* Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg;
Basel Institute for Immunology, Switzerland;
Department of Hematology, University Hospital, Heidelberg; and
Department of Applied Genetics, University of Karlsruhe, Germany
Correspondence: Margot Zöller, Department of Tumor Progression and Immune Defense, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: m.zoeller{at}dkfz.de
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: CD44 isoforms • hematopoiesis • mobilization • marrow repopulation
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The analysis of CD44 expression in hematopoiesis revealed that the latest pluripotent stem cell is CD44 positive. Also the earliest myeloid precursors are CD44high and remain CD44high when committed to the monocyte lineage, whereas commitment to granulopoiesis is accompanied by transient down-regulation. The erythroid lineage is CD44high throughout development. Expression on B and T cells also varies with the stage of maturation with down-regulation of CD44, particularly during intrathymic T cell maturation. Mature, activated, and memory T and B cells again express CD44 [4 , 5 ]. Because maturation of hematopoietic cells essentially depends on stroma cell interactions [6 ], it is interesting to note that CD44 is also expressed on stromal cells of bone marrow [7 ] and thymus [8 ]. CD44 is known to play an important role at early stages of myelopoiesis as well as lymphopoiesis [4 , 9 10 11 12 13 ]; i.e., inhibition of hematopoiesis by anti-CD44 was mainly restricted to stem cells and early progenitors, whereas anti-CD44 displayed no [9 ] or minor effects [11 ] on colony formation of committed progenitors in soft agar cultures. The molecule also is involved in the assembly of the extracellular matrix [3 , 7 ], in stem cell homing [11 , 14 ], seeding [5 , 11 ], and adhesion of progenitor cells to stromal elements [15 ] as also demonstrated by the efficacy of anti-CD44 in progenitor cell mobilization [5 , 16 ]. This does not imply that expression of the molecule is vital, since mice with a targeted deletion of CD44 show only slight alterations in hematopoiesis, i.e., particularly the egress of myeloid cells from the bone marrow appears to be disturbed [17 , 18 ].
The studies mentioned above have been largely performed with antibodies that bind to all CD44 isoforms. Because they do not distinguish between the CD44s and the CD44v molecules, we term them anti-panCD44, for the sake of clarity. Because antibodies specific for CD44 variant isoforms and, most importantly, mice with a targeted deletion of CD44 variant exons have only recently become available, far less is known of CD44v expression and function in hematopoiesis. An analysis of CD34+ human cord blood cells revealed low expression of CD44v6 and very low expression of CD44v5 [19 , 20 ]. Subpopulations of BMC also express CD44v3 and CD44v10 containing isoforms [5 , 21 , 22 ]. CD44v3 is of special importance during myelopoiesis [23 ]. It is supposed that its functional activity depends on binding of growth factors via its heparan sulfate side chains [23 ]. Furthermore, CD44v10 has been described to support B cell progenitor maturation [22 ]. CD44v6 accelerates T cell maturation in the thymus and facilitates in vitro survival of an adherent early stem cell population [24 , 25 ]. The underlying mechanisms have not yet been elucidated.
BMC transplantation after myeloablative treatment provides under selected instances an ultimate chance of curative tumor therapy [26 ]. Furthermore, there is evidence that the use of mobilized progenitor cells may be advantageous [reviewed in ref. 27 ]. Mobilization mainly is achieved by hematopoietic growth factors like granulocyte colony-stimulating factor (G-CSF), alone or in combination with myelosuppressive chemotherapy [28 ]. With increasing knowledge of the physiological egress of hematopoietic cells and of modulation of their adhesive/migratory capacity, the blockade of cytoadhesion or of signaling has become another interesting tool for stem cell mobilization [16 , 29 ]. Two of the adhesion molecules whose blockade has been repeatedly reported to support stem cell mobilization are VLA-4 (CD49d) [16 , 30 ] and CD44 [5 , 16 ].
In this study we describe that CD44v7 is expressed on stromal cells as well as on a small subpopulation of BMC. Anti-CD44v7 efficiently mobilized progenitor cells, mainly via a blockade of stromal elements. In CD44v7-/- mice we could show that it is, indeed, particularly expression of the CD44v7 exon product on stromal elements that is important for progenitor cell homing and seeding.
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Antibodies
The CD44s-specific hybridoma IM-7 (rat IgG2b) was obtained from
the American Type Culture Collection (ATCC). The anti-CD44v7 (LN7.2,
mouse IgG2a) and the anti-CD44v6 (11A6, rat IgG2a) antibodies have been
described before [31
, 32
]. Further
hybridomas were RA3-3A1 (anti-B220), E13.161 (anti-SCA-1), M1/69
(anti-HSA), 33D1 (DC specific), 331.12 (anti-µ), all obtained from
ATCC, and YTA3.2.1 (anti-CD4), YTS169 (anti-CD8), YTS154.7.7.10 (CD90),
all obtained from the European Collection of Animal Cell Cultures
(ECACC). PS/2 (anti-CD49d/VLA-4) was kindly provided by K. Miyake, Saga
Medical School, Saga, Japan [33
]. Monoclonal antibodies
(mAb) were purified by passage of culture supernatants over protein G
Sepharose 4B. Where indicated purified mAb were either biotinylated or
fluorescein isothiocyanate (FITC) labeled. For flow cytometry, purified
antibodies were used at a final concentration of 10 µg/mL. The
following monoclonal antibodies were obtained commercially: anti-CD34,
anti-CD38, anti-CD43, anti-CD117, FITC, or phycoerythrin (PE) labeled
anti-mouse and anti-rat IgG as well as streptavidin-FITC and
streptavidin-PE.
Flow cytometry
Bone marrow cells (BMC) and spleen cells (SC), stromal cells
derived from long-term bone marrow cultures (LTBMC), as well as the
stroma lines S17 [34
] and MS5 [35
]
(5 x 105 cells/well) were stained according to
routine procedures. Negative controls were incubated with an
isotype-matched control IgG and the secondary antibody. Analysis was
performed with a FACSCalibur (Becton Dickinson, Heidelberg, Germany).
Preparation of hematopoietic cells
Mice were anesthetized and bled by retro-orbital puncture,
collecting the blood in heparinized tubes. After cervical dislocation,
spleen, femora, and tibias were removed under sterile conditions. Bone
marrow was obtained by flushing the bones with 5 mL of
phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS)
using a 21G needle. Bone marrow and spleen were teased through fine
gauze. Peripheral blood mononuclear cells (PBMC) were collected after
Ficoll-Hypaque centrifugation.
Colony-forming assays [36
]
Colony-forming activity was determined by plating 5 x
104 BMC or 5 x 105 SC or 5 x
105 PBMC in 24-well plates in 0.3% semisolid agar (DIFCO)
in Iscove’s minimum essential medium (IMEM) containing 20% horse
serum (HS) and keeping the cultures at 37°C, 5% CO2 in
air. For the evaluation of granulocyte-macrophage colony-forming units
(GM-CFU) 10 ng/mL GM-CSF (colony-stimulating factor; Sigma, Germany)
and 2 mM glutamine were added. Colonies containing at least 50 cells
were counted after 7–8 days. M-CFU (macrophage) were cultured under
the same conditions, adding conditioned medium (20%) from L929 cells
as a source of M-CSF. For C-CFU (multilineage) 15% conditioned medium
from WEHI-3B cells and 5% conditioned medium from L929 cells was
added. Erythroid burst-forming units (E-BFU) were determined by
addition of 20% FCS instead of HS, 1% bovine serum albumin (fraction
V, Sigma), 15% conditioned medium of WEHI-3B cells, and 10 U/mL of
erythropoetin (Boehringer Mannheim). Colonies containing at least 500
cells were counted after 12 days. PreB-CFU (preB cell) were also
cultured with 20% FCS, 15% supernatant of an IL-7 cDNA-transfected
line (kindly provided by Dr. Rolink, Basel Institute for Immunology,
Basel, Switzerland) [37
], 2 mM L-glutamine,
and 5 x 10-4 M 2-mercaptoethanol. Colonies of at
least 50 cells were counted after 7 days.
Homing
129SV and CD44v7 knockout (129SV background) mice were lethally
irradiated and 1 day later received 5 x 106 BMC that
had been labeled with 51Cr (10 µCi/106 BMC)
for 90 min at 37°C. After washing, cells were resuspended at a
concentration of 2.5 x 107 cells/mL PBS. Mice
received 200 µL cell suspension intravenously. Animals were bled via
the retro-orbital sinus and were killed after 1–72 h. Organs (bone
marrow, spleen, thymus, liver, lung, kidney, muscle, skin) were excised
and weighed. Radioactivity was determined in a 
-counter.
Radioactivity per gram organ or per organ was calculated.
Mobilization, MRA, and reconstitution
For the mobilization of hematopoietic progenitor cells, mice
received a daily intravenous injection of 200 µg antibody three
times. Mice were killed 24 h after the last injection to collect
BMC and SC. The MRA was evaluated in vitro and in
vivo. MRA in vivo was tested by injecting intravenously
106 BMC or 5 x 106 SC into lethally
irradiated mice. Animals were killed after 8 days, and BMC were
collected and transferred into the second lethally irradiated host
(4 x 104/mouse) to determine the spleen CFUs after 12
days [38
]. Spleens were fixed in Tellesnicky’s solution
and colonies were counted after several hours of fixation. For the
in vitro evaluation of MRA the number of C-CFU was evaluated
8 days after the first transfer. The capacity for reconstitution was
evaluated by long-term survival after the transfer of titrated numbers
of BMC or SC or PBMC.
Statistical analysis
Significance of differences between control and antibody-treated
groups in MRA was calculated according to the Wilcoxon rank sum test.
Significance of differences in in vitro assays was
calculated by the two-tailed Student’s t test. All
functional assays were repeated at least three times. Mean values and
standard deviations of in vivo experiments are derived
mostly from 10 mice per group. Mean values of in vitro
studies are based on 4–10 replicates as indicated in the individual
experiments.
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Table 1. Expression of CD44v6 and CD44v7 on Hematopoietic Progenitor Cells and
Bone Marrow-Derived Stromal Cells
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Figure 1. Subpopulations of hematopoietic progenitor cells and of stromal cells
express CD44v7. (A) BMC and stroma cells from LTBMC were stained with
biotinylated anti-panCD44 or anti-CD44v7 and counterstained with
streptavidin-PE. A single-parameter overlay is shown (gray area,
negative control; black, anti-CD44v7; white, anti-panCD44). (B) BMC
were stained with biotinylated anti-CD44v7 and counterstained with
streptavidin-PE. Thereafter cells were incubated with FITC-labeled
anti-CD34, anti-CD90, anti-CD117, and anti-SCA-1. The double
fluorescence analysis is shown.
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Table 2. Subpopulations of BMC and Stromal Cells Expressing CD44v7
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Figure 2. Expression of adhesion molecules and progenitor markers is unaltered in
bone marrow cells of CD44v7-deficient mice. BMC of CD44v7-proficient
and of CD44v7-deficient 129SV mice were stained with a panel of
antibodies specific for adhesion molecules and progenitor markers.
Single-parameter overlays of stained cells (white area) and of the
negative control (black area) of the large and more granulated
population of BMC are shown.
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Figure 3. Minor impact of anti-CD44v7 treatment on expression of adhesion
and progenitor cell markers: Mice were treated for 3 days with either
control IgG or anti-CD44v7 (200 µg/mouse). BMC (A), SC (B), and PBMC
(C) were harvested and stained with antibodies for adhesion molecules
and for progenitor markers. The experiment was repeated three times for
BMC and SC and two times for PBMC; the percentages of stained cells
(mean values ± SD) are shown.
*Significance of differences.
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Table 3. Mobilization of Progenitor Cells by Anti-CD44v7 Treatment
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Progenitor homing in CD44v7-/- mice
BMC were labeled with 51Cr and were injected
into lethally irradiated mice. Mice were killed after 1–72 h, organs
were excised, and radioactivity was determined (Fig. 4
). Clearance from the blood was not severely impaired in
CD44v7-/- mice. Yet, compared with BMC from
CD44v7-competent mice, BMC from CD44v7-/- mice homed poorly
in the bone marrow. Furthermore, between 48 and 72 h after
injection, the number of radiolabeled BMC from CD44v7-proficient mice
did not decrease significantly in the bone marrow of CD44 wild-type
mice. Instead, even the low number of radioactive CD44v7-/-
BMC, which had immigrated in the bone marrow of the CD44v7-deficient
host, declined during the observation period. In the spleen, too, the
number of radioactive CD44v7-/- BMC vanished more rapidly
than in the CD44v7-competent host. Thus, homing as well as settlement
of BMC was impaired in CD44v7-/- mice. To obtain a hint as
to whether the absence of CD44v7 on progenitor cells or on stromal
cells hampered settlement in the bone marrow, BMC from CD44v7-competent
mice were injected into CD44v7-deficient mice. The CD44v7-competent
cells, indeed, homed and settled poorly in bone marrow of
CD44v7-deficient mice, indicating that expression of CD44v7 on stromal
cells was important for BMC homing. Accordingly, homing of
CD44v7-/- BMC was significantly improved when the host was
CD44v7 competent. Yet, particularly in the spleen the settlement was
not as efficient as of CD44v7-competent BMC in the CD44v7-competent
host. Trapping in the capillary beds of the liver and the lung was
independent of whether BMC or the host expressed CD44v7 (data not
shown).
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Figure 4. Homing of BMC is severely impaired in CD44v7-deficient mice. BMC from
CD44v7+/+ and from CD44v7-/- 129SV mice were
labeled with 51Cr. Cells were washed and injected into
lethally irradiated syngeneic hosts, which were either CD44v7-competent
or -deficient. Mice were killed after 1–72 h, and organs were excised
and weighed. Radioactivity was determined in a -counter. CPM ±
SD of three mice per group are given per 1 mL blood (A),
per femur (B), and per spleen (C). The experiment was repeated two
times, revealing corresponding results.
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CD44v7 on stromal cells influences marrow repopulating ability
MRA was first evaluated by the transfer of SC (5 x
106) or BMC (1 x 106) of anti-CD44v7
treated mice into the lethally irradiated host. BMC were recovered
after 8 days to monitor the presence of C-CFU or to transfer the BMC
into a second lethally irradiated host for the evaluation of CFUs
(Fig. 5
). Both the analysis of the MRA in vitro and in
vivo revealed that after anti-CD44v7 treatment C-CFU and CFUs were
strongly augmented in the spleen. To confirm mobilization of progenitor
cells by blockade of CD44v7 and to evaluate whether the mobilized
progenitors suffice for long-term reconstitution, low numbers of SC and
PBMC of anti-CD44v7-treated mice were transferred into the lethally
irradiated host to control long-term survival. The survival rate of
these reconstituted mice was significantly increased compared with
controls (Fig. 6
). The reconstitutive capacity of BMC was unaltered by anti-CD44v7
treatment of donor mice.
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Figure 5. Marrow-repopulating ability of bone marrow and spleen cells after
anti-CD44v7 treatment: Mice (three per group) were treated for 3 days
with either control IgG or anti-CD44v7 (200 µg/mouse). BMC and SC
were harvested 24 h after the last injection. Lethally irradiated
mice (10/group) were reconstituted with either 1 x
106 BMC or 5 x 106 SC. (A and B) five
mice per group were killed after 8 days to determine the number of BMC
(A) and the number of C-CFU per femur (B). (C and D) BMC from five mice
per goup were collected 8 days after the first transfer and were
re-transferred into a second, lethally irradiated host (10 mice/group).
The number of BMC (C) and the number of CFUs (D) was determined after
12 days. *Significance of differences.
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Figure 6. Anti-CD44v7 efficiently mobilizes hematopoietic progenitor cells.
CD44v7+/+ 129SV mice received three intravenous injections
of PBS, 200 µg control IgG or anti-CD44v7. BMC (A), SC (B), and PBMC
(C) were collected 24 h after the last injection and titrated
numbers were transferred into lethally irradiated CD44v7-proficient
129SV mice. The survival rate (over 3 months) is shown. Data represent
mean values of 20 mice (BMC, SC) and 15 mice (PBMC) per group (four and
three, respectively, independently performed experiments each with five
mice per group).
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For a comparative analysis of the MRA, it is important to have an estimate of the frequency of CFU, particularly of C-CFU in these mice. Evaluation of CFUs in the bone marrow (Table 4 ) revealed no significant differences between CD44v7-/- and CD44v7+/+ mice. This also accounted for the frequency of CFU in the spleen.
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Table 4. CFU in the Bone Marrow of CD44v7+/+ and
CD44v7-/- 129SV Mice
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Table 5. MRA of CD44v7-/- BMC
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Figure 7. CD44v7 deficiency on progenitor cells has no major impact on long-term
reconstitution. Lethally irradiated CD44v7+/+ (A) and
CD44v7-/- (B) 129SV mice received titrated numbers of BMC
from CD44v7+/+ or CD44v7-/- mice. The survival
rate (over 3 months) is shown. Data represent mean values of 15 mice
per group (three independently performed experiments each with five
mice per group).
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In the mouse, CD44v7 is expressed by a minority of BMC, which coexpress Thy1 and partly CD117 and SCA-1. Yet, expression of CD44v7 does not appear to have a major influence on the distribution of progenitor cells in the bone marrow, i.e. BMC subpopulations of CD44v7-/- mice do not differ significantly from those of CD44v7-competent mice. Furthermore, frequencies of CFU were comparable in CD44v7-deficient and competent mice. Finally, although the MRA of CD44v7-deficient BMC in the CD44v7-competent host was reduced, the long-term reconstitutive capacity of CD44v7-deficient BMC was not significantly impaired, i.e. when titrated numbers of CD44v7-deficient and CD44v7-competent BMC were transferred into the CD44-competent host, no significant differences in the survival rates were observed. Taken together, although some of these findings imply an involvement, they do not support an essential role of CD44v7 in the maturation of hematopoietic progenitor cells.
Instead, expression of CD44v7 on bone marrow stromal elements, although much weaker than expression of panCD44, clearly supports progenitor cell embedding in stromal niches. The importance of CD44v7 expression on stromal cells for progenitor cell homing and seeding has been demonstrated by the transfer of BMC from CD44v7-competent mice into CD44v7-deficient mice and vice versa. BMC homed poorly in CD44v7-deficient mice and the MRA was strongly reduced. Accordingly, CD44v7-deficient BMC displayed improved homing and MRA when transferred into the CD44v7-competent compared with the CD44v7-deficient host.
An additional observation deserves discussion. After treatment with anti-CD44v7 a significantly increased number of SC was recovered and the relative numbers of CFU in the spleen and the peripheral blood were strongly augmented. In addition, when SC from anti-CD44v7-treated mice were transferred into an irradiated host, they displayed a strong MRA, and lower numbers of SC and PBMC sufficed for long-term reconstitution. Notably, a comparably strong increase of progenitor cells in the spleen and the peripheral blood has not been observed when blocking panCD44 [5, 22, and O. Christ et al., unpublished results] or CD44v6 (data not shown). The findings strongly point toward functional activity of CD44v7 beyond that of an adhesive structure on bone marrow stromal cells.
One possibility could be that CD44v7 is actively involved in the process of egress. It has been argued that the egress of hematopoietic progenitor cells from the bone marrow may not be a passive but rather an active process, which can be hampered or triggered by a variety of signaling molecules [30 , 41 ]. If CD44v7 actively supported egress, we should have observed a higher frequency of progenitor CFU in the bone marrow of CD44v7-deficient mice and a lower number of CFU in the spleen. Furthermore, after anti-CD44v7 treatment an increased number of CFU in the bone marrow, but no enrichment in the periphery should have been observed. Because none of these phenomena have been seen, an active role of CD44v7 in egress does not appear very likely.
Alternatively, it has been described that via an interaction between CD44 and integrins, blockade of adhesion by anti-CD44 supports proliferation by integrin-mediated signaling [15 ]. Such cooperativity between CD44v7 and integrins at the progenitor cell could well explain the increased recovery of progenitor cells in the periphery without a concomitant depletion in the bone marrow. Furthermore, we have described before that CD44v7-deficient mice are protected from a hapten-induced colitis [31 ], which in CD44v7-competent mice can be cured by anti-CD44v7 [42 ]. The analysis of the underlying mechanisms revealed that CD44v7 likely is involved in two distinct processes. CD44v7 apparently functions as a ligand on accessory cells for a costimulatory molecule on T cells as well as an anti-apoptotic receptor on activated T cells. In fact, the increase in the overall number of progenitor cells observed by a blockade of CD44v7, as well as the observation of a superior survival rate of CD44v7-/- BMC compared with CD44v7+/+ BMC in short-term cultures (data not shown) and finally the fact that the CD44v7-/- host was more readily reconstituted by CD44v7-/- than by CD44v7+/+ BMC, all point toward functional activity of CD44v7 on progenitor cells in the process of either proliferation or survival.
Taken together, as revealed by antibody blockade and CD44v7-deficient mice, expression of CD44v7 on stromal cells strengthens adhesion of progenitor cells, such that CD44v7-competent BMC home poorly in the CD44v7-/- host. Accordingly, blockade of the molecule allows for an efficient mobilization of progenitor cells. Because blockade of CD44v7 is accompanied by a massive increase of progenitor cells in the periphery, it is likely that CD44v7 on hematopoietic progenitor cells exerts additional functions in hematopoiesis, which could be a participation in signal transduction related to proliferation or survival.
Received August 15, 2000; revised November 1, 2000; accepted November 3, 2000.
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