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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2935-2947
Abnormal Myelocytic Cell Development in Interleukin-2
(IL-2)-Deficient Mice: Evidence for the Involvement of IL-2 in
Myelopoiesis
By
Tannishtha Reya,
Nikhat V. Contractor,
Matthew S. Couzens,
Mariusz
A. Wasik,
Stephen G. Emerson, and
Simon R. Carding
From the Departments of Microbiology, Medicine, Pathology and
Laboratory Medicine, Division of Hematology and Oncology, University of
Pennsylvania School of Medicine, Philadelphia, PA.
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ABSTRACT |
Mice lacking interleukin-2 (IL-2) developed a severe hematopoietic
disorder characterized by the abnormal development of myeloid cells and
neutropenia. Analysis of the bone marrow of IL-2-deficient (IL-2 /) mice showed that the number of mature
polymorphonuclear cells was decreased by 65% to 75%, and
granulocyte/macrophage precursor cells were reduced by 50%. Bone
marrow cells from IL-2/ mice were unable to sustain
myelopoiesis in lethally irradiated mice and in long-term bone marrow
cultures (LTBMC). The addition of exogenous IL-2 to LTBMC of
IL-2/ cells partially restored hematopoietic
progenitor activity. In the bone marrow of wild-type mice, immature
(Mac-1lo) myeloid cells, including myeloblasts and
promyelocytes, constitutively expressed the  -chain of the IL-2R, and
the number of Mac-1loIL-2R+ cells was
increased by twofold to threefold in IL-2/ mice.
During culture in the presence of IL-2 and the absence of stromal
cells, Mac-1loIL-2R+ immature myeloid
cells proliferated and gave rise to mature granulocytes and
macrophages. Collectively, these observations indicate that defective
myelopoiesis in IL-2/ mice is at least in part a
consequence of their direct dependency on IL-2, and by regulating the
growth of immature myeloid cells, IL-2 plays an important role in the
homeostatic regulation of myelocytic cell generation.
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INTRODUCTION |
INTERLEUKIN-2 (IL-2) WAS initially
described as a mitogenic signal and growth factor for T
lymphocytes.1,2 However, subsequent studies have shown that
IL-2 receptors (IL-2R) are widely distributed on other
lymphoid3-5 and nonlymphoid hematopoietic cells including cells of the myeloid lineage,6-11 and that IL-2 can
modulate the growth, survival, or function of natural killer
(NK) cells, lymphokine-activated killer (LAK) cells, and B
cells.3,12-14 While the role of IL-2 in T-lymphocyte
development has been extensively investigated (reviewed in Carding and
Reya15), there is now growing evidence that IL-2 may be
able to influence the development of other lymphocytes. Recent studies
describing the expression of IL-2R on pre-B cells16,17 and our own studies identifying expression of functional IL-2Rs by
pre-pro B cells18 suggest a role for IL-2 in B-cell
development. Additionally the absence of NK cells in
IL-2R -deficient19 and IL-2R-deficient20
mice suggests that signaling through the IL-2R complex by IL-2
and/or IL-15 is also necessary for NK cell development.
In contrast to lymphoid cells, the function of IL-2R in cells of the
myeloid lineage is largely unknown. Although the ability of IL-2 to
influence the survival21 and effector
function21-26 of mature myeloid cells has been extensively
characterized, it is not clear if, or how, IL-2 influences their
development. The differential expression of IL-2Rs during the in vitro
differentiation of human myeloid cells27 and the increase
in number of circulating myeloid (colony-forming
unit-granulocyte/macrophage [CFU-GM]) progenitor cells after
treatment of cancer patients with IL-228-31 are consistent
with IL-2 having a positive effect on myelopoiesis. However, other
studies have produced contradictory findings. The addition of IL-2 to
long-term bone marrow cultures (LTBMC) or in vitro colony forming
assays has been shown to inhibit myeloid progenitor cell growth and
development.32-36 It is also not clear if the effects of
IL-2 on myeloid cell progenitors are direct or indirect. For example,
it has been shown that IL-2 can influence myelopoiesis by inducing
cytokine production by IL-2R+ T cells.35,37-39
However, the finding that the addition of recombinant IL-2 to cultures
of T-cell-depleted populations of bone marrow hematopoietic
progenitors33 and murine myeloid precursors32 promotes their growth and development is consistent with a direct role
for IL-2 in regulating hematopoietic cell activity. It is not clear,
therefore, what the mechanism(s) of action of IL-2 on developing
myeloid cells is, or if IL-2 does, in fact, normally play a role in
promoting or regulating myelopoiesis in vivo.
To address these questions and to establish the role of IL-2 in myeloid
cell development, we have determined the consequences of IL-2
deficiency on myelopoiesis in vivo. We show here that IL-2/ mice develop a profound hematopoietic
disorder characterized by the abnormal development of myeloid cells and
deficiencies in mature granulocytes. Furthermore, the finding that
immature myeloid cells can use IL-2 for their growth and
differentiation in vitro, and exogenously provided IL-2 can restore
IL-2/ hematopoietic cell proliferation in
vitro suggests that IL-2 acts directly on immature myeloid cells and
that IL-2/IL-2R interactions can regulate myeloid cell development.
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MATERIALS AND METHODS |
Mice.
C57BL.6, C57BL.6-Ptprca Pep3b/BoyJ (CD45.1) and
C57BL.6-Thy1a/Cy (Thy1.1) mouse strains were obtained from
The Jackson Laboratory (Bar Harbor, ME) and used between 4 and 6 weeks
of age. IL-2-deficient mice back-crossed for eight generations to
C57BL.6 were obtained from R. Schwartz (National Institutes of Health
[NIH], Bethesda, MD) and were bred and maintained in
filter cages at the University of Pennsylvania and used between 3 and 7 weeks of age. Sentinel animals housed with the mutant mice were
routinely screened for the presence of common mouse pathogens and none
have been detected at any time since the animals have been at the
University of Pennsylvania. The derivation and maintenance of
gnotobiotic IL-2/ mice has been described
previously.40 Animals homozygous for the mutant IL-2 gene
were identified from samples of tail DNA using the polymerase chain
reaction (PCR)-based method described by Schorle et
al.41
Cell isolation.
Blood was obtained by tail bleeding or cardiac puncture and collected
in polypropylene tubes containing heparin. Blood counts were obtained
using a Baker-1000 automated cell counter (Coulter, Hialeah, FL) and leukocyte differential counts were performed manually
on Wright-stained blood smears. Bone marrow cells were obtained from
the femurs and tibias of mice by flushing the bones with
phosphate-buffered saline (PBS) and scraping the bone fragments. T
cells for adoptive transfer experiments were isolated from mesenteric lymph nodes or spleen of 6 to 7 week old
IL-2/ and IL-2+/+ littermates
using a negative immunomagnetic selection procedure.42 Anti-B220 (clone RA3.6B2; American Type Culture Collection [ATCC], Rockville, MD), -CD11b (M1/70.15; Caltag Labs, San Francisco, CA)
antibodies and mouse Ig (Sigma, St Louis, MO) in conjunction with goat
antimouse Ig conjugated to immunomagnetic particles (BioMag Beads;
Perseptive Diagnostics, Cambridge, MA) were used to remove non-T-cell
populations. The isolated cells were routinely >98% CD3+
as determined by flow cytometric analysis.
Monoclonal antibodies, flow cytometry, and cell sorting.
The following mouse and rat monoclonal antibodies were used for the
immunochemical analysis of bone marrow cells: antimouse CD32/CD16/FcRII/III (clone 2.4G2; ATCC); CD45R/B220 (RA3-6B2; Pharmingen, San Diego, CA); CD45.1 (A20; Pharmingen); CD45.2 (104; Pharmingen); Sca-1/Ly-6A/E (E13-161.7; Pharmingen); CD90.1/Thy1.1 (HIS51; Pharmingen); CD90.2/Thy1.2 (30H.12; Life Technologies, Gaithersburg, MD); CD3 (29B; Life Technologies); CD24/HSA (M1/69; Pharmingen); CD11b/Mac-1 (M1/70.15; Caltag Laboratories, San Francisco, CA); CD43/S7 (1B11; Pharmingen); Ly-51/BP-1 (6C3; Pharmingen); Gr-1
(RB6-8C5; Pharmingen); IgM (Fab2 fragments of goat
antibody; Jackson Immunoresearch, Westgrove, PA); Ter-119 (Pharmingen). Strepavidin-phycoerythrin and strepavidin-red 670 (SA-PE;
SA-RED670; Life Technologies) were also used to detect reactivity of
biotinylated primary antibodies. All steps of the two- and three-color
staining procedure were performed using 96-well, "V"-bottomed
microtiter plates. All incubations were performed at 4°C.
Approximately 1 × 105 cells in 50 µL of staining
buffer (PBS, 5% fetal calf serum [FCS]) were incubated with 5 µg
of antimouse FcR antibody and 10 mg/mL of mouse and rat IgG (Sigma)
for 1 hour to block nonspecific binding of mouse and rat antibodies.
The cells were then incubated with the appropriate biotin-conjugated
antibodies for 45 minutes, washed twice, incubated for 30 minutes with
SA-PE or SA-RED670 and fluorochrome (fluorescein isothiocyanate
[FITC], Red 613 or PE)-conjugated antibodies, washed twice and fixed
in fixation buffer (PBS, 1% paraformaldehyde). Stained cells were run
on a FACScan or FACSCalibur (Becton Dickinson, San José, CA).
Antibody-positive populations of cells were distinguished according to
the level of staining obtained with fluorochrome-conjugated, mouse
and/or rat Ig-isotype-matched, control antibodies of
irrelevant specificity. Ten thousand events were collected and analyzed
using CellQuest software (Becton Dickinson). For cell sorting, 15 to 30 × 106 bone marrow cells were stained with Mac-1 and
anti-IL-2R antibodies as described above, using antibiotic
(penicillin/streptomycin and gentamycin, GIBCO-BRL, Gaithersburg,
MD) containing staining buffer. Stained cells were run on
a FACStar Plus cell sorter (Becton Dickinson) and the
Mac-1loIL-2R+ cells were collected and
cultured (5 × 104 cells/mL) in Dexter Media (Iscoves
Modified Dulbecco's Medium, 10% FCS, 10% horse serum, glutamine,
penicillin, streptomycin, and gentamycin) in the presence or absence of
100 U/mL of recombinant IL-2 (Boehringer Mannheim, Indianapolis, IN).
Histology.
The intact spleen and liver of IL-2/ and
control animals was fixed in formalin before embedding in paraffin and
sectioning. Ten micron tissue sections and cytocentrifuge preparations
of bone marrow cells were counterstained with Wright-Giemsa stain (Sigma) before photomicroscopy. Myeloperoxidase and acid phosphatase activity in cytocentrifuge preparations of bone marrow and blood smears
was detected using commercial cytochemical staining kits (Sigma).
In situ hybridization.
Two nonoverlapping IL-2R probes corresponding to either the 176-729 or 985-1,744-bp region of the gene43 were used to
synthesize 35S-labeled RNA probes and detect expression of
IL-2R-mRNA in cytocentrifuge preparations of sorted bone marrow
cells by in situ hybridization as described by us
previously.18
Hematopoietic progenitor cell assays.
Soft agar colony forming assays were performed by plating cells in
1.5% methylcellulose (Methocel; Dow, Midland, MI) containing, 30%
fetal bovine serum, 1% bovine serum albumin, 40 µg/mL
penicillin/streptomycin, 20 ng/mL IL-3, 20 ng/mL GM-CSF, 1 U/mL
erythropoietin, and 100 ng/mL stem cell factor. All growth factors used
were recombinant. Colonies were scored 11 days after culture. In some
experiments, 106 purified T cells from IL-2+/+
or IL-2/ mice were mixed with 5 × 104 bone marrow cells from IL-2+/+ mice before
plating in methylcellulose. LTBMC in which wild-type or
IL-2/ bone marrow cells were cultured with
wild-type bone marrow stromal cell monolayers in the presence or
absence of 100 U/mL recombinant murine IL-2 were performed essentially
as described previously.44 Fresh IL-2 was added every 3 to
4 days and at 7, 14, and 21 days the cultures were 100% depopulated of
nonadherent cells, which were subsequently evaluated for colony-forming
unit (CFU) activity in methylcellulose as described above.
Adoptive T-cell transfer.
Fifty million IL-2/ or IL-2+/+
purified T cells (described above) were injected via a tail vein into
sublethally-irradiated (650R) C57BL.6 mice. Six weeks after T-cell
transfer, the animals were euthanized and the number and distribution
of hematopoietic cells present in the bone marrow determined by flow
cytometric analysis.
Bone marrow chimeras.
Recipient B6(CD45.1) mice (n = 4 to 5 per group) were lethally
irradiated (1.1 Gy) using a 250-kV x-ray machine at 100 rad/minute. After irradiation, mice were maintained on antibiotic water containing 106 U/liter polymixin B sulfate and 1.1 g/liter of neomycin
sulfate. Two hours postirradiation 1 × 106
unmanipulated bone marrow cells were injected (200 µL/mouse) into the retro-orbital plexus of anesthetized
mice. Five groups of recipient mice (n = 4 to 5) were used; radiation
control mice injected with PBS alone, reconstitution control mice
injected with host bone marrow cells, and three groups of mice that
received IL-2/ (CD45.2) bone marrow cells
alone or IL-2/ cells mixed with host bone
marrow cells in ratios of 1:1 and 5:1 (for competitive reconstitution).
Four week old gnotobiotic IL-2/ mice were
used as the source of donor cells because at this age the cellularity
and composition of the bone marrow is comparable to that of wild-type
littermates (Table 1). At approximately 30, 60, and 90 days
postreconstitution, peripheral blood was obtained from the
retro-orbital sinus or tail vein and after lysis of erythrocytes, the
mononuclear cells were stained with antibodies specific for lineage
markers for B cells (anti-B220), myeloid cells (anti-Gr-1 and Mac-1),
or T cells (anti-CD3, CD4, and CD8) in combination with antibodies
specific for donor (CD45.2) or host (CD45.1) hematopoietic cells and
analyzed by flow cytometry. All (five of five) of the radiation control
mice died 10 to 14 days postirradiation.
Statistical analysis.
Data are presented as mean ± standard deviation (SD). Statistical
significance was assessed by Student's t-test.
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RESULTS |
Hematopoietic failure and abnormal myeloid cell development in
IL-2/ mice.
By approximately 6 weeks of age, IL-2/ mice
reared and housed in a specific pathogen-free (SPF) environment
developed a hematopoietic disorder characterized by anemia and
neutropenia (Tables 1 and 2). Although the onset and severity of
this hematopoietic disorder varied with age and among individual
homozygous animals, it was present in all (45/45) SPF
IL-2/ mice. Gnotobiotic
IL-2/ mice also developed this hematopoietic
disorder40 (Table 1), although onset was delayed by 1 to 3 weeks compared with SPF IL-2/ animals. The
reason for this delayed onset is not known, but is presumably related
to the presence of environmental microbes that may exacerbate
and/or accelerate disease. Importantly, since gnotobiotic
IL-2/ mice do not develop
colitis,40 it can be concluded that abnormal hematopoiesis
in IL-2/ mice is not secondary to the
inflammation, infection, and blood loss associated with colitis. We
were unable to detect any evidence for a similar disorder in SPF or
gnotobiotic heterozygous mice. SPF IL-2/ mice
of between 5 and 7 weeks of age were used for the majority of the
studies described below.
By 6 weeks of age, the bone marrow of IL-2/
mice showed a 35% to 50% reduction in cellularity compared with the
bone marrow of wild-type littermates (Table 1) due primarily to a
reduction in the size of the B cell (B220+) and erythroid
(Ter-119+) compartments (Fig
1). Among populations of B cells, developing pro- (S7+),
pre- (BP1+), and mature (IgM+) cells were all
affected by the absence of IL-2 (Fig 1A). Although the
proportion of granulocytic (Gr-1+) cells (Fig 1B) and
myelomonocytic (Mac-1+) cells increased as a result of this
loss of B and erythroid cells, the absolute number of these cells was
not significantly different from that present in bone marrow of normal
mice. However, morphologic analysis of viable cells recovered from the
bone marrow of 5 to 7 week old IL-2/ mice
showed that there was an increase in the number of immature myeloid
cells (Fig 2). On average, there were
twofold to threefold more blasts and promyelocytes/metamyleocytes and a
corresponding 65% to 70% decrease in the number of mature
polymorphonuclear bone marrow cells in 6 week old
IL-2/ mice compared with IL-2+/+
animals (Table 1). This left-shift in IL-2/
bone marrow cells was confirmed by indirect immunoflourescent staining
using biotin-labeled anti-Mac-1 and Gr-1 antibodies in combination
with PE-strepavidin. The amplification of staining obtained using the
indirect avidin-biotin labeling system enabled the immature
(Mac-1/Gr-1lo) and mature (Mac-1/Gr-1hi)
myeloid cell populations to be more easily resolved (compare Fig 3 with Fig 1B). The bone marrow of
IL-2/ mice contained increased numbers of
Gr-1lo and Mac-1lo cells characteristic of
immature cells of the granulocytic45 and
myelomonocytic46,47 cell lineages, respectively (Fig 3).
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| Fig 1.
Loss of hematopoietic cells in the bone marrow of
IL-2/ mice. (A) Antibodies reactive with surface
antigens that distinguish various stages of B-cell development were
used to identify pro- (S7+), pre- (BP1+),
and mature (IgM+) B cells in the bone marrow of 6-week
old IL-2/ mice. (B) FITC-Gr-1 and PE-Ter-119 were
used to distinguish cells of the myeloid and erythroid lineage,
respectively. Stained cells were run on a flow cytometer and analyzed
using CellQuest software. The level of staining obtained with
isotype-matched control antibodies was used to establish quadrant
settings for the dot plots and determine the frequency of cells stained
with B cell, erythroid and myeloid cell-specific antibodies (numbers
shown in each quadrant of dot plots in A and in histograms in B). The
results shown are representative of 10 groups of mice.
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| Fig 2.
The absence of IL-2 causes an accumulation of immature
myeloid cells in the bone marrow of IL-2/ mice.
Cytocentrifuge preparations of bone marrow cells (BM) from 6-week old
SPF wild-type (+/+) and IL-2/ (×/×) mice were
fixed and stained with Wright-Giemsa stain before photomicroscopy.
Magnification 800X. The results shown are representative of 10 groups
of mice.
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| Fig 3.
Accumulation of phenotypically-immature myeloid cells in
the bone marrow of IL-2/ mice. Bone marrow cells from
6-week old wild-type (+/+) and IL-2/ ( /)
littermates were stained with biotin-labeled Mac-1 or Gr-1 antibodies
followed by SA-PE and analyzed by flow cytometry. Note the reduction in
the size of the mature granulocytes (Gr-1hi) and
myelomonocytic (Mac-1hi) cell populations in
IL-2/ mice. The level of staining obtained with
isotype-matched control antibodies was used to determine the frequency
of cells stained with Mac-1 and Gr-1 antibodies (% values shown in
each histogram). The results shown are representative of eight
independent experiments.
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The disruption of normal bone marrow hematopoiesis in the
IL-2/ mice was accompanied by extensive
extramedullary hematopoiesis in the spleen and liver (data not shown).
While the spleens of wild-type mice showed a mild degree of
extramedullary hematopoiesis (<5% immature myeloid/erythroid cells),
the spleens of IL-2/ mice showed extensive
(70% to 95%) extramedullary hematopoiesis. This increase in
extramedullary hematopoiesis was attributable to an increase in
immature myeloid cells resulting in enlargement of the red pulp and a
relative decrease in the white pulp.
Examination of the blood of SPF IL-2/ mice
showed a progressive anemia and neutropenia (Table 2). The reduction in
mature segmented granulocytes was first evident in 3- to 5-week old
animals and appeared to precede anemia, which was detected at 6 weeks of age. Accompanying neutropenia was an absence or loss of macrophages in both the thymus48 and peritoneum (data not shown).
Peritoneal exudate cells (PEC) from 6-week old
IL-2/mice contained 60% to 80% less
glass-adherent, peroxidase-positive macrophages than wild-type
littermates. Despite this loss or absence of mature macrophages in
IL-2/ PEC, it was not possible to detect any
quantitative differences in the microbicidal activity or antigen
presentation capabilities of peritoneal phagocytic cells when compared
with those obtained from wild-type mice (TR and SRC unpublished
observations).
Decreased hematopoietic progenitor cell activity in
IL-2/ mice.
The number of bone marrow hematopoietic and myeloid progenitor cells
was evaluated by comparing in vitro colony-forming ability of bone
marrow cells from 5-week old SPF IL-2/ and
wild-type littermates. On average, the number of in vitro colony-forming cells in the bone marrow of
IL-2/ mice was reduced by about 50%
(Fig 4). In addition, the colonies generated from IL-2/ bone marrow were smaller
and contained approximately 50% less CFU-GM (Fig 4). By contrast, the
number of more primitive, multilineage progenitor cells
(granulocytic/erythroid/macrophage/megakaryocyte [GEMM])
and erythroid progenitor cells (burst-forming unit-erythroid [BFU-E]) were similar in IL-2+/+ and
IL-2/ bone marrow. These findings are
consistent with a proliferative defect in (myeloid) progenitor cells
generated in the absence of IL-2. To investigate this possibility
further, bone marrow transplant studies were undertaken.
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| Fig 4.
Reduced hematopoietic progenitor cell activity in the
bone marrow of IL-2/ mice. Bone marrow mononuclear
cells from 5-week old SPF wild-type (+/+) or
IL-2/ (/) mice were cultured in
methylcellulose containing IL-3/GM-CSF/erythropoietin/stem cell factor
for 11 days and the number of
granulocyte/erythroid/myeloid/megakaryocytic (GEMM),
granulocyte/macrophage (GM) and blast forming unit-erythroid (BFU-E)
colonies counted. * P < .005 for comparison of
IL-2/ and IL-2+/+. The results shown
were obtained from three independent experiments.
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IL-2/ bone marrow cells cannot sustain
myelopoiesis upon transfer to lethally irradiated wild-type
animals.
Whole bone marrow (106 cells) from
IL-2/ (CD45.2+) mice was injected
into lethally irradiated C57BL.6-CD45.1+ hosts; enabling
all donor cells and their progeny in chimeric animals to be
distinguished by a monoclonal antibody specific for CD45.2. Host
animals were divided into five groups (n = 4 to 5 animals per group)
depending on the source and composition of donor cells injected; those
receiving no cells (radiation control), host bone marrow
(reconstitution control), IL-2/ bone marrow
or, for competitive reconstitution experiments, a 1:1 or 1:5 mixture of
IL-2/:host bone marrow cells. Five-week old
gnotobiotic IL-2/ animals were used as bone
marrow donors because at this age, the bone marrow is similar in
cellularity and composition to wild-type bone marrow (Table 1). In
particular, the number of
Sca-1+Thy1loLin- multipotential
stem cells in IL-2/ and IL-2+/+
bone marrow was comparable (data not shown). At approximately 30, 60, and 90 days postreconstitution, blood was drawn from surviving animals
and the contribution made by donor-derived cells to the myeloid
(Gr-1+/Mac-1+), B (B220+), and T
(CD3+/CD4+/CD8+) cell lineages
determined using anti-CD45.2 or CD45.1 antibodies in conjunction with
lineage specific antibodies and flow cytometry.
All of the lethally irradiated mice receiving no cells (PBS alone) died
within 10 to 14 days after irradiation. By contrast, all (four of four)
of the animals that received IL-2+/+ bone marrow and the
majority (four of five) of mice injected with
IL-2/ bone marrow survived until euthanized
at 95 days postreconstitution. At necropsy, the lymphoid tissues and
intestines of the animals reconstituted with
IL-2/ cells were grossly and histologically
normal.
Analysis of the blood of host animals injected with
IL-2/ cells showed that while they could
contribute to short-term engraftment and multilineage reconstitution,
they were unable to sustain myelopoiesis (Fig 5). Whereas myeloid
(Mac-1+/Gr-1+) cell reconstitution by
IL-2+/+ bone marrow was almost complete (>80%) in all of
the chimeras at 90 days postreconstitution,
IL-2/-derived cells represented 25% or less
of Mac-1+/Gr-1+ cells present in the blood of
surviving chimeras. In contrast, the level of T- and B-cell
reconstitution in animals receiving IL-2/
bone marrow was high and similar to that seen in animals reconstituted with IL-2+/+ bone marrow; consistent with the presence of
lymphocyte progenitor activity in IL-2/ bone
marrow. These findings were confirmed by the results of competitive
reconstitution experiments (Fig 5). Although at 30 days
postreconstitution, IL-2/-derived cells made
up the expected frequency of myeloid cells (approximately 50% and 20%
in animals receiving host and IL-2/ cells in
a 1:1 and 5:1 ratio, respectively), their contribution declined
thereafter. By 90 days postreconstitution,
IL-2/-derived cells contributed less than
20% and 10% to the myeloid cells present in animals receiving host
and IL-2/ cells in a 1:1 and 5:1 ratio,
respectively. In contrast, IL-2/-derived
cells made up the expected frequency of B cells in the majority of
these chimeras (Fig 5). Together, the data from both the in vitro and
in vivo progenitor cell assays suggest that myeloid progenitor cells in
IL-2/ mice have an intrinsic proliferative
and/or developmental defect.
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| Fig 5.
IL-2/ bone marrow cells cannot sustain
myelopoiesis in lethally irradiated host animals. Groups (n = 4 to 5)
of irradiated host (B.6/CD45.1+) mice were injected with
autologous (IL-2+/+) bone marrow or with bone marrow
from 5-week old gnotobiotic IL-2/ mice
(IL-2/), or IL-2/ bone marrow mixed
with host bone marrow cells in 1:1 or 1:5 ratios. Approximately 30, 60, and 90 days postreconstitution, animals were bled and the mononuclear
cells stained with either antibodies specific for the host or donor
CD45 allele in combination with antibodies that identify the myeloid
(Mac-1 and Gr-1; M&G), T (CD3/CD4/CD8) and B (B220) lineages and
analyzed by flow cytometry. The results shown represent the levels (%)
of donor-derived cells in each lineage at each time point for
individual mice. The asterisk indicates that at 30 days
postreconstitution the number of T cells was too low (<1%) to allow
accurate quantitation.
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Exogenous IL-2 can restore IL-2/
hematopoietic progenitor cell activity in LTBMC.
To determine if exogenous IL-2 can overcome the intrinsic proliferative
and/or developmental defect in IL-2/
myeloid progenitor cells, the effects of adding IL-2 in vitro to LTBMC
of IL-2/ bone marrow cells was evaluated.
This in vitro approach was chosen in preference to administering IL-2
to intact IL-2/ animals due to the large
amounts of IL-2 required to treat animals, the uncertainty of the dose
and duration of treatment required, and the potential toxicity of IL-2.
Monolayers of bone marrow stromal cells were established from wild-type
bone marrow and seeded with either bone marrow cells from 3-week old,
healthy SPF IL-2/ mice or wild-type
littermates in the presence or absence of 100 U/mL IL-2. At 7, 14, and
21 days, the cultures were 100% depopulated of nonadherent cells,
which were subsequently evaluated for colony-forming ability in soft
agar colony-forming assays.
Compared with bone marrow cultures seeded with wild-type hematopoietic
cells, the number of progenitor cells continuously declined in cultures
seeded with IL-2/ cells
(Fig 6). By 3 weeks of culture, the number
of progenitor cells that could be recovered from these cultures was
reduced by more than 90%. The progenitor cell loss was particularly
apparent for both CFU-GEMM mixed-lineage progenitor population and
grnaulocyte/macrophage precursors (CFU-GM).
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| Fig 6.
Abnormal IL-2/ bone marrow
hematopoietic progenitor cell activity in LTBMC. Adherent stromal cell
layers established from bone marrow cells of normal adult C57BL.6 mice
were seeded at day 0 with 1.2 × 107 bone marrow cells
from 3-week old SPF wild-type ( ) or IL-2/ (+)
mice in the presence (dashed lines) or absence (solid lines) of 100 U/mL recombinant IL-2. At the times indicated, the cultures were 100%
depopulated and the number and type of progenitor cells present
identified by methylcellulose CFU assays as described in Materials and
Methods. The results shown are representative of three independent
experiments. CFU-GM, colony forming unit-granulocyte/macrophage; CFU-GEMM, colony forming
unit-granulocyte/erythroid/myeloid/megakaryocyte.
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This loss of hematopoietic progenitor cell activity was at least
partially restored by the addition of exogenous IL-2 to long-term cultures of IL-2/ bone marrow cells. At 14 and 21 days of culture, the number of colony-forming cells generated
from IL-2/ cells in the presence of IL-2,
although still less than that obtained from cultures of
IL-2+/+ cells, was between eightfold and 10-fold higher
than the number obtained in the absence of IL-2. Similar levels of
restored progenitor cell activity were seen for both CFU-GEMM and
CFU-GM populations. In contrast, the effect of IL-2 on wild-type
progenitor cell activity was less dramatic, resulting in a twofold to
threefold increase in the number of progenitor cells. This may be
attributable to lower levels of IL-2R-responsive immature myeloid
cells in wild-type versus IL-2/ bone marrow
(see below and Fig 7).
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| Fig 7.
Accumulation of IL-2R-expressing immature myeloid cells
in the bone marrow of IL-2/ mice. Bone marrow cells
were isolated from 5-week old wild-type (+/+) and
IL-2/ (/) littermates and stained with Mac-1
and IL-2R or isotype-matched control antibodies before analysis
using flow cytometry as described in the legend to Fig 3. (A)
IL-2R-expressing myeloid cells in the bone marrow of 6-week old
C57BL.6 mice. The boxed region represents the frequency of
IL-2R+Mac-1lo cells. Isotype-matched
control antibodies (Ctrl, right panel) were used to determine the level
of nonspecific staining (boxed region). The results shown are
representative of five independent experiments. (B) C57BL.6 (+/+)
and IL-2/ (/) bone marrow cells expressing low
levels of Mac-1 (Mac-1lo) were reanalyzed for expression of
IL-2R; the % values represent the frequency of Mac-1lo
cells that are IL-2R+. Note the accumulation of
IL-2R-expressing Mac-1lo cells in
IL-2/ mice. The results shown are representative of
six groups of mice.
|
|
These findings are consistent with the results obtained from the in
vivo bone marrow transplant studies and suggest that the abnormal
myelopoiesis in IL-2/ mice is a consequence
of an intrinsic developmental or proliferative defect in a myeloid
progenitor cell(s). They also show that in vitro, IL-2 can partially
overcome this defect and promote myeloid progenitor cell growth.
However, due to the inherent complexity and cellular heterogeneity of
LTBMC, there are many potential mechanisms by which IL-2 could enhance
myelopoiesis in LTBMC. Additional experiments were designed, therefore,
to determine if immature myeloid cells normally express functional
IL-2Rs and if IL-2 can directly influence the growth and development of
myeloid progenitors.
Immature myeloid cells express IL-2Rs.
Responsiveness to IL-2 depends on expression of a receptor for IL-2 on
the surface of the cell. Flow cytometric and in situ hybridization
techniques were used to examine expression of CD122 (IL-2R), the
signaling component of the IL-2R complex, by immature myeloid cells in
the bone marrow of 6-week old IL-2+/+ mice. Flow cytometric
analysis showed that approximately 5% of myeloid (Mac-1+)
cells in the adult bone marrow expressed IL-2R (Fig 7A). The Mac-1loIL-2R+ cells made up approximately
50% of IL-2R-expressing Mac-1+ cells (boxed region in
Fig 7A). The specificity of anti-IL-2R antibody staining was
confirmed by the lack of staining by these cells with isotype-matched
control antibodies (Fig 7A). The majority of IL-2R+
cells also expressed low levels of the IL-2R-chain (data not shown).
Analysis of IL-2/ bone marrow showed that the
number of Mac-1loIL-2R+ immature myeloid
cells was increased by more than sevenfold (Fig 7B) in
agreement with the increased number of blasts and
promyelocytes/metamyelocytes detected by histomorphologic analysis
(Table 1). Morphologic analysis of fluorescence-activated cell sorting
(FACS) isolated IL-2R+ Mac-1lo cells
confirmed that this population of cells contained myeloid progenitors
(myeloblasts and promyelocytes; Fig 8A). In
contrast, the Mac-1hiIL-2R+ population of
bone marrow cells (Fig 6) comprised more mature cells including
metamyelocytes, band cells, and mature granulocytes (Fig 8B). This
distribution of Mac-1 expression is consistent with it being a myeloid
differentiation marker that is upregulated during myeloid cell
development.46,47 To substantiate the results of the
phenotypic analysis and exclude the possibility that the anti-IL-2R
antibody staining was an artifact, in situ hybridization was used to
identify IL-2R-mRNA expression among bone marrow cells from 6-week
old severe combined immunodeficient (SCID) mice. As seen
in Fig 8C, IL-2R-mRNA was readily detected in populations of myeloid
cells that were morphologically similar to FACS-isolated IL-2R-expressing Mac-1+ cells.
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| Fig 8.
Expression of IL-2R mRNA and protein by immature cells
of the myeloid lineage. The appearance of
Mac-1loIL-2R+ (A) and
Mac-1hiIL-2R+ (B) bone marrow cells
isolated by FACS. The identity of bone marrow cells expressing the gene
encoding the -chain of the IL-2R (C and D) was determined by
hybridization in situ of 35S-labeled probes synthesized in
the antisense (C) or sense (D) orientation to cytocentrifuge
preparations of total bone marrow from adult SCID mice. All
cytocentrifuge preparations were fixed and stained with Wright-Giemsa
stain before photomicroscopy. Magnification in A 800X and B through D
1000X. The results shown are representative of four independent
experiments.
|
|
IL-2Rs expressed by immature myeloid cells are functional.
To determine whether IL-2 is capable of influencing the development of
myeloid progenitor cells, the effects of IL-2 on the growth and
development in vitro of highly purified
Mac-1loIL-2R+ cells
(Fig 9) were evaluated. Cells were isolated
from the bone marrow of adult C57BL.6 or SCID mice by FACS and cultured
in 96-well tissue culture plates for 5 to 7 days in the absence or
presence of IL-2 (100 U/mL), without any additional growth factors or
stromal cells. In three independent experiments (Fig 9), the number of myeloid cells recovered from cultures containing IL-2 (mean, 70 × 103; range, 60 to 88 × 103) was on
average sixfold to sevenfold greater than that obtained from cultures
containing media alone (mean = 10.6 × 103; range, 9 to 13 × 103). Histologic analysis of cells before and
after culture showed that the addition of IL-2 resulted in the
generation of morphologically mature myeloid cells including band cells
and segmented granulocytes, as well as macrophages (Fig 9). In
contrast, in cultures containing media alone (Fig 9) or IL-15 (data not
shown), the only other known ligand for IL-2R, the viable cells
remaining contained few if any mature granulocytes or macrophages.
These findings demonstrate that, at least in vitro, IL-2 can directly
induce the growth and development of immature
(Mac-1loIL-2R+) myeloid cells.
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| Fig 9.
IL-2 promotes the growth and differentiation of immature
myeloid cells. FACS-isolated Mac-1loIL-2R+
bone marrow cells (before culture) were cultured for 7 days in complete
media in the absence (media) or presence (+IL-2) of 100 U/mL of IL-2
after which the cells were harvested, counted, and cytocentrifuge
preparations made. Upper panel: addition of IL-2 resulted in
significant increase in the number of cells recovered at the end of
culture (P < .001). Lower panel: cytocentrifuge preparations
of Mac-1loIL-2R+ cells before (before
culture) and 7 days after culture in the absence (media) or presence
(+IL-2) of IL-2. In the presence of IL-2,
Mac-1loIL-2R+ cells give rise to
macrophages as well as mature granulocytes. Magnification 1,000X. The
results are representative of three independent experiments.
|
|
Dysfunctional IL-2/ T cells do not disrupt
normal hematopoietic progenitor cell activity.
Several studies have suggested that the hematopoietic and immune system
disorders that develop in IL-2/ mice are
caused by lymphoid hyperplasia and T-cell infiltration into various
tissues including the bone marrow.49,50 We determined, therefore, if hematopoietic failure in IL-2/
mice could be the result of indirect effects of the absence of IL-2 and
the presence of dysfunctional T cells.
The ability of IL-2/ T cells to disrupt the
activity of normal hematopoietic progenitor cells in in vivo and in
vitro assays was determined. Approximately 5 × 107 purified lymph node T cells from 6- to 7-week old
IL-2/ or wild-type C57BL.6 mice
(Thy1.2+) were injected intravenously into sublethally
irradiated Thy1.1+ C57BL.6 mice. Six weeks later animals
were euthanized and their bone marrow analyzed to determine the affects
of the transferred IL-2/ T cells. As shown in
Table 3, donor (Thy1.2+) T
cells were the predominant T-cell population in the bone marrow of host
animals. However, despite their presence, it was not possible to detect
any significant changes in the number of B (B220+),
erythroid (Ter-119+), or myeloid
(Mac-1+/Gr-1+) cells. Importantly, the
proportion of immature (Mac-1lo/Gr-1lo) and
mature (Mac-1hi/Gr-1hi) myeloid cells was also
similar to that seen in animals transfused with wild-type T cells.
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|
Table 3.
Adoptively Transferred IL-2/ T Cells
Do Not Adversely Affect the Cellularity or Composition of Host Bone
Marow
|
|
The possibility that factors produced by
IL-2/ T cells were inhibitory to
hematopoietic progenitor cell growth was also investigated. One million
purified lymph node or splenic IL-2/ or
IL-2+/+ T cells were added to 5 × 104
wild-type bone marrow cells in methylcellulose and the number of
colonies generated after 12 days counted. The number of colonies produced in the presence of IL-2/ T cells (68 ± 10) was not significantly different from the number generated in
the absence of any T cells (77 ± 6), or IL-2+/+ T cells
(81 ± 11). In addition, the type of colonies generated was also
comparable in cultures containing IL-2+/+ or
IL-2/ T cells (data not shown). Together
these results show that dysfunctional IL-2/ T
cells do not adversely affect hematopoietic cell development and are
unlikely to be the cause of abnormal myelocytic cell generation in
IL-2/ mice.
| |
DISCUSSION |
Because myeloid cells form a vital and integral part of the immune
system, it is important to understand the regulation of their
generation. A variety of experimental approaches have established that
granulocytes and macrophages are derived from CFU-GM in the presence of
hematopoietic growth factors or colony-stimulating factors
(CSF).51 Among these CSFs, G-, M-, and GM-CSF have been shown to influence the survival, proliferation, differentiation, and
function of mature granulocytes and macrophages, as well as their
precursors. However, in view of the overlapping activities of these
factors and the absence of any overt effects on myelopoiesis in some
CSF-deficient animals,52,53 it is possible that there may
be considerable redundancy among these factors,54 and other unrelated factors may be required for myeloid cell development in vivo.
The results of our studies presented here demonstrate that IL-2,
generally considered a lymphocyte growth factor, plays a role in
myelopoiesis.
In the absence of IL-2, mice develop a profound hematopoietic disorder
characterized by defective myelopoiesis and decreased numbers of mature
granulocytes. The reduced number or absence of tissue-specific
macrophages (eg, peritoneal and thymic48 macrophages) in
neonatal and adult IL-2/ mice also suggests
that IL-2 can influence macrophage development. Although we have not
been able to detect any significant difference in the microbicidal,
cytotoxicity or antigen presentation capabilities of residual
peritoneal phagocytic cells in IL-2/ mice
(T.R. and S.R.C., unpublished observations), the loss or absence of
thymic macrophages in SPF and gnotobiotic
IL-2/ mice is accompanied by abnormalities in
intrathymic T-cell progenitor development.48 The ability to
prolong the life span of neutropenic IL-2/
mice when reared and maintained under gnotobiotic
conditions40 suggests that the rapid deterioration in
health status and premature death of SPF
IL-2/ mice may be attributable, at least in
part, to the loss of mature, functional granulocytes and increased
susceptibility to (opportunistic) environmental pathogens.
Based on the inability of IL-2/ bone marrow
cells to sustain myelopoiesis in lethally irradiated mice and in LTBMC,
this hematopoietic disorder could be due to an intrinsic proliferative
and developmental defect in a hematopoietic stem or myeloid progenitor
cell population. Because inactive stem cells would prevent multilineage
reconstitution,55,56 the normal number of stem cells and
multilineage (GEMM) progenitors, and the presence of long-term lymphoid
progenitor activity in IL-2/ bone marrow
suggests that stem cells are intact and that the defective
hematopoietic cell is a myeloid progenitor cell(s). Our inability to
detect any differences in the ability of
IL-2/ bone marrow cells to generate erythroid
(BFU-E) or lymphoid progenitor cells in in vitro or in vivo
differentiation assays when compared with IL-2+/+ cells is
consistent with this interpretation.
Several lines of evidence suggest that the myeloid progenitor cell
defect in IL-2/ mice is a consequence of
their direct dependency on IL-2 for their growth and possibly
development: (1) expression of functional IL-2Rs by myeloblasts and
promyelocytes, (2) the generation of mature granulocytes (as well as
macrophages) from Mac-1loIL-2R+ immature
myeloid cells in the presence of IL-2, and (3) the accumulation of
IL-2R+ immature myeloid (Mac-1lo) cells in
IL-2/ mice. Together, these findings
demonstrate that the IL-2/IL-2R signaling pathway can directly
influence myeloid cell development in vitro. The demonstration that
IL-2 can directly promote myeloid progenitor cell growth is consistent
with, and now provides an explanation for, the observed differential
expression of IL-2Rs by myeloid cells during in vitro
differentiation27 and the ability of IL-2 to increase the
number of circulating myeloid progenitor cells in
vivo.28-31 Importantly, the requirement by myeloid
progenitor cells for IL-2 also provides an explanation for the
accumulation of IL-2R-bearing immature myeloid cells and the
concomitant loss of mature granulocytes in
IL-2/ mice. Finally, the occurrence of a
granulocyte deficiency in mice that lack the
IL-2R-chain57 similar to that which we have described in
IL-2/ mice agrees with our conclusion that
the IL-2/IL-2R signaling pathway plays an important role in regulating
myelocytic cell generation.
Although our data is most consistent with this interpretation, we
cannot exclude the possibility that IL-2 may influence myeloid cell
development in other ways. For example, IL-2 may be a competence factor
enabling immature myeloid cells to acquire responsiveness to other
growth and differentiation factors. Because IL-2 is known to be able to
influence the production of other hematopoietic growth factors by
activated T cells, and possibly by other hematopoietic cells, it may be
required to induce the synthesis of other factors essential for
hematopoiesis and stromal cell growth. The finding that IL-2 can
prevent apoptosis in cultured human blood-derived neutrophils21 suggests that IL-2 can act as a survival
factor for myeloid cells. However, we have been unable to demonstrate a
similar effect of IL-2 when included in 48-hour cultures of IL-2+/+ mouse blood or bone marrow mononuclear cells (H.B.
and S.R.C., unpublished observations). Finally, the possibility that
IL-2 has multiple effects during myelopoiesis and that the nature of its influences is dictated by the developmental stage of the
IL-2R-bearing myeloid cell cannot be ignored. This would be consistent
with the wide distribution of IL-2Rs among developing myeloid cells and
the different responses (proliferation, survival, differentiation, and
modulation of functional activity) to IL-2 that have been observed.
This mechanism of action of IL-2 would be analogous to the multiple
actions of the CSFs on CFU-GM and their progeny.51
The lymphoid hyperplasia that disrupts the bone marrow and other
tissues in IL-2/58 and
IL-2R/57 mice may exacerbate hematopoietic
failure in these animals. Indeed, the defective or abnormal B-cell
development and hemolytic anemia that occurs in
IL-2/50 and
IL-2R/57 mice has been attributed to
dysfunctional T cells. However, the fact that in progenitor cell assays
myeloid cell development is defective in the absence of T cells, the
presence of IL-2/ T cells does not interfere
with the development of normal bone marrow hematopoietic cells either
in vitro or in vivo, and the onset of the hematopoietic disorder occurs
in gnotobiotic IL-2/ mice before lymphocyte
hyperplasia40 makes it unlikely that they are the cause of
abnormal myelopoiesis. Defective myelopoiesis in gnotobiotic
IL-2/ that do not develop
colitis40 also convincingly demonstrates that this disorder
is not secondary to the inflammation, infection, and blood loss
associated with colitis. Finally, the inability to abrogate or prevent
the granulocyte defect and hematopoietic failure in
IL-2R/ mice after antibody-mediated T-cell
depletion57 is also inconsistent with the involvement of
dysfunctional T cells in the disruption of myelocytic cell development
that occurs in the absence of a functional IL-2/IL-2R signaling
pathway.
Collectively, we interpret our findings as evidence for the involvement
of IL-2 in the homeostatic regulation of myelopoiesis. We propose that
through its growth promoting activity IL-2 normally serves to expand
and maintain the size of the Mac-1loIL-2R+
myeloid progenitor cell population in the bone marrow. In its absence,
this population would be progressively depleted by the successive waves
of progenitor cell development necessary to continually replace
short-lived (on average <24 hours55) peripheral blood granulocytes. This could explain why some level of myeloid cell maturation occurs in the absence of IL-2 in
IL-2/ mice, why
IL-2/ bone marrow cells cannot sustain
long-term granulopoiesis, and why the hematopoietic defect in
IL-2/ mice is not apparent until after birth.
Alternative possibilities such as the existence of IL-2-independent
myeloid progenitors and granulocytes and a differential requirement for
IL-2 by fetal and adult myeloid progenitor cells and/or the
environments in which they develop cannot be excluded. Because the
incidence of hematopoietic failure in IL-2/
mice is coincident with, or occurs soon after, development of the bone
marrow as a definitive adult hematopoietic organ has been
completed,59 adult progenitor cells may be more dependent on IL-2 than their fetal counterparts.
In summary, the findings we report here show for the first time that
IL-2 plays an important role in regulating myelocytic cell development
in vivo. The observation that human immunodeficiency due to defective
IL-2 production60 is also associated with deficient hematopoiesis in the bone marrow (K. Weinberg, personal communication, 1996) suggests that IL-2 may have a similar role in
regulating hematopoiesis in humans.
| |
FOOTNOTES |
Submitted August 14, 1997;
accepted December 4, 1997.
Supported in part by grants from the American Cancer Society (to
S.R.C.), the Leukemia Society of America (to S.G.E. and M.S.C.), W.W.
Smith Chari Trust (to S.R.C.), and the National Institutes of
Health (to S.R.C. and S.G.E.).
Address reprint requests to Simon R. Carding, PhD, Department of
Microbiology, University of Pennsylvania, 303A Johnson Pavilion, Philadelphia, PA 19104-6076.
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.
| |
ACKNOWLEDGMENT |
We thank G. Trinchieri, M.I. Greene, and K. Weinberg for their critical
review of the manuscript, R. Schwartz for providing the IL-2-deficient
mice, and H. Pletcher for assistance with flow cytometric analyses and
cell sorting.
| |
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Abstract
Introduction
Abstract