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1996 87: 2513-2517
A critical role of VLA-4 in erythropoiesis in vivo
K Hamamura, H Matsuda, Y Takeuchi, S Habu, H Yagita and K Okumura
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A Critical Role of VLA-4 in Erythropoiesis In Vivo
By Keisuke Hamamura, Hironori Matsuda, Yumiko Takeuchi, Sonoko Habu, Hideo Yagita, and KO Okumura
Hematopoiesis requires specific interactions
with the microenvironments, and VLA-4 has been implicated
in these interactions bared on in vitro studies. To study the role of VLA4 in hematopoiesis in vivo, we performed in utero treatment
of mice with an anti-VLA-4 monoclonal antibody. Although
all hematopoietic cells in fetal liver expressed VLA4, the
treatment specifically induced anemia. lt had no effect on
the development of nonerythroidlineage cells, ineluding
lymphoids and myeloids.
In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors,
which give riseto erythroid colonies in vitro, were present.
These results indicate
that VLA-4 playsa criticalrole in erythropoiesis, while it is not critical in lymphopoiesis in vivo.
0 7 9 9 6 by The American Societyof Hematology.
D
analyzed on FACScan (Becton Dickinson, Mountain View, CA).
MoAbs against mouse cell surface markers included ACK-4 (antic-kit): RM4-5 (anti-CM) (Pharmingen, San Diego, CA). 53-6.7
(anti-CD8),I0 RM2-1 (anti-CD2),“ RA3-6B2 (anti-B220),12MlnO
(anti-Mac-]),” RB6-8C5 (anti-Gr-1),I4 TER119,’’and
PSD.3
(anti-VLA-4).6 The Lin cocktail of MoAbs against lineage markers
contained RM4-5, 53-6.7, RM2-1, RA3-6B2, MlnO, RB6-8C5, and
TERl19. Conjugation of MoAbs with FITC or biotin was performed
by standard methods. Optimal concentrations of labeled antibodies
were determined by preliminary experiments.
Immunohistochemical analysis. Fetal livers were fixed in periodate-lysine-paraformaldehyde (PLP) containing 4% padormaldehyde at 4°C overnight, embedded in OCT (Miles, Elkhart, IN), and
stored at -80°C. Frozen sections were reacted with 5pg/mL primary
at 4°C overnight. After rinsing with
antibody, PS/2.3 or WK-2:’
PBS, they were incubated with 2.5 &mL biotinylated antirat IgG
(Vector, Burlingame, CA) for 60 minutes at room temperature,
rinsed, and then reacted with avidin-biotin-peroxidase complex
(Vector) for 60 minutes at room temperature. The staining was developed by incubating the specimens in a reaction buffer (0.01% H202,
0.3 mg/mL diaminobenzidine in 50 mmoVL Tris-HCI, pH 7.6) for
5 minutes at room temperature. The specimens were washed and then
counterstained in 0.1% Mayer’s hematoxilin. In controls, primary
antibodies were substituted with PBS.
In utero treatment. Pregnant mice were administered 1 mg/d of
PS/2.3 or M/K-2 intraperitoneally from day 7 of gestation, when
hematopoiesis begins in the yolk sac of fetuses, until the day of
analysis. Trans-placental delivery of PSL2.3 to the fetuses was
checked by flow cytometric analysis for the presence of antibody
on fetal liver MNC, as detected by FITC-conjugated antirat IgG
(Caltag, San Francisco, CA). The delivery of MK-2 through the
placenta was expected based on preliminary experiments in which
isotype-matched anti-Pgp-l MoAb 1M7.8.1I6 was similarly detectable on fetal thymoctytes after administration to pregnant mice. In
both of these experiments, normal rat IgG (Sigma, St Louis, MO)
or saline were administered as controls.
Histological study. The livers from control or PSR.3-treated neonates were fixedin phosphate-buffered 10% formalin (pH 6.8),
EVELOPMENT OF hematopoietic cells depends on the
interaction with hematopoietic microenvironments
that consist of a variety of components, including stromal
cells, cytokines, and extracellular matrix (ECM) protein^."^
Among various ECM, it has been demonstrated that hematopoietic progenitor cells adhered selectively to fibronectin
(FN)but not to collagen, laminin, or proteoglycans, indicating a possibility that FN contributes to hematopoiesis! Currently known major FN receptors are VLA-4 and VLA5, which belong to the D l integrin subfamily of adhesion
molecules. Integrins not only mediate cellular adhesion, but
also transduce signals that regulate cellular response^.^ Recent studies using in vitro hematopoietic culture systems
demonstrated the importance of VLA-4 in B and T lymphopoie~is.6.~
In these studies, specific antibodies or peptides
interfering with VLA-4-mediated adhesion inhibited
lymphoid colony formation in bone marrowculture and stromal cell-dependent thymocyte differentiation. In addition,
we recently demonstrated that an anti-VLA-4 monoclonal
antibody (MoAb) inhibited stromal cell-dependent erythropoiesis in vitro? These in vitro results raise the question of
whether these observations are relevant to hematopoiesis in
vivo. To address this issue, we carried out in vivo treatment
with a MoAb against mouse VLA-4. A critical contribution
of VLA-4 to erythropoiesis in vivo was found.
MATERIALS AND METHODS
Mice. Timed pregnant C57BU6 mice were purchased from Japan SLC Inc (Shizuoka, Japan). the day of observation of a vaginal
plug was designated as day 0 of gestation.
Monoclonal antibodies. The hybridoma cells producing MoAb
against a4 subunit of VLA-4 (PS12.3) and that against mouse
VCAM-I (“2)
were kind gifts from Dr Kensuke Miyake (Department of Immunology, Saga Medical School, Japan). The MoAbs
were purified from ascites by affinity chromatography on protein GSepharose column (Pharmacia, Uppsala, Sweden).
Preparation of cells. Thymocytes were released by pressing thymic lobi between two frosted slide glasses, passed through nylon
mesh, and suspended in a-modified Eagles medium (a-MEM)
(GIBCO, Gaithersburg, MD). Livers were mashed and suspended in
a
“
,
passed through nylon mesh, and layered on lymphocyteseparating medium (JIMRO, Gumma, Japan) followed by a centrifugation at 1,200g for 10 minutes at room temperature. Mononuclear
cells (MNC) at the interface were collected, washed, and resuspended in a “ E M .
Flow cytometric analysis. Cells, lo6per sample, were incubated
at 4°C for 20 minutes with appropriate dilutions of FJTC-, PE- or
biotin-labeled MoAbs and washed twice with phosphate buffered
saline (PBS). When the cells were reacted with biotinylated antibody,
they were M e r incubated with Red613-streptoavidin at 4°C for
15 minutes, and washed twice with PBS. Immunofluorescence was
Blood, Vol 87, No 6 (March 15), 1996: pp 2513-2517
From the SecondDepartment of Intern1 Medicine, Tokyo University; the Department of Immunology. Juntendo University School of
Medicine. Tokyo: and the Department of Immunology, Tokai University School of Medicine, Isehara-shi, Japan.
Submitted April 10, 1995; accepted October 16, 1995.
Address reprint requests to KO Okwnura, MD, Department of
Immunology, Juntendo University School of Medicine, 2-1-1 Hongo,
Bunkyo-ku, Tokyo 113, Japan.
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.
0 19% by The American Society of Hematology.
0006-4971A6/8706-0025$3.at/o
2513
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2514
HAMAMURA
ET AL
Fig 1. Expmssion of VIA4
and VCAM-1 in fetal
(A) Exp d o n of VIA4 on fetal liver
MNC. Embryonic day 15 fetuses
lii.
wafedhectedfromtimedpregnant
mice,
and mononuclear
calk were isdated. They were
stained by FlTccOnjugated PS/
2.3, PEconjugated ACK45, and
the cocktail of biotinylatedMoAlw against lineage markers
(Lin). folowed by streptoavidinRedS13. and analyzed on FACSmn.
, autofluomseeme;
*--,
PS123 (anti-VIA41 staining of total liver MNC; ,
PS12.3 staining of the elecbonially gated c-kit+/Lin-cells. Ver-
-----
ticalscalerepresentsrdativodl
number and horizontalscale represents log Ruores#~lce.
(B and
C) I m m u n o h i iical staining of embryonic day 15 fetal
liver with PS123 oc WK-2. E m
bryonic day 15 fetal livers were
~ e in
d perkhe-lysinsparaformaldehyde (PLP) containing
4% paraformaldehyde. Frozen
sectiomweremactedwithaprimary antibady, PS12.3 or M/K-2,
followed by biotinylated antirat
I g G and avidin-biotin-peroxidase
complex. The staining was &
velOpea
by
diaminobenzidine,
and the n
was done
with Mayer's hematoxilin. Results
the stainingwith
pS12.3 (B) and WK-2 (C).
-
Fig 3. Histological study of newborn liver treated in utero with PS/2.3. The livers from control (A) or PS/Z.3-treated (B)neonates were
f w d in ph-hate
buffered 10% formaline (pH6.8).embeddedin paraffin, and the t i w e ~e&iom
were stainedwith 0.1% Mayer's hamoxilin
and 0.025% eosin. Arrows indicateerythroblasts, and arrow heads indicate myeloid cells.
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
2515
ROLE OF VIA-4 IN ERYTHROPOIESIS
embedded in paraffin, and the tissue sections were stained with 0.1%
Mayer's hematoxilin and 0.025% eosin.
Colony u s s q . Semi-solid methyl cellulose culture
of hematopoietic cells was performed accordingto the previously described technique" with some modifications. 'Ihe livers were isolatedfrom control or PSf2.3-treated neonates and were mashed by pressing them
betweenfrostedslideglasses.
By this manipulationhepatocytes
coaggregated, and dissociated hematopoietic cells became a single
cell suspension. These cells (2 X 104 to I X 10s) were cultured in
1 mL of Iscove's modified Dulbecco's medium (IMDM, Kyokuto,
Tokyo, Japan) containing 1.0% methyl cellulose (Dow Chemical,
Midland, MI), 30% fetal calfserum (JRHBiosciences, Lenexa, KS),
1% deionized fractionV bovine serum albumin (BSA; Sigma), and
0.1 mmoVL 2-mercaptoethanol in the presence of 6 U/mL human
recombinanterythropoietin(Chugai,
Tokyo, Japan),100 U/mL
mouse recombinant IL-3, and50 ng/mL human-recombinant granulocyte colony-stimulating factor (G-CSF; Chugai). The numbers
of
colony forming unit-erythroid (CFU-E) were scored as cell aggreLog fluorescence
gates of more than eight cells at day 3 of culture. Burst forming
fig 2. Tmnsphmtal delivory of PS12.3 to
Tho pmsenca
uniterythroid (BFU-E), colony-forming units granulocyte, macroof PSR.3 on f.M livu MNC wum analyzad by staining with FITG
phage, and granulocyte macrophage (CFU-G, CFU-M, CFU-GM),
conjug.hd.ntk.t100antibody.""-,
~ ~ : " " - , ~ n t k . t
and CFU-Mix were differentially counted at7 to
days
8 as aggregates
antiof more than40cells (200 cellsfor BFU-E) under microscopy. Some IgG staining of total liin MNCfrom contrd individuab; -,
rat IgG staining of total liin MNC from the PSI2.3-treated brdividucolonies were lifted from the cultures using micropipet, cytocentriab. Vwticd #d.
d.lrtivcall
. number and horizontaltub
fuged, and identified by Diff-Quick (Midori-Juji, Osaka, Japan) or
mprasont!s log. R
benzidine staining.
fetuses.
RESULTS AND DISCUSSION
We first evaluated the VLA-4 expression in fetal liver,
which is the main site of early hematopoiesis. Three-color
flow cytometric analysis of fetal liver MNC showed that
almost all cells strongly expressed VLA-4 (Fig lA), which
implies that VLA-4 can participate in the development of
all lineages. The c-kit+/Lin- cells, which are enriched for
stem cells: expressed VLA-4 most strongly, suggesting the
importance of VLA-4 for these cells. Immunohistochemically, VLA-4 resided on small round cells with a large nucleudcytoplasm ratio and dense chromatin; these are morphological characteristics of hematopoietic cells (Fig IB).
By contrast, VCA"1, VLA-4 ligand other than FN, resided
on the cells with irregular shape-extending cytoplasmic projections to surrounding hematopoietic cells, which are morphological characteristics of stromal cells (Fig 1C). This
implied the possibility that not only FN but also VCAM-l
could act as a VLA-4 ligand in the hematopoietic microenvironment, as suggested by the inhibitory effect of an antiVCA"1 MoAb on stromal cell-dependent lymphohematopoiesis in vitro!.'*
In vivo treatment was then performed with an a n t i - U 4 MoAb, PSL2.3, which has been demonstrated to inhibit
VLA-4-mediated cell binding to both FN andVCAM-1
and also toefficiently inhibit lymphohematopoiesisin vitro?
Fetuses were treated with P92.3 from gestational day 7 to
birth by administering the antibody into maternal peritonea.
The injected antibody was efficiently transferred to the fetuses as estimated by its deposition on fetal liver MNC.
Figure 2 shows that rat IgG was present on MNCs from
the PSL2.3-treated fetal livers, but it was absent in controls.
Because most liver MNCs from the PS/2.3-treated individuals were positive for rat IgG, we could conclude that the
amount of transplacentally delivered PSl2.3 was enough to
block VLA-4 molecules on fetal liver MNC. The neonates
that had beentreated in utero with PSL2.3 looked pale, though
they did not show any other morphological abnormality (not
shown). In these neonates, a marked anemia was defined by
red blood cell counts that were approximately one-fifth of
the controls (Table 1). Since the liver is the major site of
hematopoiesis in fetuses and neonates,I9phenotypical analysis was carried out with liver-derived MNC. The number
of TER119+ cells representing erythroid ~rogenitors'~
was
strikingly decreased. In contrast, the number of white cells
in the blood and those of Gr-l+ or Mac-l+ myeloid cells in
the liver were not significantly decreased.CD4'8', CD4+8-,
or CD4-8+ cells in the thymus and B220+ cells in the liver
were also not significantly affected. These results indicate
that the fetal anti-VLA-4 treatment interfered onlywith
erythropoiesis; it did not affect the development of other
lineages including granuloid, monocytoid, and lymphoid
cells. h addition to the erythroid cells, a significant decrease
was also noted for c-kit'&% cells in the liver and CM-8cells in the thymus. Since these cells are the most immature
cells within each organ, VLA-4 may also contribute to stem
cell homing from the yolk sac to the liver and homing of T
progenitor cells to the thymus.
We then histologically examined the liver, thymus, and
spleen of the PSL2.3-treated neonates as well as major nonhematopoietic organs including the heart,lung, kidney, and
intestine. In the control liver (Fig 3A), manyclusters of small
round cells with condensed nucleus and scant cytoplasm,
characteristic of erythroblasts (arrow), were seen.In contrast,
these cells were almost completely absent in the PSD.3treated liver, while other cells of myeloid morphology (arrow
head) were maintained (Fig 3B). The PSL2.3-treated thymus
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HAMAMURA ET AL
2516
Table 1. Effect of In Utero Treatment W*
Hematopoiesis
PW2.3 on
Cell Numbers
Source of Cells
~
Blood' (x105cells/pL)
Liver MNC
(xlOScells/organ)
Thymocyte
(x105cells/organ)
Control
PS/Z.B-treated
____~
Red cell
Whitecell
55.9 -t 7.2
11.0
0.125 t 0.0980.119
TER119'
14.3
Mac-l+
Gr-l+
B220+
17.6
c-kit'llin-
t 2.04
0.79
7.96 t 1.09
8.23
6.98 t 7.92
0.80
t 12.5
0.07
0.378
1.14 t 0.08
CD4-8CD4'8'
CD4'8CD4-8'
15.8
25.5 t 11.1
85.9 t 22.3
2.00 t 0.72
10.2 ? 5.9
IT 4.1
2 0.036
t 0.23
t 1.17
5 1.06
? 1.44
2 0.087
5 1.3
91.0 5 21.0
1.73 2 0.34
9.41 2 5.51
Neonates that hadbeen treated in utero withPS/2.3 were analyzed
on the day of birth. Two microliters of the blood
was aspirated from
the right atrium, diluted appropriately with PBS or TWk's solution,
and the numbers of red and white blood cells were counted using
Neubauer's hemocytometer. Thymocytes and splenocytes were released from each organ by pressing between two frosted slide
glasses; they were suspended in a-MEM. Thymocytes, splenocytes,
and liver MNC were stained with labeled antibodies against the indicated cell surface markers and analyzed on FACScan. Data indicate
means c SD of threeor four (*) different individuals.
and spleen did not show any histological abnormality nor
any other nonhematopoietic organs (not shown).
Next, we evaluated the colony forming capacity of the
PW2.3-treated liver to determine the stage at which the erythropoiesis was affected by the anti-VLA-4 treatment. Table
2 represents the mean colony numbers per control or treated
neonatal liver. Erythroid colonies (BFU-E and CFU-E) from
the PSR.3-treated liver were reduced to 40% of the normal
liver, but this reduction was much less prominent than the
almost complete absence of erythroblasts in the treated liver
(Fig 3). Although the reduction indicates that erythropoiesis
was arrested at the erythroid precursor stage by the antiVLA-4 treatment, erythroid progenitor cells were still present in the treated liver. A similar extent of reduction in
the colony forming capacity was also observed with other
lineages, including CFU-Mix, which represents multipotential stem cells. This suggests that VLA-4 is also involved in
the migration of stem and progenitor cells of all lineages.
This notion is consistent with the recent observations by
other investigators that VLA-4 appeared to be involved in
the lodging of CFU in murine spleens2' and that peripheralization of multiple hematopoietic progenitors was induced
by anti-VLA-4 antibody administration in primates?' However, the normalized development of myeloid and lymphoid
cells after the anti-VLA-4 treatment (Table 1) suggests that
some other VLA-4-independent pathways can compensate
for the lymphomyelopoiesis but not the erythropoiesis.
In order to determine whether the inhibitory effect of antiVLA-4 on erythropoiesis was exerted by its interference
withVLA-4/FN or VLA-4ffCAM-l interaction, we per-
formed the in utero treatment with an anti-VCAM- I MoAb
(hUK-2), which has been demonstrated to inhibit in vitro
lymphopoiesis as efficiently as PS/2.3,6 according tothe
same protocol as PW2.3. The W - 2 treatment did not cause
anemia (red blood cells, 5.31 ? 0.62 and 5.59 -t 0.72 X 10'
celVpL,mean -t SD, in the treated or control neonates,
respectively), but leukocytosis in the blood was notable
(white blood cells, 2.39 2 I .31 and 1.25 2 0.99 X lo4
cells/pL, means +- SD, in the treated or control neonates,
respectively). This effect was quite different from that of
PS/2.3 (Table l), which suggests that VCAM-I does not
play a critical role as the VLA-4 ligand for supporting erythropoiesis in vivo, although it is abundantly expressed in the
hematopoietic microenvironment (Fig 1C). VCAM- I may,
rather, contribute to the lodging as indicated by leukocytosis
after the anti-VCAM-1 treatment. Although the presence of
other VLA-4 ligands has not been excluded: FN would act
as the critical VLA-4 ligand for supporting erythropoiesis in
vivo, as previously suggested by the FN requirement for
in vitro differentiation of murine erythroleukemia cells into
reticulocytes, which was associated with erythrocyte-specific
protein induction.**
It has been demonstrated that VLA-4-mediated signaling led to cytokine gene expression and autocrine growth
in mature T lymphocytes
Interestand
ingly, VLA-4 can transmit a unique signal distinct from
that of other members of the I integrin s ~ b f a r n i l y It. ~ ~ ~ ~ ~
remains to be determinedhow VLA-4-mediated adhesion
and signaling participate in the erythropoiesis in collaboration with c-kitlstem cell factor and erythropoietin receptoderythropoietinsystems, which arealso required for
erythropoiesis.
Table 2. Colony Forming Capacity of Neonatal Liver
Treated In Utero W& PW2.3
Colony Number
Colony
BFU-E
CFU-E
CFU-GM
CFU-G
33.1
CFU-M
CFU-Mix
3.54
(XlO'
per organ1
Control
PSi2.3-Treated
9.69 t 3.94
276 t 38
6.59 -t 4.73
6.66 -C 4.32
29.0? 7.17
0.990
t 1.41
5 1.48
3.88 (40.0)
113
(40.9)
t 33
t 2.05
3.68 (55.8)
t 1.60
4.19 (62.9)
t 4.0
t(28.0)
0.018
(YoControl)
(87.6)
Semisolid methyl cellulose cultures, of the hematopoietic cells derived from new born livers from control or in utero PM.3-treated
individuals were obtained. Livers were mashed by pressing between
frosted slide glasses, and dissociated hematopoietic cells (2 x 10' to
1 x 10s) were cultured in 1 mL of IMDM containing 1.0% methyl
cellulose, 30% fetal calf serum, 1% deionized fraction V BSA. and 0.1
mmolR 2-mercaptoethanol in the presence of 6 U/mL human recombinant erythropoietin, 100 U/mL mouse recombinant 11-3. and 50 ng/
mL human-recombinant G-CSF. The numbers of CFU-E were scored
as cell aggregates of more than eightcells at day 3 of culture. BFUE, CFU-G, CFU-M, CFU-GM, and CFU-Mix were differentially counted
at days 7 to 8 as aggregates of more than40 cells (200 cells for BFUE) under microscopy. Data indicate means t SD of three different
individuals.
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ROLE OF VIA-4 IN ERYTHROPOIESIS
2517
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ACKNOWLEDGMENT
We thank Y. Kojima and Y. Murata for technical assistance on
immunohistochemistry,Drs K. Shimamura and T. Suda for advising
techniques on colonyassay, Dr K. Miyake for generous gifts of the
PSR and M/K-2 hybridoma cells, and Dr Y. Uchida for generous
support of the work.
MT.