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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2231-2239
Anti-VLA4/VCAM-1 Induced Mobilization Requires Cooperative Signaling
Through the kit/mkit Ligand Pathway
By
Thalia Papayannopoulou,
Gregory V. Priestley, and
Betty Nakamoto
From the Department of Medicine/Hematology, University of Washington,
Seattle, WA.
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ABSTRACT |
Although a large body of data on mobilization have yielded valuable
clues, the mechanism(s) dictating egress of stem/progenitor cells
during baseline hematopoiesis and after their mobilization are poorly
understood. We have previously provided functional in vivo evidence
that cytoadhesion molecules, specifically the  1
integrins, are involved in mobilization; however, the mechanism by
which this was achieved was unclear. To provide further insights into
the anti-very late antigen 4 (VLA4)/anti-vascular cell adhesion molecule 1 (VCAM-1) induced mobilization, we used these antibodies to
treat mutant mice with compromised growth factor receptor function. We
found that mobilization by anti-VLA4 does not depend on a functional granulocyte colony-stimulating factor, interleukin-7 (IL-7), or IL-3
receptor. By contrast, the functional kit receptor is required, because
W/Wv mice responded minimally, whereas
Steel-Dickie (Sl/Sld) responded normally. Both
Wv and Sl/Sld mice did not respond to
anti-VCAM-1 treatment, in contrast to their +/+ littermates and
despite normal levels of VCAM-1 expression in bone marrow cells. The
defective response to anti-VCAM-1 in W/Wv mice was
corrected after their transplantation with +/+ cells. mev/mev mice showed increased
numbers of circulating progenitors before treatment and a heightened
response after anti-VLA4 or anti-VCAM-1 treatment. Downmodulation of
kit expression was detected in normal bone marrow cells after anti-VLA4
treatment. On the strength of the above findings we conclude that (1)
anti-VLA4/VCAM-1induced mobilization likely requires signaling for
stimulation of cell migration; (2) this cooperative signaling involves
the kit/kit ligand pathway, and provides a novel example of
integrin/cytokine crosstalk; and (3) migration mediated through the
kit/kit ligand pathway may be a common contributor to different
mobilization stimuli. Dissection of the exact molecular pathways that
lead to mobilization remains a future challenge.
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INTRODUCTION |
HEMATOPOIETIC CELLS proliferate and
differentiate within a unique bone marrow microenvironment consisting
of several types of stromal cells and extracellular
matrix.1-4 Terminally differentiated cells are then
released into the circulation. In addition to circulating mature cells,
peripheral blood contains a small number of ancestor cells, ie, stem
cells and lineage-committed progenitor cells. Their existence in the
peripheral blood has been functionally shown in previous
transplantation experiments involving mice, dogs, and
primates.5 Despite barely detectable levels under basal
conditions, circulating stem/progenitor cells can be increased to
significant levels after several treatment schemes, ie, after administration of pharmacological doses of hematopoietic
cytokines/chemokines, alone and in combination, or during the recovery
period from chemotherapy.6 Although information about
circulating levels of stem progenitor cells has been available for
several decades, the molecular mechanisms that lead both to their
physiological release at basal hematopoiesis and to their enforced
emigration remain poorly understood. A large number of studies
regarding mobilization have been published in recent years, especially
after the introduction of granulocyte colony-stimulating factor
(G-CSF). However, many of these are concerned with the effectiveness of
mobilization schemes, the spectrum and characterization of mobilized
cells, and whether or not malignant cells are mobilized.6
Although these questions are of clinical importance, the suggested
schemes and observations have remained empirical and studies designed
to explore mechanisms of mobilization have been limited.6
Within the bone marrow, hematopoietic stem/progenitor cells are located
in extravascular spaces and to find their way to peripheral blood, they
must move into sinusoids and transmigrate through the basement membrane
and endothelial layer. If adhesive interactions are responsible for
their anchoring in the first place, then these same interactions have
to be severed, and the cells should acquire increased migratory
properties. A wide repertoire of cytoadhesion molecules are present on
hematopoietic cells, and their respective ligands are found on
microenvironmental cells and matrix. Several of these molecules have
been found in vitro to contribute to adhesive interactions between
hematopoietic cells and surrogate populations of bone marrow stromal
cells.7-12 However, it has not been clear to what extent
this in vitro information was relevant in vivo.
We provided the first direct in vivo evidence that a certain class of
cytoadhesion molecules, the 1 integrins, are involved in
mobilization, by reporting that anti-very late antigen 4 (VLA4) treatment of primates and mice led to mobilization of stem/progenitor cells.13 In addition to anti-VLA4, we showed that
antibodies to its ligand, vascular cell adhesion molecule 1 (VCAM-1),
present on stromal cells, can also have the same effect,14
implying that perturbations of either the hematopoietic cells
themselves or marrow stromal cells can induce mobilization. Although we
have found that the function-blocking property of the antibody was critical for response,15,16 the mechanism leading to
mobilization after antibody treatment was not immediately apparent. Is
anti-VLA4-induced mobilization the result of a simple biophysical
event, ie, deadhesion, or are additional steps required? If so, what
are the cooperating molecules? We addressed these questions by studying
first the effects of combined treatments of anti-VLA4 and
cytokine-induced mobilization. Coadministration of anti-VLA4 with
cytokines (G-CSF, kit ligand (KL), and flt3 ligand), alone or in
combination, led to enhancement of mobilization.16 Whether
this enhancement involves accentuation of a single mechanism, ie,
downregulation of VLA4, or the result of cooperation of more than one
mechanism controlled by independent actions of cytokines or anti-VLA4,
has not been clear.
To provide further insights, we have used function-blocking antibodies
and mutant mice with compromised growth factor receptor function. We
generated data which suggest that the anti-VLA4- or
anti-VCAM-1induced mobilization requires signaling, which is likely
accomplished through the kit/kit ligand pathway, providing a novel
example of integrin/cytokine interaction. Our data also raise the
possibility that signaling by the kit/kit ligand pathway may be a
common contributor to mobilization induced by other stimuli.
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MATERIALS AND METHODS |
Mice
WCB6F1 (Sl/Sld or Steel-Dickie) and
WCB6F1 (+/+) littermates, WBB6F1
(W/Wv) and WBB6F1 (+/+) littermates, C57B16
(mev/mev) and phenotypically normal littermates
(either +/+ or mev/+), and A/J mice and
(B6xA/J)F1 controls were purchased from Jackson
Laboratories (Bar Harbor, ME). The G-CSF receptor (G-CSFR) ( /)
and (+/+) controls were generously provided by Dr D. Link (Washington University School of Medicine, St Louis, MO). The interleukin-7 receptor (IL-7R) (/) and (+/+)
controls were provided by Dr Chris Clegg (Bristol-Meyers Squibb
Pharmaceutical Research Institute, Seattle, WA), and were generated by
Dr J.J. Peschon.17 All animals were housed
under specific pathogen-free (SPF) conditions in a facility at the
University of Washington approved by the American Association for the
Accreditation of Laboratory Animal Care, or at the research facility of
Bristol-Meyers Squibb.
Antibodies and Cytokines
Purified low endotoxin monoclonal rat antibodies to murine VLA4 (clone
PS/2) and VCAM-1 (clone MK-2) were a gift from Dr R. Lobb (Biogen,
Cambridge, MA). Antibodies were injected intravenously (i.v.) at 2 mg/kg body weight/d for 3 days. Cytokines used included: G-CSF,
Filgrastim Neupogen (Amgen, Thousand Oaks, CA) administered at 100 µg/kg body weight intraperitoneally twice daily; Kit Ligand (KL, SCF,
nonpegylated; a generous gift from Amgen) administered subcutaneously
(sc) at 100 µg/kg body weight/d; and Flt-3 Ligand (FL, Chinese
hamster ovary-derived; a generous gift from Immunex, Seattle, WA)
administered sc at 400 µg/kg body weight/d for either 3 or 7 days.
Collection of Tissues
Tissue sampling was performed under anesthesia with Nembutal sodium or
with Avertin. Peripheral blood was drawn from anesthetized animals
either from the retro-orbital plexus of the eye or from the vena cava
at the juncture with the portal vein. A measured volume of blood was
washed with Dulbecco's phosphate-buffered saline (DPBS) and
mononuclear cells were separated over a density gradient (Accudenz;
Accurate Chemical & Scientific, NY), and resuspended in Iscove's
Modified Dulbecco's medium (IMDM; HyClone, Logan, UT) + 0.1% bovine
serum albumin (BSA) for culture. Bone marrow cells were obtained from
donor animals anesthetized and killed by cervical dislocation. Femurs
and tibias were removed and bone marrow cells were flushed aseptically
in Hanks' Balanced Salt Solution (Hyclone) containing 0.1% BSA. Cells
were dispersed into a single-cell suspension by repeated flushing and
then allowed to settle for 1 minute to remove bone spicules.
Colony-Forming Unit-Culture (CFU-C) Assays
CFU-C assays were performed using a methylcellulose
mixture consisting of 1.2% (wt/vol) methylcellulose (Fisher
Scientific, Fairlawn, NJ), 30% fetal bovine serum (FBS; Intergen,
Purchase, NY), 1% BSA, 0.1 mmol/L 2-mercaptoethanol, 5 U/mL
recombinant erythropoietin (Genetics Institute, Cambridge, MA), 10%
(vol/vol) Mouse IL-3 Culture Supplement (Collaborative Biomedical
Products, Bedford, MA), 5% (vol/vol) pokeweed-mitogen-stimulated
spleen-cell-conditioned medium, and 50 ng/mL recombinant murine stem
cell factor (Peprotech, Rocky Hill, NJ), in IMDM. Cultures were
incubated at 37°C in 5% CO2/95% air in a humidified
chamber for 7 to 10 days. Mononuclear cells from 0.2 to 1.0 mL of
peripheral blood per mouse and/or cells from appropriately
diluted bone marrow suspensions were plated in replicate plates.
Colonies were counted on the basis of morphological criteria using a
dissecting microscope, and all colony types (burst-forming
unit-erythroid and colony-forming unit-granulocyte-macrophage) were
pooled for reporting total CFU-C.
Bone Marrow Transplantation
Bone marrow cell suspensions were obtained as described above from
WBB6F1 +/+ animals and were diluted appropriately for i.v. injection into irradiated recipients using a volume of 0.5 to 1.0 mL
per animal. W/Wv mice were used as recipients and
irradiated at 400 cGy total from a dual source (Gammacell-40; Nordion
International, Ontario, Canada) delivering between 129 and 132 cGy/min.
Fluorescence-Activated Cell Sorter (FACS) Analysis and
Immunohistochemistry
BDF1 female mice were IV injected daily for 3 days with
anti-mouse VLA4 (clone PS/2; Biogen, Cambridge, MA) 2 mg/kg body
weight, and killed 8 hours after the final injection. Low density
(<1.085 g/mL) bone marrow mononuclear cells were obtained from an
Accudenz gradient, washed with PBS + 0.1% BSA, and stained with
phycoerythrin (PE)-conjugated anti-CD117 (anti-c-kit, clone 2B8;
Pharmingen, San Diego, CA). All staining was at 1 µg
Ab/106 cells for 30 minutes on ice, followed by PBS washes.
Cells were also incubated with or without PS/2 followed by fluorescein
isothiocyanate (FITC) conjugated goat F(ab )2 anti-rat IgG (Caltag,
Burlingame, CA) to verify that the in vivo injections were at
saturating levels. Analysis was performed on a FACSCalibur cytometer
(Becton Dickinson Immunocytometry Systems, San Jose, CA).
Immunohistochemistry was performed on 6-µM frozen sections of femoral
and tibial bone marrow plugs dehydrated in 3:1 acetone: methanol fixed
in 0.2% formalin, then blocked sequentially with 10% FBS in DPBS and
levamisole to suppress endogenous alkaline phosphatase expression.
Alternate serial sections were stained with anti-mouse VCAM-1 (Clone
MK-2; Biogen) or anti-human von Willebrand Factor (DAKO, Carpinteria,
CA), followed by appropriate biotinylated secondary antibodies and
streptavidin-alkaline phosphatase (Vector, Burlingame, CA). Color was
developed with Vector alkaline phosphatase substrate kit and
counterstained with hematoxylin.
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RESULTS |
Response of Mice With Hematopoietic Growth Factor Receptor Deficiencies
G-CSFR null mice.
In previous experiments we have documented that coadministration of
G-CSF and anti-VLA4 augments the mobilization response.16 However, we could not determine whether there was exaggeration of a
single mechanism or two different mechanisms. To resolve this issue we
examined the anti-VLA4 mobilization response of mice that were null for
the G-CSFR. Generation of these mice and description of their phenotype
was previously published.18 G-CSFR null mice and controls
were administered daily i.v. injections of anti-VLA4 for 3 days and
killed the following day. The results (Fig
1) show that G-CSF null and control mice
respond similarly to the same dose of anti-VLA4 (control mice,
395 ± 37.2 CFU-C/mL blood; G-CSFR / mice, 514.2 ± 58.2
CFU-C/mL blood) and their responses were significant compared with
their pretreatment values (P < .05). Therefore, the
presence of an intact G-CSF receptor on hematopoietic cells is not
required for anti-VLA4 response.
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| Fig 1.
Five control mice and five receptor null mutant mice per
group were treated by three IV injections of anti-VLA4 (see Materials and Methods). The mobilization response, measured as CFU-C/mL blood, is
shown. All mice responded to anti-VLA4 treatment with a significant
difference from their baseline CFU-C/mL levels.
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IL-7R null mice.
IL-7R deficient mice have impaired B and T lymphopoiesis.19
It is also of interest that administration of IL-7 alone or in
combination with G-CSF in normal mice has been shown to mobilize cells.20 IL-7R null mice and controls were injected with
anti-VLA4 i.v. for 3 days and peripheral blood was drawn the fourth day to assess circulating levels of CFU-C (Fig 1). A significant
mobilization was induced by anti-VLA4 in IL-7R / mice
(P < .05) and their controls
(P < .05), although some quantitative differences
between the two sets of animals were noted (controls, 440.3 ± 103;
IL-7R /, 289.2 ± 57).
A/J mice.
A/J mice have an impaired response to IL-3 because of a defect in the
IL-3R .21 The latter is not detectable on the surface of
A/J mouse hematopoietic cells, but may be detected in the
cytoplasm.22 The defect is not absolute because A/J mast
cells in culture can generate IL-3R in the presence of IL-3 and
KL.22 A/J mice and BAF1 controls were treated
with anti-VLA4. Again, a significant mobilization response
(P < .05) was obtained (BAF1,
326 ± 30 CFU-C/mL of blood; A/J, 301 ± 48) compared with
baseline levels from both types of animals (Fig 1). However, A/J mice
were lower at baseline and after mobilization.
W/Wv and Sl/Sld mice.
W/Wv mice have a defined defect in kit kinase activity,
although the expression of kit protein on the cell surface and the binding to KL is normal.23 Control +/+ littermates
responded to anti-VLA4-induced mobilization; however, W/Wv
mice show a very blunted response (about 30% of normal), in contrast to the other mutant mice tested (11 WBB6F1 +/+ littermates,
273 ± 41 CFU-C/mL blood; 11 W/Wv mice, 93 ± 23)
(Fig 2). We were intrigued by this muted
response and proceeded to test Sl/Sld mice.
Sl/Sld mice lack expression of membrane-bound
KL,24 have a similar clinical phenotype to Wv
mice, but have normal kit receptor expression and function in their
hematopoietic cells, which can correct, by bone marrow transplantation, the defect in W/Wv mice.25 When
Sl/Sld mice were treated with anti-VLA4, they responded to
treatment similarly to their +/+ littermate controls (15 WCB6F1 +/+, 183 ± 27; 9 Sl/Sld mice,
148 ± 25), in contrast to W/Wv mice (Fig 2).
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| Fig 2.
CFU-C/mL blood from control +/+ littermate mice and
from Sl/Sld and W/Wv mice before and after
anti-VLA4 and anti-VCAM-1 treatment (see Materials and Methods for
details). Sl/Sld mice respond to anti-VLA4 similarly to
controls (P = .35), in contrast to W/Wv mice
which showed a blunted response (P < .0016). Both types of
mutant mice did not respond to anti-VCAM-1 treatment, in contrast to
their +/+ littermates. *Indicates significant differences from control responses (P < .05) and numbers above each column
indicate the number of mice treated.
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The expression of VLA4 in bone marrow cells, either lineage-positive or
lineage-negative cells, was similar in W/Wv and
Sl/Sld mice and did not differ from their +/+ littermate
controls (data not shown). Furthermore, the data could not be explained
by differences in the CFU-C content per femur, because
Sl/Sld mice, if anything, had less CFU-C per femur and are
more anemic than W/Wv mice (Table
1). We also tested whether these mice
differed in their response to anti-VCAM-1, the VLA4 ligand. Littermate
+/+ control mice of either the Sl/Sld or the
W/Wv series responded to VCAM-1. However, to our surprise,
both Sl/Sld and W/Wv mice showed no response to
anti-VCAM-1 treatment compared with their pretreatment values (5 WCB6F1 +/+ mice, 254 ± 33; 6 Sl/Sld mice,
53 ± 16 CFU-C/mL blood; 9 WBB6F1 +/+ mice,
136 ± 31; 10 W/Wv mice, 30 ± 9 CFU-C/mL blood) (Fig
2). Constitutive expression of VCAM-1 was shown on immunohistochemistry
of marrow sections, in sinusoidal endothelial cells of both mutant
strains similar to their control littermates (data not shown).
It is known that the Steel (Sl) microenvironment is defective in the
expression of membrane-bound KL and the maintenance of normal
hematopoiesis.24, 25 Because of the abnormal
microenvironment, one might have anticipated that the Sl mice might not
respond to anti-VCAM-1. This result was totally unexpected for
W/Wv mice because these mice have a functionally normal
hematopoietic stroma.25 One may argue that W/Wv
mice may respond subnormally to all mobilization treatments including anti-VLA4 or anti-VCAM-1. However, there was no difference in response
to G-CSF between Sl/Sld and W/Wv mice (see data
in following sections). Although the G-CSF response was subnormal in
both types of mice (about 50% of control responses), both strains did
mobilize progenitors to a level significantly different from baseline
level (Fig 2), unlike the almost complete lack of mobilization with
VCAM-1 treatment (W/Wv, 30 ± 9; Sl/Sld,
53 ± 16). To explain these data, we hypothesized that for either anti-VLA4 or anti-VCAM mobilization in W/Wv mice, signaling
is required but does not occur because of unresponsive hematopoietic
cells.
If our reasoning was correct, then by providing normal cells in the
W/Wv mice we should correct the mobilization defect. For
this reason we transplanted bone marrow cells from +/+ littermate
controls into sublethally-irradiated W/Wv recipients (see
Materials and Methods). Four and 8 weeks after transplantation the
hematocrits in these mice had returned to control levels (Fig
3), as would have been expected after
engraftment of normal +/+ stem/progenitor cells. W/Wv
recipients were also transplanted with W/Wv donor cells and
served as transplantation control animals. The posttransplant
hematocrit (Hct) in these mice at 4 and 8 weeks after transplantation
was at 29% and circulating progenitor cells at 4 weeks posttransplant
were much lower than those observed in the recipients of +/+ donor
cells (Fig 3). Eight weeks after transplantation all transplanted
animals were treated with anti-VCAM-1 to test mobilization response.
As seen in Figure 3, a significant response to anti-VCAM-1 is noted
after transplantation in the recipients of +/+ donor cells. This
response was similar to or better than the response of +/+ mice after
anti-VCAM-1 treatment (545.7 ± 30.5 posttransplant in
W/Wv recipients of +/+ cells; 136.2 ± 30.7 in +/+
controls postanti-VCAM-1) (Fig 2). By contrast, no response was
observed in the recipients of W/Wv donor cells. A second
group of five W/Wv recipients transplanted with +/+ donor
cells also had a normal Hct and responded well to anti-VCAM-1
treatments 8 weeks after transplantation (Hct 44.8 ± 1.15;
postanti-VCAM-1, 257.5 ± 12.6 CFU-C/mL blood). The data from
these experiments suggested that the absence of the response in
W/Wv, when administered anti-VCAM-1, is a result of the
absence of signal transmission to defective W/Wv cells. If
normal cells are administered within the same W/Wv
microenvironment, then anti-VCAM-1 treatment becomes effective.
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| Fig 3.
Transplantation of W/Wv mice with +/+
bone marrow cells ( ) or W/Wv ( ) bone marrow cells.
Eight weeks posttransplant W/Wv mice administered +/+
donor cells had normal Hct levels and responded significantly to
anti-VCAM-1, in contrast to W/Wv recipients of
W/Wv donor cells.
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mev/mev mice.
The above data suggested that a functional kit receptor is important in
the mobilization process. Mice homozygous for me locus have a severe
phenotype and die at about 20 days, but the biology of the disease has
been studied in the viable mouse variant (mev) which die
much later, at about 9 weeks.26 Mutant me mice exhibit remarkable hematopoietic defects including autoimmunity, massive expansion of myeloid cells, and splenomegaly with increased number of
CFU-E and hypersensitivity to erythropoietin.26 The me
locus encodes Shp1, and cells from these mice are expected to exhibit an augmented or prolonged activation of kit autophosphorylation, because Shp1 is a downstream negative effector of kit signaling in
vivo.27 Thus, we tested the responses of
mev/mev mice to both anti-VLA4 and anti-VCAM-1
(Fig 4). Baseline circulating levels of
CFU-C/mL are significantly higher in mev/mev
mice (650 ± 15.8), versus either mev/+, or +/+ controls
(104.7 ± 26.8). Bone marrow mononuclear cells (BM-MC) and
CFU-C/femur are similar in mev/mev and +/+
controls. (BM-MCs in +/+ mice, 13.02 ± 1.74 × 106
per femur; mev/mev mice,
10.23 ± 0.61 × 106/femur; CFU-C/femur in +/+ mice,
25,066 ± 2,296/femur; mev/mev,
25,936 ± 2,027/femur). Our data suggest that the unexplained splenomegaly in this model26 could be caused by an
increased migratory capacity and splenic redistribution of
stem/progenitor cells in mev mice. The opposite result is
observed in W/Wv mice (Fig 2), which have very low basal
numbers of circulating CFU-C (18 ± 3.4/mL blood). There is an
exaggerated response to both anti-VLA4 and anti-VCAM-1 in
mev mice compared with controls (post-anti-VLA4:
mev/mev, 3,655 ± 970; controls,
863 ± 333; postanti-VCAM-1: mev/mev,
1,942 ± 855; controls, 171.5 ± 57). Thus, constitutive
activation of kit signaling results in increased migration of cells
into the periphery and a heightened response to other mobilizing
agents. The data provide additional evidence consistent with
involvement of kit signaling in mobilization.
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| Fig 4.
Bone marrow mononuclear cells were tested for kit and
VLA4 expression after three daily injections of anti-VLA4. Labeling of
cells with 2° antibody only, or anti-VLA4 and 2° antibody, gave
superimposable profiles (not shown) indicating that cells were
saturated by anti-VLA4 in vivo. Scattergram on the left panel was
identical when untreated or treated animals were tested and the insert
depicts cells in blast window analyzed for both kit expression (using
directly conjugated anti-kit) and VLA4 expression. Note that after
anti-VLA4 treatment both kit and VLA4 expression are downmodulated
(middle and right panel). Downmodulation of kit was similar in two
other experiments comparing untreated and treated bone marrow cells.
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Response of W/Wv mice to cytokines.
To study the importance of normal kit signaling in mobilization by
other cytokines we tested the response of W/Wv mice to a
number of mobilization regimens. We confirmed published data28 that the response to G-CSF is approximately 50% of
controls (W/Wv mice, 181.8 ± 19.9; +/+ controls,
388.6 ± 36; Sl/Sld, 199.7 ± 29.6%; +/+ controls,
416.8 ± 40). Reduced responses were also observed with treatments
involving other cytokines or cytokine combinations administered for 3 days (post-G + FL for 3 days: W/Wv mice,
1,714 ± 344 CFU-C/mL; +/+ controls, 3,535 ± 346; post-FL treatment for 3 days: W/Wv, 83.7 ± 9.7; +/+ controls,
333.3 ± 26.3; post-FL + KL for 3 days: W/Wv,
146.3 ± 29.3; +/+ controls, 569 ± 79). Although there was a significant difference after 3 days of FL treatment between +/+ and
W/Wv mice, after 7 days of FL treatment there was excellent
response in W/Wv mice, with no significant differences from
controls (post-FL treatment for 7 days: W/Wv,
5,965.3 ± 1,300; +/+ controls, 3,511 ± 172). The FL
mobilization response after day 7 in W/Wv mice was
accompanied by a marked proliferative response within the bone marrow
(CFU-C/femur in W/Wv, 607,300 ± 56,910; +/+ controls,
210,322 ± 19,473; P < .05).
The normal responses of W/Wv mice to FL at day 7 stand in
contrast to the subnormal responses previously observed with G-CSF or
combinations of G + FL or KL + FL or FL alone for 3 days. The data with
FL at 7 days would suggest that either W/Wv mice may not
respond early on as readily as control mice, or that the FL response is
an exception to the responses with other cytokines. A subnormal
response was noted by other investigators with different doses of
G-CSF28; however, very high doses have not been tested. If
with high doses the subnormal response persists, one may suggest that
the response to FL is qualitatively different from that of other
cytokines. FL signals via binding to its tyrosine kinase receptor,
flt3, present on hematopoietic stem/progenitor cells. Thus, it is
theoretically possible that the mobilizing defect in the kit tyrosine
kinase receptor of W/Wv mice may be compensated for by
signaling through the flt3 alternative tyrosine kinase.
Downmodulation of c-kit expression after anti-VLA4 treatment.
To test whether any changes could be detected in kit expression after
anti-VLA4 treatment, we treated normal
B6D2F1 mice for 3 days with
anti-VLA4 (2 mg/kg/d). Eight hours after the last injection femurs were
removed for preparation of bone marrow cell suspensions. Bone marrow
mononuclear cells were labeled either with goat anti-rat FITC or with
anti-VLA4 (rat anti-mouse 4) followed by secondary antibody (goat
anti-rat FITC). The FACS histograms of these two populations were
virtually superimposable (data not shown), indicating that the cells
were saturated in vivo by anti-VLA4 antibody. Cells labeled with
anti-VLA4 plus 2° antibody (as above) were also labeled
with anti-kit PE antibody. A separate bone marrow aliquot was also
labeled with anti-kit PE only and all samples analyzed by FACS. Control
bone marrow cells from mice that were not treated with anti-VLA4 were
processed and labeled similarly, then run concurrently with the other
samples. Before anti-VLA4 treatment most of the population of cells
within the blast window (Fig 5, left panel)were doubly labeled with anti-kit and anti-VLA4 (Fig 5, middle panel).
After in vivo anti-VLA4 treatment (Fig 5, right panel), a significant
proportion of kitlo or kitneg cells was
observed, along with reduction of anti-VLA4 brightly labeled cells. The
data suggest that bone marrow cells display downmodulation of both kit
and VLA4 after anti-VLA4 treatment.
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| Fig 5.
A model to explain anti-VLA4/VCAM-1induced
mobilization. Normally there is strong adhesion of hematopoietic cells
to stromal endothelial cells through the VLA4/VCAM-1 pathway, which
overrides any positive effects of kit on migration of stem/progenitor
cells expressing kit. Consequent to antifunctional antibody treatment, there is deadhesion, which, either indirectly (by relieving the negative pressure on kit) or directly through communicating molecules, activates kit with ensuing stimulation of migration. During this process cells that are ready to egress downmodulate both 4 and kit.
It is likely that increased migration via a kit-dependent pathway is
achieved by activation/deactivation of another integrin to provide
mechanistic support of cell movement. This effect may be strengthened
by alleviation of a transdominant effect that VLA4 may exert on other
integrins.
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DISCUSSION |
Mechanisms of Anti-VLA4/VCAM-1Induced Mobilization
Adhesion/migration phenomena of mature leukocytes and the sequence of
events from their activation to their extravasation and final
recruitment in tissue sites of inflammation have been studied
extensively and described in detail, especially using in vitro
experimental models.29 Largely on the basis of in vitro information, several in vivo studies have been conducted in animal inflammation models using either function blocking antibodies or
anti-adhesive peptides or small molecules.30 Detailed
analyses of the antibodies' mechanism of action in vivo, their
specific tissue requirements, or their cooperative molecules have not
been described and extrapolations have been made from in vitro data. However, in vivo the situation may be very complex, with a coexistent network of negative and positive mediators and a cascade of events triggered after the initial stimulus, so that the in vivo setting cannot be faithfully mimicked with in vitro modeling. Consequently, the
in vivo behavior of specific molecules may not be predicted from in
vitro data. For example, although anti-2 integrin
antibodies inhibit adhesive interactions of CD34+ cells in
vitro,10 in vivo administration of anti-2
has not been effective in mobilizing CD34+ cells, in contrast to
anti-4 antibody and despite the similar effects of these antibodies
in vitro.13
In the case of anti-VLA4 administration in vivo, a simplistic view is
that it can cause disruption of adhesive phenomena within the bone
marrow, similar to its mode of action in vitro in long-term bone marrow
cultures.9 Weakening of the adhesion between bone marrow
cells and their microenvironment provides a biophysical mechanism
whereby physiologically important connections of stem cells with their
microenvironment are now severed. For mobilization to occur,
hematopoietic cells need to migrate to intravascular spaces through
basement membrane and endothelial cells, and such a migration is a
prerequisite for their egress from the bone marrow to the circulation.
Although changes in integrin-mediated adhesion per se can promote
directed migration, adhesion-based mechanisms modulating cell
locomotion are particularly complex and are usually driven by a
multistep cascade of reactions involving several adhesion molecules.31-34 Events leading to mature leukocyte
transmigration involve activation of several classes of adhesion
molecules by ligands, activating antibodies, or peptide mimetic
molecules.29 Our adhesion-blocking antibody abrogates
4-mediated adhesion in vitro, but is not known to behave like
anti-1 activating antibodies initiating signaling. One
then would ask: Is a single deadhesion step in vivo sufficient to
initiate subsequent events leading to mobilization, or are signaling
and additional cooperative molecules required for mobilization?
1 integrins are considered as "locomotive" adhesion receptors and it is possible that antibody treatment per se
can influence cell motility. Recently an antibody was described which
inhibits neutrophil adhesion and increases motility.35 Although this mode of action may be a theoretical possibility in the
anti-VLA4-induced mobilization process, the data presented herein
support alternative possibilities.
One can propose that the initial deadhesion step leads to engagement of
several other intracellular signaling molecules, some of which are
interconnected with growth factor signaling pathways. In hematopoietic
stem/progenitor cells, the functional association between kit and
integrins is of extreme interest. c-kit occupies a unique place in
hematopoiesis because it affects proliferation, differentiation, and
migration of multiple cell lineages, including hematopoietic
stem/progenitor cells.23 Because migration is dependent on
adhesive interactions with extracellular matrix, c-kit could mediate
some of its function through inside-out signaling and modulation of
integrins.36-40 Mast cells and eosinophils exhibit rapid
induction to fibronectin adherence with KL stimulation, dependent on
kit kinase activity.37, 39 Cells from hematopoietic cell
lines with a progenitor cell phenotype also show a transient modulation
of integrin avidity after KL stimulation.40 In addition to
c-kit, another tyrosine kinase receptor, the platelet-derived growth
factor receptor, has also been reported to influence integrin-mediated
adhesive phenomena.41 However, the relevance of the
short-lived in vitro effects of kit ligand to its delayed in vivo
mobilization effect is unclear.
In the present study we provide evidence that kit receptor kinase
activity modulates the effectiveness of anti-4 integrin-induced mobilization. W/Wv cells with compromised kit kinase
activity have a blunted mobilization, whereas cells from
Sl/Sld mice with a similar clinical phenotype but normal
kit kinase activity in stem/progenitor cells respond like normal
control mice to anti-4-induced mobilization. Because
W/Wv mice do not respond appropriately to other mobilizing
stimuli,28 one could argue that hyporesponsiveness is a
general phenomenon for W/Wv and as such may not be directly
associated with kit function. However, hyporesponsiveness to cytokines,
specifically G-CSF, is not a unique feature of W/Wv because
it was observed in Sl mice.27 On the other hand, other receptor mutant mice, like the G-CSF null mice, IL-7R null mice, or
mice with compromised IL-3 function (A/J mice) respond normally to
anti-4 mobilization. Although testing of additional mutant mice (ie,
Mpl / mice) would have been of interest, the data thus far do
suggest that the association between kit function and anti-4
mobilization may be causative and may be involved in changes in cell
motility. Furthermore, both W/Wv and Sl mice did not
respond to anti-VCAM-1induced mobilization, in contrast to their +/+
littermates and despite normal expression of VCAM-1 in bone marrow.
Collectively, the data would imply that signals initiated by
anti-VCAM-1 cannot be transmitted to hematopoietic stem/progenitor
cells, either because their kit function is compromised, as in
W/Wv mice, or because of the absence of membrane-bound KL
in the microenvironment of Sl mice. The finding that W/Wv
mice 8 weeks posttransplant of +/+ donor bone marrow cells mobilized normally after anti-VCAM-1 treatment supports this view. Bone marrow
mononuclear cells after anti-VLA4 treatment display (by FACS analysis)
downmodulation of kit, which may be consequent to their prior
activation, and is similar to what has been reported for human
cells.42 The increased mobilization in
mev/mev mice with constitutive activation of
kit is also compatible with this hypothesis. The increased spleen size
and increased CFU-C content in mev mice noted
previously26 may result from a heightened migratory capacity of stem/progenitor cells leading to high levels of ongoing mobilization and spleenic seeding.
Mobilization: How Many Mechanisms? Any Emerging Theme?
Because several cytokines with diverse actions and different cognate
receptor distribution mobilize a wide spectrum of stem/progenitor cells, one may suggest that some of the events triggered by cytokine administration may be common. We would like to propose that a major
common pathway involved in mobilization, integrating signaling through
integrins and initiating cell migration, is the kit/kit ligand pathway.
Merging information from the present study and the
previously published studies,6 the following
general mechanistic model for mobilization is proposed. Mobilization is
a multistep process, usually accomplished in two phases: (1) the
initiation phase, which encompasses the initial triggering event, and
(2) the amplification phase, which includes several steps implemented
through integrin/cytokine crosstalk or integrin/other adhesion
molecules cross-influence and proliferative effects. The triggering
event could be applied to cells themselves or components of their bone
marrow microenvironment (stromal cells, extracellular matrix
components). For example, anti-VLA4 or anti-VCAM-1 treatment are
examples of effects on hematopoietic stem/progenitor cells or
microenvironment cells, respectively. Such triggering events could act
alone, leading to a short-lived mobilization (ie,
post-IL-8,43 Mip1,44 IL-1, or early
postendotoxin6). Activation of signaling pathways by
subsequent steps could lead to sustained and more efficient mobilization. Cooperative signaling could be mediated through growth
factor receptors with tyrosine kinase activity (kit, flt3) and/or through integrin cross-talk.45 In the case
of hematopoietic growth factors, this effect is also compounded by an
increase in proliferation of cells within the bone marrow because of
growth factor synergy affecting proliferation.
Do all the different growth factors have their own triggering or
initiating event? Growth factors like G-CSF, which also affect end
stage cells, could initiate that event in neutrophils, so that if
neutrophils are functionally incompetent or not present, they do not
respond and no mobilization ensues (ie, as in G-CSF /
mice,17 or in chronic granulomatous disease
patients.46) With KL or FL, the situation is even more
speculative. The receptors for these ligands are present mostly on
early cells and on certain cells within the microenvironment. Thus,
endothelial cells expressing kit, or macrophages and/or
dendritic cells with flt3 receptors could be affected in addition to
primitive hematopoietic cells. Downmodulation of kit on cells may
provide a stimulus for migration. Profound proliferative effects within
the bone marrow resulting from many cytokines could impact existing
pools of cells within bone marrow spaces or have secondary effects on
blood flow.47 A delayed response for KL or FL would be
compatible with the notion that proliferative events precede
mobilization. However, proliferative effects themselves may not
necessarily initiate mobilization.17
It has recently been reported that G-CSFR-deficient mice do not
respond to IL-8 or cyclophosphamide-induced mobilization, suggesting
that these stimuli act indirectly through G-CSF signalling. W/Wv mice with deficient kit signaling and mpl-deficient
mice respond subnormally to G-CSF.28 Given the fact that
many signaling molecules are shared by these hematopoietic growth
factor receptors, it is conceivable that if signaling is required for
mobilization, and if in these mice suboptimal stimuli are generated,
mobilization may not be optimal. The net effect of mobilization is also
dependent on proliferation events. Absence of synergistic actions
between cytokines compound the magnitude of mobilization. In addition, one may predict that incompetence of cells to respond to the triggering event, either through receptor deficiency or deficiency of several relevant signaling proteins, may cause defects in mobilization. Thus,
one can test whether mice deficient in signaling molecules (ie, Lck)
are capable of responding to known mobilizing stimuli. These and other
murine models should help further dissect the pathways responsible for
mobilization.
| |
FOOTNOTES |
Submitted November 17, 1997;
accepted December 30, 1997.
Address reprint requests to Thalia Papayannopoulou, Hematology, Box
357710, University of Washington, Seattle, WA 98195.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to Dr D. Link for allowing us to study his G-CSF null
mice and to Drs J.J. Peschon and Chris Clegg for the IL-7R null mice.
The generous gift of anti-VLA4 and anti-VCAM-1 antibodies by Dr R. Lobb (Biogen) and flt3 ligand by Dr S. Lyman (Immunex) is greatly
appreciated. We also thank Dr J. Harlan for helpful discussions and
critical reading of the manuscript and Sherri Brenner for her skillful
secretarial assistance.
| |
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Abstract
Introduction
Abstract