|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 140-148
Heparan Sulfate Proteoglycans Mediate Interleukin-7- Dependent B
Lymphopoiesis
By
Lisa A. Borghesi,
Yoshio Yamashita, and
Paul W. Kincade
From the Oklahoma Medical Research Foundation, Immunobiology and
Cancer Program, Oklahoma City.
 |
ABSTRACT |
Heparin/heparan sulfate proteoglycans (HSPGs) have the potential to
bind and directly regulate the bioactivity of hematopoietic growth
factors including interleukin-7 (IL-7), a cytokine critical for murine
B-cell development. We examined the consequence of manipulating soluble
heparin and cell-surface heparan sulfate to IL-7-dependent responses
of B-cell precursors. Soluble heparin was found to inhibit production
of lymphoid, but not myeloid, cells in long-term bone marrow cultures.
Analysis of pro-B cells lacking plasma membrane HS suggests that this
glycosaminoglycan is required for efficient binding and responsiveness
to IL-7. By contrast, responses of hematopoietic cells to other
cytokines were not influenced by heparin addition or HS removal.
Therefore, HSPGs on B-lineage precursors may function as IL-7 receptor
components similar to HSPGs known to be important for the bFGF
receptor. Other experiments suggest that HSPGs on the surface of
stromal cells provide a weakly associating docking site for IL-7,
possibly controlling availability of this cytokine to B-cell
precursors. Together these data demonstrate a direct role for
heparinlike molecules in regulating the IL-7-dependent stages of
murine B lymphopoiesis.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
GLYCOSAMINOGLYCANS (GAGs) present in the
bone marrow (BM) are well situated to participate in the regulation of
lympho-hematopoiesis. Proteoglycans tethered to the plasma membrane of
lymphocytes and stromal cells, as well as GAGs synthesized as part of
the extracellular matrix, may influence hematopoietic processes. Recent
attention has been focused on heparin/heparan sulfate proteoglycans
(HSPGs) as potential regulators of hematopoietic cell behavior. By
mediating adhesive interactions and modulating cytokine bioactivity
these glycosaminoglycans may contribute to the biological activity of specific BM microenvironments.
Heparin/HSPG-dependent interactions between hematopoietic precursors
and the microenvironment may be important for cell anchorage as well as
maturation processes. For example, heparan sulfate (HS) has been shown
to be involved in adhesion and long-term maintenance of hematopoietic
cells.1,2 Cell-surface molecules such as CD45, Mac-1,
PECAM-1, and Thy-1, which are known to be heparin-binding proteins, may
contribute to this interaction.3-5 Similarly, heparin/HSPGs may influence proliferation and differentiation of various
hematopoietic lineages. Association with BM-derived HS induced
morphological changes characteristic of differentiation in myeloid
leukemic cells.6 Furthermore, interaction of pre-B cells
with the STIM-Ig fusion protein, a stromal-derived molecule shown to
augment interleukin-7 (IL-7)-driven proliferation, was blocked by
heparin.7
Heparinlike GAGs are recognized to bind and potentially modulate the
bioactivity of several hematopoietic regulatory factors including IL-7,
granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-3,
and transforming growth factor-
(TGF-).8-13 Interaction with heparin/HSPGs may serve to
compartmentalize individual growth factors to specific niches of the
microenvironment, regulate degradation of bioactive factors during
transport, and provide an immobilized matrix upon which such factors
can be presented to target cells.11,12,14 Heparin/HSPGs may
also serve as coreceptors that facilitate interaction of growth factors
with their receptors. For example, basic fibroblast growth factor
(bFGF) does not bind to its high-affinity receptor in
HS-deficient mutants but binding can be restored by addition of
exogenous heparin.15 The syndecan family of cell-surface
HSPGs, which has been shown to promote functional FGF:FGF-receptor
complexing and signaling,16,17 may serve as such
coreceptors. Interestingly, not all growth factors are positively
influenced by interaction with GAGs. Binding of interferon-
(IFN-) to heparin competitively inhibits IFN-:IFN--receptor complex formation,18 highlighting the potential for
selective regulation of cytokine bioactivity.
Murine B-lymphocyte development is critically dependent on IL-7
availability. Early B-cell precursors that do not receive this
growth signal rapidly undergo apoptosis.19 Low
concentrations of IL-7 may then favor the maturation of more
differentiated cells.20 Despite observations that
heparinlike molecules can bind to IL-7,8,9 the role of
these GAGs in regulating IL-7 bioavailability/bioactivity at this point
in development is unknown. Therefore, we examined the
contribution of soluble heparin and cell surface HS to the IL-7-dependent stages of B lymphopoiesis.
| |
EXPERIMENTAL PROCEDURES |
Animals.
Male BALB/c mice 6 to 8 weeks old were obtained from the Oklahoma
Medical Research Foundation Laboratory Animal Resources Center. Animals
were housed in a room apart from other colonies and were maintained on
a 12:12 light:dark cycle with food and water available ad libitum.
Media and reagents.
RPMI-1640, Fischer's medium, minimal essential media (MEM)-
(-MEM), Dulbecco's phosphate-buffered saline (PBS;
free of Ca2+ and Mg2+), and horse serum were
purchased from GIBCO-BRL (Grand Island, NY). Fetal bovine serum (FBS)
tested to be permissive for B-lineage lymphocyte growth was from
Intergen Co (Purchase, NY). Heparin from two different sources (Sigma,
St Louis, MO, and ICN Pharmaceuticals Inc, Costa Mesa, CA) was found to
have similar activity and data using the Sigma preparation are
presented. Chondroitin sulfate A was from Sigma. Recombinant murine
GM-CSF and IL-7 were from R&D Systems (Minneapolis, MN).
Cell lines.
The cell lines DW34,21 2E8,22 F10 (T. Shimozato
and P.W. Kincade, manuscript in preparation), BC7.12
(derived in our laboratory from BALB/c BM, unpublished),
DA/GM and NFS-60 (responsive to GM-CSF and G-CSF, respectively; kindly
provided by Dr Donna Rennick, DNAX Research Institute, Palo Alto, CA),
FDC-P1,23 and CTLL-2 (clone TIB 214; American Type Culture
Collection [ATCC], Rockville, MD) were maintained in RPMI-1640
complete medium (5% FBS, 2 mmol/L L-glutamine, 100 U/mL penicillin,
100 µg/mL streptomycin, and 5 × 10 5 mol/L
-mercaptoethanol). The rat lymphoma Nb2 (a gift of Dr Li-Yuan
Yu-Lee, Baylor College of Medicine, Houston, TX) was maintained in
Fischer's medium containing 10% horse serum, 104
mol/L -mercaptoethanol, 0.5% gentamicin, 2 mmol/L L-glutamine. Factor-dependent cell lines were maintained in the excess of purified recombinant (r) GM-CSF, conditioned medium (CM) from IL-7-transfected NIH-3T3 cells, CM from IL-3-transfected Chinese hamster ovary (CHO)
cells, or lung CM as a source of G-CSF24 as indicated (see
Fig 2).
Long-term BM cultures (LTBMCs).
Whole BM (WBM) cells were cultured under
lymphoid-permissive25 or myeloid-permissive26
conditions. In lymphoid-supportive cultures, WBM cells resuspended in
RPMI-1640 complete medium were aliquotted at 8.0 × 106 cells/6 mL/flask into 25-cm2 tissue culture
flasks (Costar Corp, Cambridge, MA) and cultured at 37°C in 7%
CO2. For establishment of myeloid-supportive cultures, WBM
cells resuspended in -MEM containing 20% horse serum,
106 mol/L hydrocortisone sodium succinate, 100 U/mL
penicillin, and 100 µg/mL streptomycin were aliquotted at 12.0 × 106 cells/8 mL/flask and maintained at 33°C in
5% CO2. For establishment of switch
cultures,27 flasks maintained under myeloid-supportive conditions for 5 weeks were gently rinsed and the medium replaced with
lymphoid-supportive medium. Cultures were subsequently maintained under
lymphoid-permissive conditions. In each LTBMC system, half of the
medium was replaced with fresh medium weekly. Heparin or chondroitin
sulfate A was added at the weekly feedings as described in the figure
legends. Lineage identity of nonadherent cells was confirmed using
fluorescently labeled antibodies specific to Gr-1 for
myeloid-permissive cultures and CD19 or CD45 for lymphoid-permissive cultures (data not shown). Viability in all cultures was typically greater than 98%.
Removal of cell-surface HS.
Digestion with heparitinase (Seikagaku, Ijamsville, MD) was performed
as previously described28 except that the reaction was
performed in PBS (pH 7.0) containing 0.1% bovine serum albumin (BSA).
The efficacy of heparitinase treatment was confirmed by flow cytometric
analysis using the monoclonal antibodies (MoAbs) 10E4 and 3G10 (both
from Seikagaku), which react with native HS and heparitinase-digested
HS, respectively.29 SB/14 (rat IgG2a), a newly
characterized antibody to the IL-7 receptor chain (Y. Yamashita and
P.W. Kincade, manuscript in preparation), was similarly used to analyze treated cells.
Immunofluorescence analysis of IL-7 binding.
Staining of cells with biotinylated IL-7 (kit no. NF700; R&D Systems)
was performed according to the manufacturer's instructions. In brief,
washed cells were incubated with biotinylated IL-7 or the negative
control reagent (biotinylated soybean trypsin inhibitor) for 45 minutes
followed by incubation with avidin-fluorescein for another 30 minutes
on ice. The specificity of biotinylated IL-7 was confirmed using the
supplied neutralizing antibody. We also determined that biotinylated
IL-7 retained biological activity as measured by the capacity to
stimulate proliferation of F10 lymphocytes (data not shown). Identical
procedures were followed for staining with biotinylated IL-2 (kit no.
NF200; R&D Systems).
Proliferation assays.
WBM cells resuspended in RPMI-1640 complete medium or factor-dependent
cells resuspended in the complete medium used to maintain each line
were aliquotted at 1.0 to 2.0 × 104 cells/well to
96-well plates. Nonsaturating amounts of growth factor (as determined
in titration experiments for each cell population) were pre-incubated
with heparin for 30 minutes at room temperature after which the mixture
was added to wells, in triplicate. The final concentration of heparin
was 100 µg/mL in a total volume of 200 µL/well.
For proliferation experiments involving heparitinase, mock- or
enzyme-digested cells were resuspended in RPMI-1640 complete medium and
aliquotted at 1.0 to 2.0 × 104 cells/well. Fresh
heparitinase (0.01 U/mL) or vehicle was added in the presence or
absence of growth factor to a final volume of 100 µL. Flow cytometric
analysis of a separate aliquot of cells confirmed the continued
efficacy of heparitinase for at least 50 hours (data not shown).
In all assays, plates were incubated at 37°C in 7% CO2
for 3 days. [3H]thymidine (65 Ci/mmol; ICN
Pharmaceuticals Inc) was added at 1.0 µCi/well during the last 6 hours of incubation. Incorporated radioactivity was detected using a
Beckman LS 6000SE liquid scintillation counter.
Determination of soluble sulfated glycosaminoglycan.
Confluent monolayers of BMS2,22 BMS2.4,30 or
OP4231 stromal cells were cultured for 5 to 7 days in
RPMI-1640 complete medium. Supernatant was collected, passed through a
0.2-µm filter to remove debris, and concentrated 10× using a
spin freezer concentrator. Samples were assayed for sulfated
glycosaminoglycan content using the Blyscan kit (Accurate Chemical & Scientific Corp, Westbury, NY) as described by the manufacturer except
that the assay was scaled to 300 µL final volume. Aliquots of
concentrated culture supernatant or concentrated RPMI-1640 complete
medium were added to 300 µL of the Blyscan dye reagent and vortexed
for 30 minutes. In some experiments, before addition of the Blyscan
dye, concentrated supernatants were treated with 0.1 U/mL heparitinase
for 30 minutes at 37°C after which another 0.1 U/mL heparitinase
was added and the incubation repeated. Mock-treated controls received
diluent during the incubation period. Samples were centrifuged at
8,000g for 10 minutes and the pellet solubilized in 300 µL of
the dissociation reagent. After 30 minutes of vortexing optical density
was read at 656 nm. The glycosaminoglycan concentration of samples was determined using a standard curve established with
chondroitin-4-sulfate.
Bioassay of stromal cell-bound IL-7.
Stromal cells derived from IL-7 knockout mice (kindly provided by Dr
Pamela Witte, Loyola University Medical Center, Maywood, IL) were
resuspended in RPMI-1640 complete medium and seeded at  5.0 × 104/0.4 mL/well in 24-well tissue-culture plates. Cultures
were incubated at 37°C in 7% CO2 for 1 day before use.
Adherent stromal cells treated with or without heparitinase as
described above were then incubated with purified murine rIL-7 (5 ng/well) for 30 minutes at 37°C followed by gentle washing to
remove unbound cytokine. BC7.12 pro-B cells were added at 2 × 104 cells/well and cultures were incubated at 37°C in
7% CO2 for 3 days after which viable lymphocytes were
enumerated by trypan blue exclusion.
| |
RESULTS |
Heparin abrogates lymphopoiesis but not myelopoiesis in LTBMC.
The capacity of heparin/HS to interact with essential growth factors
such as IL-7 and the CSFs points to a specific mechanism by which these
glycans can participate in hematopoiesis. Therefore, we investigated
the ability of heparin to impact hematopoietic development using LTBMCs
under conditions described for lymphopoiesis (Whitlock-Witte
type)25 or myelopoiesis (Dexter type).26 LTBMCs established from BALB/c BM were treated with heparin (50 µg/mL) beginning at culture initiation and thereafter weekly. While control (vehicle alone) Whitlock-Witte cultures commenced lymphocyte production by 3 weeks, heparin-treated flasks did not produce detectable numbers
of lymphocytes by 5 weeks (Fig 1A, left
panel, solid lines). Lymphocyte development was not perturbed in LTBMCs
treated with an equivalent amount of chondroitin sulfate A, a GAG shown
not to block IL-7 bioactivity.8,9 In contrast to the
inhibitory effect on lymphocyte outgrowth, heparin did not impair
establishment of myeloid-permissive cultures (Fig 1A, left panel,
dashed lines).
View larger version (16K):
[in this window]
[in a new window]
View larger version (74K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
| Fig 1.
Effect of heparin on lymphopoiesis and myelopoiesis in
LTBMCs. (A, left) WBM cells were seeded using lymphoid-supportive
(solid line) or myeloid-supportive (dashed line) conditions as
described in Experimental Procedures. Heparin ( ; 50 µg/mL),
chondroitin sulfate ( ; 50 µg/mL), or vehicle ( ) was added
weekly beginning at culture initiation. (Right) LTBMCs cultured under
myeloid-permissive conditions for 5 weeks were switched (arrow) to
lymphoid-permissive conditions. Heparin (50 µg/mL) or vehicle was
added weekly throughout the entire assay. (B) LTBMCs maintained under
lymphoid-supportive conditions were cultured in the absence (left) or
presence (right) of 25 µg/mL heparin for 6 weeks. Original
magnification × 100. (C) Lymphoid-permissive LTBMCs were established
for 4 to 6 weeks in the absence of treatment and then treated weekly
with 50 µg/mL heparin or vehicle. The data are presented relative to
the number of lymphocytes recovered from flasks receiving vehicle alone
and results from three independent experiments are shown.
|
|
As shown in Fig 1B, Whitlock-Witte LTBMCs receiving heparin were
completely devoid of lympho-hematopoietic foci. Despite the paucity of
B-cell precursors, microscopic examination of heparin-treated cultures
showed a normal adherent cell monolayer. This adherent layer was found
to be competent to support propagation of foster lymphocytes, albeit at
a reduced level (data not shown).
The differential effects of heparin on lymphopoiesis versus
myelopoiesis were even more striking in a switch culture. In this culture system, BM cells propagated under myeloid-permissive conditions for 5 weeks were subsequently switched to lymphoid-supportive conditions. Heparin (50 µg/mL) or vehicle was added weekly throughout the entire experiment. Although heparin treatment had no discernible effect on myelopoiesis, lymphopoiesis in those same cultures was completely inhibited (Fig 1A, right panel). These data show that soluble heparin inhibits B lymphopoiesis in LTBMCs.
Temporal effects of heparin on lymphopoiesis.
Although heparin inhibited lymphopoiesis in flasks treated with this
GAG beginning at the first week of culture (Fig 1A and B), heparin did
not block LTBMCs that were well established before treatment. LTBMCs
cultured for 4 to 6 weeks before commencement of weekly heparin
treatment had normal-to-elevated levels of lymphopoiesis (Fig 1C).
Heparin likely affects several qualitative aspects of the cell layer
and the consequence to lymphopoiesis may vary, for example, according
to whether the marrow is well established or remodeling (eg, during
fetal development or regenerating after wounding).
Differential effects of heparin on factor-dependent proliferation.
In addition to lymphocytes and myeloid cells, hematopoietic tissue
supports the outgrowth of several other cytokine-dependent lineages. We
explored the effects of heparin using a panel of factor-responsive
cells of hematopoietic origin (Fig 2). WBM
cells or the indicated cell lines were stimulated with limiting amounts of growth factor in the presence of heparin or vehicle. While heparin
blocked the growth of each IL-7-dependent population tested, this GAG
did not noticeably affect the proliferation of cells dependent on
several other growth factors including IL-3, G-CSF, GM-CSF, and
lactogen. These data point to a regulatory effect of heparinlike GAGs
that may be specific to IL-7.
View larger version (25K):
[in this window]
[in a new window]
| Fig 2.
Comparison of heparin effects on growth factor-dependent
cells. WBM (105 cells/well) or cell lines (104
cells/well) were stimulated with the indicated growth factors in the
presence or absence of 100 µg/mL heparin and proliferation was
assessed by [3H]thymidine incorporation. Growth factors
were added in an amount that produced half-maximal stimulation for each
population. The data are presented as mean ± SD of triplicate
wells.
|
|
IL-7 binds to native cell surface HS on B-cell precursors.
A prominent reservoir of heparinlike GAGs exists tethered to the plasma
membrane of BM cells in the form of HS.32,33 Cell-surface HS present on lymphocytes and stromal cells could potentially participate in regulation of IL-7 bioavailability/bioactivity. To
directly examine whether this cytokine interacts with GAGs on
B-lymphocyte precursors, plasma membrane HS was destroyed using heparitinase. The efficacy of enzyme treatment was assessed using the
MoAbs 10E4 and 3G10 which recognize native HS and a neo-epitope revealed by heparitinase treatment, respectively. Before enzymatic digestion, 60% of F10 lymphocytes were stained by 10E4 while less than
1% displayed the 3G10 epitope. After digestion, 99% of this population was stained by 3G10 indicating essentially complete removal
of HS (Fig 3A and B).
View larger version (30K):
[in this window]
[in a new window]
| Fig 3.
Immunofluorescence analysis of cytokine binding to
lymphocyte cell-surface HS. (A) Mock- (control) or (B)
heparitinase-digested (+ h'ase) F10 cells were stained and analyzed
by two-color flow cytometry. Biotinylated 10E4 (followed by
streptavidin-red 613) recognizes native cell-surface HS while 3G10
(FITC) detects heparitinase-cleaved HS. (C) The capacity of these
B-cell precursors to bind biotinylated IL-7 or the negative control
reagent, biotinylated soybean trypsin inhibitor (STI), followed by
avidin-fluorescein, was subsequently assessed. (D) Binding of
biotinylated IL-2 to control or heparitinase-treated CTLL-2 cells; the
binding profiles directly overlap. (E) Control or heparitinase-digested
F10 lymphocytes were stained with an antibody to the IL-7 receptor
chain (SB/14) or rat IgG2a followed by goat anti-rat
Ig-FITC.
|
|
The bright staining of control F10 pro-B cells by biotinylated IL-7 was
significantly diminished after digestion with heparitinase (Fig 3C).
Heparitinase treatment similarly reduced IL-7 binding to BC7.12 (pro-B)
and DW34 (pre-B) cells, as well as primary B-lymphocyte precursors
derived from Whitlock-Witte LTBMCs (Table
1). Despite the dramatic decrease in IL-7 binding, expression of the
IL-7 receptor chain on F10 cells was only slightly diminished after digestion with heparitinase (Fig 3E). Heparitinase treatment did not
dramatically affect expression of the CD19, CD45, CD43, BP-1, or M1/69
antigens, confirming the specificity of this enzyme for HS (data not
shown). In contrast to effects on IL-7 binding, the capacity of
biotinylated IL-2 to interact with CTLL-2 cells was not affected by
heparitinase (Fig 3D). These data suggest that membrane-associated HS
may play an essential role in IL-7-dependent interactions.
Loss of cell-surface HS diminishes responsiveness to IL-7.
A reduction in the capacity of lymphocytes to bind to IL-7 may have
direct biological implications. To investigate if the interaction of
IL-7 with cell-surface HS is necessary for efficient cytokine
stimulation, we examined the capacity of lymphocytes which lack plasma
membrane HS to respond to limiting amounts of IL-7. F10 cells treated
in the presence or absence of heparitinase were cultured for 3 days
with IL-7 and proliferation was assessed (Fig 4, solid
bars). Digestion with heparitinase reduced IL-7-driven lymphocyte
proliferation 50% to 75% compared with controls, a magnitude of
inhibition similar to the effects of heparin. By contrast, heparitinase
treatment had no discernible effect on the IL-3-dependent responses of
FDC-P1 cells (Fig 4, hatched bars). These data directly demonstrate the
importance of cell-surface HS to IL-7-dependent stages of B
lymphopoiesis.
View larger version (18K):
[in this window]
[in a new window]
| Fig 4.
Cytokine responsiveness of heparitinase-treated
hematopoietic cell lines. Mock- or heparitinase-digested F10 or FDC-P1
cells were cultured with the indicated growth factor in the presence or
absence of heparin (100 µg/mL) and proliferation was assessed after 3 days by [3H]thymidine incorporation. The data are
reported as the average ± SD of triplicate samples and are
representative of five (F10) or two (FDC-P1) independent experiments.
|
|
IL-7 binds to native cell-surface HS on stromal cells.
Stromal cells may support B lymphopoiesis in part by regulating local
growth factor availability, for example, through selective compartmentalization.11,12 GAGs tethered to the plasma
membrane as well as those released by stromal cells have the potential to regulate bioavailability of IL-7. Sulfated GAGs were detected in the
CM of several different stromal cell lines (µg/mL sulfated GAG:
cell-free medium, 0.29 ± 0.24; OP42 CM, 3.52 ± 0.06; BMS2.4 CM,
4.79 ± 0.14; BMS2 CM, 4.23 ± 0.03). However, only
20% to 30% was sensitive to heparitinase (heparitinase-digested BMS2
CM, 3.02 ± 0.18). Therefore, we focused on the potential of IL-7 to interact with membrane-associated HS.
Fluorescence analysis showed that biotinylated IL-7 readily bound to
BM-derived lymphocyte-supportive stromal cells (ie, BMS2 and OP42)
(Table 1). By contrast, the myeloid cell line FDC-P1 was not detectably
stained by IL-7. These results are particularly striking in that
neither BMS2 nor OP42 expresses the IL-7 receptor chain (Fig 5A,
and data not shown), and point to a unique regulatory role for GAGs on
the surface of these stromal cells. However, the binding of IL-7 to
stroma appears to be of low avidity as much of the biotin-labeled
cytokine could be removed by extensive washing (data not shown).
To directly confirm that cell-surface HS enabled IL-7 binding to
stroma, BMS2 cells were treated with heparitinase. Before enzymatic
treatment, BMS2 cells displayed a bimodal binding profile when stained
with biotinylated IL-7 while subsequent to digestion, only a single dim
fluorescent peak was observed (Fig 5B). A
similar reduction in the capacity to bind IL-7 was observed upon
heparitinase digestion of the OP42 stromal cell line (Table 1). The
specificity of the heparitinase was confirmed in that no significant
changes in CD44 or CD9 expression were detected upon treatment with
this enzyme (data not shown).
View larger version (17K):
[in this window]
[in a new window]
| Fig 5.
Immunofluorescence analysis of IL-7 binding to stromal
cell plasma membrane HS. (A) BMS2 stromal cells were stained with an
antibody (SB/14) to the IL-7 receptor chain or rat
IgG2a followed by goat anti-rat Ig-FITC; the binding
profiles directly overlap. (B) Mock- (control) or heparitinase-digested
(+ h'ase) BMS2 cells were stained with biotinylated IL-7 or the
negative control reagent (STI) as described in the legend to Fig 3.
Control BMS2 cells displayed a bimodal profile (rightmost peaks).
Binding of biotinylated IL-7 was readily blocked using a neutralizing
antibody to IL-7 and efficacy of the digestion procedure was confirmed
using the 10E4 and 3G10 MoAbs (data not shown).
|
|
Stromal cell-bound IL-7 stimulates B-lymphocyte
precursors.
The bioactivity of stromal cell-bound IL-7 was assessed using the
IL-7-dependent indicator cell line BC7.12. Stromal cells were
incubated with 5 ng of purified rIL-7, gently washed to remove unbound
cytokine, and cocultured with BC7.12 pro-B lymphocytes. Stromal cells
that had been washed of excess IL-7 retained low but detectable amounts
of bioactive cytokine (Fig 6). After 3 days
of culture, 61,000 ± 4,500 BC7.12 lymphocytes were recovered in
wells containing mock-digested IL-7-laden stroma versus only 37,000 ± 4,100 in wells containing mock-digested vehicle-treated stroma
(P = .006; mean ± SD of four independent experiments). This
capacity of stromal cells to retain bioactive IL-7 was significantly reduced by treatment with heparitinase (49,500 ± 6,400 BC7.12 lymphocytes; P = .0347 v mock-digested controls).
Maximal proliferation was obtained in cultures of BC7.12 lymphocytes
incubated with stroma and IL-7 in the absence of washing, indicating
that only a fraction of the available soluble cytokine is bound by the
stromal cell plasma membrane. These data indicate that the low avidity interaction of IL-7 with stromal cells is partially dependent on
cell-surface HS.
View larger version (15K):
[in this window]
[in a new window]
| Fig 6.
Bioactivity of stromal cell-bound IL-7. Stromal cells
(5 × 104/well) derived from IL-7 knockout mice were
treated in the presence or absence of heparitinase and susbequently
incubated with rIL-7 (5 ng/well) for 30 minutes at 37°C. Cells were
washed to remove unbound IL-7 and then 2 × 104 BC7.12
lymphocytes were added to each well. Cultures were incubated for 3 days
after which viable lymphocytes were enumerated. To assess maximal IL-7
bioactivity, the washing step was omitted in one treatment group
(central bar). The results are presented as the average ± SD of four
independent experiments.
|
|
| |
DISCUSSION |
Local availability of IL-7 reflects not only synthesis and stability of
this growth factor but also compartmentalization to specialized sites
within the BM. Such niches are shaped by the molecules displayed on
stroma and stroma-derived matrix, as well as those present on
hematopoietic cells. We examined the ability of heparinlike molecules
to regulate the bioavailability and bioactivity of IL-7. We
demonstrated lineage-specific effects of heparin manipulation in
long-term culture of normal lympho-hematopoietic cells. In addition, we
specifically focused on the role of native HSPGs on the plasma membrane
of BM cells. Not only did HSPGs present on both B-lymphocyte
progenitors and lymphocyte-supportive stroma direct binding of IL-7 to
the cell surface, but this association was critical for efficient
proliferative stimulation of B-cell precursors. This study establishes
a direct role for HS in regulating the IL-7-dependent stages of B
lymphopoiesis.
Heparinlike GAGs have been shown to serve as coreceptors for targeting
growth factors such as FGF to plasma membrane
receptors.15,34 In this case, interaction with GAGs is
essential for binding of FGF to its high-affinity receptor. That HSPGs
may similarly participate in directing IL-7 to the plasma membrane of
target cells is suggested by two lines of evidence: the competitive
affinity of IL-7 for heparin and heparan sulfate (kd of 25 nmol/L and 70 to 80 nmol/L,9 respectively, v kd of
6 to 11 nmol/L35 for binding of IL-7 to the IL-7 receptor),
and the ability of each GAG to regulate IL-7 bioactivity. Our
observations directly demonstrate that cell-surface HS is required for
efficient labeling of B-cell precursors by IL-7. Removal of plasma
membrane HS was associated with a dramatic inhibition of IL-7 binding
and a consequent reduction in proliferative stimulation. There is some
size variation in the IL-7 receptor that might be
glycosylation-dependent,35 but HS has not been reported on
the or chains of the IL-7 receptor. During development in the
BM, B-lineage precursors express different proteoglycans at different
stages of development. For example, the HSPG syndecan-1 is expressed on
B-cell precursors but is lost before extravasation to the peripheral
compartment.36 Therefore, we consider it possible that
HSPGs on B-lymphocyte precursors and/or stromal cells
participate in IL-7-dependent events.
Positioning or "compartmentalization" of growth factors in the BM
microenvironment is recognized to directly influence hematopoiesis. For
example, localization of GM-CSF to GAGs on marrow-derived extracellular
matrix drives granulocyte-macrophage colony formation,12 and adsorption of GM-CSF and IL-3 on matrix HS serves to present these
factors in biologically active form to multipotent
precursors.11 Studies with T-lymphocyte lineage cells
indicate that matrix immobilized IL-7 can deliver proadhesive
signals,37,38 as can hepatocyte growth factor and
MIP-1.39,40 Our finding that heparinlike molecules
enable binding of IL-7 not only to B-cell precursors, but also to
BM-derived stromal cells implies an additional mechanism by which
specific microenvironments may be generated at the plasma membrane.
HSPG-dependent targeting of cytokines to the surface of
lymphocyte-supportive stromal cells may enable immobilization and presentation of specific growth factors to dependent hematopoietic cells. We found that IL-7 could bind to marrow-derived stromal cells
(OP42, BMS2) as well as adherent cells derived from other tissues (TEC,
NIH-3T3). These data correlate with our previous observations that
several types of adherent cells could support the short-term survival
of B-lymphocyte precursors,41 and suggest one mechanism by
which these positive effects might occur. Interestingly, BMS2.4, a cell
line shown to suppress the outgrowth of B-cell precursors,30,41 similarly binds IL-7. It will be
interesting to determine if these stromal cells have an equivalent
capacity to present IL-7 to dependent B-cell precursors or if, in some instances, the stroma-bound IL-7 is retained in a form unavailable to
lymphocytes.
That the interaction of IL-7 with heparinlike molecules is unique is
suggested by our observations that (1) heparin abrogates lymphopoiesis
but not myelopoiesis in LTBMCs; (2) heparin inhibits IL-7-dependent
proliferation but not that mediated by other hematopoietic cytokines
including IL-3, G-CSF, and GM-CSF; (3) removal of HS with heparitinase
blocks binding of IL-7 but not, for example, IL-2 to the plasma
membrane; and (4) cells lacking HS have significantly diminished
proliferative responses to IL-7 whereas responses to IL-3 are
unaffected. It is important to note that while our studies used soluble
heparin as a surrogate GAG, the specificity of the interaction between
IL-7 and heparinlike molecules may be further refined in the marrow by
local expression of different species of
proteoglycans6,33,42,43 as well as by the fine structure of
HS on each proteoglycan core. The MS-5 murine stromal cell, for
example, synthesizes at least seven different HSPGs including syndecans.33 The proteoglycan core of syndecans can be
uniquely sulfated by individual cells in a manner that alters the
functional capacity of the molecule.44 These differences
may determine whether the interaction of IL-7 with a particular
cell-surface GAG is inhibitory, enhancing, or neutral to cytokine
bioavailability/bioactivity.
In this study we defined the contribution of heparinlike molecules to
the IL-7-dependent stages of B-cell development and described one way
in which specific microenvironments may be created within the BM.
Clearly, the potential exists not only for HSPGs to regulate additional
hematopoietic processes within the marrow, but also for such mechanisms
to operate in other tissue compartments.
| |
ACKNOWLEDGMENT |
We thank Dr Ralph Sanderson for helpful advice on removing cell-surface
heparan sulfate; Viji Dandapandi and John Rummage for excellent
technical work; and Shelli Wasson for secretarial assistance.
| |
FOOTNOTES |
Submitted June 5, 1998;
accepted September 2, 1998.
Supported by Grants No. AI20069 and AI33085 from the National
Institutes of Health awarded to P.W.K. L.A.B. was supported by Training
Grant No. HL07207-21.
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 Paul W. Kincade, PhD, Immunobiology and
Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th St,
Oklahoma City, OK 73104; e-mail: paul-kincade{at}omrf.ouhsc.edu.
| |
REFERENCES |
1.
Siczkowski M, Clarke D, Gordon M:
Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.
Blood
80:912, 1992[Abstract/Free Full Text]
2.
Gupta P, McCarthy JB, Verfaillie CM:
Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.
Blood
87:3229, 1996[Abstract/Free Full Text]
3.
Coombe DR, Watt SM, Parish CR:
Mac-1 (CD11b/CD18) and CD45 mediate the adhesion of hematopoietic progenitor cells to stromal cell elements via recognition of stromal heparan sulfate.
Blood
84:739, 1994[Abstract/Free Full Text]
4.
Watt SM, Williamson J, Genevier H, Fawcett J, Simmons DL, Hatzfeld A, Nesbitt SA, Coombe DR:
The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes.
Blood
82:2649, 1993[Abstract/Free Full Text]
5.
Hueber A-O, Pierres M, He H-T:
Sulfated glycans directly interact with mouse Thy-1 and negatively regulate Thy-1-mediated adhesion of thymocytes to thymic epithelial cells.
J Immunol
148:3692, 1992[Abstract]
6.
Luikart S, Kenney P, Oregema T:
Human bone marrow heparan sulfate induces leukemia cell differentiation.
Connect Tissue Res
31:99, 1995
7.
Oritani K, Kincade PW:
Identification of stromal cell products that interact with pre-B cells.
J Cell Biol
134:771, 1996[Abstract/Free Full Text]
8.
Kimura K, Matsubara H, Sogoh S, Kita Y, Sakata T, Nishitani Y, Watanabe S, Hamaoka T, Fujiwara H:
Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor.
J Immunol
146:2618, 1991[Abstract]
9.
Clarke D, Katoh O, Gibbs R, Griffiths S, Gordon M:
Interaction of interleukin 7 (IL-7) with glycosaminoglycans and its biological relevance.
Cytokine
7:325, 1995[Medline]
[Order article via Infotrieve]
10.
Ramsden L, Rider CC:
Selective and differential binding of interleukin (IL)-1, IL-1, IL-2 and IL-6 to glycosaminoglycans.
Eur J Immunol
22:3027, 1992[Medline]
[Order article via Infotrieve]
11.
Roberts R, Gallagher J, Spooncer E, Allen T, Bloomfield F, Dexter T:
Heparan sulphate bound growth factors: A mechanism for stromal cell mediated haemopoiesis.
Nature
332:376, 1988[Medline]
[Order article via Infotrieve]
12.
Gordon MY, Riley GP, Watt SM, Greaves MF:
Compartmentalization of a haemopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment.
Nature
326:403, 1987[Medline]
[Order article via Infotrieve]
13.
McCaffrey T, Falcone DJ, Du B:
Transforming growth factor-1 is a heparin binding-protein: Identification of putative heparin-binding regions and isolation of heparins with varying affinity for TGF-1.
J Cell Physiol
152:430, 1992[Medline]
[Order article via Infotrieve]
14.
Bruno E, Luikart SD, Long MW, Hoffman R:
Marrow-derived heparan sulfate proteoglycan mediates the adhesion of hematopoietic progenitor cells to cytokines.
Exp Hematol
23:1212, 1995[Medline]
[Order article via Infotrieve]
15.
Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM:
Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor.
Cell
64:841, 1991[Medline]
[Order article via Infotrieve]
16.
Steinfeld R, Van Den Berghe H, David G:
Stimulation of fibroblast growth factor receptor-1 occupancy and signaling by cell surface-associated syndecans and glypican.
J Cell Biol
133:405, 1996[Abstract/Free Full Text]
17.
Bernfield M, Hooper KC:
Possible regulation of FGF activity by syndecan, an integral membrane heparan sulfate proteoglycan.
Ann NY Acad Sci
638:182, 1991[Medline]
[Order article via Infotrieve]
18.
Sadir R, Forest E, Lortat-Jacob H:
The heparan sulfate binding sequence of interferon- increased the on rate of the interferon--interferon- receptor complex formation.
J Biol Chem
273:10919, 1998[Abstract/Free Full Text]
19.
Grabstein K, Waldschmidt T, Finkelman F, Hess B, Alpert A, Boiani N, Namen A, Morrissey P:
Inhibition of murine B and T lymphopoiesis in vivo by an anti-interleukin 7 monoclonal antibody.
J Exp Med
178:257, 1993[Abstract/Free Full Text]
20.
Rolink A, Kudo A, Karasuyama H, Kikuchi Y, Melchers F:
Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo.
EMBO J
10:327, 1991[Medline]
[Order article via Infotrieve]
21.
Nishikawa S-I, Ogawa M, Nishikawa S, Kunisada T, Kodama H:
B lymphopoiesis on stromal cell clone: stromal cell clones acting on different stages of B cell differentiation.
Eur J Immunol
18:1767, 1988[Medline]
[Order article via Infotrieve]
22.
Pietrangeli CE, Hayashi S-I, Kincade PW:
Stromal cell lines which support lymphocyte growth: Characterization, sensitivity to radiation and responsiveness to growth factors.
Eur J Immunol
18:863, 1988[Medline]
[Order article via Infotrieve]
23.
Dexter T, Garland J, Scott D, Scolnick E, Metcalf D:
Growth of factor-dependent hemopoietic precursor cell lines.
J Exp Med
152:1036, 1980[Abstract/Free Full Text]
24.
Nicola NA, Metcalf D, Matsumoto M, Johnson GR:
Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells: Identification as granulocyte colony-stimulating factor.
J Biol Chem
258:9017, 1983[Abstract/Free Full Text]
25.
Whitlock C, Witte O:
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.
Dexter T, Allen T, Lajtha L:
Conditions controlling the proliferation of haematopoietic stem cells in vitro.
J Cell Physiol
91:335, 1977[Medline]
[Order article via Infotrieve]
27.
Dorshkind K:
In vitro differentiation of B lymphocytes from primitive hemopoietic precursors present in long-term bone marrow cultures.
J Immunol
136:422, 1986[Abstract]
28.
Stanley MJ, Liebersbach BF, Liu W, Anhalt DJ, Sanderson RJ:
Heparan sulfate-mediated cell aggregation.
J Biol Chem
270:5077, 1995[Abstract/Free Full Text]
29.
David G, Bai XM, Van Der Schueren B, Cassiman J-J, Van Den Berghe H:
Developmental changes in heparan sulfate expression: In situ detection with mAbs.
J Cell Biol
119:961, 1992[Abstract/Free Full Text]
30.
Kincade P, Medina K, Pietrangeli C, Hayashi S-I, Namen A:
Stromal cell lines which support lymphocyte growth II. Characteristics of a suppressive subclone, in
Gupta S
(ed):
Mechanisms of Lymphocyte Activation and Immune Regulation III. New York, NY, Plenum, 1991, p 227.
31.
Smithson G, Medina K, Ponting I, Kincade PW:
Estrogen suppresses stromal cell dependent lymphopoiesis in culture.
J Immunol
155:3409, 1995[Abstract]
32.
Del Rosso M, Cappelletti R, Dini G, Fibbi G, Vannucchi S, Chiarugi V, Guazzelli C:
Involvement of glycosaminoglycans in detachment of early myeloid precursors from bone-marrow stromal cells.
Biochim Biophys Acta
676:129, 1981[Medline]
[Order article via Infotrieve]
33.
Drzeniek Z, Siebertz B, Stöcker G, Just U, Ostertag W, Greiling H, Haubeck H-D:
Proteoglycan synthesis in hematopoietic cells: Isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5.
Biochem J
327:473, 1997
34.
Rapraeger AC, Krufka A, Olwin BB:
Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation.
Science
252:1705, 1991[Abstract/Free Full Text]
35.
Goodwin RG, Friend D, Ziegler SF, Jerzy R, Falk BA, Gimpel S, Cosman D, Dower SK, March CJ, Namen AE, Park LS:
Cloning of the human and murine interleukin-7 receptors: Demonstration of a soluble form and homology to a new receptor superfamily.
Cell
60:941, 1990[Medline]
[Order article via Infotrieve]
36.
Sanderson RD, Lalor P, Bernfield M:
B lymphocytes express and lose syndecan at specific stages of differentiation.
Cell Regul
1:27, 1989[Medline]
[Order article via Infotrieve]
37.
Kitazawa H, Muegge K, Badolato R, Wang J-M, Fogler WE, Ferris DK, Lee C-K, Candéias S, Smith MR, Oppenheim JJ, Durum SK:
IL-7 activates 41 integrin in murine thymocytes.
J Immunol
159:2259, 1997[Abstract/Free Full Text]
38.
Ariel A, Hershkoviz R, Cahalon L, Williams DE, Akiyama SK, Yamada KM, Chen C, Alon R, Lapidot T, Lider O:
Induction of T cell adhesion to extracellular matrix of endothelial cell ligands by soluble or matrix-bound interleukin-7.
Eur J Immunol
27:2562, 1997[Medline]
[Order article via Infotrieve]
39.
Adams DH, Harvath L, Bottaro DP, Interrante R, Catalano G, Tanaka Y, Strain A, Hubscher SG, Shaw S:
Hepatocyte growth factor and macrophage inflammatory protein 1: Structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.
Proc Natl Acad Sci USA
91:7144, 1994[Abstract/Free Full Text]
40.
Tanaka Y, Adams DH, Hubscher S, Hirano H, Siebenlist U, Shaw S:
T-cell adhesion induced by proteoglycan-immobilized cytokine MIP-1.
Nature
361:79, 1993[Medline]
[Order article via Infotrieve]
41.
Borghesi L, Smithson G, Kincade PW:
Stromal cell modulation of negative regulatory signals that influence apoptosis and proliferation of B lineage lymphocytes.
J Immunol
159:4171, 1997[Abstract]
42.
Wight T, Kinsella MG, Keating A, Singer JW:
Proteoglycans in human long-term bone marrow cultures: Biochemical and ultrastructural analysis.
Blood
67:1333, 1986[Abstract/Free Full Text]
43.
Morris A, Turnbull J, Riley G, Gordon M, Gallagher J:
Production of heparan sulphate proteoglycans by human bone marrow stromal cells.
J Cell Sci
99:149, 1991[Abstract/Free Full Text]
44.
Sanderson RD, Turnbull JE, Gallagher JT, Lander AD:
Fine structure of heparan sulfate regulates syndecan-1 function and cell behavior.
J Biol Chem
269:13100, 1994[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
E. Faller, J. Kakal, R. Kumar, and P. MacPherson
IL-7 and the HIV Tat protein act synergistically to down-regulate CD127 expression on CD8 T cells
Int. Immunol.,
March 1, 2009;
21(3):
203 - 216.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. den Dekker, S. Grefte, T. Huijs, G. B. ten Dam, E. M. M. Versteeg, L. C. J. van den Berk, B. A. Bladergroen, T. H. van Kuppevelt, C. G. Figdor, and R. Torensma
Monocyte Cell Surface Glycosaminoglycans Positively Modulate IL-4-Induced Differentiation toward Dendritic Cells
J. Immunol.,
March 15, 2008;
180(6):
3680 - 3688.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. D. Milne, S. A. Corfe, and C. J. Paige
Heparan Sulfate and Heparin Enhance ERK Phosphorylation and Mediate preBCR-Dependent Events during B Lymphopoiesis
J. Immunol.,
March 1, 2008;
180(5):
2839 - 2847.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Chung, E. P. Dudl, D. Min, L. Barsky, N. Smiley, and K. I. Weinberg
Prevention of graft-versus-host disease by anti IL-7R{alpha} antibody
Blood,
October 15, 2007;
110(8):
2803 - 2810.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Fluur, A. De Milito, T. J. Fry, N. Vivar, L. Eidsmo, A. Atlas, C. Federici, P. Matarrese, M. Logozzi, E. Rajnavolgyi, et al.
Potential Role for IL-7 in Fas-Mediated T Cell Apoptosis During HIV Infection
J. Immunol.,
April 15, 2007;
178(8):
5340 - 5350.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Lai, R. A. Zeff, and I. Goldschneider
A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells
Blood,
March 1, 2006;
107(5):
1776 - 1784.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. E. Johnson, N. Shah, A. Panoskaltsis-Mortari, and T. W. LeBien
Murine and Human IL-7 Activate STAT5 and Induce Proliferation of Normal Human Pro-B Cells
J. Immunol.,
December 1, 2005;
175(11):
7325 - 7331.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Bradl, J. Wittmann, D. Milius, C. Vettermann, and H.-M. Jack
Interaction of Murine Precursor B Cell Receptor with Stroma Cells Is Controlled by the Unique Tail of {lambda}5 and Stroma Cell-Associated Heparan Sulfate
J. Immunol.,
September 1, 2003;
171(5):
2338 - 2348.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Oritani, S. Hirota, T. Nakagawa, I. Takahashi, S.-i. Kawamoto, M. Yamada, N. Ishida, T. Kadoya, Y. Tomiyama, P. W. Kincade, et al.
T lymphocytes constitutively produce an interferonlike cytokine limitin characterized as a heat- and acid-stable and heparin-binding glycoprotein
Blood,
January 1, 2003;
101(1):
178 - 185.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kouro, V. Kumar, and P. W. Kincade
Relationships between early B- and NK-lineage lymphocyte precursors in bone marrow
Blood,
November 15, 2002;
100(10):
3672 - 3680.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Iwata, L. Graf, N. Awaya, and B. Torok-Storb
Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein
Blood,
July 30, 2002;
100(4):
1318 - 1325.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-Q. Xia and K.-J. Kao
Heparin Induces Differentiation of CD1a+ Dendritic Cells from Monocytes: Phenotypic and Functional Characterization
J. Immunol.,
February 1, 2002;
168(3):
1131 - 1138.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Bechard, T. Gentina, M. Delehedde, A. Scherpereel, M. Lyon, M. Aumercier, R. Vazeux, C. Richet, P. Degand, B. Jude, et al.
Endocan Is a Novel Chondroitin Sulfate/Dermatan Sulfate Proteoglycan That Promotes Hepatocyte Growth Factor/Scatter Factor Mitogenic Activity
J. Biol. Chem.,
December 14, 2001;
276(51):
48341 - 48349.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Shah, L. Oseth, H. Tran, B. Hirsch, and T. W. LeBien
Clonal Variation in the B-Lineage Acute Lymphoblastic Leukemia Response to Multiple Cytokines and Bone Marrow Stromal Cells
Cancer Res.,
July 1, 2001;
61(13):
5268 - 5274.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Kouro, K. L. Medina, K. Oritani, and P. W. Kincade
Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators
Blood,
May 1, 2001;
97(9):
2708 - 2715.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Salek-Ardakani, J. R. Arrand, D. Shaw, and M. Mackett
Heparin and heparan sulfate bind interleukin-10 and modulate its activity
Blood,
September 1, 2000;
96(5):
1879 - 1888.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Yamashita, K. Oritani, E. K. Miyoshi, R. Wall, M. Bernfield, and P. W. Kincade
Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes
J. Immunol.,
May 15, 1999;
162(10):
5940 - 5948.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
