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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 940-949
Hyaluronate-Enhanced Hematopoiesis: Two Different Receptors Trigger the
Release of Interleukin-1 and Interleukin-6 From Bone Marrow
Macrophages
By
Sophia Khaldoyanidi,
Jürgen Moll,
Svetlana Karakhanova,
Peter Herrlich, and
Helmut Ponta
From Forschungszentrum Karlsruhe, Institute of Genetics,
Karlsruhe, Germany.
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ABSTRACT |
The glycosaminoglycan hyaluronate (HA) is part of the extracellular
environment in bone marrow. We show here that HA activates signal
transduction cascades important for hemopoiesis. In myeloid and
lymphoid long-term bone marrow cultures (LTBMC), treatment with
hyaluronidase (HA'ase) results in reduced production of both progenitor and mature cells. Exogeneous HA added to LTBMC had the
opposite effect: it enhanced hematopoiesis. The effect of HA is mediated through two different HA receptors on bone marrow macrophage-like cells, one of which is CD44 while the other is unknown.
HA induces bone marrow macrophages to secrete IL-1 (CD44-dependent) and IL-6 (CD44-independent). The two receptors address different signal
transduction pathways: CD44 links to a pathway activating p38 protein
kinase while the other yet unknown receptor induces Erk activity. There
was no difference of the effect of HA and HA'ase on hematopoiesis in
LTBMC and on cytokine production by macrophages in CD44-deficient mice
compared with wild-type mice, indicating that the CD44 hyaluronate
receptor and its signal transduction can be compensated for. Our data
suggest a regulatory role for the extracellular matrix component HA in
hematopoiesis and show the induction of signal transduction by HA receptors.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE BEHAVIOR OF CELLS, reflected by their
program of gene expression, is guided and controlled by
microenvironmental cues. The bone marrow represents an instructive
example of such microenvironmental influences. Hematopoiesis in bone
marrow depends on the mutual interaction of hematopoietic stem cells
and progenitor cells with stromal cells, and on the interaction of
these cells with components of the extracellular matrix
(ECM).1-3 The ECM in bone marrow consists mainly of
fibronectin, hemonectin, thrombospondin, collagens, laminin, and the
glycosaminoglycans (GAGs) heparan sulfate, dermatan, chondroitin
sulfate (CS), and hyaluronic acid (HA).4,5
The development of hematopoietic cells in the bone marrow can be
mimicked in vitro, at least in part, in so-called long-term bone marrow
culture (LTBMC).6 LTBMC contains the cellular constituents of bone marrow and supports the generation of progenitors and their
differentiation into certain lineages. During culture, ECM is produced
mainly by stromal cells, and components of the ECM such as heparan
sulfate, CS, and fibronectin are required for the development of
hematopoietic progenitors.7-10 HA has also been detected in
LTBMC but it is not known whether it plays a role in
hematopoiesis.11
This study has been motivated by our interest in the function of CD44
proteins which are the major known HA receptors on the surface of
cells.12 Hematopoietic progenitor cells in the bone marrow
and progenitor cell lines carry one or several different CD44 proteins
on their surface,13,14 all of which carry an HA-binding
motif in the N-terminal domain.15,16 As CD44 is required
for myelopoiesis in LTBMC,17,18 it is conceivable that HA
binding is an essential step in hematopoiesis. In this study we examine
whether HA addition or its depletion by treatment with hyaluronidase
(HA'ase) influences hematopoiesis in LTBMC. We report that HA enhances
and HA'ase inhibits both myelopoiesis and lymphopoiesis. HA exerts
this effect by binding to a subpopulation of adherent stromal cells and
by inducing the production of interleukin-1 (IL-1) and IL-6.
Enriched bone marrow-derived macrophages (BMDM) respond to HA by the
release of both cytokines. Antibodies directed against the HA-binding
motif of CD44 inhibit IL-1 secretion but not IL-6 release by
macrophages, indicating that BMDM carry two HA receptors, both of which
mediate signal transfer in response to HA. The signal transduction
pathway from CD44 to IL-1 involves p38 MAP kinase phosphorylation,
clearly distinguished from the other HA induced pathway leading to Erk
activation and IL-6 release. Our data support the idea that, in
addition to other extracellular matrix components, HA controls
hematopoiesis at an early progenitor stage before commitment toward the
myeloid and lymphoid lineages.
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MATERIALS AND METHODS |
Cell lines.
Rat fibrosarcoma (RFS) cells19 were cultured
in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal calf serum (FCS). Their supernatant was
used as a source of HA.19 Wehi-3B cells20 were
cultured in RPMI supplemented with 10% FCS. The conditioned medium
(CM) was used as a source of IL-3.20 L929
cells21 were cultured in DMEM supplemented with 10% FCS. Their CM was used as a source of macrophage colony-stimulating factor
(M-CSF).21 An IL-7 producer cell line was
kindly provided by Dr H. Karasuyama (Basel Institute for
Immunology, Basel, Switzerland)22 and cultured in RPMI with
10% FCS. The murine T-cell lymphoma cell line HA923 was
provided by Dr D. Naor (Jerusalem). It was used as a
positive control for CD44-dependent HA binding.
Mice.
C57Bl/6, DBA, (C57Bl/6 × DBA)F1, BALB/c, and 129 mice were
purchased from Jackson Laboratories (Bar Harbor, ME). Mice were used
for the experiments at the age of 6 to 12 weeks.
CD44 / mice were obtained from Dr T.W.
Mak (University of Toronto, Toronto, Canada)24
and back-crossed into a C57Bl/6 background. All animals were kept under
specific pathogen-free conditions.
LTBMC.
Myeloid LTBMC were established as described.6 In brief,
mice were killed by cervical dislocation, and femurs and tibias were
removed. Bone marrow cells were flushed out of the bones with
phosphate-buffered saline (PBS) containing 2% FCS. Bone marrow cells
(1 × 106 cells/mL) were cultured in DMEM supplemented
with 20% horse serum (Linaris, Bettingen, Germany) and
hydrocortisone (106 mol/L; Sigma, St Louis,
MO) in 6-well plates at 33°C in a humid atmosphere
containing 5% CO2. Cultures were fed weekly by changing half of the culture medium. Nonadherent cells were collected from the
culture medium, counted, and used for flow cytometry or assayed for
colony-forming units (CFU). HA or HA'ase, respectively, was added to
LTBMC medium before culture set-up and before medium changes.
Lymphoid LTBMCs were performed as described.25 Briefly,
bone marrow cells were cultured in RPMI supplemented with 5% FCS, 5 × 105 2-mercaptoethanol (2-ME) and 2 mmol/L
L-glutamine, at 37°C and 5% CO2 in a humid atmosphere.
Cultures were fed weekly by changing half of the medium.
The source of HA was either rooster comb HA from Sigma or HA isolated
from the supernatant of RFS cells.19 HA'ase was obtained from Calbiochem (La Jolla, CA) (isolated from
Streptomyces hyaluronlyticus). HA determinations were performed
as described.26
BMDM cultures.
BMDM cultures were obtained using a protocol previously
described.27 Bone marrow cells were flushed from femurs and
tibias with PBS containing 2% FCS. The bone marrow cells were
dispersed at 1 × 106 cells per mL in DMEM containing
2 mmol/L glutamine, 0.37% (wt/vol) NaHCO3, 10% (vol/vol)
heat-inactivated FCS, and 10% (vol/vol) L929 cell-conditioned medium.
Cultures were maintained at 37°C in a 5% CO2
atmosphere for 5 days. The medium was changed and cells were used
between days 6 and 8 thereafter. Ninety-nine percent of cells in the
monolayer were esterase-positive and were phagocytic for latex
particles. The cells were starved for 12 hours before stimulation with HA.
CFU assay.
Bone marrow cells or nonadherent cells from LTBMC were plated at a
concentration of 1 × 104 cells/mL in semisolid
methylcellulose supplemented with 30% FCS, 1% bovine serum albumin
(BSA), 104 mol/L 2-ME, 2 mmol/L L-glutamine
(StemCell Technologies, Vancouver, Canada), and 15%
Wehi-3B CM as a source of IL-3.28 Plates were cultured at
37°C in 5% CO2 atmosphere. Colonies were counted under the microscope after 7 days of culture. Other cytokines used for determination of lineage-specific progenitors were
granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and
erythropoietin (Boehringer Mannheim, Mannheim, Germany),
M-CSF (supernatant of L929 cells) and IL-7 (supernatant of a producer
cell line, see Cell lines).
Flow cytometry.
The monoclonal antibody (MoAb) KM 81 was obtained from American Type
Culture Collection (ATCC; Rockville, MD; no. TIB241), phycoerythrin (PE)-labeled anti-mouse CD11b/CD18 was obtained from
Cedarlane (Ontario, Canada). Rooster comb HA (Sigma) and chondroitin sulphate A (CS-A; Fluka, Buchs, Switzerland)
were fluorescein isothiocyanate (FITC)-labeled as previously
described.29 For flow cytometry, 5 × 105
cells were incubated with PE- or FITC-labeled antibodies at a concentration of 10 µg/mL at 4°C for 30 minutes and washed twice with FACS-buffer (PBS, 3% FCS, 0.01% NaN3). Fluorescence
was analyzed on a FACStar Plus flow cytometer (Becton Dickinson,
Heidelberg, Germany).
Immunohistochemistry.
For examination of the HA binding ability of adherent cells from LTBMC,
cells were fixed with 3% paraformaldehyde, washed with PBS, and
incubated with FITC-labeled HA in PBS at 4°C for 30 minutes.
Cultures were washed and inspected by fluorescence microscopy. The
cytoskeleton was stained as follows: Cells of LTBMC were washed twice
with PHEM-buffer (60 mmol/L PIPES, 25 mmol/L HEPES, 10 mmol/L EGTA, 2 mmol/L MgCl2, pH 6.9), fixed with 2.5% glutaraldehyde
(Serva, Heidelberg, Germany) in stabilizing buffer (20 mmol/L HEPES, 5 mmol/L EGTA, 2 mmol/L phenylmethylsulfone fluorid
(PMSF), 1 mmol/L MgSO4, 1 g/L
Na-tosyl-L-argininemethylester, 20% glycerol, pH 6.8) at 37°C for
15 minutes. Thereafter, cells were treated with 50 to 100 µL staining
solution (3.3 µmol/L phalloidine-rhodamine in methanol 1:10 in
PHEM-buffer) at 37°C for 2 hours. The stained probes were evaluated
by fluorescence microscopy.
Enzyme-linked immunosorbent assay (ELISA).
LTBMC or BMDMC were stimulated by HA (100 µg/mL), and CM was
collected and kept frozen in aliquots at  80°C until use. CM samples were checked by ELISA (Endogen, Woburn, MA) for
IL-1 , IL-1, IL-3, IL-4, IL-6, IL-10, tumor necrosis factor-
(TNF-), and GM-CSF according to the instructions of the manufacturer.
Protein analysis.
Western blot analysis of signal transduction kinases was performed
according to standard procedures. Cells were lysed in sample buffer
(20% glycerol, 4% sodium dodecyl sulfate [SDS], 0.16 mol/L Tris pH
6.8, 4% 2-ME, 0.05% bromphenol blue). Lysates were sonicated and
heated at 100°C for 5 minutes. Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to
Immunobilon-PVDF membrane (Millipore, Bedford, UK). Immunostaining was
performed using anti-phospho-p38 and anti-phospho-Erk antibodies (New
England Biolabs, Beverly, MA; cat. nos. 9210 and 9101, respectively). Horseradish peroxidase-linked anti-rabbit antibodies (DAKO, Hamburg, Germany) were used as secondary antibodies. The staining
was visualized using the enhanced chemiluminescence (ECL)
reaction (Amersham, Freiburg, Germany). To control for protein loading,
the membranes were stripped and incubated with anti-p38 and anti-Erk
antibodies (Santa Cruz Biotech, Santa Cruz, CA; SC535 and SC1647).
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RESULTS |
HA'ase treatment inhibits hematopoiesis in LTBMC.
To assess a role for HA in hematopoiesis, we examined the effect of HA
deprivation on the production of nonadherent cells in myeloid and
lymphoid LTBMC. To achieve HA deprivation, LTBMC were cultured in the
presence of HA'ase. The production of nonadherent cells in both
lymphoid (Table 1) and myeloid cultures
(Fig 1A) was severely inhibited by HA'ase
treatment throughout the course of the culture. The inhibition was
dose-dependent (see inset in Fig 1A).
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| Fig 1.
Influence of HA'ase on myelopoiesis in LTBMC. (A)
Nonadherent cells from myeloid LTBMC in the absence of HA'ase
(control) or treated with HA'ase (Streptomyces HA'ase, Calbiochem;
0.1 U/mL) were harvested once per week, and their number was counted.
Standard error (SE) was calculated from results obtained with 3 independent cultures. The insert shows the dose-dependent effect of
HA'ase on the production of nonadherent cells in LTBMC after 4 weeks
in culture. The bars indicate SE calculated from 3 independent wells
each. (B) The production of clonogenic cells in either control LTBMC or
in LTBMC treated with HA'ase (0.1 U/mL) was measured by the CFU assay
(Materials and Methods). Nonadherent cells were harvested from LTBMC
and plated in semisolid culture in the presence of IL-3 (Materials and
Methods). SE was calculated from results obtained from 3 independent
wells.
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HA'ase treatment did not alter the morphology of adherent stromal
cells, but the ratio of progenitors to stromal cells rapidly changed
after the first week of culture. Areas of hematopoiesis, "cobblestone areas," appeared much later and were significantly smaller in HA'ase-treated cultures (Fig 2,
see different time points in C, F, and I) than in control cultures (Fig
2, A, D, and G). To quantitate this observation, we compared the
proliferative capacity of adherent cells in control and HA'ase-treated
LTBMC by measuring 3H-thymidine incorporation. HA'ase
treatment reduced thymidine incorporation to less than 10% (not
shown), a reduction that matched the reduction of proliferating cells
within the cobblestone areas of the adherent cell layer (compare Fig 2D
and F). Consistant with the decreased number of hematopoietic areas,
the number of clonogenic cells in the LTBMC as measured by CFU assay
was dramatically reduced by HA'ase treatment (Fig 1B and Table 1).
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| Fig 2.
Influence of HA and of HA'ase on formation of the
adherent layer. Primary bone marrow cells were cultured in 6-well
plates without any addition (A, D, and G) or with the addition of 100 µg/mL of HA (B, E, and H) or with 0.1 U/mL HA'ase (C, F, and I).
Photographs were taken after 1 (top panel), 4 (middle panel), and 8 (bottom panel) weeks of culture. Only adherent cells are shown. In (D),
(E), (G), and (H), typical cobblestone-like cells are visible, which
are increased in number in (E) and (H), whereas in (F) and (I) such
cells are hardly detectable. The magnification was 400-fold.
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To define whether HA'ase acted directly on proliferation of the
progenitor cells, we examined the effect of HA'ase on colony formation
of primary bone marrow progenitor cells in methylcellulose. Depending
on the type of progenitor to be determined, different cytokines were
added. For polypotent and bipotent myeloid progenitors, IL-3 and
GM-CSF, respectively, were added. For erythroid burst-forming colonies,
IL-3 together with erythropoietin was added. For monopotent macrophage
and granulocyte progenitors, M-CSF and G-CSF, respectively, were added,
and for lymphoid progenitors, IL-7 was added. Colony formation by any
of these treatments was not influenced by the presence of HA'ase (not
shown), suggesting that HA'ase does not act directly on progenitor
cells. Furthermore, this result shows that HA'ase exerted no toxic
effects on progenitors, but rather specifically inhibited hematopoiesis
at the level of stromal cells.
Exogeneously added HA increases cell production in LTBMC.
The HA'ase data speak for a regulatory role of HA in hematopoiesis.
This conclusion is further supported by the effects we observed when HA
was added exogenously to LTBMC. Both commercially available HA
(Fig 3) and HA from CM of HA-producing RFS
cells (not shown)19 stimulated the yield of myeloid (Fig
3A) and lymphoid (Table 1) nonadherent cells in LTBMC in a
dose-dependent manner. The optimal HA concentration required to
increase the number of nonadherent cells in LTBMC was 100 µg/mL
(inset in Fig 3A). Measurement of the endogenous concentration of HA in
the supernatant of LTBMC by staining of HA with stain-all26
was less than 10 µg/mL (below detection level). After Proteinase K
treatment of LTBMC and vigorous shaking, HA could be measured in the
supernatant (15 to 20 µg/mL). Exogenously added HA was easily
detected and within the 7-day period between medium change no change in
HA concentration was measurable. The total number of clonogenic cells
in LTBMC was also increased (Fig 3B and Table 1). Both the number of
cells attached to plastic and the number of cells forming areas of
hematopoiesis were significantly higher in the HA-treated cultures than
in control cultures (see Fig 2B, E, and H in comparison with A, D, and
G). The HA-dependent increase was detectable as early as 7 days after the start of the cultures. To rule out any contamination of the HA
preparations with cytokines, we heat-treated the HA preparations before
addition to LTBMC. The heat treatment had no influence on the results
(Fig 3A inset, and not shown).
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| Fig 3.
HA stimulates myelopoiesis in LTBMC. (A) Nonadherent
cells from myeloid LTBMC in the absence of HA (control) or treated with
HA (100 µg/mL; rooster comb HA from Sigma) were determined each week.
SE was calculated from results obtained from 3 independent wells. The
inset shows the dose-dependent effect of HA on the production of
nonadherent cells in myeloid LTBMC after 4 weeks in culture. The
asterisk indicates the use of heat-treated HA (1 hour at 95°C). The
bars indicate the SE determined from results obtained from 3 independent wells. (B) Clonogenic cells in myeloid LTBMC grown in the
absence of HA (control) or presence of HA (100 µg/mL) were determined
at the time indicated by plating of nonadherent cells in semisolid
methylcellulose culture medium in the presence of IL-3 (Materials and
Methods). SE was calculated from results obtained from 3 different
wells.
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Consistent with the data obtained by HA depletion, HA appeared to act
on stromal cells in that HA did not influence colony outgrowth from
committed progenitors in methylcellulose supplied with any of the
cytokines specified above (not shown). Addition of CS-A, another GAG
found in the ECM of bone marrow, did not further stimulate
hematopoiesis in vitro over a wide range of concentrations (data not
shown), demonstrating the specificity of HA action. Thus, complementing
the data obtained by HA'ase treatment, addition of exogenous HA
promotes both myelopoiesis and lymphopoiesis.
HA upregulates IL-1 and IL-6 production in LTBMC.
Because HA did not directly act on progenitors during growth in
methylcellulose but rather appeared to influence hematopoiesis via
stromal cells, its mode of action could be either to stimulate stromal
cells to act directly on progenitor cells, or to produce and/or release
an activity that promotes progenitor proliferation. The first
assumption seemed unlikely, because in transwell assays where we
separated nonadherent cells (including progenitors) from stromal cells
by a membrane, HA treatment of stromal cells still resulted in an
increased number of nonadherent cells in the transwell (not shown). To
test for the latter possibility, CM from control cultures and from
HA-treated cultures were examined for the ability to stimulate myeloid
colony formation in methylcellulose of primary bone marrow cells in the
presence of suboptimal concentrations of IL-3. CM from HA-treated
cultures indeed enhanced both the colony size (not shown) and the
number of colonies (Fig 4A) compared with
control CM. We conclude that CM from HA-treated cultures contained a
soluble colony-promoting activity produced by stromal cells.
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| Fig 4.
HA stimulates the production of a colony promoting
activity in LTBMC. (A) LTBMC was treated with HA (100 µg/mL). CM was
harvested at day 28 and tested for colony-promoting activity. Freshly
isolated bone marrow cells were added to methylcellulose cultures and
the colony formation by myelopoietic progenitors was determined in the
presence of suboptimal doses of IL-3 (5% CM of Wehi-3B cells) and the
addition of 20% of CM from either control LTBMC (lanes 1 and 3) or of
CM from LTBMC treated with HA (lanes 2 and 4). After 7 days of culture,
the numbers of colonies were counted. In lanes 3 and lane 4, hematopoietic progenitors were determined similarly to lanes 1 and 2, but antibodies directed against IL-6 (100 µg/mL) were added in
addition. SE was calculated from 3 independent assays. (B) CM of
control LTBMC or LTBMC cultured for 4 weeks in the presence of HA (100 µg/mL) or in the presence of HA'ase (0.1 U/mL) was assayed for IL-6
by ELISA.
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This activity could represent one or several of the cytokines known to
act on bone marrow stem cells and early hematopoietic progenitors.28 Therefore, the HA-CM was examined by ELISA
for the presence of GM-CSF and IL-1, -1, -3, -4, -6, and -10. The level of IL-6 was upregulated 4- to 5-fold in LTBMC derived fom one of
several strains of mice (C57Bl/6xDBA)F1, DBA, BALB/c, or 129) upon treatment with HA (Fig 4B). Among the other cytokines, only
IL-1 levels were slightly but consistantly enhanced upon HA
treatment (not shown). To prove that the colony-promoting activity of
CM from HA-treated cultures was indeed caused by the production of
IL-6, we added cytokine-neutralizing antibodies. The effect of the
HA-CM on CFU formation was reduced to the level of control CM by
IL-6-specific antibodies (Fig 4A). Hence, we can conclude that HA
induces in LTBMC the release of IL-6 (and weakly of IL-1) from
adherent stromal cells, which promotes the proliferation of
hematopoietic progenitors.
HA binds to the surface of a subpopulation of stromal cells in LTBMC.
To investigate which cell type responded to HA by cytokine production,
we first examined LTBMC cells for binding FITC-labeled HA. To this end,
the cells to be examined were incubated with FITC-HA either directly or
after treatment with HA'ase to remove endogenously produced HA that
might preoccupy putative receptors, and thus to increase the binding
sensitivity. Incubation with FITC-HA was done at room temperature or at
4°C for 30 minutes. Nonadherent cells from LTBMC in steady state
(after 4 weeks in culture) showed no staining as determined by flow
cytometry (not shown). The adherent cells were paraformaldehyde-fixed
before incubation with FITC-HA at 4°C. In contrast, patchy staining
was detected with stromal cells with a typical macrophage morphology (Fig 5). Cobblestone areas, however, showed
no staining (data not shown). Based on this observation, we focused our
attention on the stromal population, because we considered these cells
candidates for HA-induced signaling. The macrophage nature of the
HA-stained cells was confirmed by the presence of CD11b antigen (data
not shown).
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| Fig 5.
Binding of HA to macrophage cells. After 4 weeks, LTBMC
nonadherent cells were washed out, adherent cells were treated for 30 minutes at 37°C with HA'ase (5 U/mL), and then fixed with 3%
paraformaldehyde and incubated with FITC-labeled HA at 4°C for 30 minutes. Immunofluorescence-staining of a representative cell is shown.
Magnification was 1,000-fold. Similar staining was observed without
HA'ase treatment.
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HA induces cytokine release from BMDM.
To prove that BMDM were the targets of HA and the source of HA-induced
cytokine release, bone marrow cells were cultured in the presence of
M-CSF to enrich for macrophages. Enriched macrophages were stained by
FITC-labeled HA similarly to the monocytic cells in LTBMC (not shown).
HA caused upregulation of cytokine release, both of IL-6 and of
IL-1, with a lag period of 2 to 4 hours as measured by ELISA
(Fig 6 and Fig 9). BMDM from all mice
strains tested [(C57Bl/6xDBA)F1, DBA, BALB/c, or 129] were inducible
for both cytokines, and the induction of IL-1 was considerably
stronger in the enriched macrophage preparation than in LTBMC.
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| Fig 6.
HA treatment of BMDM upregulates IL-6 production. BMDM
were isolated as described in Materials and Methods and were then
treated with HA (100 µg/mL). CM was collected at the indicated times
and tested for the presence of IL-6 by ELISA. The error bars indicated
SE calculated from results obtained from 3 independent wells.
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HA-induced cytoskeletal rearrangement in BMDM.
The brief response kinetic for cytokine release (Fig 6) is compatible
with a direct action of HA on signal transduction triggering cytokine
synthesis and release. Further to this, we detected a second type of
HA-induced response in BMDM which may also be the result of a signal
transduction process. At 4 hours after HA addition, the BMDM were
stained with phalloidine-rhodamine, which showed that the actin
cytoskeleton was rearranged (Fig 7). Such
changes in the cytoskeleton are indicative of signal
transduction.30 The changes in the cytoskeleton are
comparable to those that occur during macrophage activation (not
shown).
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| Fig 7.
HA-induced actin rearrangement in LTBMC adherent cells.
Adherent cells of LTBMCs after 4 weeks in culture were incubated with
HA (100 mg/µL) for 10 minutes, 4 hours, and 16 hours, respectively.
Staining of the actin cytoskeleton by phalloidine-rhodamine (Materials
and Methods) is shown. Magnification was 1,000-fold.
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Two hyaluronate receptors mediate signal transduction.
The action of HA on enriched BMDM raises two questions: Which is the
relevant surface receptor for HA, and which signal transduction pathway
is this receptor connected to? Because CD44 is a plausible receptor
candidate, we investigated the effect of an antibody that blocks the
HA-binding motif of CD44 (KM81), on cytokine production of BMDM.
Because antibodies may bind to the Fc-receptor on macrophages and
thereby activate the cells, we used F(ab')2 fragments
of KM81 antibodies for the blocking studies. Interestingly, KM81
F(ab')2 fragments reduced HA-induced IL-1 release,
but not that of IL-6 (Fig 8). The KM81
F(ab')2 fragments were functional in that they blocked the binding of HA to the cell line HA9 (a derivative of the
murine lymphoma LB17 selected for HA binding to CD4423), and they were functional throughout the experiment (24 hours) because
the medium from the macrophages treated with HA and KM81 F(ab')2 for 24 hours still blocked the binding of HA
to the HA9 cell line (data not shown). These results indicate the
presence of at least two relevant HA receptors on BMDM: CD44 and an yet unknown receptor.
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| Fig 8.
Influence of KM81 MoAb on cytokine production by
macrophages upon HA treatment. BMDM were treated with HA (100 µg/mL)
for the times indicated either in the presence or absence of
F(ab')2 fragments of the pan CD44 antibody KM81 (20 µg/mL). The concentration of IL-6 (A) and IL-1 (B) was determined
in the supernatant by ELISA. SE was calculated from results obtained
from 3 independent wells each.
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The two different HA receptors likely feed into different signal
transduction pathways that activate protein kinases and, finally,
different transcription factors. We explored whether HA could activate
members of the proline-directed protein kinases that phosphorylate
transcription factors. Indeed, BMDM responded to HA treatment with the
activation of proline-directed protein kinases as detected by
antibodies recognizing the phosphorylated forms of Erk and p38
(Fig 9). The lag period of activation for both kinases was between 30 and 60 minutes and activation persisted beyond 120 minutes. The question of whether the two postulated HA
receptors link into different pathways was addressed by blocking CD44
with antibody KM81 F(ab')2 fragments. KM81
F(ab')2 fragments blocked specifically the HA-induced
activation of p38 but not that of Erk (Fig 9), indicating that indeed
at least 2 different HA receptors mediate HA signaling and that they
stimulate 2 different signal transduction pathways. The antibody KM81
thus defines a pathway from CD44 through p38 (or through a coactivated
protein kinase not yet detected) to IL-1. However, IL-6 secretion
depends on stimulation of another HA receptor and parallels the
activation of Erk.
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| Fig 9.
HA-induced activation of proline-directed protein kinases
in BMDM. BMDM were stimulated with HA (100 µg/mL) for the indicated
times, then lysed, and protein samples were analyzed by Western
blotting for the presence of phosphorylated p38 kinase and
phosphorylated Erk. The same membranes were stripped and stained with
anti p38 or Erk antibodies, respectively. Where indicated, cells were
preincubated for 15 minutes at 4°C with KM81
F(ab')2 fragments (20 µg/mL) before HA
stimulation.
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Function of CD44 in bone marrow is compensated for in CD44-deficient
mice.
The dependence of IL-1 induction by HA on CD44 suggests that this
function might be missing in CD44 knock-out mice. Therefore, we
investigated whether LTBMC originating from bone marrow of CD44/ mice showed any deficiency in
hematopoiesis and whether bone marrow macrophages could respond to HA.
CD44/ mice were obtained by gene
disruption.24 Bone marrow cells from wild-type and from
CD44/ mice were cultured in the presence or
absence of HA. In both wild-type and CD44/
LTBMC, exogeneous HA stimulated the generation of nonadherent cells as
well as of clonogenic cells (Fig 10).
Stromal cells from CD44/ LTBMC also bound
FITC-HA to an extent similar to that of stromal cells from wild-type
bone marrow (not shown). Conversely, treatment of LTBMC from both
sources with HA'ase led to dramatic inhibition of hematopoiesis (Fig
10). The number of both nonadherent cells and hematopoietic precursors
was reduced. HA also induced the release of IL-6 and IL-1 in
cultures of macrophages derived from CD44/
bone marrow (not shown). As expected, IL-1 induction was not inhibited by KM81 F(ab')2 fragments in macrophages
derived from CD44/ mice (not shown).
View larger version (17K):
[in this window]
[in a new window]
| Fig 10.
Influence of HA and HA'ase on myeloid LTBMC from CD44
knock-out mice. Myeloid LTBMC from CD44-deficient mice were untreated
or treated with HA or HA'ase as described in the legends to Figs 1 and
3. The number of nonadherent cells (A) and hematopoietic progenitors
(B) were measured once per week. SE was calculated from results
obtained from 3 independent wells.
|
|
| |
DISCUSSION |
This report addresses the role of the extracellular matrix component HA
of the bone marrow microenvironment in hematopoiesis. We show that HA
is not only a passive structural component, but a necessary and a
specific signal-inducing molecule for hematopoiesis in the bone marrow.
Specifically, HA'ase treatment of murine LTBMC abrogates lymphopoiesis
and myelopoiesis, whereas addition of HA to LTBMC enhances
lymphopoiesis and myelopoiesis. HA regulates a decisive step before the
commitment of polypotent progenitor cells.
The mode of action of HA is to induce the release of cytokines from
BMDM, particularly of IL-6 and IL-1, but not of GM-CSF, IL-3, IL-4,
IL-10, and TNF-. IL-1 has been reported to be released in
response to HA from both peripheral and bone marrow
macrophages.31,32 To our knowledge, this manuscript reports
for the first time an activation of IL-6 release by a GAG. HA binding
to macrophages led to changes in the cytoskeleton and to the activation
of p38 kinase and Erk kinase, both of which are key enzymes in
different signal transduction pathways.33
F(ab')2 fragments of antibodies directed against the
HA-binding domain of the HA receptor CD44 reduce the IL-1 induction,
indicating a role for CD44-mediated signaling to IL-1. This
observation is in agreement with a previous report.32 The
IL-6 induction by HA on the same bone marrow macrophages was not
inhibited by the KM81 F(ab')2 fragments, suggesting
that a different receptor for HA is involved. Antibody blocking of the
protein kinase activation confirms the existence of two different receptors, both of which interact with HA as p38 kinase was activated in a CD44-dependent manner while Erk kinase activation was independent of CD44. Thus, we conclude that on BMDM, HA binding to CD44 leads to
activation of p38 kinase and to induction of IL-1. On the other
hand, the binding of HA to a still unknown receptor leads to the
activation of Erk kinase and the stimulation of IL-6 release.
The induction kinetics of Erk and p38 after HA ligation by both CD44
and the unknown HA receptor are quite slow compared with signaling
activation by receptor tyrosine kinases. Recently, however, the
activation of two receptor tyrosine kinases, DDR1 and DDR2, has also
been described to be slow, similar to the kinetics we observed here.
Strikingly, DDR1 and DDR2 are also receptors for extracellular matrix
components, namely for collagens.34,35
The induction of cytokines may explain the stimulatory effect of HA on
hematopoiesis. IL-6, together with CSFs, is known to increase
proliferation and to maintain primitive hematopoietic progenitor
cells.36-39 Furthermore, IL-6 also stimulates
megakaryocytopoiesis and the formation of monocytic colonies from
hematopoietic progenitor cells.40-42 In agreement with
these findings, IL-6-deficient mice have reduced the number of early
progenitors in the bone marrow and in blood, suggesting that
proliferation and differentiation of hematopoietic progenitors are
impaired in these mice.43 IL-1, in contrast, seems to
have only minor effects on hematopoiesis44,45 and no
profound effect on cell number, morphology, and colony-forming cells in
LTBMC.46 In line with these results, IL-1-deficient mice have no hematopoietic phenotype.47 Thus, we conclude
that the decisive step promoting hematopoiesis by HA in LTBMC, and by
analogy in bone marrow, is the induction of IL-6 in BMDM.
It has been shown that CD44 is involved in the regulation of
hematopoiesis, although the mechanism of its action is not yet well
understood. The application of panCD44 MoAbs led to an inhibition of hematopoiesis in LTBMC from different
species.17,18,48 CD44 antibodies capable of
stimulating hematopoiesis in LTBMC have also been
described.49,50a These data suggest that CD44 proteins are
engaged in steps and mechanisms other than mediating an HA effect (see
also the discussion in ref 18).
The best-characterized HA receptor on the surface of cells is CD44.
ICAM-1 on rat liver endothelial cells has been described as capable of
binding HA,51 but recent studies showed that this observation was based on an artefact.52 Recently, an HA
receptor termed RHAMM on Ras-transformed fibroblasts was defined as
part of the HARC complex.53,54 An MoAb directed against a
portion of the published cDNA sequence of RHAMM recognized an
intracellular HA binding protein but not a cell-surface
protein.55,56 Therefore, RHAMM was not considered as a
relevant receptor in our experimental context. Interestingly, the
presence of two HA binding receptors on the same cell has also been
described on a brain endothelial cell line,57 one of which
is CD44, while the other is yet unknown.
From the fact that HA-dependent IL-1 induction in BMDM is mediated
by CD44, one could expect that this induction does not occur in CD44
knock-out mice. This is not the case, suggesting that the CD44 HA
binding function is compensated for in the knock-out mice. The
compensating molecule is yet unknown. The assumption of existence of a
compensatory mechanism is further corroborated by the finding that
hematopoiesis is by and large normal in CD44 knock-out
mice,24 whereas a functional relevance of CD44 proteins has
been deduced from biochemical studies.17,18 An explanation for this apparent contradiction between the results obtained with knock-out animals and molecular biological analysis might be that CD44
substitutes can only be established during early embryogenesis. Consistent with this assumption is the observation that a conditional antisense-mediated elimination of CD44 from keratinocytes showed a
striking skin phenotype,58 whereas in knock-out animals the skin develops perfectly normally.24
Similarly, a defined limb phenotype in embryogenesis was created by
antibody interference with CD44 function in the apical ectodermal
ridge, which resulted in repression of mesenchyme
proliferation59 while CD44-deficient mice develop normal
limbs.24
Our experiments have identified a new function for the ECM component HA
in the activation of cytokine release through two different signaling
pathways and have added evidence to the increasing number of examples
that the components of the ECM are not only space-filling and
structural molecules, but also have specific regulatory functions in
gene expression.
| |
NOTE ADDED IN PROOF |
During the preparation of this manuscript, a paper was
published60 which reported that HA stimulates IL-6
expression in dendritic cells.
| |
ACKNOWLEDGMENT |
We thank Martin Hegen, Andreas Herrlich, Axel Knebel, Harald
König, and Carsten Wei for fruitful discussion and technical advice. The CD44 knock-out mice were kindly provided by R. Schmits (Homburg, Germany) and T.W. Mak (Toronto, Canada).
| |
FOOTNOTES |
Submitted September 16, 1998; accepted March 31, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Helmut Ponta, PhD,
Forschungszentrum Karlsruhe, Institute of Genetics, PO Box 3640, D-76021 Karlsruhe, Germany; e-mail: genetik{at}igen.fzk.de.
| |
REFERENCES |
1.
Quesenberry PJ:
Stroma-dependent hematolymphopoietic stem cells.
Curr Top Microbiol Immunol
177:151, 1992[Medline]
[Order article via Infotrieve]
2.
Chabannon C, Torok-Storb B:
Stem cell-stromal cell interactions.
Curr Top Microbiol Immunol
177:123, 1992[Medline]
[Order article via Infotrieve]
3.
Morrison SJ, Shah NM, Anderson DJ:
Regulatory mechanisms in stem cell biology.
Cell
88:287, 1997[Medline]
[Order article via Infotrieve]
4.
Verfaillie C, Hurley R, Bhatia R, McCarthy JB:
Role of bone marrow matrix in normal and abnormal hematopoiesis.
Crit Rev Oncol Hematol
16:201, 1994[Medline]
[Order article via Infotrieve]
5.
Klein G:
The extracellular matrix of the hematopoietic microenvironment.
Experientia
51:914, 1995[Medline]
[Order article via Infotrieve]
6.
Dexter TM, Allen TD, Lajtha LG:
Conditions controlling the proliferation of haemopoietic stem cells in vitro.
J Cell Physiol
91:335, 1977[Medline]
[Order article via Infotrieve]
7.
Gordon MY, Riley GP, Clarke D:
Heparan sulfate is necessary for adhesive interactions between human early hemopoietic progenitor cells and the extracellular matrix of the marrow microenvironment.
Leukemia
2:804, 1988[Medline]
[Order article via Infotrieve]
8.
Siczkowski M, Clarke D, Gordon MY:
Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.
Blood
80:912, 1992[Abstract/Free Full Text]
9.
Spooncer E, Gallagher JT, Krizsa F, Dexter TM:
Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of haemopoiesis by beta-D-xylosides.
J Cell Biol
96:510, 1983[Abstract/Free Full Text]
10.
Verfaillie CM, Benis A, Iida J, McGlave PB, McCarthy JB:
Adhesion of committed human hematopoietic progenitors to synthetic peptides from the C-terminal heparin-binding domain of fibronectin: Cooperation between the integrin alpha 4 beta 1 and the CD44 adhesion receptor.
Blood
84:1802, 1994[Abstract/Free Full Text]
11.
Minguell JJ:
Is hyaluronic acid the "organizer" of the extracellular matrix in marrow stroma? [editorial; comment].
Exp Hematol
21:7, 1993[Medline]
[Order article via Infotrieve]
12.
Lesley J, Hyman R, Kincade PW:
CD44 and its interaction with extracellular matrix.
Adv Immunol
54:271, 1993[Medline]
[Order article via Infotrieve]
13.
Trowbridge IS, Lesley J, Schulte R, Hyman R, Trotter J:
Biochemical characterization and cellular distribution of a polymorphic, murine cell-surface glycoprotein expressed on lymphoid tissues.
Immunogenetics
15:299, 1982[Medline]
[Order article via Infotrieve]
14.
Kansas GS, Muirhead MJ, Dailey MO:
Expression of the CD11/CD18, leukocyte adhesion molecule 1, and CD44 adhesion molecules during normal myeloid and erythroid differentiation in humans.
Blood
76:2483, 1990[Abstract/Free Full Text]
15.
Peach RJ, Hollenbaugh D, Stamenkovic I, Aruffo A:
Identification of hyaluronic acid binding sites in the extracellular domain of CD44.
J Cell Biol
122:257, 1993[Abstract/Free Full Text]
16.
Yang B, Yang BL, Savani RC, Turley EA:
Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein.
EMBO J
13:286, 1994[Medline]
[Order article via Infotrieve]
17.
Miyake K, Medina KL, Hayashi S, Ono S, Hamaoka T, Kincade PW:
Monoclonal antibodies to Pgp-1/CD44 block lympho-hemopoiesis in long-term bone marrow cultures.
J Exp Med
171:477, 1990[Abstract/Free Full Text]
18.
Moll J, Khaldoyanidi S, Sleeman JP, Achtnich M, Preuss I, Ponta H, Herrlich P:
Two different functions for CD44 proteins in human myelopoiesis.
J Clin Invest
102:1, 1998[Medline]
[Order article via Infotrieve]
19.
Underhill CB, Chi-Rosso G, Toole BP:
Effects of detergent solubilization on the hyaluronate-binding protein from membranes of simian virus 40-transformed 3T3 cells.
J Biol Chem
258:8086, 1983[Abstract/Free Full Text]
20.
Lee JC, Hapel AJ, Ihle JN:
Constitutive production of a unique lymphokine (IL 3) by the WEHI-3 cell line.
J Immunol
128:2393, 1982[Abstract]
21.
Yamamoto-Yamaguchi Y, Tomida M, Hozumi M:
Effect of mouse interferon on growth and differentiation of mouse bone marrow cells stimulated by two different types of colony-stimulating factor.
Blood
62:597, 1983[Abstract/Free Full Text]
22.
Karasuyama H, Melchers F:
Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.
Eur J Immunol
18:97, 1988[Medline]
[Order article via Infotrieve]
23.
Vogt Sionov R, Naor D:
Hyaluronan-independent lodgment of CD44+ lymphoma cells in lymphoid organs.
Int J Cancer
71:462, 1997[Medline]
[Order article via Infotrieve]
24.
Schmits R, Filmus J, Gerwin N, Senaldi G, Kiefer F, Kundig T, Wakeham A, Shahinian A, Catzavelos C, Rak J, Furlonger C, Zakarian A, Simard JJ, Ohashi PS, Paige CJ, Gutierrez-Ramos JC, Mak TW:
CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity.
Blood
90:2217, 1997[Abstract/Free Full Text]
25.
Whitlock CA, Witte ON:
Long-term culture of B lymphocytes and their precursors from murine bone marrow.
Proc Natl Acad Sci USA
79:3608, 1982[Abstract/Free Full Text]
26.
Homer KA, Denbow L, Beighton D:
Spectrophotometric method for the assay of glycosaminoglycans and glycosaminoglycan-depolymerizing enzymes.
Anal Biochem
214:435, 1993[Medline]
[Order article via Infotrieve]
27.
Riches DW, Underwood GA:
Expression of interferon-beta during the triggering phase of macrophage cytocidal activation. Evidence for an autocrine/paracrine role in the regulation of this state.
J Biol Chem
266:24785, 1991[Abstract/Free Full Text]
28.
Testa JE, Molineux:
Hemopoiesis. A Practical Approach. Oxford, UK, Oxford University Press, 1993.
29.
de Belder AN, Wik KO:
Preparation and properties of fluorescein-labelled hyaluronate.
Carbohydr Res
44:251, 1975[Medline]
[Order article via Infotrieve]
30.
Cowin P, Burke B:
Cytoskeleton-membrane interactions [published erratum appears in Curr Opin Cell Biol 8:244, 1996].
Curr Opin Cell Biol
8:56, 1996[Medline]
[Order article via Infotrieve]
31.
Hiro D, Ito A, Matsuta K, Mori Y:
Hyaluronic acid is an endogenous inducer of interleukin-1 production by human monocytes and rabbit macrophages.
Biochem Biophys Res Commun
140:715, 1986[Medline]
[Order article via Infotrieve]
32.
Noble PW, Lake FR, Henson PM, Riches DW:
Hyaluronate activation of CD44 induces insulin-like growth factor-1 expression by a tumor necrosis factor-alpha-dependent mechanism in murine macrophages.
J Clin Invest
91:2368, 1993
33.
Treisman R:
Regulation of transcription by MAP kinase cascades.
Curr Opin Cell Biol
8:205, 1996[Medline]
[Order article via Infotrieve]
34.
Vogel W, Gish GD, Alves F, Pawson T:
The discoidin domain receptor tyrosine kinases are activated by collagen.
Mol Cell
1:13, 1997[Medline]
[Order article via Infotrieve]
35.
Shrivastava A, Radziejewski C, Campbell E, Kovac L, McGlynn M, Ryan TE, Davis S, Goldfarb MP, Glass DJ, Lemke G, Yancopoulos GD:
An orphan receptor tyrosine kinase family whose members serve as nonintegrin collagen receptors [in process citation].
Mol Cell
1:25, 1997[Medline]
[Order article via Infotrieve]
36.
Ikebuchi K, Wong GG, Clark SC, Ihle JN, Hirai Y, Ogawa M:
Interleukin 6 enhancement of interleukin 3-dependent proliferation of multipotential hemopoietic progenitors.
Proc Natl Acad Sci USA
84:9035, 1987[Abstract/Free Full Text]
37.
Wong GG, Witek-Giannotti JS, Temple PA, Kriz R, Ferenz C, Hewick RM, Clark SC, Ikebuchi K, Ogawa M:
Stimulation of murine hemopoietic colony formation by human IL-6.
J Immunol
140:3040, 1988[Abstract]
38.
Suda T, Yamaguchi Y, Suda J, Miura Y, Okano A, Akiyama Y:
Effect of interleukin 6 (IL-6) on the differentiation and proliferation of murine and human hemopoietic progenitors.
Exp Hematol
16:891, 1988[Medline]
[Order article via Infotrieve]
39.
Miura N, Okada S, Zsebo KM, Miura Y, Suda T:
Rat stem cell factor and IL-6 preferentially support the proliferation of c-kit-positive murine hemopoietic cells rather than their differentiation.
Exp Hematol
21:143, 1993[Medline]
[Order article via Infotrieve]
40.
Lotem J, Shabo Y, Sachs L:
Regulation of megakaryocyte development by interleukin-6.
Blood
74:1545, 1989[Abstract/Free Full Text]
41.
Quesenberry PJ, McGrath HE, Williams ME, Robinson BE, Deacon DH, Clark S, Urdal D, McNiece IK:
Multifactor stimulation of megakaryocytopoiesis: effects of interleukin 6.
Exp Hematol
19:35, 1991[Medline]
[Order article via Infotrieve]
42.
Jansen JH, Kluin-Nelemans JC, Van Damme J, Wientjens GJ, Willemze R, Fibbe WE:
Interleukin 6 is a permissive factor for monocytic colony formation by human hematopoietic progenitor cells.
J Exp Med
175:1151, 1992[Abstract/Free Full Text]
43.
Bernad A, Kopf M, Kulbacki R, Weich N, Koehler G, Gutierrez-Ramos JC:
Interleukin-6 is required in vivo for the regulation of stem cells and committed progenitors of the hematopoietic system.
Immunity
1:725, 1994[Medline]
[Order article via Infotrieve]
44.
Warren DJ, Moore MA:
Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells.
J Immunol
140:94, 1988[Abstract]
45.
Warren MK, Conroy LB, Rose JS:
The role of interleukin 6 and interleukin 1 in megakaryocyte development.
Exp Hematol
17:1095, 1989[Medline]
[Order article via Infotrieve]
46.
Delikat S, Harris RJ, Galvani DW:
IL-1 beta inhibits adipocyte formation in human long-term bone marrow culture.
Exp Hematol
21:31, 1993[Medline]
[Order article via Infotrieve]
47.
Zheng H, Fletcher D, Kozak W, Jiang M, Hofmann KJ, Conn CA, Soszynski D, Grabiec C, Trumbauer ME, Shaw A, Kostura MJ, Stevens K, Rosen H, North RJ, Chen HY, Tocci MJ, Kluger MJ, van der Ploeg LHT:
Resistance to fever induction and impaired acute-phase response in interleukin-1 beta-deficient mice.
Immunity
3:9, 1995[Medline]
[Order article via Infotrieve]
48.
Khaldoyanidi S, Schnabel D, Fohr N, Zoller M:
Functional activity of CD44 isoforms in haemopoiesis of the rat.
Br J Haematol
96:31, 1997[Medline]
[Order article via Infotrieve]
49.
Rossbach HC, Krizanac-Bengez L, Santos EB, Gooley TA, Sandmaier BM:
An antibody to CD44 enhances hematopoiesis in long-term marrow cultures.
Exp Hematol
24:221, 1996[Medline]
[Order article via Infotrieve]
50.
Ghaffari S, Dougherty GJ, Eaves AC, Eaves CJ:
Diverse effects of anti-CD44 antibodies on the stromal cell-mediated support of normal but not leukaemic (CML) haemopoiesis in vitro.
Br J Haematol
97:22, 1997[Medline]
[Order article via Infotrieve]
50a. Khaldoyanidi S, Sleeman J, Moll J, Herrlich P, Ponta H:
Selective activation of cytokines by CD44 variant specific antibodies
in macrophages triggers hematopoiesis. (in preparation)
51.
McCourt PA, Ek B, Forsberg N, Gustafson S:
Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan.
J Biol Chem
269:30081, 1994[Abstract/Free Full Text]
52.
McCourt PA, Gustafson S:
On the adsorption of hyaluronan and ICAM-1 to modified hydrophobic resins.
Int J Biochem Cell Biol
29:1179, 1997[Medline]
[Order article via Infotrieve]
53.
Turley EA, Austen L, Vandeligt K, Clary C:
Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells.
J Cell Biol
112:1041, 1991[Abstract/Free Full Text]
54.
Hardwick C, Hoare K, Owens R, Hohn HP, Hook M, Moore D, Cripps V, Austen L, Nance DM, Turley EA:
Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility [published erratum appears in J Cell Biol 118:753, 1992].
J Cell Biol
117:1343, 1992[Abstract/Free Full Text]
55.
Hofmann M, Fieber C, Assmann V, Göttlicher M, Sleeman J, Plug R, Howells N, von Stein O, Ponta H, Herrlich P:
Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein.
J Cell Sci
111:1673, 1998[Abstract]
56.
Assmann V, Marshall JF, Fieber C, Hofmann M, Hart IR:
The human hyaluronan receptor RHAMM is expressed as an intracellular protein in breast cancer cells.
J Cell Sci
111:1685, 1998[Abstract]
57.
Rahmanian M, Pertoft H, Kanda S, Christofferson R, Claesson-Welsh L, Heldin P:
Hyaluronan oligosaccharides induce tube formation of a brain endothelial cell line in vitro.
Exp Cell Res
237:223, 1997[Medline]
[Order article via Infotrieve]
58.
Kaya G, Rodriguez I, Jorcano JL, Vassalli P, Stamenkovic I:
Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation.
Genes Dev
11:996, 1997[Abstract/Free Full Text]
59.
Sherman L, Wainwright D, Ponta H, Herrlich P:
A splice variant of CD44 expressed in the apical ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is required for limb outgrowth.
Genes Dev
12:1058, 1998[Abstract/Free Full Text]
60.
Haegel-Kronenberger H, de la Salle H, Bohbot A, Oberling F, Cazenave JP, Hanau D:
Adhesive and/or signaling functions of CD44 isoforms in human dendritic cells.
J Immunol
161:3902, 1998[Abstract/Free Full Text]
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|
|
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M. R. Horton, M. A. Olman, C. Bao, K. E. White, A. M. K. Choi, B.-Y. Chin, P. W. Noble, and C. J. Lowenstein
Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages
Am J Physiol Lung Cell Mol Physiol,
October 1, 2000;
279(4):
L707 - L715.
[Abstract]
[Full Text]
[PDF]
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M. E. Mummert, M. Mohamadzadeh, D. I. Mummert, N. Mizumoto, and A. Takashima
Development of a Peptide Inhibitor of Hyaluronan-Mediated Leukocyte Trafficking
J. Exp. Med.,
September 18, 2000;
192(6):
769 - 780.
[Abstract]
[Full Text]
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S. Bruno, M. Fabbi, M. Tiso, B. Santamaria, F. Ghiotto, D. Saverino, C. Tenca, D. Zarcone, S. Ferrini, E. Ciccone, et al.
Cell activation via CD44 occurs in advanced stages of squamous cell carcinogenesis
Carcinogenesis,
May 1, 2000;
21(5):
893 - 900.
[Abstract]
[Full Text]
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T. Vincent, M. Jourdan, M.-S. Sy, B. Klein, and N. Mechti
Hyaluronic Acid Induces Survival and Proliferation of Human Myeloma Cells through an Interleukin-6-mediated Pathway Involving the Phosphorylation of Retinoblastoma Protein
J. Biol. Chem.,
April 27, 2001;
276(18):
14728 - 14736.
[Abstract]
[Full Text]
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ABSTRACT
INTRODUCTION