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HEMATOPOIESIS
From the University Medical Clinic, Section for
Transplantation Immunology and Immunohematology, Tübingen,
Germany; Department of Pathology, University of Cambridge, Cambridge,
England; Transplantation Biology, Clinical Research Division, Fred
Hutchinson Cancer Research Center, Seattle, WA; and Nikolaus Fiebinger
Center for Molecular Medicine, University of Erlangen-Nuremberg,
Germany.
Laminins are a family of disulfide-linked heterotrimeric proteins
consisting of 3 different subunits termed The hematopoietic microenvironment of the bone
marrow plays a fundamental role in regulating proliferation,
differentiation, and migration of the developing blood
cells.1-3 An essential part of the bone marrow
microenvironment is its complex extracellular matrix (ECM), which is
synthesized by nonhematopoietic stromal cells.4,5 ECM
molecules including glycoproteins, proteoglycans, and the collagen
family have been shown to be directly involved in controlling cell
adhesion and proliferation of maturing hematopoietic cells as well as
presentation of cytokines to hematopoietic progenitor cells.6-10
The laminins represent a large gene family of ECM molecules found
predominantly in basement membranes of epithelial cells but also in
interstitial tissues and embryonic mesenchyme.11,12 The
heterotrimeric laminin molecules consist of 3 different subunit chains,
which are termed Laminins are expressed in the bone marrow
microenvironment.22 Early studies did not distinguish
between laminin isoforms because the used polyclonal antisera against
LN-1 identified both the A dominant feature of all laminin molecules is that they can act as
strong adhesive substrates for many cell types. However, several
studies indicate that hematopoietic cell types do not seem to adhere to
LN-1.25-27 Another laminin isoform, LN-10/11, isolated
from human placenta, on the contrary, is strongly adhesive for mouse
and human hematopoietic cells.23,28 Interactions with
laminins can be mediated by different cellular receptors, including
members of the integrin family and dystroglycan.29 The
identified interactions of human hematopoietic cells with LN-10/11
seemed to be mediated by In the present study we have analyzed the expression pattern of 11 different laminin chains in human bone marrow. This was performed in
vitro using long-term bone marrow cultures (LTMCs) and established
stromal cell lines from the marrow microenvironment but also in
cryostat sections of native bone marrow tissue. Furthermore, different
laminin preparations representing different laminin isoforms were used
to study their functional interactions with human hematopoietic
progenitor cells. Because cell adhesion can be directly linked to
induction of cell proliferation, both mitogenic and adhesive properties
of the different laminin isoforms for human hematopoietic cells have
been tested.
Cell cultures
The human hematopoietic cell lines KG1a, U937, KU.812, and K562 were
used in previous studies.6,8,32 Two B-cell lines, BOB and
COX, were obtained after Epstein-Barr virus infection of peripheral B
lymphocytes following standard procedures (C.A.M., unpublished data,
1982). The plasmocytoma cell lines, U266 and NCI-H929, and the
pre-B-leukemic cell line, BV173, were from the German Collection of
Microorganisms and Cell Cultures, Braunschweig, Germany. The
interleukin-3-dependent CD34+ TF-1 cell line, kindly
provided by Dr Kitamura (University of Tokyo), was cultured with 2%
conditioned medium from the carcinoma cell line, 5637, as an exogeneous
source of interleukin-3.
Long-term bone marrow cultures
Isolation of CD34+ cells by MiniMACS CD34+ progenitor cells were purified from bone marrow mononuclear cells using the MiniMACS cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, 5 × 107 mononuclear cells were resuspended in phosphate-buffered saline (PBS) containing human immunoglobulin and monoclonal hapten-conjugated anti-CD34 antibody. Labeled cells were washed, centrifuged, and resuspended in a buffer containing super-paramagnetic MACS Microbeads conjugated to an antihapten antibody. Magnetic separation was performed by applying labeled cells on positive selection columns. Columns were washed repeatedly, and CD34+ cells were eluted by removing the column from the magnetic field. The purity of the isolated CD34+ cells was determined by fluorescence-activated cell sorter analysis.ECM components and antibodies Different laminin preparations were used in the cell binding and proliferation studies. Murine laminin isolated from the Engelbreth-Holm-Swarm tumor, which consists mainly of the LN-1 isoform, was obtained from Biozol (Eching, Germany). Human merosin, purified from human placenta and consisting of LN-2/4,33 was purchased from Life Technologies (Eggenstein, Germany). Another laminin preparation isolated from human placenta by mild pepsin digestion followed by affinity chromatography on monoclonal antibody (mAb) 4C7-coupled Sepharose was also obtained from Life Technologies. The composition of this human placental laminin preparation has been determined previously as LN-10/11 by Ferletta and Ekblom.34Rabbit antiserum against LN-1 was that used in previous
studies.17,35 The antiserum against human LN-10/11 was
commercially available from Life Technologies. The rat mAb 4H8-2 was
used to detect laminin Purification of bone marrow laminin isoforms Conditioned media of LTMCs were incubated overnight with DEAE Sephacel anion exchanger (Amersham-Pharmacia Biotech, Freiburg, Germany). After washing the DEAE Sephacel, bound proteins were eluted using 10 mmol/L Tris, pH 8.5, containing 1 mol/L NaCl. Contaminating fibronectin was removed by incubation with gelatin Sepharose 4B, and laminin isoforms were then purified by affinity chromatography on NHS-activated Sepharose to which the isolated laminin 2 and  1
chain antibodies, C4 and D18, had been coupled. Bound laminin isoforms
were eluted using a buffer containing 40 mmol/L sodium phosphate, pH
11.75, and 150 mmol/L NaCl. After immediate neutralization, the
laminin-containing solution was concentrated by ultrafiltration. The
protein content of the concentrated laminin preparation was determined
by Micro BCA protein assay (Pierce, Rockford, IL).
Reverse transcriptase and polymerase chain reaction Total RNA was isolated from the different cell types by acid guanidinium isothiocyanate phenol chloroform extraction.39 Possible DNA contaminations of the RNA preparations were eliminated by deoxyribonuclease treatment. Expression of messenger RNA (mRNA) for human laminin 1-5, 1-3, and 1-3 chains were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) methodology. Based on published sequences for the human laminin chains
(EMBL accession numbers for 1, 2, 3A,
3B, 4, 5: X58531, Z26653, X85107, L34156, S78569,
Z95636; for 1-3: M61916, S77512, L25541; and for 1-3:
J03202, Z15008, AF041835), specific primer pairs were designed with the
aid of the HUSAR/GCG program from EMBL (Heidelberg, Germany).
Reverse transcription was performed with Moloney murine leukemia virus RT (Gibco) with 1.0 µg oligo(dT)12-18. PCR
conditions with 4 units of Taq polymerase (Perkin-Elmer, Weiterstadt,
Germany) and 0.3 µmol/L of each primer included denaturation at
94°C for 40 seconds, annealing at 60°C for 1 minute, and
polymerization at 72°C for 1 minute. A total of 35 cycles were run.
Control experiments were performed using total RNA preparations without
reverse transcription. The PCR products of the expected sizes were
analyzed by gel electrophoresis in 2.0% agarose gels, purified using
the PCR purification kit (Qiagen, Hilden, Germany), and sequenced with
the ABI Prism DyeDeoxy terminator kit (Perkin-Elmer).
Northern blotting Total RNA from bone marrow stromal cells were electrophoresed on agarose gels containing 2% formaldehyde. After transfer to Hybond N+ membranes (Amersham-Pharmacia), the RNAs were cross-linked to the membranes by UV irradiation. The membranes were prehybridized at 42°C in DIG Easy Hyb solution buffer according to the manufacturer's suggestions (Roche). Hybridization was performed overnight in the same solution with the following complementary DNA (cDNA) probes: a 2.5-kb EcoRI fragment of cDNA clone A1 corresponding to human laminin1 chain,40 a 3.2-kb
EcoRI fragment of clone M10-22 corresponding to human
laminin 2 chain,41 a 3.6-kb EcoRI fragment
of clone HL-40 corresponding to human laminin 1
chain,42 and a 1.4-kb human GAPDH cDNA probe as control.
Complementary DNA probes for the human laminin 2, 3, 4, 5,
2, 1, 2, and 3 chains were generated by subcloning the PCR
amplification products using the Topo cloning kit (Invitrogen, NV Leek,
the Netherlands). Isolated inserts were labeled with digoxigenin (DIG)
using the DIG DNA labeling and detection kit (Roche). After
hybridization the membranes were washed, blocked with 2% Boehringer
blocking reagent, and developed with the chemiluminescence detection
system using alkaline phosphatase-conjugated anti-DIG antibodies and CSPD as substrate for the phosphatase (Roche).
Immunohistochemistry Native bone marrow samples taken from the sternum of hematologically healthy donors during thoracic surgery were frozen in Tissue-Tek embedding medium (Sakura, Zouterwoude, the Netherlands). Sections measuring 5 µm, prepared on a cryostat, as well as the long-term bone marrow stromal cells grown on glass coverslips, were fixed in methanol for 5 minutes at 20°C. After washing, the fixed
cells or tissue sections were incubated with the primary antibodies for
1 hour. Bound antibodies were detected after washing by incubation with
Cy3-conjugated or fluorescein isothiocynate-conjugated secondary
antibodies (Dianova, Hamburg, Germany). Control stainings were
performed by omitting the primary antibodies.
Immunoblotting Conditioned media and cell extracts of human bone marrow stromal cells were analyzed by immunoblotting for laminin isoform chain composition. Media and cell extracts were boiled for 5 minutes with loading buffer containing dithiothreitol, separated on 5%-to-15% sodium dodecyl sulfate-polyacrylamide gradient gels, and transferred to nitrocellulose. After blocking, the filters were incubated with laminin chain-specific antibodies. Bound antibodies were detected either with alkaline phosphatase-conjugated antibodies (Dako Diagnostika, Hamburg, Germany) followed by colorimetric reaction with the Fast BCIP/NBT system (Sigma), or with horseradish peroxidase-conjugated antibodies (Dako) and the enhanced chemiluminescence reagent (Amersham-Pharmacia).Cell proliferation assays To analyze mitogenic activities of different laminin preparations, a nonradioactive cell proliferation assay (Roche) based on formazan formation was used. Using 96-well flat-bottom microtiter plates (Costar, Wiesbaden, Germany), 5 × 103 freshly isolated CD34+ cells per well or 5 × 104 bone marrow mononuclear cells per well were cultured in a final volume of 100 µL per well RPMI 1640 culture medium supplemented with 10% FCS containing various amounts of different laminin preparations. After 6 days of incubation, the cell proliferation reagent WST-1 (Roche) was added to each well. After 1, 2, or 4 hours of incubation, the absorbance of the samples was measured against a background control at 440 nm. The optical density determined in each well was directly correlated with the number of viable cells in the assay. All experiments were carried out in triplicate.Cell adhesion and inhibition assays The assay was carried out as described previously.8 Briefly, serial dilutions of the different laminin preparations were immobilized onto plastic by air drying. Nonspecific cell binding to plastic was blocked by subsequent incubation of the culture dishes with 1% human serum albumin in PBS. Cells in serum-free medium were permitted to adhere to the immobilized laminin preparations for 1 hour at 37°C. Nonadherent cells were removed by gently rinsing the dishes with PBS. Specific cell attachment was evaluated under an Axiovert microscope (Zeiss, Oberkochen, Germany).To inhibit cell adhesion to immobilized laminin preparations, cells under study were preincubated with different anti-integrin antibodies (diluted 1:50) for 30 minutes under constant rotation. On the other hand, the immobilized laminin preparations were preincubated for 30 minutes with polyclonal antilaminin antisera. After these preincubation periods, the adhesion assays were performed in the presence of the respective antibodies as described above.
Detection of individual laminin chains in human bone marrow stroma To determine which laminin chains are expressed by the heterogeneous bone marrow stromal cell population, RT-PCR analysis of the 5 human laminin chains, the 3 chains, and the 3 chains were performed. Using RNA from primary LTMCs, specific amplification products were obtained for all analyzed laminin chains except the 3
chain and the 3 chain (Figure 1).
Similar results were obtained with RNA preparations from the
established human bone marrow stromal cell lines L87/4 and HS-5 (data
not shown). The identities of the amplified products were confirmed by
DNA sequencing. These results suggested that all laminin isoforms, with
the exception of LN-5 and LN-12, which contain the 3 or the 3
chains, respectively, could be present in the human bone
marrow.
Northern blot analysis, however, revealed a more restricted expression
pattern. Under stringent conditions, no hybridization signal for
laminin
Immunofluorescence stainings of the adherent stromal layer of LTMCs
with chain-specific antibodies against the laminin
Expression of the
Localization of laminin chains in human bone marrow Immunostaining of human bone marrow cryostat sections with an antiserum against LN-1 showed widespread expression of laminins in the marrow. Strong staining signals could be observed in arteriolar walls, sinusoidal endothelial basement membranes, intersinusoidal interstitial connective tissue, and basement membranes of adipocytes (Figure 5F). Similar staining patterns were observed with mAbs against the laminin1 and 1 chains, although
at slightly different staining intensities (Figure 5C,E). A completely
different expression pattern was observed for the laminin 5 and 2
chains. Expression of laminin 2 seemed to be restricted to the
arteriolar walls, whereas laminin 5 could be found in arteriolar
walls and sinusoidal cells (Figure 5B,D). The laminin 4 chain was
detected mainly in intersinusoidal spaces (Figure 5A).
Double labeling of a bone marrow section with the antiserum against the
laminin
The codistribution of laminin
Mitogenic response of bone marrow cells to LN-10/11 To determine whether laminins have a proliferation-inducing influence on developing hematopoietic cells, 4 different laminin preparations were tested in a nonradioactive proliferation assay. LN-1 (111) and LN-2/4 (211/221) did not show any
mitogenic effect on freshly isolated bone marrow mononuclear cells
(Figure 8A). A laminin preparation
isolated from LTMC supernatants by affinity chromatography as well as
the LN-10/11 isoform, however, showed a strong mitogenic effect on bone
marrow mononuclear cells that was dose-dependent. A more than 6-fold
increase in cell proliferation could be obtained at a concentration of
1 µg/mL LN-10/11, which was further enhanced at higher concentrations
(Figure 8A). Analyzing the kinetics of this proliferative response, a
constant increase in cell numbers could be observed with increasing
time in the presence of these 2 laminin preparations (data not shown).
To determine the responsive cells in the heterogeneous bone marrow mononuclear cell preparation, CD34+ hematopoietic
progenitor cells were isolated by MiniMACS technology. This highly
enriched CD34+ cell population also responded to LN-10/11
and to the bone marrow laminin preparation with a proliferative burst
(Figure 8B), indicating that the proliferative activity of the early
hematopoietic progenitors can be influenced by interactions with
laminin isoforms present within the human bone marrow.
Adhesive interactions of hematopoietic cells with LN-10/11 are
mediated by
The specificity of binding to LN-10/11 was confirmed by inhibition with
an antiserum against LN-10/11. Preincubation of the LN-10/11-coated
plastic with the antiserum raised against LN-10/11 completely blocked
adhesion of the hematopoietic cell types (Figure 10). The adhesive interactions of U266
and KG1a cells with LN-10/11 were mediated by integrins of the
The adhesive capacity of the LN-10/11 isoform was then tested using a
variety of myeloid and lymphoid cell lines. In addition to the
CD34+ KG1a cell line, the myeloid cell lines TF-1, U937,
and K562 also adhered to LN-10/11, whereas the megakaryoblastic cell
line KU.812 did not. The Epstein-Barr virus-transformed B-lymphoid
cell lines, BOB and COX, and the pre-B-leukemic cell line, BV173, did
not adhere to LN-10/11, but both plasmocytoma cell lines (U266,
NCI-H929) strongly adhered to LN-10/11 (Table
3). This differential adhesion pattern of
hematopoietic cell lines to LN-10/11 also demonstrated the specificity
of the observed adhesive interactions.
The bone marrow provides a specified microenvironment for the
development, growth, and differentiation of hematopoietic cells. Our
present study provides new insights into the complex nature of laminin
isoforms expressed in human bone marrow. We could demonstrate that
human bone marrow stromal cells do not express laminin isoforms containing LTMCs are a widely used model to study myeloid
development.1 In this in vitro system, proliferation and
differentiation of the hematopoietic progenitor cells are strongly
dependent on the formation of a heterogeneous adherent stromal cell
layer. Analysis of the stromal cells of human LTMCs by RT-PCR revealed amplification products of all analyzed laminin chains, with the exception of the The ability of the human stromal cells to synthesize all 5 laminin The laminin isoform expression pattern in human bone marrow stromal
cells as detected by Northern blotting was confirmed by immunofluorescence stainings and Western blotting using laminin chain-specific antibodies. Immunoblotting of stromal cell lysates with
an antiserum against human laminin Remarkably, the laminin isoforms secreted by the stromal cells were not
deposited in an extracellular meshwork, as is the case for many other
ECM molecules, including fibronectin, tenascin, perlecan, or various
collagen types.6,8,44,45 The reason for this is not clear,
but it may have functional implications suggesting that laminin
isoforms secreted by bone marrow stromal cells might be important for
cell proliferation or cell migration of hematopoietic cells. Secretion
into the cell culture supernatants rather than an extracellular
deposition facilitates isolation of laminin isoforms. The human bone
marrow stromal cell line HS-5 may be regarded as a suitable source for
isolation of human LN-8/9 because it only synthesizes the In native human bone marrow, laminin isoforms were found in the marrow
cord, in arteriolar walls, and in basement membranes of endothelial
sinusoidal cells and adipocytes. The laminin The finding that the laminin Using identical cell adhesion conditions for LN-1, LN-2/4, and LN-10/11, we demonstrated that LN-10/11 was a strong adhesive substrate for the 2 hematopoietic cell lines, KG1a and U266, which represent 2 distinct cell types present in bone marrow. LN-1, however, did not show any adhesive activity; LN-2/4 isoforms showed an intermediate activity. A similar finding has recently been reported for sickle red blood cells. Whereas normal red blood cells do not adhere to any laminin isoform, sickle red blood cells strongly adhered to LN-10/11 but not to human LN-2/4 or murine LN-1.53 The specificity of hematopoietic cell interactions with LN-10/11 was further shown by using lymphoid cell lines that represent different stages of B-lymphocyte development. BOB and COX cells representing mature B cells outside the bone marrow did not adhere to LN-10/11, whereas plasmocytoma cell lines, which represent plasma cells found in the marrow microenvironment, strongly adhered to LN-10/11.28 Inhibition assays revealed that the adhesive interactions of the human
hematopoietic cells with LN-10/11 are mediated by integrins of the As shown recently for tenascin-C, ECM molecules can directly stimulate hematopoietic cell proliferation.10 Laminin isoforms can also induce a mitogenic response, as shown previously for nonhematopoietic cells with LN-1.55 The mitogenic effect observed in the present study for LN-10/11 on bone marrow mononuclear cells was also very striking. As expected from the expression pattern in the human bone marrow, LN-1 and LN-2/4 did not show any mitogenic effect on developing hematopoietic cells. Since bone marrow mononuclear cells represent a heterogeneous cell population, it is important to determine the individual cell types that can respond to the mitogenic stimuli. Whereas the early CD34+ hematopoietic progenitor cells were not able to give a mitogenic response to tenascin-C,10 these cells could respond to LN-10/11 with increased cell proliferation, indicating an important function for LN-10/11 in early hematopoietic progenitor cell development. A major challenge for the future will be to determine the receptors that transduce the mitogenic signals to the hematopoietic cells. Since the laminin preparations used have been purified from tissues or cell culture supernatants, it is possible that they are contaminated with growth factors that could be responsible for the observed mitogenic effects. Although we cannot exclude this possibility, the fact that the proliferation-inducing LN-10/11 isoform and the inactive LN-2/4 isoform were both isolated from human placenta makes this possibility unlikely. Furthermore, LN-1 was isolated from tumor tissue with high content of growth factors and did not show any mitogenic activity. Taken together, the adhesive and mitogenic response of
hematopoietic cells to LN-10/11 suggests important functions for this laminin isoform during hematopoiesis. Although there are
species-dependent differences in laminin isoform expression in the bone
marrow, the LN-10 isoform is expressed both in mouse and human. A
careful examination of hematopoietic cell development in laminin
The authors thank Ingrid Teufel and Jutta Hildenbrand for expert technical assistance. We are grateful to Drs Jeffrey Miner (Washington University, St Louis, MO), Rupert Hallmann (University of Erlangen), and Karl Tryggvason (Karolinska Institut, Stockholm, Sweden) for their kind donation of antibodies and cDNA probes. We also thank Dr Christoph Faul (University of Tübingen) for a constant supply of bone marrow samples.
Submitted January 24, 2000; accepted August 25, 2000.
Supported by grants from the Deutsche Forschungsgemeinschaft (Kl 709/2-3) and from the fortüne-program of the Medical Faculty of the University of Tübingen (grant 761-0-0).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Gerd Klein, University Medical Clinic, Dept II, Section for Transplantation Immunology and Immunohematology, ZMF (Center for Medical Research), Waldhörnlestrasse 22, 72072 Tübingen, Germany; email: gerd.klein{at}uni-tuebingen.de.
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Top
Abstract
Introduction
6 mol/L hydrocortisone, and
antibiotics (Dexter conditions).
).
The size of the detected laminin chain mRNAs was estimated using a
DIG-labeled molecular weight marker whose largest fragment runs at 7 kb
(
). Expression of laminin 
