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(Journal of Leukocyte Biology. 2001;69:343-352.)
© 2001 by Society for Leukocyte Biology

Importance of CD44v7 isoforms for homing and seeding of hematopoietic progenitor cells

Oliver Christ*, Ursula Günthert{dagger}, Rainer Haas{ddagger} and Margot Zöller*

* Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg;
{dagger} Basel Institute for Immunology, Switzerland;
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The adhesion molecule CD44 consists of many isoforms of which particularly CD44v7 is of major importance in hematopoietic progenitor cell homing. An increase of progenitor cells in the periphery was observed after treating mice with a CD44v7-specific antibody, concomitant with a substantially augmented marrow-repopulating ability (MRA). Because CD44v7 is expressed on a fraction of bone marrow cells (BMC) as well as on long-term bone marrow culture-derived stromal cells, we aimed to differentiate between the functional relevance of CD44v7 on either cell type by transferring CD44v7+/+ BMC into CD44v7-/- mice and vice versa. CD44v7+/+ BMC homed poorly in the bone marrow of CD44v7-/- mice and their MRA was severely impaired. CD44v7-/- BMC, instead, exhibited an improved MRA when transferred into the CD44v7+/+ host, although their MRA remained below that of CD44v7+/+ BMC. Thus, it is predominantly, but not exclusively, expression of CD44v7 on stromal cells which supports progenitor cell homing.

Key Words: CD44 isoforms • hematopoiesis • mobilization • marrow repopulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CD44 comprises a family of adhesion molecules with binding domains for hyaluronate (HA), fibronectin, laminin, type IV collagen, and glucosaminoglycans [1 ]. The hematopoietic or standard isoform (CD44s) is expressed rather ubiquitously. A multitude of so-called variant isoforms (CD44v), created by the insertion of up to 10 additional exons between exon 5 and 6 of CD44s [2 ], are expressed in a tissue-related and developmentally restricted manner [3 ]. In addition to other functions CD44 isoforms are known to be involved in hematopoiesis [4 , 5 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
129SV and CD44v7 knockout mice (129SV background) [31 ] were bred at the central animal facilities of the German Cancer Research Center. Mice were kept under specific pathogen-free conditions. They were used for experiments at the age of 8–10 weeks. Where indicated, animals were irradiated with 9 Gy using a whole-body irradiation chamber with a 137Cs source.

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 {gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of CD44v7 on hematopoietic progenitor cells and BM-derived stromal cells
Expression of CD44v7 was evaluated in freshly harvested BMC, in LTBMC-derived stromal cells, and in the two stromal lines MS5 and S17, which support myeloid and lymphoid progenitor cell maturation, respectively (Table 1 , Fig. 1A ). The vast majority of BMC and of stromal cells in LTBMC as well as both stromal lines, S17 and MS5, express panCD44 at a high level. CD44v7 isoforms have been detected on less than 20% of BMC and 40% of BM-derived stromal cells, which are composed of different elements such as fibroblasts, reticulocytes, lipocytes, monocytes, and others. We did not observe staining of spleen or thymic stromal elements (data not shown). The stromal line S17 was stained to over 60% by anti-CD44v7. Expression of CD44v7 on the stromal line MS5 was weaker.


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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.

 
To further characterize BMC expressing CD44v7, double fluorescence staining was performed with antibodies specific for CD24 (HSA), CD34 (Sgp90), CD43 (sialophorin), CD45R (B220), CD49d (VLA-4), CD54 (ICAM-1), CD90 (Thy1.2), CD117 (c-kit), and antibodies recognizing SCA-1 and a dendritic cell (DC) marker (Table 2A , Fig. 1B ). All these populations were stained by a panCD44-specific antibody. Anti-CD44v7 stained over 90% of CD90+ cells, over 50% of dendritic cells and of CD117+ cells, and 40% of SCA-1+ cells in the bone marrow. Within the tested panel of stromal cell markers, over 50% of CD62P+ cells were stained with anti-CD44v7 (Table 2B) .


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  		Table 2. Subpopulations of BMC and Stromal Cells Expressing CD44v7

 
We also compared the distribution of progenitor markers and adhesion molecules in CD44v7-competent versus CD44v7-deficient mice (Fig. 2 ). Expression of progenitor markers did not differ in dependence of CD44v7 expression. This accounted, in addition, for CD11b, CD45R, CD54, and a DC marker (data not shown). With the exception of CD44v7, expression of other adhesion molecules, notably of panCD44 and of VLA4, two major adhesion markers of BMC, was unimpaired on BMC of CD44v7-/- mice. Expression of CD44v6 and CD44v10 also was unaltered in CD44v7-/- mice. We also did not observe differences between stromal cells of LTBMC from CD44v7-/- and CD44v7+/+ mice (data not shown).



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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.

 
Mobilization of progenitor cells by blockade of CD44v7
By the rather selective expression of CD44v7 on hematopoietic progenitor cells and stromal cells [13 ], it became tempting to speculate that blocking of CD44v7 could be an efficient means for the mobilization and peripheralization of hematopoietic progenitor cells. Mobilization was approached by three intravenous injections of 200 µg purified anti-CD44v7 in 24-h intervals. Application of the antibody led to down-modulation of the adhesion molecules CD44v7 and VLA4 in the bone marrow as well as in the spleen and in PBMC. Expression of panCD44 was unaltered (Fig. 3 ). With the exception of an increased frequency of SCA-1+ BMC and SC and a decreased frequency of CD117+ cells in bone marrow, peripheral blood, and spleen, expression of progenitor markers was also unaffected (Fig. 3) .



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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.

 
Yet, and most impressively, a significantly higher number of spleen cells and a distinctly increased number of PBMC, which exerted a strong proliferative activity, were recovered after anti-CD44v7 treatment (Table 3A ). Furthermore, SC, PBMC and, to a lower degree, also BMC contained higher numbers of C-CFU, M-CFU, and GM-CFU. In addition, the number of E-BFU was significantly increased in the peripheral blood (Table 3B) . Thus, a blockade of CD44v7 appeared to mobilize progenitor cells.


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  		Table 3. Mobilization of Progenitor Cells by Anti-CD44v7 Treatment

 
By the surprising enrichment of spleen cells in anti-CD44v7-treated mice, as well as by the increase of CFU in the spleen and the peripheral blood, it became interesting to see whether expression of CD44v7 on BMC or on stromal cells influenced progenitor homing and/or egress. To control the hypothesis, homing of BMC from CD44v7-/-- and CD44v7-competent mice was evaluated in a comparative manner.

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 {gamma}-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.

 
As shown above, expression of panCD44 was not affected by deletion of the variant exon. Thus, differences in BMC homing and settlement can be assigned to the absence of CD44v7 exclusively. The findings support the interpretation that CD44v7 on bone marrow stromal elements serves as a major target for BMC adhesion. Expression of CD44v7 on hematopoietic progenitor cells contributes to progenitor homing, although to a lesser degree. To reinforce the argument of a major contribution of CD44v7 expression on bone marrow stromal cells, MRA was compared between CD44v7-competent and CD44v7-/- mice.

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).

 
Hence, a blockade of CD44v7 apparently was accompanied by an increased recovery of progenitor cells in the periphery. Because, on the other hand, the reconstitutive capacity of BMC was neither decreased nor increased, we interpreted the data in the sense that the antibody affected stromal cells rather than progenitor cells. The hypothesis of CD44v7 on bone marrow stromal cells being an important ligand for hematopoietic progenitor cells could be controlled by testing the MRA ability of CD44v7+/+ BMC when transferred into CD44v7-/- mice.

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

 
The recovery of C-CFU per femur after the transfer of BMC from CD44v7-proficient into CD44v7-deficient mice was reduced to less than 20% compared with controls (Table 5 ). Accordingly, the number of CFUs was reduced by over 80%. The MRA of CD44v7-deficient BMC in the CD44v7-deficient mouse revealed a further, but less prominent decrease in the number of C-CFU and of CFUs compared with the transfer of BMC from CD44v7-competent into CD44v7-deficient mice. Because BMC of CD44v7-/- and CD44v7+/+ mice contained comparable numbers of C-CFU, the finding supports our interpretation that it is mainly expression of CD44v7 on bone stromal elements that influences hematopoiesis. If this holds true, the MRA of CD44v7-/- BMC should be significantly improved after the transfer into CD44v7-competent mice. This has, indeed, been observed, although the number of C-CFU per femur and the number of CFUs were still lower than after the transfer of 129SV BMC into 129SV mice.


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  		Table 5. MRA of CD44v7-/- BMC

 
Additional support for our interpretation that CD44v7 serves as a target for progenitor adhesion is derived from long-term reconstitution experiments. No significant differences in survival rates were observed between CD44v7-competent mice that received graded numbers of CD44v7+/+ or CD44v7-/- BMC. The same observation did hold true for the transfer of CD44v7+/+ and CD44v7-/- BMC into the CD44v7-deficient host, although after the transfer of low numbers of BMC, the CD44v7-/- host displayed a reduced survival rate compared with the CD44v7+/+ host. This accounted particularly for the transfer of CD44v7+/+ BMC (Fig. 7 ).



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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).

 
Thus, CD44v7-containing isoforms on bone marrow stromal cells serve as a major target for progenitor cell homing and seeding.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We and other groups have described recently that a blockade of panCD44 facilitates mobilization of hematopoietic progenitor cells [5, 16, and O. Christ et al., unpublished results]. Because panCD44 is abundantly expressed on many tissues and cells, whereas expression of CD44-variant isoforms, particularly CD44v6 and CD44v7, is mostly restricted to epithelial stem cells and to hematopoietic cells at defined stages of maturation and activation [39 , 40 ], we speculated that a blockade of these CD44v isoforms may provide more efficient targets for mobilization. By either antibody blockade of CD44v7 or by the use of CD44v7-deficient mice we could demonstrate that it is the CD44v7 isoform that serves as a target for progenitor cell homing and seeding.

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.


   ACKNOWLEDGEMENTS
 
This investigation was supported by the Deutsche Forschungsgemeinschaft, Grant Zo40/5-3 (M. Z.) and the Tumorzentrum Heidelberg/Mannheim (M. Z., R. H.). The Basel Institute for Immunology was founded and is supported by Hoffmann LaRoche, Inc., Basel, Switzerland. We cordially thank Drs. J. Andersson and A. J. Potocnik, Basel Institute for Immunology, Basel, Switzerland, and Dr. S. Matzku, Merck Ag, Darmstadt, Germany, for discussions and helpful suggestions during preparation of the manuscript.


   FOOTNOTES
 
Current address of Rainer Haas: Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Germany.

Received August 15, 2000; revised November 1, 2000; accepted November 3, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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