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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 75-88
Impaired Ability of Bone Marrow Stromal Cells to
Support B-Lymphopoiesis With Age
By
Robert P. Stephan,
Colette R. Reilly, and
Pamela L. Witte
From the Departments of Cell Biology, Neurobiology, and Anatomy and
Microbiology and Immunology, Loyola University Chicago, Maywood, IL.
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ABSTRACT |
B-lymphopoiesis decreases with age. We studied how aging affects
bone marrow stromal cells, because they provide the growth factors and
cell contacts required for B-lymphopoiesis. No differences were noted
in the cell-surface phenotype of young and old primary-cultured stromal
cells. Fluorescence-activated cell sorter-purified
stromal cells from old mice were deficient in the ability to support
the proliferation of interleukin-7 (IL-7)-specific B-lymphoid cell
lines. The kinetics of this response indicated that IL-7 was not
immediately available from stromal cells of either age and was further
delayed on aged stromal cells. The levels of IL-7 protein within
stromal cells were equivalent between young and old animals, suggesting
that the production of IL-7 was not altered by aging. Negligible
amounts of IL-7 were found either freely secreted or in the
extracellular matrix of cultures of young and old marrow. Contact
between the lymphoid cells and the primary stromal cells was required
for detectable proliferation, suggesting that cell contact was required
for the release of IL-7. We propose that stromal cells regulate
B-lymphopoiesis by limiting the amount of IL-7 available to the
developing precursors. Therefore, we conclude that the age-related
decrease in the function of bone marrow stromal cells is related to the
impaired release of IL-7.
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INTRODUCTION |
BONE MARROW stromal cells have been
called "the essential cells of the hematopoietic
microenvironment" and the "keys to hematopoietic
development."1,2 Stromal cell lines have been a rich
source for the discovery of new growth factors and
cytokines.2 It has been assumed that stromal cells regulate
hematopoiesis by controlling the availability of these growth factors
to the developing precursors. However, the hypothesis that stromal
cells regulate hematopoiesis has not been thoroughly tested and the
mechanism of this regulation is unknown. Stromal cell lines can be
induced to increase the transcription of certain growth
factors.2 It is also possible that stromal cells have the
capacity to limit the secretion and availability of the protein
products. Therefore, the hypothesis that stromal cells regulate
hematopoiesis has not been rigorously tested and an understanding of
the mechanism(s) of any regulatory process is lacking.
Complicating the study of stromal cells is the fact that the stroma of
the marrow consists of many different cell types. A predominant type of
stromal cell is the adventitial reticular cell (hereafter the only cell
we term stromal cell). This cell is alkaline phosphatase positive and
has extensive processes that adhere to many
hematopoietic cells.3 One important adhesion molecule on
stromal cells is vascular cell adhesion molecule-1
(VCAM-1) (CD106).4,5 Stromal cells are the
only cells in the marrow that produce interleukin-7
(IL-7).6 Because both stromal cell contact and IL-7 are
essential for B-lymphopoiesis, stromal cells are required for
B-lymphopoiesis.7,8 Therefore, the study of the production
of IL-7 by stromal cells provides a unique opportunity to determine if
stromal cells regulate B-lymphopoiesis by controlling the availability
of IL-7 or if stromal cells are just the constitutive source for this
growth factor.
Stromal cells in the marrow are rare (~15,000 cells per femur, or
0.125% of the marrow cellularity).6 In a marrow cell
suspension, stromal cells are mostly contained in tight aggregates, in
which early stem cells and selected hematopoietic precursors are
sequestered.5,6,9 Therefore, it is very difficult to study
the function of freshly isolated stromal cells. However, long-term bone
marrow culture systems have been developed that mimic certain
characteristics of the marrow microenvironment.10-12 The
long-term bone marrow culture system for B-lymphocytes (LTBMC-B), as
developed by Cheryl Whitlock and Owen Witte,11,12 has a
stromal layer consisting of macrophages and stromal
cells.13-15 These primary stromal cells are the culture
equivalent of the adventitial reticular cells of the marrow in that
they express alkaline phosphatase activity, VCAM-1, and  -actin and
are the only source of IL-7 within LTBMC-B.15 Because
stromal cells can be easily purified from LTBMC-B in sufficient numbers
to study their function, we have used the LTBMC-B system as a model to
determine if stromal cells regulate B-lymphopoiesis by limiting the
availability of IL-7.
One physiologic example of how stromal cells may participate in the
regulation of B-lymphopoiesis is aging. It has recently been
appreciated that B-lymphopoiesis, like T-cell production in the thymus,
decreases with age.16-22 In contrast, myelopoiesis and
erythropoiesis appear to remain constant as an animal
ages.23,24 Our previous work showed that one component of
the mechanism for the age-related decrease in B-lymphopoiesis is a
reduction in the ability of stromal cells to support the proliferation
of pro-B cells.17 We hypothesized that the production of
IL-7 by stromal cells is the underlying mechanism for this functional
impairment. One group has already suggested that there is an
age-related decrease in the amount of IL-7 in LTBMC-B.25
However, those investigators only examined the conditioned media from
intact LTBMC-B, which precludes knowing if the decreases found were due
to changes in the amount of IL-7 produced per stromal cell, the number
of stromal cells present in the cultures, or changes in the uptake of
IL-7 by the B-cell precursors present. It is therefore imperative to
examine the activities of purified stromal cells, because recently we
reported age-related changes in the number of stromal cells per LTBMC-B
and in the response of B-cell precursors from LTBMC-B to
IL-7.26 Most importantly, the investigators from that
previous study never showed that the response by the indicator line to
LTBMC-B supernatant was specific to IL-7. Synergistic factors, such as
insulin-like growth factor-1 (IGF-1), stem cell factor (SCF), and
flt3-ligand (FL), act on B-cell precursors only in combination with
IL-7.27-30 Therefore, it is unknown if the activity
measured in their assays was that of IL-7 alone or of IL-7 in
combination with another factor. These weaknesses prevent accurate
conclusions about changes in the production of IL-7 by bone marrow
stromal cells with increasing age.
The overall purpose of the current study is to directly test if the
production of IL-7 by stromal cells is altered with age. The results
suggest that the mechanism for the age-related decline in stromal cell
function involves the release of IL-7 rather than its production. IL-7
does not appear to be constitutively secreted by stromal cells from
young or old animals. Instead, it appears that the secretion of IL-7 by
aged stromal cells is delayed in comparison with young stromal cells,
and the availability of IL-7 requires cell-cell contact with B-cell
precursors. This finding suggests that stromal cells have the ability
to limit the release of IL-7, which we propose is how stromal cells
regulate B-lymphopoiesis.
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MATERIALS AND METHODS |
Animals.
Female BALB/c mice 1 and 24 months of age were obtained from the
National Institute on Aging (Bethesda, MD). IL-7  / mice
and their wild-type, age-matched controls (IL-7 +/+) were obtained from
Dr R. Murray (DNAX, Palo Alto, CA). Upon receipt, the animals were
housed at the Animal Research Facility at Loyola University (Chicago,
IL). The animals were killed between 4 and 10 days after receipt. All
animals were visually inspected for splenomegaly, lymph node
enlargement, and tumors. Mice presenting these conditions were removed
from these studies.
Preparation of Whitlock-type LTBMC-B.
Whitlock-type LTBMC-B11,12 were initiated from pooled
femoral bone marrow of at least 7 female mice 1 or 24 months of age and
were grown in RPMI 1640 supplemented with 5% fetal bovine serum
(selected lot no. 11112473; Hyclone, Logan, UT), L-glutamine,
penicillin and streptomycin, and 5 ×
10 5 mol/L  -2-mercaptoethanol. By 4 weeks, a
complex adherent layer, consisting of stromal cells and macrophages,
had formed and was supporting ongoing B-lymphopoiesis. In some
cultures, murine recombinant IL-7 (rIL-7; Genzyme,
Cambridge, MA) was added at all culture feedings beginning at 2 weeks
after initiation at a final concentration of 0.5 ng/mL. Supplementation
with IL-7 began 2 weeks after culture initiation to prevent the
maintenance and expansion of already committed B-cell progenitors in
the marrow preparations. The final concentration of 0.5 ng/mL was
chosen because it will stimulate B-cell progenitors but is fourfold
lower than the minimum level needed to stimulate macrophage
progenitors.31 Lymphoid cells were removed by washing with
0.02% EDTA and enumerated by trypan blue exclusion and
phase-microscopy.
Discrimination of stromal cells by flow cytometry.
Stromal cells in LTBMC-B were differentiated as described
before15 based on differential forward versus side scatter
and the uptake of acetylated low-density lipoprotein
(acLDL), labeled with
1,1 -dioctadecyl-1-2.3.3.3-tetramethyl-indocarocyanine
perchlorate (DiI; Biomedical Technologies [Stoughton, MA] or
Molecular Probes [Eugene, OR]). LTBMC-Bs were incubated for 3 hours
with 5 µg acLDL at 37°C and 7.5% CO2. Lymphoid cells
were removed by the addition of 0.02% EDTA. The adherent cells
(stromal cells and macrophages) were then harvested by treatment with
either 0.25% trypsin-EDTA (Life Technologies, Grand Island, NY; in
cell sorting experiments) or a second treatment with 0.02% EDTA (for
phenotypic analyses), followed by gently scraping with a silicon rubber
policeman. The live stromal cell population was discriminated on a
FACStar Plus (Becton Dickinson, San Jose, CA) using 488 nm excitation
from an argon laser and detected using a 575-nm band pass filter. All
analyses were performed 4 to 7 weeks after culture initiation.
Phenotypic examination of stromal cells.
After acLDL treatment and harvesting as described above, aliquots of
the adherent cells from LTBMC-B were incubated on ice with the
following antibodies: MK/2 (monoclonal anti-VCAM-1, rat IgG1; ATCC,
Rockville, MD), polyclonal rabbit anti-SCF (Genzyme), or monoclonal
hamster anti-murine stromal cell antibodies. The hamster anti-stromal
cell monoclonal antibodies were derived from a fusion of splenocytes
from Armenian hamsters, which had been immunized with stromal cells,
and mouse myeloma P3X63Ag8.653. The antibody-bound adherent cells were
then incubated with the appropriate biotinylated secondary antibody,
donkey anti-rat IgG, donkey anti-rabbit IgG, or goat anti-hamster IgG
[all F(ab)2; purchased from Jackson ImmunoResearch,
West Grove, PA], followed by streptavidin-allophycocyanin (APC; Becton
Dickinson or Pharmingen [San Diego, CA]). The APC was excited at 647
nm from a krypton laser and emission was detected through a 670-nm band
pass filter. All data were gated on the live stromal cell population.
Proliferation of IL-7-dependent pre-B-cell lines.
Four independently derived pre-B-cell lines
(cµ+sµ; BC76, BC77, BC715, and 2E8;
all gifts of P.W. Kincade, Oklahoma Medical Research Foundation,
Oklahoma City) were used throughout these experiments.
These cells were derived from LTBMC-B initiated with marrow from BALB/c
mice. For the characterization of growth factor responsiveness of these
cells, 5 × 104 cells were cultured for 72 hours in
100 µL final volume of media with the following recombinant
cytokines: IL-7, SCF, FL (all from Genzyme), or IGF-1 (Biosource
International, Camarillo, CA). Where noted, conditioned media from
stromal cells (as described below) were titrated into the usual media.
One microcurie of 3H-thymidine (20 Ci/mmol; NEN Research,
Boston, MA) was added to each well 4 hours before harvesting onto glass
fiber filter strips (Cambridge Technology, Watertown, MA). The amount
of 3H-thymidine incorporated was determined with a 1900CA
Tri-Carb liquid scintillation analyzer (Packard, Meriden, CT).
Stromal cell-mediated proliferation of IL-7-dependent pre-B-cell
lines.
Ten thousand primary stromal cells were directly fluorescence-activated
cell sorter (FACS)-sorted from LTBMC-B into 96-well
flat-bottom plates. Three days after the stromal cells were sorted, all
of the media was removed (stromal cell conditioned media [SCCM]) and
stored at 20°C. IL-7-dependent pre-B cells (1 ×
104; as described above) were added to the stromal cells in
100 µL of media and cocultured with the stromal cells for 4 days. For
experiments testing the necessity of cell-cell contact for stromal cell
activity, 24-well plates were used with either 25,000 or 50,000 stromal
cells per well. An equal number of IL-7-dependent pre-B cells were
added, either in direct contact with the stromal cells or separated by
0.45-µm pore size membrane (Fisher, Pittsburgh, PA). The stromal cell
lines RS2 and RS4 were derived in our laboratory by FACS sorting
stromal cells from LTBMC-B at limiting dilution. In experiments using
these stromal cell lines, the stromal cells were plated 24 hours before
the addition of the IL-7-dependent pre-B cells. In some experiments,
either murine rIL-7 (Genzyme) or a neutralizing mouse anti-human IL-7
monoclonal antibody (M25; cross-reactive against mouse IL-7; Genzyme)
was added to the coculture at the time of culture initiation. The cells
were harvested by treatment with trypsin and the lymphoid cells were
identified and enumerated by trypan blue exclusion and phase
microscopy. For cell cycle analysis, the cells were fixed with 200 µL
phosphate-buffered saline (PBS), 200 µL heat-inactivated fetal bovine
serum, and 1.2 mL 70% cold ethanol for 1 to 4 days at 4°C. The
cells were then washed twice and stained with 0.5 mg/mL propidium
iodide with 0.1 nmol/L EDTA and 0.05 ng/mL RNAse A. Cells were stained
in the dark at room temperature for 1 to 3 hours before FACS analysis.
Measurement of secreted mouse IL-7 by enzyme-linked immunosorbent
assay (ELISA).
We developed an ELISA for mouse IL-7 using commercially available
antibodies. Immulon 4 microtiter plates (Dynatech Laboratories,
Chantilly, VA) were coated overnight at 4°C with 5 µg/mL
monoclonal mouse anti-human/mouse IL-7 antibody (M25; Genzyme) in 50
µL per well. The plates were washed with PBS using a microtiter plate
washer (Elcatech, Winston-Salem, NC) and patted on paper towels to
remove excess liquid. Nonspecific binding was diminished by blocking
each well with 200 µL of 20% (vol/vol) fetal calf serum in PBS for 2
hours at 37°C. The plates were then washed as described previously.
For the standard curve, twofold serial dilutions were made with murine
rIL-7 (Genzyme) diluted in culture media for final IL-7 concentrations
ranging from 25 to 0.049 ng/mL. Forty microliters of each concentration
was added per well. Culture medium alone served as a negative control.
For each sample to be tested, 40 µL of supernatant or extract was
placed into each of three wells. Plates were incubated at 37°C for
2 hours and then washed with PBS containing 0.05% (vol/vol) Tween 20.
Fifty microliters of goat anti-murine IL-7 antibody (lot no. BH03; R&D
Systems, Minneapolis, MN) at a final concentration of 5 µg/mL was
added to each well and incubated at 37°C for 45 minutes. The plate
was then washed with PBS containing 0.05% Tween 20 and patted dry.
This pairing and order of anti-IL-7 antibodies gave the best results
of six combinations tried (data not shown). The goat anti-murine IL-7
antibody was detected with 50 µL of 2 µg/mL of mouse anti-goat IgG
conjugated with biotin (Jackson Immunoresearch). The plate was
incubated at 37°C for 45 minutes and washed with PBS containing
0.05% Tween 20 and patted dry. Fifty microliters of a 1:1,000 dilution
of streptavidin-alkaline phosphatase (Southern Biotechnology,
Birmingham, AL) was added to each well. The plate was then incubated at
37°C for 30 minutes. The plate was washed with PBS containing
0.05% Tween 20 and patted dry, followed by a wash with PBS alone and
patted dry. P-nitrophenyl phosphate (Sigma, St Louis, MO) was mixed at
1 mg/mL with 10 mmol/L diethanolamine, 0.5 mmol/L MgCl2, pH
9.5, and the solution was used to show the streptavidin-alkaline
phosphatase. In some experiments, the sensitivity was further enhanced
with GIBCO/BRL ELISA Amplification System as substrate (Life
Technologies). The optical density of each well was read at 405 nm with
a Vmax Kinetic Microplate Reader (Molecular Devices, Menlo Park, CA),
and the data were analyzed on Softmax version 2.3 (Molecular Devices)
for Macintosh. The instrument was set up for automatic
plate shaking. Standard curves were read on a four-parameter curve.
Experimental samples were plotted on the resulting standard curve and
were considered to be quantitative if found on the linear portion of
the curve. Those on the tail of the curve were determined to be
qualitatively positive for IL-7. Those below the tail of the curve were
determined to be negative for IL-7.
Extraction of IL-7 from the extracellular matrix of LTBMC-B.
The extraction of IL-7 from the matrix of LTBMC-B was based on the
method described by Clarke et al.32 LTBMC-B were grown in
100-mm2 tissue culture dishes. All supernatant was
aspirated from the cultures and replaced with 2 mL of sterile 0.6 mol/L
NaCl in Dulbecco's PBS without calcium and magnesium. The cultures
were sealed with Parafilm (American National Can,
Neeneh, WI) and incubated on ice for 2 hours with gentle rocking. The
salt solution (extract) was removed under sterile conditions and 1 mL
was placed into two siliconized 1.7-mL microcentrifuge tubes. The
extract was centrifuged at 13,000 rpm for 5 minutes. The supernatant
was transferred to a clean siliconized microcentrifuge tube and stored
at 20°C until ready to be tested in an IL-7 ELISA.
IL-7-dependent proliferation of freshly isolated pro-B cells.
B220+CD43+IgM pro-B cells
were isolated from fresh marrow of young mice by flow cytometry as
described previously.17 The following antibodies were used:
rat-antimouse CD45R/B220 (RA3-6B2) conjugated with
phycoerythrin, rat-antimouse CD43 (S7) conjugated with
biotin (both from Pharmingen), and fluorescein-conjugated
affinity-purified donkey antimouse IgM (µ-chain specific; Jackson
ImmunoResearch, West Grove, PA). The anti-CD43 was shown with
streptavidin-APC that was purchased from Becton Dickinson. Ten thousand
sorted pro-B cells (<2% contaminating sIgM+) were
cultured in the presence of IL-7 and stromal cell conditioned media in
100 µL total volume at 37°C and 7.5% CO2 for 4 days.
The lymphoid cells were harvested and enumerated using trypan blue
exclusion and phase microscopy.
Immunofluorescent staining for cytoplasmic IL-7 analyzed using flow
cytometry.
The procedure for cytoplasmic FACS staining for IL-7 was based on that
of Prussin and Metcalf.33 AcLDL labeling and harvesting of
adherent cells with trypsin were performed as above, except that 10
µg/mL Brefeldin A (Sigma) was included during the labeling protocol
and the labeling procedure was extended to 4 hours. The adherent cells
were fixed with 4% cold paraformaldehyde for 10 minutes. All
subsequent washes contained 0.1% saponin (Sigma) and 0.01 mol/L HEPES
(Life Technologies). The nonspecific uptake of IgG staining by the
cells was blocked with 10% donkey serum and 100 µg/mL mouse Ig
(Jackson Immunoresearch) for 10 minutes at room temperature. Cells were
incubated with either 1 µg rabbit anti-human IL-7 antibody
(affinity-purified, cross-reactive with mouse IL-7; Biosource) or
purified rabbit IgG (Jackson Immunoresearch). The rabbit anti-IL-7
antibody and rabbit IgG were centrifuged at 100,000g for 1 hour
to remove antibody aggregates. After incubation for 30 minutes, 5
µg/mL of biotin-conjugated donkey antirabbit Ig
[F(ab)2; Jackson Immunoresearch] was then added to
the cells for 30 minutes. Streptavidin-APC (Becton Dickinson or
Pharmingen) was used to show the biotinylated antibodies and analyzed
on a FACStar Plus. The flow cytometry data were collected from the
stromal cell-gated population. No specific staining for IL-7 was found
without permeabilization of the stromal cells (data not shown). The
levels of staining for cytoplasmic IL-7 were reduced by preincubating
the rabbit anti-human/mouse IL-7 antibodies with a nonsaturating amount
of murine IL-7 for 1 hour (data not shown).
Measurement of macrophage colony-stimulating factor (M-CSF) activity
in stromal cell conditioned media (SCCM).
SCCM was collected as described above and used in a soft agar colony
assay for macrophages (colony-forming units-macrophage
[CFU-M]). Freshly harvested bone marrow cells from
young (1 to 3 months of age) mice were placed in 0.3% soft agar with
20% SCCM as previously described.15 Colonies (>20 cells)
with macrophage morphology were scored with an inverted microscope
after 7 days of culture at 37°C. Recombinant M-CSF (Genzyme) was
used as a positive control.
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RESULTS |
Previously, we found an age-related decline in the ability of bone
marrow stromal cells to support pro-B-cell
proliferation.17 This alteration in stromal cell function
consistently occurred only between 12 and 24 months of age. Therefore,
the present study compares stromal cells from young (1 month old, at
the peak of B-lymphopoiesis) and old (24 months old) BALB/c mice. Our
work has documented the relative homogeneity of the primary-cultured
stromal cells in cell surface phenotype and production of
cytokines.15 Because of the variation among stromal cell
lines in both phenotype and function (Deryugina and
Müller-Sieburg2 and personal unpublished
observations), only freshly isolated stromal cells
from primary Whitlock-type LTBMC were used throughout this study.
Bone marrow stromal cells do not change in composition or expression
of cell-surface molecules with increasing age.
The cell surface phenotype of the stromal cells was examined to
determine whether (1) the expression of any specific molecules was
altered or (2) the composition of the stromal cell population was
changed with increasing age. An adhesion molecule expressed on stromal
cells in vitro and in vivo that is essential for the adhesion of B-cell
progenitors to stromal cells is VCAM-1.4,5 The expression
pattern and intensity of VCAM-1 on the stromal cells did not change
with age (Fig 1A). SCF delivers a
synergistic growth signal to B-cell progenitors in the presence of IL-7
and can participate in the adhesion between B-cell progenitors and
stromal cells.30 One isoform of SCF is also expressed on
the surface of the stromal cells.34,35 Examination of
membrane-bound SCF by flow cytometry showed that the relative levels of
SCF also did not change with age (Fig 1B). We also tested a panel of 11
new monoclonal antibodies directed against antigens on stromal cells
that are currently being characterized. Each of these antibodies binds
75% to 100% of the stromal cells from LTBMC-B. The expression and
distribution of each of the stromal cell antigens detected by these
monoclonal antibodies were unaffected by the aging process (data not
shown). Therefore, neither the composition of the stromal cells nor
their cell-surface phenotype is greatly affected by aging.
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| Fig 1.
The cell surface phenotype of primary-cultured stromal
cells does not change with age. FACS histograms of the distribution of
(A) VCAM-1 and (B) SCF on primary cultured stromal cells. Gates were
set to include only live stromal cells based on FSC, SSC, and acLDL
uptake. Eleven other less well-characterized monoclonal antibodies
against stromal cells were also tested and no differences in either
surface intensity or cellular distribution were found with increasing
age (data not shown).
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Age-related changes in the ability of stromal cells to support the
proliferation of IL-7-specific cell lines.
IL-7 is the primary lymphopoietic growth factor necessary for early
B-cell proliferation,8 and it is produced solely by the
stromal cells in Whitlock-type bone marrow cultures and in the bone
marrow.6,15 Furthermore, the neutralization or absence of
IL-7 activity results in a phenotype remarkably similar to that seen in
the bone marrow of aged mice.7,8 Therefore, we wanted to
determine if IL-7 production by stromal cells was changed with
increasing age. As described below, very little or no IL-7 was found in
stromal cell conditioned media. Therefore, we determined the amount of
IL-7 produced by stromal cells by measuring the proliferation of
IL-7-dependent, B-lineage indicator cell lines when directly in
contact with stromal cells.
To accurately measure cytokines with a bioassay, the responder cell
line cannot proliferate to any other factor present in the experimental
system. Four independently derived pre-B-cell lines were initially
used to minimize the chances that another stromal-derived growth
factor(s) was participating with IL-7 in driving proliferation. Stromal
cells from LTBMC-B produce SCF, IGF-1, and FL, which synergize with
IL-7 to increase the proliferation of certain B-cell progenitors. We
determined that the IL-7-dependent cell lines were unresponsive to
SCF, IGF-1, and FL, either alone or in combination with IL-7 (Table
1). Furthermore, none of the cell lines
appeared to respond to any other soluble factors secreted by stromal
cells (see below). The proliferation of the responder cell lines on
stromal cells was completely inhibited by 0.1 µg/mL of neutralizing
antibody against IL-7, demonstrating the IL-7 dependency of the cells
(data not shown). However, it was still possible that the
IL-7-dependent cell lines could respond to another growth factor
secreted by stromal cells or present on the surface of stromal cells.
Such a factor might elicit a proliferative response by
itself or synergize with IL-7 to increase proliferation. Therefore, we
tested these possibilities with stromal cells from IL-7 /
mice (IL-7 / stromal cells). None of the IL-7-dependent
cell lines proliferated in response to the IL-7 / stromal
cells, confirming the requirement for IL-7 and showing that no other
stromal cell-derived factor can induce an independent response (Fig
2). Three of the cell lines (BC715, BC77,
and 2E8) showed no evidence for proliferation to a stromal cell-derived
synergistic factor when cultured in the presence of a suboptimal
concentration of rIL-7 (Fig 2A). Therefore, the growth of these cells
on stromal cells is a measure of IL-7 alone. On the other hand, culture
of the BC76 cell line with IL-7 / stromal cells and
exogenous rIL-7 did increase the amount of proliferation (Fig 2B),
suggesting that BC76 is responsive to an unknown stromal-derived factor
that synergizes with IL-7. Therefore, proliferation of BC76 on stromal
cells measures a combined activity of the stromal cells and is not an
accurate measure of the amount of IL-7 produced by the stromal cells.
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Table 1.
Characterization of Four IL-7-Dependent Pre-B-Cell
Lines: Lack of Response to Stromal Cell-Derived Growth Factors Other
Than IL-7
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| Fig 2.
Determination of the responsiveness of four
IL-7-dependent pre-B cell lines to other stromal cell-derived factors.
The response of the cells to IL-7 +/+ and / stromal cells was
compared in the presence of suboptimal concentrations of rIL-7. Ten
thousand pre-B cells were cocultured with 10,000 FACS-purified stromal
cells for 4 days. The pre-B cells were then harvested and enumerated.
(A) The response of BC715. (B) The response of BC76. The response of
BC715 represents one of three cell lines that did not respond to any
other growth factor besides IL-7. This was concluded because the
presence of the IL-7 / SC does not appear to affect the amount of
proliferation. Similar results were obtained with the pre-B-cell lines
BC77 and 2E8. The response of BC76 represents a cell line that
responded to another growth factor in combination with IL-7 because, in
the presence of rIL-7, the amount of proliferation is greater on the
IL-7 / SC than without any stromal cells. ( ) No SC; ( ) IL-7
+/+ SC; ( ) IL-7 / SC.
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The IL-7-specific cell lines were used as a relative measure of the
amount of IL-7 produced from stromal cells obtained from young and old
mice. The growth of each of the indicator cell lines was significantly
less on the stromal cells from old mice when compared with the stromal
cells from young mice (Fig 3). The ability
of the stromal cells from 24 month-old-mice to support IL-7-dependent
proliferation averaged only 52% of that of the stromal cells from
1-month-old mice. These data indicate that the ability of stromal cells
to produce and/or secrete IL-7 is diminished with aging.
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| Fig 3.
Impaired ability of stromal cells from aged mice to
support the proliferation of IL-7-dependent pre-B-cell lines. The
IL-7-responsive pre-B-cell lines were used to compare the ability of
stromal cells to support IL-7-mediated proliferation. Ten thousand
pre-B cells were cocultured with 10,000 FACS-purified stromal cells
(SC) from 1-month-old mice ( ) or 24-month-old mice ( ) for 4 days.
The pre-B cells were then harvested and enumerated. (A) The results
from one of four or five experiments with each cell line. (B) The
relative ability of aged SC to support IL-7-mediated proliferation as
compared with young SC. The amount of proliferation supported by the
young SC for each experiment was set as 1.00. The results shown are the
average ± SD from four or five experiments with each pre-B-cell
line. *P < .02; **P < .05.
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If available IL-7 is deficient in aged marrow, then the replacement of
IL-7 in aged marrow should boost the numbers of lymphoid cells. This
was tested in two ways. First, the addition of 0.025 to 0.1 ng/mL of
rIL-7 to purified stromal cells from aged marrow resulted in levels of
proliferation of IL-7-dependent cells approximately equal to that on
stromal cells from young mice (data not shown). As a separate
experimental system, we supplemented LTBMC-B with rIL-7. Whitlock-type
LTBMC-B of aged (24 months old) marrow produced threefold fewer
B-lymphoid cells per culture than cultures initiated from young (1
month old) marrow.26 The numbers of lymphoid cells within
the supplemented cultures of aged marrow increased 17-fold to greater
levels than that found in the unsupplemented LTBMC-B of young marrow
(Fig 4). Identical supplementation of
LTBMC-B of young marrow induced only a threefold increase (Fig 4). In
vivo, the primary stage of B-cell deficiency in the aged marrow is the
pre-B-cell stage.17 Supplementing cultures of aged marrow
with IL-7 increased the numbers of
cµ+sµ pre-B cells 32-fold, whereas
the pre-B cells in supplemented cultures of young marrow increased only
2-fold. Therefore, we conclude that, in LTBMC-B from aged mice, one
reason for the reduced yield of B-cell progenitors produced by these
cultures is insufficient available IL-7. We also predict that a
diminished availability of IL-7 is one reason for the reduced number of
pre-B cells in aged mice.17
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| Fig 4.
Supplementation of LTBMC-B of marrow from young and old
mice with IL-7 increases the number of B-lineage cells produced per
culture. The addition of rIL-7 at 0.5 ng/mL for 2 weeks increased the
number of B-lineage cells produced in LTBMC-Bs of young and aged
marrow. The total number of lymphoid cells per culture was determined
using trypan blue exclusion and phase microscopy. The results were
calculated from 2 to 5 pooled cultures. () No added IL-7; ()
added IL-7.
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Evaluation of IL-7 and soluble inhibitory factors secreted by stromal
cells.
IL-7 is found only in a secreted form.36 Therefore, we next
attempted to compare the levels of IL-7 that were secreted by the
stromal cells from young and old mice. To detect secreted IL-7, we
developed a sensitive ELISA for mouse IL-7. Detectable limits were 0.1
ng/mL of IL-7, and quantitation was derived from the linear portion of
the standard curve, which fell at concentrations greater than 0.782
ng/mL (Table 2). IL-7 was detected in the
media in 21 of 39 individual LTBMC-B cultures. However, in only 6 of
these cultures were the levels great enough to be quantifiable (Table
2). The paucity of IL-7 in the LTBMC-B did not appear to be solely due
to its rapid uptake by the lymphoid progenitors present in the LTBMC-B,
because IL-7 was detected in the conditioned media from FACS-purified
stromal cells only occasionally (2 of 5 experiments) and was below
quantitative limits (Table 2).
It was recently reported that IL-7 can bind to sulfated
glycoaminoglycans of the extracellular matrix.32 The
binding of IL-7 to heparin or heparin sulfate can be dissociated by
treatment with 0.6 mol/L NaCl.32 In control experiments, we
found that treatment with 0.6 mol/L NaCl efficiently eluted at least 30
ng of rIL-7 that was bound to 0.25 mg/mL of heparin (data not shown).
We then used this elution procedure to determine if IL-7 was bound in
the matrix of intact LTBMC-B. Even after concentration of the eluate,
IL-7 was usually undetectable or present only in the qualitative range
(<0.782 ng/mL; Table 2). To investigate the possibility that IL-7 was
rapidly degraded in the stromal cell cultures, 10 ng/mL of rIL-7 was
added to FACS-purified stromal cells for up to 3 hours. The
concentration of recoverable IL-7 was reduced by 19% in the first 15
minutes, but did not significantly change thereafter. This suggests
that the lack of IL-7 in the supernatant is not due to rapid
degradation, binding to the matrix, or uptake by stromal cells.
As an alternative method, we developed a bioassay using the
IL-7-dependent cell lines to compare the amount of IL-7 in the
conditioned media from purified stromal cells. Although this approach
is extremely sensitive (routinely detectable at  1 pg/mL and
quantitative at  10 pg/mL IL-7), IL-7 activity in the conditioned
media from FACS-purified stromal cells from 1- or 24-month-old mice was
below the level of detectability (Fig 5A).
Furthermore, it appears that the absence of IL-7 activity in the SCCM
is not solely due to the general degradation of proteins in the SCCM,
because M-CSF activity is detected in SCCM from both age groups (Fig
5B). These results suggest that quantitative amounts of IL-7 are not
freely secreted by primary cultured stromal cells.
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| Fig 5.
Examination of SCCM. (A) Measurement of IL-7 secreted by
stromal cells from young and old mice. A bioassay for IL-7 was used to
measure the amount of IL-7 secreted by FACS-purified stromal cells in
72 hours. SCCM was used at a final concentration of 10%. The results
shown are the average ± SD of 3 to 4 replicate wells using the
pre-B-cell line BC715; similar results were found using two other
pre-B-cell lines, BC76 and 2E8. Similar results were also obtained
when 20% and 30% SCCM were used and with SCCM from 4 separate
collections (data not shown). The responses to 0.001 and 0.0025 ng/mL
rIL-7 are shown for comparison. (B) Production of secreted M-CSF
activity by stromal cells. The amount of M-CSF activity present in SCCM
from FACS-purified stromal cells was determined by a CFU-M assay. The
number of macrophage colonies having greater than 20 cells was
determined using a dissecting microscope after culture in semisoft agar
for 7 days. The data are reported as the average ± SD of 3 to 4
plates per group. The results shown are from one representative
experiment of three performed with different collections of SCCM after
72 hours. The results shown used SCCM at 20%; no age-related
differences were observed when the SCCM was used at 10% (data not
shown). The responses to 2.5 and 7.5 U/mL rM-CSF are shown for
comparison.
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B-lymphopoiesis is regulated by both positive and negative regulators
(reviewed in Kincade37). Therefore, the reduced amount of
proliferation on the stromal cells from aged mice could also be due to
an increase in the concentration of these inhibitors. This possibility
was tested by examining the activity of the stromal cell conditioned
media on the proliferation of the IL-7-dependent pre-B-cell lines in
the presence of rIL-7. The addition of stromal cell conditioned media
(up to 30%) did not affect the proliferation of the IL-7-dependent
cells in the presence of IL-7 (Fig 6A).
Because the IL-7-dependent cell lines used here do not respond to
other known positive growth factors, it could be argued that the
absence of detectable inhibitory factors was due solely to the use of
cell lines that are no longer sensitive to these factors. Therefore, to
ensure that the detection of an inhibitory factor was not missed,
FACS-purified pro-B cells
(B220+CD43+IgM) were used in
place of the IL-7-dependent cells. Stromal cell conditioned media from
either age group also did not inhibit the proliferation of freshly
isolated pro-B cells in the presence of IL-7 (Fig 6B). Unlike the
IL-7-dependent pre-B-cell lines, the freshly isolated pro-B cells
respond to the synergistic factors produced by stromal cells.
Therefore, the increase in activity of the stromal cell conditioned
media over IL-7 alone is probably due to the presence of other
synergistic growth factors produced by the stromal cells. The absence
of inhibitory activity of the stromal cell conditioned media was found
regardless of the concentration of IL-7 used (range, 0.05 to 5.0 ng/mL;
data not shown). Furthermore, the activity of exogenously added rIL-7
in cocultures of stromal cells and the IL-7-dependent pre-B cells was
not inhibited by the presence of stromal cells from aged mice (data not
shown), suggesting that neither soluble nor cell-membrane bound
inhibitory factors were involved. Therefore, the reduced ability of
stromal cells from aged mice to support the proliferation of
IL-7-dependent B-cell precursors is not due to an increased production
of inhibitory factors.
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| Fig 6.
Absence of soluble inhibitory activities present in SCCM.
SCCM from FACS-purified stromal cells does not inhibit the
proliferation of (A) IL-7-dependent BC715 cells or (B) freshly
isolated pro-B cells in the presence of 1.0 ng/mL rIL-7. In (B),
FACS-purified pro-B cells
(B220+CD43+IgM) from young
mice were used to maximize the detection of any factors present in the
SCCM. The additional activity of the SCCM with IL-7 on freshly isolated
pro-B cells is probably due to the presence of synergistic factors that
are not active on the IL-7-dependent pre-B-cell lines. No inhibitory
activities were found in SCCM with 0.1 to 5.0 ng/mL of IL-7, at 20%
and 30% final volumes of SCCM, and on the other IL-7-dependent
pre-B-cell lines (data not shown). At least 3 separate collections of
SCCM have been tested with the freshly isolated pro-B cells and with
each cell line.
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Comparison of the amount of IL-7 within stromal cells.
Because the amount of IL-7 secreted by stromal cells appears to
decrease with aging, we asked if this was due to decreased production
of IL-7 by the stromal cells. To examine the production of IL-7, we
determined the cytoplasmic levels of IL-7 protein within the stromal
cells. Previously, we used immunocytochemistry to show the presence of
IL-7 within both primary-cultured and freshly isolated stromal
cells.6,15 Flow cytometric analysis of cytoplasmic IL-7
using the same anti-IL-7 antibodies offered a semiquantitative
approach for the comparison of IL-7 levels within stromal cells from
young and old mice. Justification of this methodology is shown in Fig
7. Two stromal cell lines were derived from
LTBMC-B in our laboratory by limiting-dilution FACS sorting. The
stromal cell line RS2 supported a greater amount of proliferation of
the IL-7-dependent cell lines than the RS4 stromal cell line (Fig 7A).
When analyzed for content of cytoplasmic IL-7 by flow cytometry, RS2
cells showed a higher specific staining with anti-IL-7 antibody (Fig
7B). The net mean fluorescent intensity (MFI) specific for IL-7 was
calculated by subtracting the MFI obtained with staining with normal Ig
from the MFI obtained with staining with anti-IL-7 antibody
(representative histograms from staining RS2 cells are shown in Fig
7C). The net specific MFI for IL-7 showed that the relative content of
IL-7 protein in the RS2 cells is 2 to 3 times higher than in the RS4
cells (Fig 7D). Therefore, cytoplasmic flow cytometry provides an
accurate relative comparison of the levels of IL-7 protein within
stromal cells and correlates with the ability to support the
proliferation of IL-7-dependent pre-B cells in coculture.
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| Fig 7.
Justification of cytoplasmic FACS as a method to compare
the amount of IL-7 within the cytoplasm of stromal cells. (A)
Comparison of the ability of 2 stromal cell clones to support
IL-7-mediated proliferation of BC715 pre-B cells. (B) Histograms
showing the levels of cytoplasmic IL-7 staining of the same 2 stromal
cell clones as determined by flow cytometry. (C) Histograms comparing
the levels of cytoplasmic staining of RS2 cells using normal Ig and
anti-IL-7. (D) Net specific IL-7 staining for the same 2 stromal cell
clones calculated as the difference in MFIs between staining with
anti-IL-7 and control Ig antibodies. (), RS2; ( ), RS4.
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FACS analysis was used to compare the intracytoplasmic levels of IL-7
protein within stromal cells from primary cultures of marrow from 1-
and 24-month-old mice. Representative histograms comparing the
cytoplasmic staining of stromal cells from young and old mice with
normal Ig and anti-IL-7 antibody are shown in Fig
8A and B. This confirms our previous work
using immunocytochemistry that the primary stromal cells are relatively
homogenous in IL-7 expression at early times of culture.15
There was no significant difference between stromal cells from young
and old marrow in the net specific MFI for cytoplasmic IL-7 (Fig 8C).
Therefore, although stromal cells from aged mice are impaired in their
ability to support the proliferation of IL-7-dependent cell lines, the
level of IL-7 protein within the stromal cells does not appear to
change with age.
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| Fig 8.
Comparison of the relative amount of IL-7 within the
cytoplasm of stromal cells from young and old mice. The relative amount
of IL-7 within stromal cells was determined by cytoplasmic flow
cytometry as described in Fig 7 and in Materials and Methods. The
histograms compare the levels of cytoplasmic staining for normal Ig and
anti-IL-7 antibody in stromal cells from (A) 1-month-old mice and (B)
24-month-old mice. (C) Net specific IL-7 staining was calculated as the
difference in MFIs between staining with anti-IL-7 and control Ig
antibodies. The average ± SD was calculated from four separate
experiments using stromal cells from two independent culture
initiations is shown. In each experiment, two to four pooled LTBMC-Bs
were used as the source of stromal cells.
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Induction of the response to IL-7 is delayed on stromal cells from
aged mice.
We hypothesized that the mechanism for the age-related dysfunction of
stromal cell from aged mice is the impaired secretion of IL-7. If this
hypothesis is correct, one may predict that the detection of IL-7
secreted by aged stromal cells would be delayed when compared with
young stromal cells. To test this prediction, we determined when we
could first detect IL-7 activity after culturing on stromal cells.
Changes in the percentage of the IL-7-dependent cell lines in the
S/G2/M stages of the cell cycle were used as a measure of IL-7 activity
to allow for more accurate assessment at early times of culture when
only small changes in cell numbers have occurred. The detection of IL-7
activity from stromal cells was defined as when the percentage of cells
in S/G2/M was greater than the percentage of cells in cell cycle that
were cultured without stromal cells and without IL-7 (0 IL-7). With
stromal cells from young mice, IL-7 activity was routinely first
detected within the first 24 to 48 hours. In contrast, the onset of
detectable IL-7 activity was not observed until about 24 hours later
when cultured with stromal cells from old mice (Fig
9). The delay in the increase in cells
actively cycling on aged stromal cells was not due to the death of the
responder cells, because few cells in the cocultures were undergoing
apoptosis through the first 72 hours of culture (Fig 9). Instead, it
appeared that the IL-7-specific cells were accumulating in the G0/G1
stages of the cell cycle (data not shown). These results suggest that
the age-related deficiency in stromal cell function is due to the
delayed secretion of IL-7.
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| Fig 9.
Kinetics of the responsiveness of IL-7-specific cell
lines to stromal cells. (A and B) The kinetics of the secretion of IL-7
by FACS-purified stromal cells was determined by measuring changes in
the percentage of responding IL-7-specific cells in S/G2/M stages of
the cell cycle. (C and D) Age-related differences in the kinetics of
the responses to stromal cells are not due to the death of the
responding cells. () 0 ng/mL IL-7; ( ) 0.001 ng/mL IL-7; ( ) 1.0
ng/mL IL-7; () 1-month-old SC; () 24-month-old SC. (A) and (C)
show representative results with the IL-7-specific cell line BC77; (B)
and (D) show representative results with BC715. Each cell line was
tested in two or three separate experiments. In all panels, the
responses of the IL-7-specific cells to 0.001 and 1.0 ng/mL of rIL-7
are shown solely for comparison.
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IL-7 activity produced from stromal cells requires direct cell
contact with B-cell precursors.
We next examined if direct cell-cell contact between the stromal cells
and the IL-7-specific B-cell precursors is required for the
proliferation of the IL-7-dependent cell lines. Direct cell-cell
contact between the stromal cells and the B-cell precursors was
prevented using 0.45-µm membrane inserts. We found
that minimal proliferation of the IL-7-dependent cell lines occurred
without direct cell-cell contact between the pre-B cells and the
stromal cells (Fig 10A). Normal levels of
proliferation occurred when rIL-7 was exogenously added to the wells
(Fig 10B). Furthermore, proliferation of the IL-7-dependent cell lines
occurred in the presence of rIL-7 regardless of whether the IL-7 was
added to the stromal cell side or the B-cell precursor side of the
transwell chamber, indicating that IL-7 could pass through the
transwell membrane if it was freely secreted by the stromal cells (data
not shown). Therefore, direct cell-cell contact between B-cell
precursors and stromal cells is required for stromal-derived IL-7 to
induce proliferation.
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| Fig 10.
Requirement for direct cell-cell contact for the
secretion of IL-7 from stromal cells. Cell contact between the
IL-7-dependent pre-B cells and the stromal cells was prevented by use
of a 0.45-µm membrane (no contact). (A) IL-7-dependent proliferation
did not occur without direct cell contact with the stromal cells. (B)
Proliferation of IL-7-dependent pre-B cells to rIL-7 was not affected
by the presence or absence of cell contact with stromal cells. The
results shown in (B) used rIL-7 at a concentration of 0.25 ng/mL;
similar results were found with IL-7 at 1.0 ng/mL (data not shown). The
results shown in (A) and (B) used the pre-B-cell line BC77; similar
results were found with BC715. () No SC; () 1-month-old SC; ()
24-month-old SC.
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DISCUSSION |
Although most processes of bone marrow hematopoiesis are unaffected by
aging, it has recently become appreciated that there is a significant
age-related decrease in B-lymphopoiesis.16-22 In our
previous work, we found functional alterations in both the stromal
cells and in the B-cell precursors.17 Because the activity
of IL-7 in the marrow is directed mainly on lymphoid development, as is
the age-related decrease in lympho-hematopoiesis, we hypothesized that
the production of IL-7 by stromal cells would be the underlying
deficiency of the stromal cells from aged mice. One previous study
suggested that a decrease in the relative quantity of IL-7 protein in
LTBMC-B occurs with increasing age.25 However, it is
unclear if the activity measured in that study was IL-7 alone or IL-7
in combination with another factor. Our use of four independently
derived IL-7-dependent cell lines, three of which appear to be
uniquely IL-7-specific, clearly proves that the stromal cells from
aged mice are impaired in their ability to support IL-7-mediated
proliferation. However, because a decrease in the amount of IL-7
protein within stromal cells does not occur with increasing age and
because the response of the IL-7-specific cells to stromal cells from
old mice is delayed when compared with stromal cells from young mice,
the functional alteration of the stromal cells appears to be at the
level of IL-7 secretion. Because of the modest level of IL-7 in stromal
cell conditioned media or within the extracellular matrix and the
requirement of the stromal cells for cell contact with the
IL-7-dependent cells for the release of IL-7 activity, we propose that
the regulation of B-lymphopoiesis by stromal cells occurs by modulating
the availability of IL-7 at the level of protein secretion, not
production.
Our results, along with the published work of a number of other
investigators, have led us to propose a model whereby IL-7 is stored
inside stromal cells and secreted only in a tightly regulated and
directed manner into discrete microenvironments. IL-7 is absolutely
required for marrow B-lymphopoiesis,7,8 and stromal cells
are the only cells in the marrow and in the LTBMC-B that produce
IL-7.6,15 Although we can detect IL-7 protein within
stromal cells6,15 (Fig 8), we cannot detect substantial
amounts of secreted IL-7. We find that the anti-IL-7 monoclonal
antibody M25 stains the stromal cells in a very punctate pattern, as if
the IL-7 is within vesicles (personal observations).
Using electron microscopy, Jacobson et al38 found that CD29
(1 integrin), as identified by the antibody KMI6, is localized on
the stromal cells only to places of lymphoid contact in vivo.
Therefore, the concept of lymphopoietic-specific stromal niches has
been shown. We predict that a B-cell precursor in one of these
specialized stromal niches signals the stromal cell to locally release
IL-7 directly to that B-cell precursor. This precursor then leaves the
niche after receiving the IL-7 to undergo cell division. This explains
why the majority of cells responsive to IL-7 alone (CFU-IL-7) are not
in close contact with stromal cells in native marrow.5 The
capacity of B-cell progenitors to induce signals within stromal cells
has been shown by Jarvis and LeBien,39 and Sudo et
al40 reported the activation of a stromal cell line by
contact with a pre-B-cell line. Recently, Tang et al41
suggested that IL-7 secretion by human stromal cells can be negatively
regulated, supporting the hypothesis that the secretion of IL-7 is not
a constitutive process. All of these findings can be used in support
for a model of directed secretion of IL-7 by bone marrow stromal cells.
Special note needs to be made of the cell-cell contact specificity in
our system. The proliferation of the IL-7-specific cell lines in the
presence of rIL-7 is not affected by the presence of stromal cells.
This finding suggests that the direct cell-cell contact between the
B-cell progenitors and the stromal cells influences stromal cell
function. Because the release of IL-7 from aged stromal cells is
impaired even though the amount of IL-7 protein within stromal cells
does not change with age and because we cannot detect constitutive
secretion of IL-7 in SCCM, we believe that cell-cell contact is
required to induce the secretion of IL-7 from the stromal cells.
However, we cannot exclude the possibility that the amount of IL-7
secreted is so low that stromal cells must localize secreted IL-7 to
reach a biologically active threshold, thereby preventing IL-7 from
crossing the transwell membrane and preventing our detection of IL-7 in
SCCM. Further experiments are needed to distinguish between these
possibilities. Furthermore, in seemingly direct contrast with our
results, Hardy et al42 showed contact-independent
production of IL-7 activity by stromal cells. The important difference
is that they used a stromal cell line while we used primary-cultured
stromal cells. Using our bioassay, we readily detect IL-7 in the SCCM
from our stromal cell lines (data not shown), suggesting that these
stromal cell lines also secrete IL-7 in a contact-independent manner.
This difference stresses the importance of using nontransformed cells
to study the natural regulatory processes of a specific cell type.
Furthermore, it may also suggest that the regulation of IL-7 secretion
is a delicate process that is easily disrupted during a cell's
immortalization.
A recent study by Selleri et al43 showed the potent effects
of small amounts of locally secreted cytokines produced by stromal
cells. In their study, stromal cells were stably transfected to produce
interferon- (IFN-). The transfected stromal cells had a 100-fold
greater IFN- activity than what would be predicted by the amount of
IFN- that was detected in the stromal cell conditioned media. Thus,
negligible amounts of IL-7 might be detected in the culture
supernatant, whereas the amount of IL-7 may be relatively high within
the lymphopoietic niches provided by the stromal cells. Furthermore,
because a majority of stromal cells in vivo produce IL-7,6
the lymphopoietic stromal niche may also be a means of limiting
B-lymphopoiesis, which would allow the other hematopoietic lineages to
develop in the same tissue.
Why does such regulation of B-lymphopoiesis by the stromal cells need
to occur? One advantage of stromal cell regulation is its ability to
explain a mechanism for positive selection in B-lymphopoiesis. The
pre-B-cell receptor is composed of a functional heavy chain with the
surrogate light chain (VpreB and  5) that is required for
B-lymphopoiesis.44 One popular hypothesis is that the
pre-B-cell receptor has a ligand on stromal cells.45 This
predicted ligand-receptor pair may play a role in inducing the
secretion of IL-7. This could explain a mechanism for positive
selection by precluding the IL-7-mediated expansion of those B-cell
precursors that are unable to express a functional heavy chain and
therefore cannot express the pre-B-cell receptor. The regulation of
the availability of IL-7 also minimizes any inhibitory effects that
IL-7 possesses. Grawunder et al46 reported that the
differentiation of pre-B-I cells into sIgM+ cells in vitro
does not occur when stromal cells and a relatively high level of IL-7
(500 to 5,000 U/mL) are present in cultures. In their system,
differentiation can proceed when the exogenous IL-7 is
removed.46 However, other laboratories, including our own,
observe the differentiation of sIgM pro-B cells
(equivalent to pre-B-I) into sIgM+ cells when stromal cells
are used as the only source of IL-7.17,42 Therefore, by
limiting the availability of IL-7, stromal cells may limit the amount
of proliferation that a precursor undergoes, allowing each cell the
chance to further differentiate into an sIgM+ B cell. The
diminished availability of IL-7 when associated with stromal cells from
aged mice may explain why the percentage of sIgM+ re |
Abstract
Introduction
Abstract