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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Exp Hematol. Author manuscript; available in PMC 2009 September 21.
Published in final edited form as:
PMCID: PMC2747096

Altered SDF-1/CXCR4 axis in patients with primary myelofibrosis and in the Gata1low mouse model of the disease



To assess whether alterations in the SDF-1/CXCR4 occur in patients with primary myelofibrosis (PMF) and in Gata1low mice, an animal model for myelofibrosis, and whether these abnormalities might account for increased stem/progenitor cell trafficking.

Materials and Methods

In the mouse, SDF-1 mRNA levels were assayed in liver, spleen and marrow. SDF-1 protein levels were quantified in plasma and marrow and CXCR4 mRNA and protein levels were evaluated on stem/progenitor cells and megakaryocytes purified from the marrow. SDF-1 protein levels were also evaluated in plasma and in marrow biopsy specimens obtained from normal donors and PMF patients.


In Gata1low mice, the plasma SDF-1 protein was 5-times higher than normal in younger animals. Furthermore, SDF-1 immuno-staining of marrow sections progressively increased with age. Similar abnormalities were observed in PMF patients. In fact, the plasma SDF-1 levels in PMF patients were significantly higher (by 2-fold) than normal (p<0.01) and SDF-1 immuno-staining of marrow biopsiy specimens demonstrated increased SDF-1 deposition in specific areas. In two of the patients, SDF-1 deposition was normalized by curative therapy with allogenic stem cell transplantation. Similarly to what already has been reported for PMF patients, the marrow from Gata1low mice contained fewer CXCR4posCD117pos cells and these cells expressed low levels of CXCR4 mRNA and protein.


Similar abnormalities in the SDF-1/CXCR4 axis are observed in PMF patients and in the Gata1low mice model of myelofibrosis. We suggest that these abnormalities contribute to the increased stem/progenitor cell trafficking observed in this mouse model as well as patients with PMF.

Keywords: Gata1, SDF-1, megakaryocytes, primary myelofibrosis


Primary myelofibrosis (PMF) is characterized by constitutive mobilization of CD34+ hematopoietic stem/progenitor cells, as well as endothelial progenitor cells13. Increased stem/progenitor cell trafficking has also been described in the Gata1low mouse model of this disease. Mice harboring the hypomorphic Gata1low mutation, that impairs expression of this gene in megakaryocytes4, are born thrombocytopenic5,6. The megakaryocytes in the marrow from these mutants present the same morphological abnormalities as those observed in the marrow of PMF patients79. The mutants slowly develop with age all the hallmarks of the disease, including increased stem/progenitor cell trafficking and extramedullary hematopoiesis in liver10,11. The disease is associated with a profound disruption of the structural architecture of the marrow. We hypothesized that the abnormal cell trafficking observed both in the patients and in the mouse model might be the consequence of stem/progenitor cell dislodgment due to structural alterations of the stem cell niche. Alternatively, anatomical abnormalities may not be sufficient to alter the balance of events that result in the retention of stem/progenitor cells within the marrow. Other parameters, such as changes in chemokine levels10,1214 and/or disruption of adhesive interactions favoring retention of stem/progenitor cells within the marrow15, may be operational.

SDF-1 is a chemokine primarily produced by the liver16. In addition to the liver, SDF-1 is produced in the marrow by several cell types, including osteoblasts17. Recent genetic studies have demonstrated that osteoblasts are critical cellular elements for stem cell maintenance and development1820. Stem cells are believed to reside in close proximity to the endosteal surface of the bone, where osteoblasts are also located21,22. Interactions between SDF-1, produced by the osteoblasts21, and its receptor, CXCR4, expressed by the stem/progenitor cells23,24, is thought to play a major role in stem cell trafficking in embryogenesis17,24 and in maintaining the stem cells within their niche in the marrow25,26. A great deal of evidence indicates that alterations in SDF-1 concentrations in marrow and blood result in stem/progenitor cell mobilization. In fact, under steady-state conditions, the levels of soluble SDF-1 present in the blood of younger animals are low. SDF-1 is very sensitive to protease degradation2730.

A significant source of SDF-1 in the blood and marrow is present within megakaryocytes and platelets. These cells, in fact, express CXCR431,32 and are capable of capturing SDF-1, which is stored within their cytoplasm and released in response to external stimuli33. The concentration of soluble SDF-1 in the blood, however, increases in association with many of the conditions that induce stem/progenitor cell mobilization. Both sustained elevations of SDF-1 in NOD/SCID mice delivered by an adenoviral vector delivery system3436, and chronic administration to the animals of SDF-1 peptide analogs37,38, result in mobilization of stem/progenitor cells. In addition, increased plasma SDF-1 levels are also observed during mobilization induced by sulfated glycans39. Although G-CSF-induced stem/progenitor cell mobilization is not associated with increased levels of SDF-1 in the blood, this treatment reduces the levels of SDF-1 in the marrow and alters CXCR4 expression by stem/progenitor cells40,41 by inducing a proteolytic environment42. By contrast, increased SDF-1 production is observed in the marrow following DNA induced damage and has been suggested to improve bone marrow recovery and facilitate stem cell engraftment post-trasplantation43. On the other hand, inactivation of CXCR4 function with neutralizing antibodies impairs stem/progenitor cell engraftment in the bone marrow35.

The aim of this study was to determine whether abnormalities in the SDF-1/CXCR4 axis exist and might play a role in stem/progenitor cell trafficking in PMF. To achieve this goal, we analyzed the expression of SDF-1 (both mRNA and protein) in tissues from Gata1low mice during different stages of the disease. The results provide evidence for abnormalities in the SDF-1/CXCR4 axis both in the mouse model of the disease and in patients with PMF. In the latter, it was possible to directly attribute these abnormalities to the defective PMF stem cell clone by comparing results obtained in two patients before and following curative therapy with stem cell transplantation. Overall, alterations in the SDF-1/CXCR4 axis observed with disease progression may at least participate in, if not be primarily responsible for, abnormal stem/cell progenitor cell trafficking in PMF.



Mice with the hypomorphic Gata1low mutation5,10 were bred at the Istituto Superiore di Sanità and maintained under good animal care practice conditions. Wild type littermates at the Gata1 locus were used as controls. Mice were divided into two age groups: 8–9-month (younger adult mice), i.e. at the time when they presented increased stem/progenitor cell trafficking, and 12–23-month (older adult mice), i.e. when they had developed extramedullary hematopoiesis in the liver (10,11 and Table 1). All the experiments were performed according to protocols approved by the Institutional Animal Care Committee.

Table I
Number of platelets (Ptl) in the blood and frequency of stem/progenitor cells in the blood, femur and liver from wild type and Gata1low littermates. Gata1low mice were divided into adult and old animals, as indicated. The presence of erythroblasts (TER119 ...

Hematological parameters

Blood was collected from the retro-orbital plexus into ethylen-diamino-tetracetic acid-coated microcapillary tubes (20–40 µL/sampling). Platelet (plt) counts were determined manually12.

Histology and immunohistochemistry of murine tissues

Mouse tissues were fixed in 10% (v/v) phosphate-buffered formalin (Sigma, St, Louis, MO, USA), paraffin embedded and cut into 2,5-3 µM section according to standard procedures. SDF-1β (Rb anti-rat SDF-1b, Torrey Pines Biolabs, Huston, TX) staining was done in frozen sections using the ABC/Complex/HRP kit from Dako (Carpinteria, CA), stained with 0.05% diaminobenzidine and counterstained with Mayer’s Hematoxylin. All the preparations were stained at the same time and under the same conditions. Negative controls were represented by preparations incubated with irrelevant antibodies (data not shown). Slides were permanently mounted and photomicrographs were taken with a Nikon COOLPIX995 digital camera with an adapter fitting into the eyepiece of an Olympus BH-2 microscope with a 20X objective (Images were transferred directly to the computer and Photoshop 7.0 was used to adjust brightness, contrast and color balance).

Progenitor cell determination

The frequency of progenitor cells was determined by plating either total blood (2 µL/mL), or the mononuclear cell fraction of marrow (2×104 cells/mL) or liver (5×104 cells/mL) from representative normal and GATA-1low littermates in standard methylcellulose cultures (0.9% w/v) containing fetal bovine serum (30% v/v, Sigma) and recombinant growth factors [rat stem cell factor (SCF, 100 ng/mL), mouse interleukin-3 (IL-3, 10 ng/mL), granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF) (50 ng/mL each) (all from Sigma) and human erythropoietin (EPO, 2 U/mL; Boehringer Mannheim, Mannheim, Germany)]11. The cultures were incubated at 37 °C in a humidified incubator containing 5% CO2 in air and colonies derived from more or less mature progenitor cells scored at day 8.

Flow cytometry analysis and cell purification

Cells were incubated for 30 min on ice with fluorescein isothiocyanate (FITC)-CXCR4 in combination either with phycoerythrin (PE)-CD34 and allophycocyanin (APC)-CD117 (that recognizes c-Kit) or biotinaled-streptavidin-CD61 and phycoerythrin (PE)-CD41. All the antibodies were purchased from PharMingen (San Diego, CA, USA) and used at a concentration of 1 µg/106 cells. In addition to the Pharmigen antibody, a polyclonal purified rabbit anti-rat CXCR4 (Partec, Münster, Germany) followed by a FITC-coniugated goat antirabbit IgG (Immunotech, Miami, FL) was also used. Cell fluorescence was analyzed with a FACS ARIA (Becton Dickinson, Franklin Lakes, NJ). Non-specific fluorescent signals were gated out with appropriate fluorochrome-conjugated isotype controls and dead cells were excluded by propidium iodide staining. Cells in the stem/progenitor cell (CD117pos) or megakaryocyte (CD61pos) gates were purified by sorting, as previously described44,45 and were >90% pure upon FACS reanalysis.


Bone marrow washes were prepared by gently flushing the cavity of two femurs with 400 µL of phosphate buffered saline containing 0.1% Tween. Blood was collected from the retro orbital plexus of the animals with a heparin-coated Pasteur pipette and platelet-poor plasma prepared by centrifugation at 5000 rpm (platelet contamination of the samples <9 %). SDF-1 was measured with a commercial ELISA kit (R&D Systems, Inc. Minneapolis, MN, USA).

RNA isolation and quantitative RT-PCR

Total RNA was prepared from mononuclear cell fractions of marrow, spleen and liver following direct lysis with Trizol (Gibco BRL). Total RNA was also prepared from the entire femur by pulverizing the organ with a pestle under liquid nitrogen before dissolution in Trizol. RNA (1 µg) was retrotrascribed with random primers using the SuperScript III kit (Invitrogen Life Technologies, Bethesda, MD). Gene expression was quantified with a Taqman PCR kit (PE Applied Biosystems, Foster City, CA) using the ABI PRISM 7300 Sequence Detection System (Applied Biosystem). GAPDH was also quantified in each reaction. Quantitative values were obtained from the threshold cycle number (Ct), by subtracting the average Ct of the target gene from that of Gapdh and expressed as 2− ΔCt.

Human Subjects

The normal donors (13), patients with PMF (27) and polycythemia vera (PV, 10) included in this study have been previously described46. Two of the patients with PMF underwent allogenic stem cell transplantation and were studied again 12 months after the transplant47. At the time of the analyses, the transplanted PMF patients had normocellular bone marrows with few reticulin fibers and near normal number of CD34+ in peripheral blood.

Human Immuno-histochemistry

Bone marrow biopsies were fixed with zinc formalin, decalcified and embedded in paraffin. For immunochemical staining, the tissue sections were deparaffinized in xylene and hydrated in graded alcohol. Heat-induced antigen retrieval was performed with citrate buffer for 20 minutes at 95 degrees in a steamer. The sections were then stained with a monoclonal anti-human SDF-1 antibody at 1:200 dilution (R&D Systems, Inc., Minneapolis, MN, USA), followed by incubation with a peroxidase-labeled polymer conjugated to a goat antimouse immunoglobulin. Bound antibody was stained with a histostainer (DAKO) using a peroxidase detection kit with the chromogen 3,3’-diaminobenzidine tetrahydrochloride. The slides were counterstained with Light Green. All staining procedures were performed utilizing an isotypic antibody as a negative control.


Levels of SDF-1 mRNA in the femurs of Gata1low mice are reduced with disease progression

Although SDF-1 mRNA was detectable in all organs analyzed, the major site of SDF-1 mRNA production in wild type mice was the liver. In fact, levels of SDF-1 mRNA in the liver were 1-log higher than those in the marrow and 2-logs higher than those in the spleen (Figure 1A). No difference between levels of SDF-1 mRNA expressed by the liver and the spleen was observed in wild type and Gata1low littermates, regardless of the age of the animals (Figure 1A, and analyses not shown). In contrast, marrow cells from Gata1low mice expressed levels of SDF-1 mRNA that were significantly (p<0.01) lower than those expressed by normal mice.

Figure 1
Reduced expression of SDF-1 mRNA in the bone marrow of Gata1low mice

Since osteoblasts, the cells primarily responsible for producing SDF-1 in the marrow, are poorly represented in cells recovered from the bone cavity, the levels of SDF-1 mRNA in the entire mouse femur were analyzed (Figure 1B). Levels of SDF-1 mRNA in the entire femur from wild type littermates, indicated by shaded area in Figure 1B, did not change with age (analyses not shown). In contrast, the levels of SDF-1 mRNA present in the entire femur from Gata1low mice significantly decreased with age and was lower than those of wild type littermates (Figure 1B).

Adult Gata1low mice contain elevated peripheral blood levels of soluble SDF-1 protein

The amount of SDF-1 present in the marrow cells of wild type and Gata1low mice was generally low (<100–400 pg/mL) (Figure 2). Marrow washes from Gata1low mice contained levels of SDF-1 that were not statistically significantly greater than normal mice (Figure 2A). There was also no difference in the SDF-1 content of marrow washes from younger and older Gata1low animals (Figure 2A).

Figure 2
The plasma from younger (≤9 month-old) Gata1low mice contains significantly higher SDF-1 levels than those present in the plasma of wild type littermates

In the blood, SDF-1 is present as a soluble protein or within platelets. Approximately 2 ng of soluble SDF-1 was detected in ~1 mL of blood from wild type mice (Figure 2B). By contrast, approximately ~350 pg of SDF-1 was detected per 105 platelets. The average number of platelets in the blood is 7.5×105/µL and the total blood volume of a mouse is ~3 mL48,49. Based on these assumptions, we calculated that ~24 µg of SDF-1 was present in the platelets (1000 times more than the soluble form) (Table II).

Table II
Absolute values of soluble and Ptl-associated SDF-1 in the blood of Wild Type and Gata1low mice. Gata1low mice were divided into adult and old animals, as indicated.

The levels of soluble SDF-1 in the plasma of older Gata1low mice were similar to normal controls (~8 ng), but the levels in the plasma of younger (≤9 month) mutant mice were 4-times higher than normal (~24 µg, p<.01, Figure 2B and Table II). No difference was observed in the SDF-1 content of platelets prepared from wild type and Gata1low mice, at all ages studied (Figure 2C). Since the blood from Gata1low mice contains 5-times fewer platelets than normal controls (Table 1), the amount of SDF-1 within platelets in the mutant mice is, therefore, estimated to be 5-times lower than normal (Table II).

Increased levels of SDF-1 protein by immuno-histochemistry in the marrow of Gata1low mice with disease progression

SDF-1 was detectable in marrow sections from wild type mice (Figure 3A). The staining was localized within megakaryocytes and did not increase with age (Figure 3A). SDF-1 staining was also localized within megakaryocytes in marrow sections from younger Gata1low mice. The intensity of the staining of the mutant megakaryocytes, however, was greater than that of the corresponding normal cells (Figure 3A). The SDF-1 staining of marrow sections from older animals (>10-month) was clearly stronger than that of wild type animals and younger mutant mice (Figure 3A). In this case, SDF-1 staining was not only localized within the megakaryocytes, but was also associated with structures that resemble collagen fibers (Figure 3A).

Figure 3Figure 3
Increased SDF-1-immunostaining of marrow and spleen sections from Gata1low mice with age

Also the spleen sections of Gata1low mice reacted more strongly with the SDF-1 antibody than those from normal animals (Figure 3b). In this case, the staining was localized within the megakaryocytes of younger mutant mice and both in megakaryocytes and extracellular areas of older mice (Figure 3B). No difference was observed in SDF-1 staining of liver sections from wild type and Gata1low mice.

Stem/progenitor cells from the marrow and the blood of Gata1low mice express lower levels of CXCR4 than normal controls

The frequency of megakaryocytes (CD41pos/CD61pos cells) in the marrow of Gata1low mice is 3–4-times greater than normal (Figure 4A and 10,45). Six percent of both wild type and Gata1low megakaryocytes expressed CXCR4 on their surface (Figure 4A). No difference was observed in the intensity of CXCR4 expressed (calculates as AFI) by megakaryocytes from the marrow of wild type and Gata1low littermates.

Figure 4Figure 4
Marrow megakaryocytes (CD41pos/CD61pos) from Gata1low mice express normal levels of CXCR4 while marrow stem/progenitor cells (CD117pos) from the same mice express reduced levels of CXCR4. On the other hand, CD117pos cells from the marrow and blood of ...

Stem/progenitor cells present in the marrow of normal mice expresses CD11750 and are further divided by the CD34 marker, into specific cell subpopulations51,52. CD117 expression identifies also stem/progenitor cells in the marrow of mice with the Gata1low mutation11,44. CD34, however, is not an effective marker for identifying functional subpopulations from Gata1lowCD117pos cells45. Therefore, for the purpose of this study, the levels of CXCR4 expression were analyzed on the entire CD117pos cell population present in the marrow of wild type and Gata1low littermates.

The frequency of stem/progenitor cells (CD117pos) in the marrow of Gata1low mice was similar to that of normal animals (Figure 4B, Table I and 11,44,45). However, because of a 5-fold reduction in marrow cellularity observed in these mice with disease progression, the total number of stem/progenitor cells in the marrow was calculated to be 5-fold lower than normal (Table I and 11,44). CXCR4 was detectable in ~70% of the CD117pos cells present in the marrow of wild type mice (Figure 4B, C). Since wild type CD117posCXCR4pos cells purified by sorting retained the sorting profile upon re-analyses (Figure 5A), these cells represent a distinct subpopulation of the normal stem/progenitor cells present in the marrow. In contrast, both the frequency (~40%), and the level of expression (AFI/cell = 430 vs 710 in mutant and wild type cells, respectively), of CXCR4 expressed by the CD117pos cells in the marrow of Gata1low mice were significantly lower than in wild type animals (Figure 4B, C).

Figure 5
In the marrow from Gata1low mice, megakaryocytes (CD61pos) express normal levels of CXCR4 mRNA while the CXCR4pos fraction of the stem/progenitor cells (CD117pos/CD34neg and CD34pos) express levels of CXCR4 mRNA lower than normal

The levels of CXCR4 mRNA expressed by megakaryocytes (CD61pos), by the entire stem/progenitor cell population (CD117pos) and by the subpopulation of stem/progenitor cells expressing CXCR4 (CD117posCXCR4pos) purified from the marrow of wild type and Gata1low littermates are shown in Figure 5A–B. CXCR4 mRNA was expressed at comparable levels by CD61pos (megakaryocytes) cells purified from the marrow of wild type mice and Gata1low littermates; (Figure 5B). By contrast, CD117posCXCR4pos cells purified from the marrow of Gata1low mice expressed levels of CXCR4 mRNA significantly lower than those expressed by wild type CD117posCXCR4pos (Figure 5B).

In conclusion, the CD117pos cells in the marrow of Gata1low mice express less CXCR4 than their wild type counterpart in terms of frequency of CXCR4pos cells and the amount of surface receptor and mRNA expressed by CXCR4pos cells.

To further characterize the trafficking abnormalities of the Gata1low mice, we compared the frequency of progenitor cells in the blood of younger and old Gata1low and wild type littermates. These experiments also evaluated the frequency of erythroblasts (Ter119pos/CD71pos) and of progenitor cells in the liver, which was used as a control for development of extramedullary hematopoiesis. Since it has been suggested that the proteolytic marrow microenvironment of PMF might lead to trafficking by cleaving surface proteins on stem/progenitor cells, the frequency of progenitor cells in the blood was determined both by functional assay flow cytometrically (CD117pos cells). The expression of CXCR4 on the surface of the circulating CD117pos cells was analyzed as well. As previously described12, the blood from Gata1low mice contains greater numbers of progenitor cells than the blood from wild type littermates (Table 1 and Figure 4B). The contour plots of the CXCR4 staining of the CD117pos cells in the blood and in the marrow of Gata1low mice were identical (Figure 4B). By contrast, wild type CD117pos cells present in the blood express lower levels of CXCR4 than CD117pos cells present in the marrow (53 and Figure 4B).

PMF patients have higher blood levels of SDF-1 higher than normal

SDF-1 levels ranging from 800 to 1800 pg/mL were detectable in the plasma of normal volunteers. However, plasma levels of SDF-1 in PMF patients were increased (p<0.01, Figure 6). This increase in plasma SDF-1 levels was not unique to patients with PMF, since the plasma of patients with PV also contained significantly higher levels of SDF-1 than controls (p<0.05, Figure 6).

Figure 6
The plasma from PMF and PV patients contains SDF-1 levels significantly higher that those present in the plasma of normal donors

The excessive expression of SDF-1 in the marrow of PMF patients is restored by marrow transplantation

We assessed SDF-1 expression in BM biopsy specimens of both normal subjects (5) and PMF (10) patients using immunohistochemical staining. In two PMF patients, SDF-1 expression was analyzed before and 12 months after successful allogenic stem cell transplantation. In the 5 normal BM biopsies examined, SDF-1 was present primarily along the osteoblasts lining the BM endosteal region and endothelial cells lining both the small and large blood vessels including the periarterial regions as well as the blood capillaries of the bone (Figure 7A). By contrast, the extent of and intensity of SDF-1 staining was significantly increased in each of the 10 PMF BM biopsy specimens examined (Figure 7A). The staining for SDF-1 was present in the dilated BM sinusoids that characterize PMF, around the vessel walls within the BM which were increased in PMF, and within the extracellular matrix. The increased SDF-1 deposition was seen in BM biopsies from patients with the prefibrotic forms of PMF as well as those with marked BM fibrosis (Figure 7A). The increased distribution of SDF-1 was no longer observed in two patients who had received curative treatment with allogenic stem cell transplants (Figure 7B). These data indicate that the increased distribution of SDF-1in PMF is a consequence of an abnormal clone and can be normalized by the replacement of this malignant clone with normal HSC and their progeny.

Figure 7Figure 7
A. SDF-1 immunohistochemistry of bone marrow sections from two normal controls and 2 PMF patients


Disruption of the SDF-1/CXCR4 axis has been reported to lead to stem cell mobilization in several animal models (see 1821). The comparison of data obtained using an animal model and by studying patients with PMF provides evidence that alterations in CXCR4 expression or SDF-1 levels may be, at least partially, responsible for the abnormal stem/progenitor cell trafficking observed in PMF. The majority (~90%) of SDF-1 protein detected in the blood from wild type animals was present within platelets (Figure 2). The SDF-1 content of the blood platelets from Gata1low mice was normal. However, since these mice have 3-times fewer platelets than normal in the blood, the overall content of platelet-associated SDF-1 in the circulation of these animals was calculated to be lower than normal (Table I). By contrast, the levels of soluble SDF-1 in the blood of younger Gata1low mice were 4-fold higher than normal (Figure 2). A two-fold increase in the amount of soluble SDF-1 present in the blood was also observed in patients with PMF (Figure 6). These increases were not specific to this myeloproliferative disorder as high levels of SDF-1 were also observed in the plasma of patients with PV (Figure 6). The elevation of SDF-1 observed in the blood of the animal model and of the PMF patients were modest (~2–4-fold) (Figure 2 and and6)6) when compared to the levels required to experimentally induce stem/progenitor cell mobilization in NOD/SCID mice (25-fold)34,35 and are, therefore, unlikely to be responsible for a gradient driven mobilization of stem/progenitor cells.

Both in wild type and Gata1low mice, the major source of SDF-1 mRNA is the liver (Figure 1A). Significant levels of SDF-1 mRNA, however, were also expressed by cells flushed from the bone cavity and in the entire femur (i.e. marrow cells plus osteoblasts). The levels of SDF-1 expressed by the cells in the marrow from Gata1low mice (at all ages) were significantly lower than normal while those expressed by the entire femur progressively decreased with age. These results may reflect a progressive reduction with age of specific cell populations, possibly represented by the hemopoietic cells, responsible for producing SDF-1 in the femur. In fact, it has been reported that human stem/progenitor cells and megakaryocytes produce SDF-1 mRNA54,55. However, CD117pos cells (wild type and Gata1low cells alike) express negligible levels of SDF-1 mRNA (2−ΔCt < 2×10−4) while some SDF-1 mRNA expression (2−ΔCt < 1×10−3) was observed in CD61pos cells from normal mice but not in those from Gata1low mice.

Marrow sections both from Gata1low mice (Figure 3A and 7,10) and from PMF patients8 are characterized by an increased frequency of megakaryocytes which react strongly with the SDF-1 antibody (Figure 3 and and7).7). In the marrow of Gata1low mice, the immunostaining was mainly localized within the megakaryocytes (Figure 3A). The fiber deposition observed in older animals was also associated with strong SDF-1-staining of the extracellular matrix (Figure 3A). These high levels of SDF-1 detected by immuno-histochemistry are not in contradiction with the near normal levels of SDF-1 detectable by ELISA in marrow washes from the same animals (Figure 2A). It is possible, in fact, that the mild detergent solution used to wash the marrow was incapable of removing SDF-1 from the extracellular matrix. High levels of SDF-1 immunostaining were also observed in marrow megakaryocytes of PMF patients (Figure 7A). Indirect proof that increased SDF-1 staining both of the altered megakaryocytes and of the extracellular matrix was due to the abnormal PMF clone is provided by the observation that SDF-1 staining of marrow biopsies from 2 PMF patients that had undergone curative allogenic stem cell transplantation was normal (Figure 7B). Since wild type and Gata1low megakaryocytes expressed similar levels of CXCR4 (both mRNA and protein, Figure 5), the increased SDF-1 uptake of Gata1low megakaryocytes revealed by immuno-staining is not due to increased numbers of receptors on the surface. Megakaryocytes from Gata1low mice and from PMF patients express, however, the same intrinsic abnormalities (i.e. higher frequency and higher retention in the marrow because of maturation retardation68,56 and increased localization near the endosteum, in close spatial relationship with the osteoblasts57. All these factors can contribute to the increased megakaryocyte up-take of SDF-1. Since production of SDF-1 in the marrow of Gata1low mice is not higher than normal (Figure 1), entrapment of SDF-1 within the increased number of mutant megakaryocytes reduces the amount of SDF-1 available for other cells in the marrow. Furthermore, since SDF-1 is very sensitive to protease degradation2730 and both the full length and its proteolytic products58 are recognized by immunostaining, it is possible that the high levels of SDF-1 observed in the marrow both of Gata1low mice and PV patients represent proteolytically cleaved SDF-1. In summary, it is possible that, in spite of high levels of SDF-1 immunostaining, the marrow of the animal model and of the PMF patients is deprived of functional SDF-1 because of increased SDF-1 uptake by the high numbers of defective megakaryocytes and presence of inactive degradation products of SDF-1 bound to the extracellular matrix.

Stem/progenitor cells purified from the marrow of Gata1low mice expressed reduced levels of CXCR4 mRNA and barely detectable levels of CXCR4 antigen on their surface (Figure 4 and and5).5). Low levels of CXCR4 expression were also reported in mobilized human CD34+ cells in PMF patients59. The biological consequences of reduced CXCR4 expression on these cells were identified by experiments showing that the CD34+CXCR4- cells from PMF patients had reduced capability to migrate in vitro toward a gradient of SDF-160. The fact that reduced CXCR4 expression is implicated in abnormal cell trafficking in PMF is further suggested by the inverse correlation existing between CXCR4 expression and the number of circulating CD34+ cells that has been recently reported59.

The mechanism responsible for reduced CXCR4 expression by stem/progenitor cells in the animal model and in the PMF patients remains unclear. It has been suggested that it may be due to increased cleavage of the extracellular domain of CXCR4 because of the proteolytic environment created by the pathological neutrophil emperipolesis within the megakaryocytes15. In this context, the normal levels of CXCR4 expressed by the megakaryocytes present in the marrow of Gata1low mice are of interest. If proteolytic mechanisms are responsible, one might suggest this is not the case in mutant mice. On the other hand, since mRNA levels were decreased, it is likely that altered CXCR4 expression on the stem/progenitor cells is a direct consequence of the mutation that induces the formation of the PMF clone. This hypothesis is supported by the transcriptosomal analysis of the genes abnormally expressed in PMF that has indicated that reduced CXCR4 mRNA expression on CD34+ cells from PMF patients is directly associated with the presence of the V617FJAK2 mutation61, the genetic abnormality recently demonstrated to be frequently associated with myeloproliferative disorders6264. These observations are similar to the dose dependent alteration of CXCR4 expression on stem/progenitor cells induced by p210, the BCR-ABL mutation determining chronic myelogenous leukemia65,66 The availability of the Gata1low model should allow one to demonstrate whether altered CXCR4 expression in the stem/progenitor cells is a direct consequence of the mutation responsible for the development of PMF and, if so, gaining a greater understanding of the molecular mechanism that determines the altered CXCR4 expression on the abnormal PMF clone.

In conclusion, these results suggest that abnormal stem/progenitor cell trafficking in PMF is accompanied by down regulation of CXCR4 expression by the stem/progenitor cells by a mechanism yet to be defined.


This study was supported by a grant from the National Cancer Institute (P01-CA108671), and by the National Institute of Health (grant HL58734)


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