,
Karl Tryggvason,
Jenny Persson,
Peter Ekblom,
Sten-Eirik Jacobsen, and
Marja Ekblom
From the Department of Medical Biochemistry and
Biophysics, Karolinska Institute and BioStratum AB, Stockholm,
Sweden; Department of Cell and Molecular Biology; Stem
Cell Laboratory, Department of Laboratory Medicine; both of University
of Lund, Sweden; and Department of Hematology, University
Hospital, Lund, Sweden.
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Abstract |
Laminins are    heterotrimeric extracellular proteins that
regulate cellular functions by adhesion to integrin and nonintegrin receptors. Laminins containing 4 and 5 chains are expressed in
bone marrow, but their interactions with hematopoietic progenitors are
unknown. We studied human bone marrow cell adhesion to laminin-10/11 (511/521), laminin-8 (411), laminin-1
(111), and fibronectin. About 35% to 40% of
CD34+ and CD34+CD38 stem and
progenitor cells adhered to laminin-10/11, and 45% to 50% adhered to
fibronectin, whereas they adhered less to laminin-8 and laminin-1.
Adhesion of CD34+CD38 cells to laminin-10/11
was maximal without integrin activation, whereas adhesion to other
proteins was dependent on protein kinase C activation by
12-tetradecanoyl phorbol-13-acetate (TPA). Fluorescence-activated cell-sorting (FACS) analysis showed expression of integrin 6 chain
on most CD34+ and CD34+CD38
cells. Integrin 6 and 1 chains were involved in binding of both
cell fractions to laminin-10/11 and laminin-8. Laminin-10/11 was highly
adhesive to lineage-committed myelomonocytic and erythroid progenitor
cells and most lymphoid and myeloid cell lines studied, whereas
laminin-8 was less adhesive. In functional assays, both laminin-8 and
laminin-10/11 facilitated stromal-derived factor-1 (SDF-1)-stimulated transmigration of CD34+ cells, by an
integrin 6 receptor-mediated mechanism. In conclusion, we
demonstrate laminin isoform-specific adhesive interactions with human
bone marrow stem, progenitor, and more differentiated cells. The
cell-adhesive laminins affected migration of hematopoietic progenitors,
suggesting a physiologic role for laminins during hematopoiesis.
(Blood. 2003;101:877-885)
© 2003 by The American Society of Hematology.
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Introduction |
Hematopoietic cell development occurs within the
bone marrow microenvironment, where adhesive interactions between
progenitor cells and their extracellular matrix ligands are essential
for normal cell proliferation and differentiation and for maintenance of the hematopoietic stem cell.1 Binding of extracellular
matrix ligands to cell surface adhesion receptors activates
receptor-mediated signal transduction pathways and thereby regulates
cellular functions. Evidence for convergence of intracellular signaling
pathways initiated by ligand binding to growth factor and cell adhesion
receptors indicates that cooperation between these 2 signaling pathways is essential for normal cell development and functions (reviewed by
Levesque and Simmons2).
More than 20 different adhesion receptors have been identified on
hematopoietic progenitors (reviewed by Verfaillie3). Of
the receptors for extracellular matrix molecules, integrins are the
most extensively studied. Integrins are dimeric proteins composed of
and chains. The extracellular domain mediates cell-cell or
cell-extracellular matrix interactions, and the cytoplasmic domain
provides a link with cytoskeletal proteins and is involved in the
transmission of intracellular signals. Different integrins have
specific functions in intracellular signal transduction, activation of
growth factor receptors, and association with other transmembrane
proteins (reviewed by Giancotti and Ruoslahti4).
The role of fibronectin binding to integrins 41 and 51 in
hematopoietic cell-matrix adhesive interactions is well established. Primitive long-term in vivo repopulating cells, multipotent-, and
single-lineage clonogenic progenitors5-8 adhere to
different domains of fibronectin. Adhesion to fibronectin or
its cell-binding fragments stimulates proliferation and migration of
stem and progenitor cells9-10 and proliferation of
lineage-committed and differentiated cells.11-13 In
agreement with the in vitro findings, studies with integrin 4 null
chimeric mice have shown impaired embryonal and postnatal development
of erythroid, myeloid, and B cells, most likely attributable to
impaired cellular interactions of the 41 integrin receptor with
fibronectin.14-15
Laminins are large extracellular matrix proteins that regulate
survival, proliferation, differentiation, and specialized functions of
several types of cells. All laminins are heterotrimeric proteins composed of , , and polypeptide chains. So far, 5 (1-5), 3 (1-3), and 3 (1-3) chain variants have been
characterized in mammalians, and 15 different heterotrimers have been
proposed (reviewed by Colognato and Yurchenco16).
Expression of laminin isoforms shows a high degree of developmentally
regulated and tissue-specific variation. In vitro studies, gene
targeting experiments, and studies of mutated genes have indicated
different functions for different laminins (reviewed by
Timpl17). Major cell-binding domains of laminins are
located in the carboxyterminus of chains, and consequently the
cell-binding activities of laminin isoforms are largely determined by
the chains. Several integrins (11, 21, 31,
61, 64, 71, 91, v3) bind to laminins but with variable binding affinities to different
isoforms.18-22
Most studies on hematopoietic cell interactions with laminins have been
performed by using laminin-1 (111), isolated from Engelbreth-Holm-Swarm (EHS) tumor, which can be transplanted
into a mouse, or from placental laminin containing several laminin isoforms. Mature granulocytes,23-24
lymphocytes,25 mononuclear phagocytes,26
activated macrophages,27 and eosinophils28 adhere to these laminins in vitro. Adhesion to these laminins influences survival and maturation of eosinophils29 and
proliferation of macrophages and T lymphocytes.27,30 In
contrast to mature blood cells, human bone marrow progenitor cells have
not been found to adhere to laminin-1.31
Laminin 5 chain is found in most adult basement membranes, including
endothelia.32-33 Laminin 4 chain is synthesized by
endothelial and mesenchymal cells and is also expressed at specific
sites of loose connective tissue.34 In the bone marrow,
laminin 4 and 5 chains, assembled with 1 and 1 chains to
form laminin-8 (411) and laminin-10 (511), are
present in sinusoidal subendothelial basement membranes, and 4
laminins are in addition located in the intersinusoidal spaces. In
contrast, laminin-1 has not been found in bone marrow in
vivo,35-36 in agreement with its expression mainly in
a subset of epithelial basement membranes.37-38
Because of their expression in bone marrow, the 4 and 5 laminins
might affect hematopoietic cell development and functions. To define
the role of different laminins for primitive and developing human
hematopoietic cells, we isolated CD34+CD38
and CD34+ cell fractions and tested their adhesion to
several laminin isoforms and fibronectin. This revealed that both stem
and progenitor cells bound to laminin-10/11 nearly as efficiently as to
fibronectin. The cells adhered also to laminin-8, although with a lower
binding affinity than to laminin-10/11. Both these laminin isoforms
promoted migration of CD34+ cells in vitro. The 61
integrin was identified as a major laminin receptor for both
laminin-10/11 and laminin-8 in these cells. These findings suggest that
the interactions with bone marrow laminins could be functionally
important during stem and progenitor cell development.
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Materials and methods |
Reagents and antibodies
Recombinant laminin-8 was produced in a mammalian expression
system by triple transfection of human laminin 4 and 1 chains and
mouse laminin 1 chain.21 Human laminin-10/11, purified by immunoaffinity column with monoclonal antibody 4C7 against laminin
5 chain,39 was from Gibco (Täby, Sweden). This
laminin contains laminin-10 (511) and laminin-11
(512).40 However, based on amino acid sequencing,
placental laminin-10/11 purified by 4C7 contains mainly
laminin-10.41 Human plasma fibronectin and mouse laminin-1
were purchased from Gibco. Fluorochrome-conjugated antibodies against
human CD34 (anti-HPCA-2, clone 8G12), CD38 (clone HB7), integrin 6
(GoH3), integrin 3 (C3 II.1), and the isotype standards were from
Becton Dickinson (San Jose, CA). The MIKd 2 antibody against
integrin 3 was from Cymbys Biotechnology (Hants, United Kingdom).
Mouse antihuman integrin 3 antibody ASC-6 was from Chemicon
(Temecula, CA). The purified GoH3 antibody was from Becton Dickinson
and Immunotech (Marseille, France). Mouse antihuman integrin 1
antibody P4C10 was from Gibco. Rat immunoglobulin G (IgG)
antibody 193 against mouse laminin 1 chain has been previously
described.42
Cell lines
The human hematopoietic cell lines were grown in RPMI 1640 (Gibco) and 10% fetal calf serum (Gibco) in a humidified
environment at 37°C and 5% CO2.
Isolation and analysis of bone marrow cell fractions
Bone marrow samples were obtained from healthy volunteers, after
informed consent, using guidelines approved by the Ethical Committee, Lund University. Mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden). CD34+ cells were isolated by 2 passages through
magnetic columns (MidiMacs; Miltenyi Biotec, Bergish Gladbach, Germany)
by using a hapten-conjugated CD34 antibody (Qbend/10) and an antihapten
antibody conjugated to magnetic beads (CD34+ isolation kit;
Miltenyi Biotec). CD34 expression was analyzed by immunostaining with a
FACSCalibur flow cytometer (Becton Dickinson) by using the CellQuest
program (Becton Dickinson) and was usually more than 90%.
For isolation of CD34+CD38 and
CD34+CD38+ cell fractions, CD34+
cells were first enriched by magnetic columns and thereafter incubated with phycoerythrin (PE)-anti-CD38 antibody and fluorescein
isothiocyanate (FITC)-anti-CD34 antibody or isotype-matched control
antibodies. The cells were sorted with a FACS VantageSE flow
cytometer (Becton Dickinson).43 A total of 3% to 5% of
the cells with lowest CD38 expression
(CD34+CD38) and 10% of the cells with
highest CD38 expression (CD34+CD38+) were isolated.
For analysis of expression of integrin 3 and 6 chains,
CD34+ cells were enriched by the magnetic column and
thereafter stained with FITC-anti-CD34, allophycocyanin
(APC)-anti-CD38, PE-conjugated GoH3 antibody against integrin 6
chain or, alternatively, PE-conjugated antibody against integrin 3
chain, and corresponding isotype controls. The expression of integrin
3 and 6 chains in CD34+ and
CD34+CD38 cells was analyzed by FACSCalibur
(Becton Dickinson).
Cell adhesion assay
The 96-well nontissue culture plates (Sigma, Stockholm, Sweden)
were coated overnight at 4°C with 50 µL extracellular matrix proteins diluted with phosphate-buffered saline (PBS) (Gibco). The proteins were used at 10 to 30 µg/mL. As negative controls, PBS
was used for coating. Thereafter, the wells were washed 3 times with
PBS and blocked with 2% heat-denatured fatty acid-free bovine serum
albumin (BSA) (Sigma) in PBS for 1 hour in a humidified environment at 37°C and 5% CO2. The wells were washed
twice with PBS and once with Iscove modified Dulbecco medium
(IMDM) (BioWhittaker, Walkersville, MD). The cells were resuspended in
IMDM and added to the wells in a volume of 50 to 100 µL per well. In
some experiments, 300 ng/mL stromal-derived factor-1
(SDF-1) (R&D Systems, Oxon, United Kingdom) was added to the cell
suspension, or the cells were incubated with 5 ng/mL 12-tetradecanoyl
phorbol-13-acetate (TPA) (Sigma) for 1 to 4 hours before
incubation in the wells.
The cell-adhesion assay was performed at 37°C, 5% CO2 in
humidified atmosphere for 1 hour. The nonadherent cells were detached by shaking the plate and by 1 to 3 washes with IMDM. The adherent cells
were fixed with methanol for 10 minutes and thereafter stained with
0.1% Giemsa (Sigma) for 10 to 30 minutes. Thereafter, the plates were
washed with large volumes of deionized water. The adherent cells from
the entire bottom area of the wells were counted by using a Zeiss
Axioskop2 microscope (Carl Zeiss Mikroscopie, Jena, Germany), and the
percentage of adherent cells were counted as follows: (no. of adherent
cells/no. of cells plated) × 100. In experiments using cell lines,
the adherent cells were fixed with 96% ethanol for 10 minutes and
stained with 0.1% crystal violet in water for 30 minutes. Thereafter,
the plates were washed with deionized water, and adherent cells were
lysed with 0.2% Triton X-100 (Sigma). Absorbance was measured at 595 nm with a DigiScan microplate reader by using DigWin software (Asys
Hitech, Eugendorf, Austria). For antibody inhibition experiments, the antibodies (P4C10 at a dilution of 1:50, other antibodies at 25 µg/mL) were added to cell suspension and the cells were incubated at
37°C for 10 minutes before they were added to the wells. The role of
divalent cations on cell adhesion was tested by using 10 mM EDTA
(ethylenediaminetetraacetic acid).
Progenitor assays
The 96-well tissue culture plates (Falcon, Becton
Dickinson) were coated with 10 µg/mL extracellular matrix
proteins or PBS as a control, blocked with BSA, and washed as described
in the adhesion assay. CD34+ cells were incubated in the
wells as described above. Thereafter, the nonadherent cells were
collected, and the adherent cells were detached with vigorous
pipetting. The cells in adherent and nonadherent fractions were
cultured in triplicate (600/mL) in 1 mL 0.8% methylcellulose culture
medium containing cytokines (Methocult GF+ H4435; StemCell Technologies, Vancouver, BC, Canada). After 14 days of culture in a
humidified environment at 37°C and 5% CO2, the colonies
were counted by using an inverted microscope.
Cell migration assay
Transwell inserts with 5 µm pore size (Costar, Cambridge, MA)
were coated with extracellular matrix proteins, blocked with BSA, and
washed as described for the adhesion assay. For control, the inserts
were coated with PBS instead of protein solutions. The cells (200 000
cells in 100 µL per well) were resuspended in migration buffer (IMDM,
0.2% BSA) and added into the upper chambers; 0.6 mL migration buffer
containing 100 ng/mL SDF-1 (R&D Systems) was added to the bottom
chambers. After incubation for 4 hours at 37°C in a humidified
environment containing 5% CO2, the cells from the lower
chamber were collected, and adherent cells from the lower surface of
the Transwell inserts and the lower chamber were collected after
treatment with 0.6 mL trypsin. For antibody perturbation experiments,
azide-free rat antihuman antibody GoH3 against integrin 6 chain
(Immunotech) or control monoclonal rat IgG antibody was added to the
cells in migration buffer, and the cells were incubated for 10 minutes
at 37°C before they were added to the Transwell inserts.
Statistical analysis
Results are expressed as mean ± SD of triplicate assays or as
indicated. Statistical significance was determined using the unpaired t test.
RNA isolation and RT-PCR
RNA from cells was isolated with Trizol reagent (Gibco).
Complementary DNA synthesis was performed by using a cDNA synthesis kit
(Gibco). The primers used for the integrin 6 cDNA PCR were 5'-ATCTCTCGCTCTTCTTTCCG-3' and 5'-GACTCTTAACTGTAGCGTGA-3', covering the
alternatively spliced region present in integrin 6A but not in 6B
mRNA.44 Amplified cDNA was analyzed on a 1% agarose.
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Results |
Expression of laminin receptors in stem and progenitor
cells
Integrins 61, 64, and 31 have been found to
mediate cell adhesion to laminin-10/11 and
laminin-8.20-22,35 The 1 integrin chain is ubiquitously
expressed in hematopoietic cells, but expression of integrin 6 and
3 chains during early human hematopoiesis has been unclear. We
therefore analyzed their expression in bone marrow stem and progenitor
cells by flow cytometry (Figure 1). The
CD34+ stem and progenitor cells were isolated by magnetic
separation (Figure 1A). Immunostaining by using 2 different monoclonal
antibodies C3 II.1 (Figure 1B) and MIKd 2 (data not shown) did not show
expression of integrin 3 in CD34+ cells or
CD34+CD38 cells. In contrast, integrin 6
chain was found in more than 80% of CD34+ cells and 90%
of CD34+CD38 cells (Figure 1B), suggesting
that integrin 61 receptor might mediate adhesion of bone marrow
stem and progenitor cells to laminins. In agreement with the high
expression of integrin 6 chain in CD34+ cells, more than
80% of CD34+CD38+ cells also expressed
integrin 6 chain (not shown).
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| Figure 1.
Expression of integrin 6 chain and integrin 6A and
6B mRNA splice variants in bone marrow stem and progenitor cells.
(A) Fluorescence-activated cell-sorting (FACS) analysis after
immunostaining with anti-CD38 and anti-CD34 antibodies of
CD34+-enriched bone marrow cells. The vertical and
horizontal bars were set on the basis of isotype-matched negative
control profiles (99.3% of cells negative). The numbers indicate
percentages of cells in each gated area. The purity of
CD34+-enriched cells is more than 95%. (B)
CD34+ cells and CD34+CD38 cells
were gated, and expression of integrin 3 and integrin 6 was
studied by antibodies (C3II.1 and GoH3) against integrin 3 (3)
and 6 (6) (shaded histograms). Immunostaining with
isotype-matched control antibodies is shown as open histograms. Shown
is 1 representative of 2 experiments. (C) RT-PCR analysis for integrin
6A and 6B mRNA splice variants of bone marrow
CD34+CD38 cells, CD34+ cells, and
mononuclear CD34 cells. MW indicates molecular weight
markers showing positions of 600 bp and 500 bp markers on the gel;
Control, PCR reaction without cDNA. The approximately 550 and 420 bp
fragments corresponding to integrin 6A and 6B splice variants
were seen in all 3 cell populations.
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The integrin 6 polypeptide chain is expressed in 2 functionally
different variants, 6A and 6B, with different cytoplasmic domains
generated by alternative mRNA splicing.44 The expression of the integrin 6 mRNA splice variants was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) by using
primers covering the 130 base pair (bp) alternatively spliced segment. Both the 550 bp and 420-bp cDNA fragments corresponding to 6A and
6B mRNAs, with a higher expression of the larger splice variant, were detected in CD34+CD38,
CD34+, and CD34 fractions. (Figure 1C).
Adhesion of bone marrow stem and progenitor cells to
laminin isoforms and fibronectin
We studied adhesion of bone marrow stem and progenitor cells to
laminin-1, laminin-8, and laminin-10/11 and compared cell binding to
fibronectin, the so far best characterized bone marrow extracellular
adhesion protein. For these assays we isolated bone marrow
CD34+ progenitor cells and
CD34+CD38 cells, consisting of a highly
enriched primitive stem and progenitor cell population.45
Because integrin receptors can exist in different functional stages
with low or high binding capacity for particular ligands, experiments
were performed both without and after activation of integrins with the
protein kinase C activator TPA, which rapidly up-regulates
integrin-ligand binding affinity.46
About 35% to 40% of CD34+ cells and
CD34+CD38 cells adhered to laminin-10/11
(Figure 2A,B). Adhesion of both stem and
progenitor cell fractions to laminin-10/11 was almost as high as to
fibronectin. Adhesion of CD34+CD38 cells to
laminin-10/11 was maximal without treatment with TPA, indicating
steady-state receptor activation in these cells. In contrast, adhesion
of CD34+CD38 cells to fibronectin and
laminin-8 and adhesion of CD34+ cells to all studied
proteins was dependent on protein kinase C activation, as shown by
greatly enhanced cell adhesion after TPA treatment. Laminin-8 and
laminin-1 were less adhesive substrates to CD34+ cells than
laminin-10/11, indicating isoform-specific differences in the
cell-adhesive interactions of laminins with hematopoietic stem and
progenitor cells. However, about 25% of
CD34+CD38 cells were adhesive to laminin-8,
and adhesion of CD34+ cells to laminin-8 also clearly
exceeded adhesion to PBS-coated wells, suggesting physiologically
significant interactions of bone marrow stem and progenitor cells with
both laminin-10/11 and laminin-8.
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| Figure 2.
Adhesion of bone marrow stem and progenitors cells to
laminin isoforms and fibronectin.
Adhesion of human bone marrow CD34+ cells (A,D),
CD34+CD38 cells (B), and
CD34+CD38+ cells (C) to fibronectin (FN),
laminin-1 (LN-1), laminin-8 (LN-8), and laminin-10/11 (LN-10/11). PBS
indicates cell adhesion to wells coated with PBS instead of proteins;
light columns, cell adhesion without treatment with TPA (n = 9); dark
columns, cell adhesion after 1 hour of treatment with TPA (PBS,
n = 6; other columns, n = 9; values are shown as means ± SDs);
asterisks, a significant difference from the corresponding control
value (percentage of adherent cells in wells coated with PBS);
ns, not significant; *P < .05; **P < .01;
***P < .001. (D) CD34+ cell adhesion to wells
coated with 10 µg/mL and 30 µg/mL proteins. The difference in cell
adhesion to each protein at 10 µg/mL and 30µg/mL was not
significant (P > .05).
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Of the CD34+ cells with high expression of CD38
(CD34+- CD38+), consisting of a more mature
progenitor cell population than the CD34+CD38
cells, approximately 35% adhered to laminin-10/11 and fibronectin after cell activation with TPA (Figure 2C). Less than 20% of
CD34+CD38+ cells adhered to laminin-8.
Receptors involved in adhesion to laminins
We used function-blocking monoclonal antibodies to analyze the
role of integrin 6 and 1 chains in adhesion of stem and
progenitor cells to 4 and 5 laminins (Figure
3). TPA-induced adhesion of the
CD34+ cells to laminin-10/11 and laminin-8 was completely
or largely inhibited by antibodies against integrin 6 and 1
chains (Figure 3A,B). Adhesion of CD34+CD38
cells to laminin-10/11 was largely inhibited by an antibody against integrin 6 chain (Figure 3C) and almost completely inhibited by an
antibody against integrin 1 chain (Figure 3D). Adhesion of
CD34+CD38 cells to laminin-8, studied
after TPA treatment (Figure 3E), was also partially inhibited by
antibodies against integrin 6 and integrin 1 chains.
Binding was fully inhibited with EDTA, indicating that adhesion is
dependent on the presence of divalent cations. Hence, although also
other laminin receptors might be involved, integrin 61 is a
ubiquitous receptor for laminin-8 and laminin-10/11 in most
CD34+ and CD34+CD38 cells.
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| Figure 3.
Inhibition of adhesion of bone marrow stem and
progenitor cells to laminin-10/11 and laminin-8 by monoclonal
antibodies against integrin 6 and 1 chains.
Adhesion of CD34+ cells (A,B) and
CD34+CD38 cells (C-E) to laminin-10/11
(LN-10/11; A,C,D) and laminin-8 (LN-8; B,E). PBS indicates cell
adhesion to wells coated with PBS instead of proteins. Adhesion assay
was performed in the presence of antibodies GoH3 against integrin 6
and P4C10 against 1 chain (int.6 and int.1, respectively),
isotype-matched control rat or mouse monoclonal antibodies (rIgG and
mIgG, respectively), or 10 mmol EDTA. No ab indicates cell adhesion to
laminin-10/11 or laminin-8 without antibody addition. The results are
shown as percentages of adhesion to laminin-10/11 or laminin-8
without antibody addition. There was a significant difference in cell
adhesion in the presence of anti-integrin antibodies or EDTA compared
with adhesion to laminins without antibody addition
(**P < .01; ***P < .001). Shown are means
of 1 experiment representative of 2 or more experiments performed
in triplicate.
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Adhesion of bone marrow-committed progenitor cells to laminins
and fibronectin
To analyze the adhesion of committed progenitors to laminins and
fibronectin, adherent and nonadherent CD34+ cells were
separately cultured in methylcellulose in the presence of cytokines.
Most myeloid (granulocyte, macrophage colony-forming units [CFU-GMs])
(Figure 4A), erythroid (erythroid
burst-forming units [BFU-Es]) (Figure 4B), and multipotent
(granulocyte, erythroid, macrophage, megakaryocyte CFUs (CFU-GEMMs])
(data not shown) progenitors were adherent to laminin-10/11, whereas
less than 40% of the myeloid and erythroid colony-forming cells
(CFCs) were adherent to fibronectin and other laminin
isoforms. This result shows that the hematopoietic progenitor cells
efficiently adhere to laminin-10/11 without prior integrin activation,
whereas they adhered less to laminin-1 and -8 or fibronectin.
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| Figure 4.
Adhesion of clonogenic progenitor cells CFU-GM and BFU-E
to laminins and fibronectin.
Bone marrow CD34+ cells were plated in 96-well plates
coated with PBS, fibronectin (FN), laminin-1 (LN-1), laminin-8 (LN-8),
or laminin-10/11 (LN-10/11). After incubation for 1 hour at a 37°C,
5% CO2, humidified environment, the nonadherent and
adherent cells were collected separately and plated in methylcellulose
progenitor assay. The percentages of adherent progenitors were
calculated as follows: (no. of adherent CFCs/no. of adherent CFCs + no. of nonadherent CFCs) × 100. The total numbers of CFCs adherent
to wells coated with PBS were 3672 ± 730, fibronectin 8664 ± 990,
laminin-1 4690 ± 340, laminin-8 8408 ± 522, and laminin-10/11
22 553 ± 1635; the values are mean ± range of 2 measurements
for laminin-8 and mean ± SD of 3 measurements for other proteins and
PBS. Asterisks indicate a significant difference from the control value
(percentage of adherent colonies in wells coated with PBS).
*P < .05; **P < .01;
***P < .001.
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Adhesion of hematopoietic cell lines to laminins and
fibronectin
Hematopoietic cell lines of the B-lymphocytic (CO, BJAB, NALM/6,
CA-46, DAUDI, DG-75, KM-3), plasma cell (LP-1), early myeloid (KG-1),
erythroid-megakaryocytic (K562, HEL), promyelocyte (NB-4, HL-60) or
monocyte-macrophage (Monomac, U-937) lineages were used in adhesion
assays. The assays were performed without and after cell activation
with TPA. Fourteen of the 15 cell lines were adhesive to fibronectin
(Table 1), whereas adhesion to laminins
was highly isoform-specific. Thirteen of the cell lines adhered to
laminin-10/11. Only 4 cell lines adhered to laminin-8, and 2 adhered to
laminin-1. Thus, laminin-10/11, like fibronectin, was a ubiquitous
adhesive protein for differentiated precursors of both B-lymphocytic,
erythroid, megakaryocytic, and myelomonocytic cell lineages, whereas
adhesion to laminin-8 and laminin-1 was restricted to a few cell
lines.
The effect of laminins and fibronectin on migration of
CD34+ cells
The ability of laminin-10/11 and laminin-8 to promote
SDF-1-stimulated transmigration of bone marrow CD34+
progenitor cells was studied by using Transwell inserts coated with the
extracellular matrix proteins. CD34+ cell migration was
greatly enhanced through membranes coated with both laminin-10/11 and
laminin-8 (Figure 5A). Migration
stimulated by each laminin isoform was similar when protein
concentrations at 30 µg/mL (data not shown) or at 10 µg/mL were
used. In agreement with previous reports, fibronectin was found to
promote SDF-1-stimulated migration of CD34+ cells (not
shown). Migration of CD34+ cells through Transwell inserts
coated by both laminin-8 (Figure 5B) and laminin-10/11 (Figure 5C) was
effectively inhibited by the GoH3 antibody against integrin 6
chain.
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| Figure 5.
Effects of laminin-8 and laminin-10/11 on
SDF-1-stimulated migration of bone marrow progenitor cells across
Transwell inserts.
(A) Migration of CD34+ cells across Transwell inserts
coated with PBS, laminin-8 (LN-8), or laminin-10/11 (LN-10/11). Shown
are means ± SDs of 1 experiment representative of 2 experiments
performed in triplicate. (B,C) Inhibition of migration of
CD34+ cells across Transwell inserts coated with laminin-8
(B) and laminin-10/11 (C) by monoclonal antibody GoH3 against integrin
6 chain. Migration was studied in the absence of antibodies (no ab),
in the presence of the GoH3 antibody against integrin 6 (int.6),
and in the presence of irrelevant rat monoclonal antibody (rIgG).
Asterisks indicate a significant difference from the control value
(migration across Transwell inserts coated with PBS in panel A;
migration in the absence of antibodies in panels B and C).
***P < .001.
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Adhesion of stem and progenitor cells to laminins in the
presence of SDF-1
Protein kinase C signal transduction pathway has been found to be
involved in SDF-1/CXCR-4 signaling.47 We therefore
studied whether SDF-1, like TPA, enhances adhesion of progenitor
cells to laminins. SDF-1 did not stimulate adhesion of
CD34+CD38 or the more mature progenitor cells
to laminin-8 or laminin-10/11 (Figure
6).
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| Figure 6.
Adhesion of stem and progenitor cells to laminins in the
presence of SDF-1.
Adhesion of CD34+ (A), CD34+CD38
(B), and CD34+CD38+ cells to laminin-1 (LN-1),
laminin-8 (LN-8), and laminin-10/11 (LN-10/11) in the presence of 300 ng/mL SDF-1 (dark columns) and without SDF-1 (light columns). The
differences in cell adhesion in the presence or absence of SDF-1
were not significant (P > .05; LN-1, n = 3; LN-8,
n = 9; LN-10/11, n = 12). Data are expressed as means ± SDs.
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Discussion |
Laminin 4 and 5, 2 recently characterized laminin chains,
are widely expressed in tissues and are major components of basement membranes of blood vessels. Gene deletion of laminin 5 chain results
in embryonally lethal phenotype with multiple defects,48 whereas 4 chain null mutant mice have abnormal motoneuron synapses, impaired angiogenesis, extensive bleeding, and anemia in neonatal stage.49-50 In vitro studies on several types of cells,
including epithelial and endothelial cell lines, have established an
important cell-adhesive role for laminin-10/11 20,51 and
laminin-8.21,22 We have previously shown that mouse
multipotent hematopoietic FDCP-mix cells adhere to
laminin-10/11,35 suggesting that these laminins might also
interact with hematopoietic progenitors. Recent studies have shown
binding of monoblastic, T-, and B-lymphoid cell lines and
CD4+ lymphocytes to laminin-8 and laminin-10/11, platelets
to laminin-8, and mouse integrin 2-deficient granulocytes to
laminin-10/11,41,52-55 suggesting specific roles for
different laminin isoforms in interactions with lineage-differentiated
and mature hematopoietic cells. However, the cellular
interactions of 4 and 5 laminin isoforms with human hematopoietic
stem and progenitor cells have been unclear.
Here we demonstrate that defined stem and progenitor cell populations
from normal human bone marrow adhere to bone marrow laminin isoforms,
suggesting a significant role for these laminins for early
hematopoietic development. CD34+CD38 cells
represent only 0.05% to 0.1% of the nucleated human bone marrow cells
and are highly enriched in stem cells, defined by their ability to
maintain long-term hematopoiesis on irradiated stroma in
vitro56 or to reconstitute long-term multilineage hematopoiesis in myeloablated recipients in vivo.57
Our present results show that CD34+CD38 cells
adhere to laminin-10/11 with nearly similar binding affinity as to the
so far best characterized bone marrow adhesion protein, fibronectin.
The CD34+CD38 cells adhered also to
laminin-8, suggesting a biologically significant interaction. Our
results raise the possibility that the most primitive long-term
repopulating stem cells are adhesive to laminins. It will therefore be
interesting to use experimental stem cell transplantation models or
long-term stroma culture-based assays to test whether the long-term
reconstituting stem cells adhere to laminin isoforms.
The CD34+ marker, expressed in 1% to 2% of bone marrow
mononuclear cells, defines a cell population consisting of multipotent, lineage-committed, and lineage-differentiated progenitor cells, which
express early myeloid, erythroid-megakaryocytic, and T- and
B-lymphocytic markers. A large proportion, 40% to 50%, of CD34+ cells adhered to laminin-10/11 and fibronectin, and
nearly 15% adhered to laminin-8 after maximal cell activation with
TPA. The CD34+ cell fraction with high CD38 expression
(CD34+CD38+), which contains more mature
progenitor cells, also adhered to laminins and fibronectin. We
therefore studied by colony assays whether the committed progenitors
were adherent to these proteins. Most multipotent, myeloid (CFU-GM),
and erythroid (BFU-E) progenitors were adherent to laminin-10/11. A
much lower fraction of committed myeloid and erythroid progenitors were
adherent to fibronectin, laminin-8, and laminin-1. However, maximal
adhesion of CD34+ and CD34+CD38+
progenitor cells to fibronectin and laminins was dependent on prior
protein kinase C activation. Because the cells were not exposed to TPA
before colony assays, the lower binding of CFU-GMs and BFU-Es to
fibronectin than laminin-10/11 may be due to different activation
stages of the receptors for individual matrix proteins in the progenitors.
Integrins can exist in different functional states with low or high
binding capacity to particular ligands.58 Such
conformational changes can be triggered by extracellular signals,
including divalent cations, activating antibodies, or physiologically
by ligand binding to the receptor. An important physiologic mechanism
is activation of integrins by intracellular signals, induced by
physiologic agonists. Adhesion of hematopoietic progenitor cells to
fibronectin and activation of integrin receptors are modulated by a
variety of cytokines and chemokines, and such modulation might be a
major regulatory mechanism influencing stem and progenitor cell
proliferation, transendothelial or stromal migration, and
homing.2,10,59,60 To activate integrin receptors, we used
phorbol ester TPA, which activates the protein kinase C pathway and
mimics the effect of physiologic agonists. Adhesion of most stem and
progenitor cell fractions to laminin-10/11 and laminin-8 was stimulated
by TPA, suggesting that specific cytokines and chemokines modulate
progenitor cell adhesion to laminins.
Most studied lineage-differentiated cells lines expressing
B-lymphocytic, erythroid-megakayocytic, monocyte-macrophage, or myeloid
differentiation markers adhered to fibronectin, and most adhered also
to laminin-10/11, in agreement with reported findings.41 In line with previous studies with cell lines,22,41
laminin-8 and laminin-1 were less adhesive to lineage-differentiated
cells than laminin-10/11. The cell lines studied have been established from patients with hematologic malignancies, and the findings may thus
reflect biologically relevant interactions of primary leukemic and
lymphoma cells with their physiologic environment. However,
lineage-differentiated cell populations from healthy donors should be
used to study the interactions of nontransformed cells with laminin
isoforms present in bone marrow, vascular endothelia, lymph nodes, and thymus.
Integrins 61, 64, and 31 have been identified
as receptors for both laminin-10/11 and
laminin-8.21-22,35,51 In addition, laminin-10/11 binds
cells via nonintegrin receptors, -dystroglycan on endothelial,
muscle, and epithelial cells,18,61 and Lutheran and LW
glycoproteins on erythroid cells.62 The 1
integrin chain is ubiquitously expressed in hematopoietic cells,
whereas integrin 6 receptor chain expression has been found in
lineage-differentiated precursors and mature cells, including
erythroid, megakaryocytic, monocyte-macrophage lineage cells, and a
subset of lymphoid cells. Expression of integrin 3 chain has been
reported in hematopoietic cell lines41 but is unclear for
most hematopoietic cell subsets, including stem and progenitor cells.
Integrin 3 chain mRNA in mouse stem and progenitor cells was shown
in one study by PCR, but the proportion of cells expressing it is not
known.63
The present results using FACS analysis showed that integrin 3 chain
is not expressed in human CD34+ cells or the
CD34+CD38 cell fraction. In contrast,
integrin 6 chain was found in most of both CD34+ and
CD34+CD38 cells. This is in agreement with
the reported high expression of integrin 6 chain in mouse stem and
progenitor cells.64 The present findings by using
functionally active antibodies indicate that integrin 61 is a
major laminin receptor in bone marrow stem and progenitor cells.
Furthermore, the inhibition of cell adhesion with antibodies against
the corresponding receptor confirmed the specificity of ligand-receptor
interaction.65,66
We detected integrin 6 mRNA expression also by RT-PCR in
CD34+ and CD34+CD38 cells, in
agreement with the immunostaining results. The 2 described integrin
6 mRNA variants, generated by alternative mRNA splicing, could be
amplified in both cell fractions. The 2 splice variants have different
cytoplasmic domains and different functional properties. In adult
tissues, the larger variant, integrin 6A, is assocated with the 1
subunit in lymphocytes, macrophages, and platelets, whereas both
6A1 and 6B1 variants have been found in endothelial cells
(reviewed by de Melker et al67,68). Transfection and gene
deletion studies have shown that the 2 integrin 6 variants can
equally associate with the integrin 1 subunit and have similar ligand binding specificity and affinity. However, the 6A, in contrast to 6B, triggers protein kinase C-dependent activation of
mitogen-activated protein (MAP) kinases and is more active than 6B in promoting migration of cells, as also shown by impaired in vitro migration of 6A-deficient lymphocytes on
laminin.69 The presence of the integrin 6A splice
variant in CD34+ and CD34+CD38
cells indicates that interactions of laminins with integrin 6 receptor could be involved in hematopoietic stem and progenitor cell
migration and mobilization.
Small numbers of stem and progenitor cells are present in peripheral
blood during steady-state hematopoiesis, suggesting that continuous
stem cell mobilization and homing into bone marrow is a physiologic
process.70 The mechanisms involved in stem cell
mobilization and homing are not yet clear. It is apparently a multistep
process directed by chemoattractants and mediated by cell-adhesive
interactions with stromal cells and matrix components of the bone
marrow environment.71 In vitro studies have shown that
migration of human bone marrow CD34+ cells through bone
marrow endothelial cell layer involves interaction of several cell
surface adhesion molecules including 1 and 2 integrins, platelet
endothelial cell adhesion molecule-1 (PECAM-1), and
O-glycosylated proteins.72 In vivo experiments
using blocking antibodies and gene-deleted mice suggested that the
integrin 1 chain and integrin 41 receptor in hematopoietic
stem and progenitor cells, and stromal vascular cell adhesion
molecule-1 (VCAM-1), are involved in the homing and
mobilization of stem cells and progenitors.73-76 However,
studies with chimeric mice with homozygous deletions of integrin chains have not settled the role of individual integrin chains in
stem cell homing, and it is possible that multiple receptors are
involved.14,77-78
In vitro studies have shown a migration promoting activity for both
laminin-10/11 and laminin-8 in tumor cell li |
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Abstract
Introduction