Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells

Experimental Cell Research 299 (2004) 383 – 392 www.elsevier.com/locate/yexcr

Bone marrow stromal proteoglycans regulate megakaryocytic differentiation of human progenitor cells Sonja Zweegmana,*, Jakob van den Bornb, Adriana M.C. Musa, Floortje L. Kesslera Jeroen J.W.M. Janssena, Tanja Netelenbosa, Peter C. Huijgensa, Angelika M. Dr7gera b

a Department of Hematology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1081 HV Amsterdam, The Netherlands

Received 24 March 2004, revised version received 6 June 2004 Available online 23 July 2004

Abstract Adherence of hematopoietic progenitor cells (HPCs) to stroma is an important regulatory step in megakaryocytic differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We examined the role of sulfated glycoconjugates, such as proteoglycans (PGs), on human bone marrow stroma (hBMS). To this end, PG structure was altered by desulfation or enzymatic cleavage. PGs participated in adhesion of human HPC, as desulfation resulted in about 50% decline in adhesion to hBMS. Heparan sulfate proteoglycans (HSPGs) were found to be responsible by showing about 25% decline in adhesion after pre-incubation of HPC with heparin and about 15% decline in adhesion after enzymatic removal of HSPGs from hBMS. Furthermore, PGs were involved in binding cytokines. Both desulfation and enzymatic removal of stromal HSPGs increased release of megakaryocytopoiesis-inhibiting cytokines, that is, interleukin-8 (IL-8, 1.9-fold increase) and macrophage inflammatory protein-1a (MIP-1a, 1.4-fold increase). The megakaryocytic output of HPC grown in conditioned medium of desulfated stroma was decreased to 50% of the megakaryocytic output in CM of sulfated stroma. From these studies, it can be concluded that PGs in bone marrow, in particular HSPGs, are involved in binding HPC and megakaryocytopoiesis-inhibiting cytokines. Bone marrow stromal PGs thus reduce differentiation of HPC toward megakaryocytes. D 2004 Elsevier Inc. All rights reserved. Keywords: Bone marrow; Megakaryocytopoiesis; Proteoglycan

Introduction Megakaryocyte maturation and thrombopoiesis have been shown to require relocation of hematopoietic progenitors (HPCs) from stroma to the vascular niche [1]. Indeed, adhesion of megakaryocytic progenitors to stromal cells has been found to inhibit megakaryocytic differentiation [2–5]. Adherence of megakaryocytic progenitors to stroma therefore is an important regulatory step in megakaryocytic

* Corresponding author. Department of Hematology, VU University Medical Center, BR 240, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Fax: +31 20 4442601. E-mail address: [email protected] (S. Zweegman). 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.06.018

differentiation. However, the mechanisms through which megakaryocytic progenitors are inhibited by stroma are poorly understood. We propose that stromal sulfated glycoconjugates, such as proteoglycans (PGs), participate in this process. In bone marrow, PGs can bind both HPCs and cytokines, and can thus expose HPCs to high local concentrations of cytokines [6,7]. Accordingly, membrane-associated heparan sulfate proteoglycans (HSPGs) have been found to bind and present growth factors to HPCs, which resulted in inhibition of both differentiation and proliferation of HPCs [8–10]. PG structure is characterized by a core protein to which glycosaminoglycan (GAG) chains are covalently attached. The structural heterogeneity of GAGs is caused by variation

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in sugar moieties, their linkages, chain length, and pattern of sulfation. The precise positioning of sulfate groups determines the specificity of GAG–protein interactions and biological activities [11–14]. To test the hypothesis that PG structure can affect megakaryocytopoiesis, we altered human bone marrow stromal PGs by desulfation or enzymatic cleavage. We show here that this reduced adhesion of HPCs to hBMS, increased release of cytokines, and reduced megakaryocytic differentiation. These data indicate that in bone marrow obtained from healthy individuals, PGs participate in megakaryocytic differentiation of HPCs.

described [2]. Bone marrow aspirates were performed in patients undergoing cardiac surgery after informed consent. Mononuclear cells were cultured in long-term bone marrow culture (LTBMC) medium (1–2  106 cells/ml): alpha medium supplemented with 12.5% horse serum, 12.5% fetal calf serum, 10 4 M h-mercaptoethanol, 10 6 M hydrocortisone sodium succinate, glutamine, penicillin, and streptomycin. After 3–4 weeks of culturing, the adherent stromal cell layer was harvested using trypsin (all obtained from Gibco, Grand Island, NY), irradiated with 10 Gy, and replated in 24-well plates (Costar, Cambridge, MA) at 2  105/ml supplemented IMDM (see Culture conditions section for preparation).

Materials and methods

Heparan sulfate on hBMS The presence of HS on hBMS was shown by staining with the 3G10 mAb [16] after degradation of HS-GAGs. The 3G10 mAb recognizes an epitope in the HS-stub that is only exposed after HS degradation by heparitinase and can be used as a pan-HS marker (Fig. 4A).

Hematopoietic progenitor cells (CD34-positive cells) Source and isolation HPC were isolated from leukapheresis material, obtained after informed consent, from patients with acute myeloid leukemia in first remission, relapsed Non Hodgkin’s lymphoma without marrow involvement, or solid tumors without marrow involvement. For isolation of CD34-positive cells, the CliniMacs CD34 isolation kit (Miltenyi, Bergisch Gladbach, Germany) was used as described previously [2]. Purity was always more than 90% as determined by flow cytometry using a phycoerythrin (PE)-labeled anti-CD34 mAb (HPCA2, Becton Dickinson, Mountain View, CA). To avoid false-positive staining with GPIIb/IIIa antibodies due to nonspecific binding of platelets [15], cells were washed with PBS/0.5% BSA containing 5 mM EDTA. Chromium-labeling of HPC CD34-positive cells (1.0  106) were labeled with 100 ACi of Na251CrO4 (Amersham, Arlington Heights, IL) for 2 h at 378C and 5% CO2 and then washed twice with Iscove’s Modified Dulbecco Medium (IMDM) and resuspended at 0.6  106/ml in LCM. 51Cr-labeled cells were added to untreated, sodium chlorate-, or hepari(ti)nase-treated human bone marrow stroma (hBMS) in a total volume of 500 Al. In some experiments, 51Cr-labeled cells were incubated with unfractionated heparin (100 Ag/ml) for 1 h, before addition to untreated hBMS. Cells were allowed to adhere for 2 h at 378C and 5% CO2. Nonadherent cells were removed by two gentle washes with IMDM. Adhered cells were lysed with 1 M NaOH/1% SDS for 10 min. The radioactivity of each lysate was measured in a Wizard 140 automatic gamma counter and related to the radioactivity obtained from cultures from which nonadherent cells had not been removed.

Desulfation of hBMS Human BMS layers were treated with 30 mM sodium chlorate (Sigma co, St Louis) during 24 h (after which adhesion of 51Chromium-labeling of HPC was investigated) or during the entire culture period of 7 days (Sigma) to inhibit incorporation of sulfate into GAGs [17]. The effectiveness of the chlorate treatment was controlled at days 1 and 7 by mAb JM403 [18]. This antibody recognizes an epitope in HS that is dominated by N-unsubstituted glucosamine units. Staining increases under chlorate conditions, due to lack of sulfation of the deacetylated glucosamine residues (Figs. 4B and C showing 7-day-old hBMS). The effects of sodium chlorate were negated by raising the sulfate concentration through simultaneous addition of 10 mM sodium sulfate (Fig. 4D) (Sigma Aldrich chemie, Steinheim, Germany) [19]. Fluorescence of cells was visualized with a Nikon Eclipse E800 microscope. Images were captured with a Nikon DXM1200 RGB-camera. Image analysis was performed using analySIS software (Soft Imaging System GmbH, Germany). The sum of the intensity of all pixels was calculated per image. It was ruled out that sodium chlorate treatment affects the cellular composition of hBMS. The percentage of macrophages/monocytes and fibroblasts was investigated, by using the mAbs M0P9 (BD Biosciences, San Diego, CA) and ASO2 (Dianova, Hamburg, Germany), respectively. No significant changes were induced by prolonged sodium chlorate treatment (data not shown).

Human bone marrow stroma

Culture conditions

Culture of human bone marrow stroma Human primary bone marrow stromal feeder layers were established from human mononuclear cells as previously

Liquid culture CD34-positive cells (1.0  105/ml) were cultured for 7 days in serum-free IMDM with or without sodium chlorate,

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supplemented with 1% deionized BSA (Cohn fraction V), iron-saturated human transferrin (300 Ag/ml), insulin (100 ng/ml), a mixture of sonicated lipids (20 Al/ml), prepared as previously described [3] (all obtained from Sigma Co), Stem Cell Factor (50 ng/ml), and TPO (10 ng/ml) (both from PeproTech EC Ltd, London, UK). It was ruled out that sodium chlorate affected the expression of cytokine and chemokine receptors on CD34positive cells, using mAbs against the interleukin-3 receptor (PE-conjugated anti-CD123, 9F5, Pharmingen, San Diego, CA), the granulocyte macrophage-colony stimulating factor (GM-CSF) receptor (FITC-conjugated anti-CD116, M5D12, Pharmingen), the macrophage inflammatory protein-1a (MIP-1a) receptor CCR5 (FITC-conjugated anti-CD195, 2D7, Pharmingen), and the stromal derived factor 1a (SDF1a) receptor CXCR4 (12G5, R&D systems)(n = 2, data not shown). To investigate whether the presence of sodium chlorate affected the expression levels of CD41 and CD42a on megakaryocytic cells, CD34-positive cells were cultured for 7 days without sodium chlorate to obtain megakaryocytic cells. Subsequently, cells were kept for 24 h without or with 30 mM of sodium chlorate. The expression of CD41 and CD42a was unaffected by the presence of sodium chlorate (n = 2, data not shown). Contact culture Irradiated stromal cells (2  105/ml) were plated to obtain a confluent layer in a 24-well plate. CD34-positive cells (1  105/ml) were cultured for 7 days directly on stroma, using the same supplemented medium as in liquid cultures (LCs), with or without sodium chlorate. Supernatants with nonadherent cells were removed. Subsequently, remaining adherent cells were harvested after treatment with trypsin for 5 min. Noncontact culture A transwell insert with a 0.4-Am microporous membrane (Costar, Corning Incorporated, NY) was placed above the irradiated stromal layer. CD34-positive cells (1.0  105/ml) were cultured in the upper well. The same supplemented medium was used as in liquid and contact cultures, with or without sodium chlorate. Cells were removed from the transwell inserts by vigorous washing. Conditioned medium Irradiated stromal cells were plated in LTBMC medium to obtain a confluent layer in a 24-well plate. Supplemented medium with or without sodium chlorate, as described above, was added after 24 h. After 7 days, conditioned medium was harvested. Subsequently, freshly isolated CD34-positive cells (1  105/ml) were cultured in conditioned medium for 7 days. In case conditioned medium was used for the determination of cytokines, TPO was added in a concentration of 1 ng/ml. To remove sodium chlorate, supernatants of hBMS were subjected to a desalting procedure against supplemented serum-free IMDM medium

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without chlorate. To this end, PD10 columns were used, according to the instructions of the manufacturer (Amersham Pharmacia Biotech, Mijdrecht, the Netherlands). Burst forming unit-megakaryocyte Megakaryocytic clonogenic assays were performed as previously described [3]. To assess the number of Burst Forming Unit-Megakaryocyte (BFU-Mk), cultures were studied after 21 days [20]. Colonies were stained by an indirect immuno-phosphatase-alkaline labeling (APAAP) technique using the anti-GPIIb/IIIa mAb (anti-CD41, 5B12, Dako, Mijdrecht, the Netherlands). All cultures were performed in duplicate. PGs and GAGs Monoclonal antibodies For detection of HS, CS, and DS epitopes, the following antibodies were used: JM403 [18], JM13 [21], and 10E4 for HS, MC21C for CS, and 6-B-6 for DS (the latter three from Seikagaku, Tokyo, Japan). Enzymatic degradation of HS Cleavage of HS-GAGs on hBMS was achieved by treatment for 2 h at 378C and 5% CO2 with a cocktail of 10 mU/ml of each of the following enzymes: heparinase (EC 4.2.2.7), heparitinase I (EC 4.2.2.8), and heparitinase II (EC 4.2.2.8) (all from Seikagaku). Medium 199 with 10 mM HEPES and 2 mM CaCl2 was used in control experiments. Immunophenotyping by flow cytometry The expression of surface antigens was measured using directly labeled antibodies. Nonspecific binding was blocked with human immunoglobulins (CLB, Amsterdam, the Netherlands). Each sample was incubated at room temperature with mouse mAbs in the appropriate dilution for 15 min and after two wash steps with PBS/0.1% BSA analyzed with a flow cytometer (Facs-Calibur, Becton-Dickinson, Cell Quest program). Appropriate mouse immunoglobulin isotype controls were used (FITC-conjugated IgG1, Dako). Megakaryocytes were quantified using a mAb against GPIIb/IIIa (FITC-conjugated anti-CD41, 5B12, Dako) and against GPIX (anti-CD42a, FMC25, CLB). Expression of HS, CS, and DS on CD34-positive cells was quantified using mAbs mentioned under PGs and GAGs. Concomitantly, CD34-APC was added (Becton Dickinson) for 15 min at room temperature. A second step with F(ab’)2 goat antimouse/anti-IgM-PE (Immunotech, Marseilles, France) or rat-anti-mouse-PE for DS staining was performed for another 15 min at RT. The CD34-positive population was determined by lower scatter proportions and expression of CD34. ELISA Levels of interleukin-8 (IL-8), interleukin-6 (IL-6), MIP1a, transforming growth factor-h (TGF-h), thrombopoietin

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(TPO), interleukin-3 (IL-3), and GM-CSF were determined using commercially available ELISA kits (R&D systems, Abingdon, UK). Platelet-factor 4 (PF4) was determined by an ELISA kindly donated by Diagnostica Stago, Roche (Almere, the Netherlands). It was ruled out that the presence of sodium chlorate affected the determination of cytokine levels. Addition of 30 mM sodium chlorate to the standard curve gave similar results to the standard curve without addition of sodium chlorate (data not shown). Statistical analysis Numbers are given as mean F standard error of the mean. Statistical significance of differences was determined using the Wilcoxon matched-pairs signed-ranks test in case of paired experiments in which hBMS of similar donors was used. The Mann–Whitney U test was used in cases of nonpaired experiments, using hBMS of different donors, as is indicated in the text. A P value b0.05 was considered to indicate a statistically significant difference.

Results Desulfation of stroma decreases adhesion of CD34-positive cells Since sulfated glycoconjugates have been described to promote adhesion of HPC by the presentation of chemokines and subsequent activation of integrins [7,22], the effect of stroma desulfation on the adhesion of HPC was investigated by using 51Cr-labeled CD34-positive cells. Approximately half of the CD34-positive cells adhered to normal hBMS (n = 6, 49.0 F 7.2%). Blocking sulfation of hBMS resulted in a 50% decrease of adhered CD34-positive cells (Fig. 1). HSPG was identified as a sulfated glycoconjugate, as pre-incubation of the 51Cr-labeled CD34-positive cells with heparin, as well as enzymatic removal of HSPG decreased adhesion to hBMS with 27.8 F 5.6% and 15.7 F 5.5%, respectively (Fig. 1). The number of BFU-Mk grown out of 5000 cells adhered to untreated hBMS was similar to the megakaryocytic clonogenic outgrowth out of 5000 cells adhered to desulfated hBMS (data not shown). Since sodium chlorate treatment resulted in a 50% decrease in adhesion of CD34positive cells, an equivalent decrease in the absolute number of adherent megakaryocytic progenitor cells occurred. Stroma-based megakaryocytic development of CD34-positive cells is sulfation dependent To investigate the effect of blocking the functional properties of glycoconjugates on megakaryocytic differentiation, CD34-positive cells were cultured on hBMS in the absence or presence of sodium chlorate for 7 days. Blocking the sulfation in this culture system led to a significant decrease of approximately 50% in the relative as

Fig. 1. Adhesion of CD34-positive cells is decreased by desulfation, by preincubation of CD34-positive cells with heparin, and by enzymatic cleavage of HSPG from hBMS. 51Cr-labeled CD34-positive cells were allowed to adhere to untreated, sodium chlorate-, and hepari(ti)nase-treated hBMS for 2 h. In additional experiments, 51Cr-labeled CD34-positive cells were preincubated with heparin for 1 h. The percentage of adhered cells was determined by measuring radioactivity of remaining cells after washing. n = 3, all performed in duplo. *P = 0.037.

well as in the absolute numbers of megakaryocytes, quantified by mAb against CD41 and CD42a. When CD34-positive cells were cultured separated from hBMS in a transwell, a similar decrease was observed (Table 1). To investigate whether the decrease in megakaryocytic development of CD34-positive cells was explained either by desulfation of glycoconjugates present on CD34-positive cells or by desulfation of glycoconjugates present on hBMS, the following experiments were performed. Sulfation of CD34-positive cells plays a minor role in megakaryocytic development Firstly, we investigated whether CD34-positive cells expressed PGs. As shown in Fig. 2, HSPG was found to be present. Sodium chlorate treatment of CD34-positive cells resulted in an increase in expression of JM403, recognizing unsubstituted glucosamine (Fig. 2, lower right), indicating that sulfation of PGs on CD34-positive cells was impaired by sodium chlorate. No expression of dermatan sulfate and trace expression of chondroitin sulfate were detected (data not shown). To determine whether PGs on CD34-positive cells were physiologically important for megakaryocytic development, sodium chlorate was added in liquid cultures in the absence of hBMS. Although a significant decrease (14.4 F 8.9%, n = 12, P b 0.01) in megakaryocytic output was observed, the decrease in megakaryocytic output was significantly less as compared to the decrease in stromabased cultures (Table 1; 33.3 F 10.4% decrease in contact cultures and 49.1 F 16.9% decrease in noncontact cultures). This indicates that although sulfated glycoconju-

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Table 1 Decrease in megakaryocytic output upon desulfation hBMS contact chlorate CD41 (%) CD41 TN (105) CD42a (%) CD42a TN (105)

29.8 2.0 15.3 0.7

F F F F

24.1 1.5 19.1 0.8

hBMS noncontact + chlorate 17.8 1.1 6.9 0.3

F F F F

11.2* 1.0* 6.3* 0.3*

chlorate 38.1 1.9 20.1 1.1

F F F F

12.4 0.7 12.9 0.8

+ chlorate 18.6 0.9 7.4 0.3

F F F F

4.9^ 0.3^ 5.7^ 0.3^

CD34-positive cells were cultured for 7 days in contact hBMS (n = 16) or noncontact hBMS cultures (n = 6) in the absence or presence of 30 mM sodium chlorate. A significant decrease in megakaryocytic output was observed after sodium chlorate treatment (*P b 0.01, ^P b 0.05). TN, total number.

gates expressed on CD34-positive cells slightly affect megakaryocytic development out of CD34-positive cells, the decrease in megakaryocytic output in stroma-based cultures cannot be explained by desulfation of glycoconjugates on CD34-positive cells alone. Cytokines are released upon stroma desulfation The second hypothesis ascribes the decrease in megakaryocytopoiesis to the desulfation of stromal glycoconjugates. This could be the result of release of inhibitory cytokines from desulfated stromal cells, which is supported by the similar decrease in megakaryocytes in noncontact cultures as compared to contact cultures (Table 1). To separate the effect of sodium chlorate treatment on sulfated glycoconjugates present on CD34-positive cells from the effect on sulfated glycoconjugates on stroma, we

used conditioned medium obtained after culturing hBMS for 1 week in the presence or absence of sodium chlorate. Thereafter, conditioned medium was subjected to PD10 desalting columns to remove sodium chlorate. Subsequently, freshly isolated CD34-positive cells were cultured in these conditioned media without sodium chlorate for another 7 days. Again, less megakaryocytes were obtained from cultures using conditioned medium of desulfated stroma (23.3 F 21.5% inhibition compared to conditioned medium of untreated stroma, n = 5, P b 0.05). This indicates that upon desulfation of hBMS, stromal factors are released, which inhibit megakaryocytic development out of CD34positive cells whose sulfated glycoconjugates were intact. To characterize the factors released from desulfated stromal cultures, levels of IL-8, MIP-1a, PF4, TGF-h, IL6, and TPO were determined in 7-day-old conditioned medium of sodium chlorate-treated and untreated hBMS.

Fig. 2. Expression of HS on CD34-positive cells. Representative flow cytometric analysis of CD34-positive cells stained with antibodies recognizing epitopes in HS (upper left: 10E4, upper right: JM13, lower left: JM403). When CD34-positive cells were incubated overnight with 30 mM sodium chlorate, an increase in staining of the JM403 mAb was observed (lower right).

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Fig. 3. Release of cytokines upon desulfation of hBMS and reversal by addition of sodium sulfate. IL-8, MIP-1a, and IL-6 levels were determined in conditioned medium of 7-day-old untreated ( C S) and sodium chlorate (+C S)-treated hBMS. Treatment with 30 mM sodium chlorate resulted in a significant increase in all cytokine levels as compared to conditioned medium of untreated cultures. Addition of sodium sulfate (+C +S) abrogates the effects of sodium chlorate.

Levels of IL-8, MIP-1a, and IL-6 were significantly higher in conditioned medium of sodium chlorate-treated as compared to untreated hBMS (Fig. 3; 1.89 F 0.52-fold increase, 1.43 F 0.42-fold increase, and 3.81 F 4.47-fold increase, respectively). The increase in cytokine levels already occurred after 24 h of sodium chlorate treatment (data not shown). Concomitant addition of sodium sulfate,

knowing to counteract the blocking of sulfation by sodium chlorate, reversed these increases (Fig. 3). In three out of five experiments, TPO levels were also higher in conditioned medium of sodium chlorate-treated as compared to untreated hBMS (0.39 F 0.02 vs. 0.25 F 0.04 ng/ml, n = 3). In two experiments, levels were below the detection level ( b 0.08 ng/ml). TGF-h levels were almost

Fig. 4. Immunofluorescence staining of HS and sulfated glycoconjugates expressed on hBMS layers. (A) HS is abundantly present on hBMS as indicated by staining the HS-stub after enzymatic degradation of GAG-side chains of HS, using mAb 3G10. (B) HS staining of untreated hBMS using mAb JM403, which recognizes an epitope in HS that is dominated by N-unsubstituted glucosamine units (mean fluorescence, 66 arbitrary units). (C) HS staining of sodium chlorate-treated hBMS, positively staining with JM403 (mean fluorescence, 210 arbitrary units). The increase in JM403 staining after sodium chlorate treatment indicates that GAGs become desulfated upon treatment with sodium chlorate. (D) Reversal of staining with JM403 by concomitant addition of sodium sulfate (mean fluorescence, 80 arbitrary units), which indicates that the effects of sodium chlorate were negated by raising the sulfate concentration.

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similar in conditioned medium harvested from sodium chlorate-treated as compared to untreated hBMS (5.54 F 0.54 vs. 5.01 F 1.47 ng/ml, respectively, n = 4). PF4 was below the detection level in both untreated and sodium chlorate-treated hBMS (b6.25 U/ml). Therefore, we conclude that cytokines, particularly IL-8, MIP-1a, and IL-6, were at least partly bound to sulfated glycoconjugates and released by desulfation. HS, being abundantly expressed on hBMS (Fig. 4), therefore is a likely candidate for such a sulfated macromolecule. To prove that HS was indeed involved in cytokine binding, hBMS was also treated with HS-degrading enzymes. If HS would bind cytokines, the levels of released cytokines in the supernatant are expected to be higher as compared to untreated hBMS, which appeared to be the case. After removing the conditioned medium of 7-day-old hBMS, a cocktail of hepari(ti)nases or medium without hepari(ti)nases were added. After 2 h of incubation, levels of IL-8 and IL-6 were measured in the supernatants of untreated and hepari(ti)nase-treated hBMS. These levels were significantly higher in the supernatants of enzymetreated hBMS as compared to supernatants of nontreated hBMS, indicating that at least a fraction of these cytokines was bound to HS (Fig. 5). To investigate if HS-bound cytokines inhibit megakaryocytic differentiation, CD34-positive cells were cultured in medium of hBMS layers from which HSPGs were enzymatically degraded and compared to cultures in medium of untreated hBMS layers. Since HS is continuously produced, it was not possible to degrade HS with the use of enzymes for the culture period of 7 days. Therefore, conditioned medium of 7-day-old untreated stromal cultures was collected. Subsequently, hepari(ti)nases were added to

Fig. 5. Release of cytokines upon hepari(ti)nase digestion of HS on hBMS. IL-8 and IL-6 levels were determined in hepari(ti)nase-digested supernatants and compared to non-hepari(ti)nase-digested supernatants (1.46fold higher IL-8 levels and 1.34-fold higher IL-6 levels, both n = 7). *P = 0.028, compared to heparinase. ^P = 0.028, compared to heparinase.

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hBMS layers or to control wells without hBMS. After treatment for 60 min, these enzyme-degraded products were added to the previously harvested conditioned medium. Freshly isolated CD34-positive cells were cultured for 7 days in these media. Irrespectively of whether enzymatic treatment of stroma was performed or not, similar numbers of megakaryocytes developed out of CD34-positive cells (32.1 F 19.2 vs. 29.5 F 15.6 from heparinase treated vs. untreated hBMS, n = 5, P = 0.35). This is likely explained by the fact that the levels of IL-8 and IL-6 liberated upon HS digestion increased the levels already present in 7-day-old conditioned medium with 10% only (data not shown).

Discussion Here we show that stromal sulfated glycoconjugates, HSPGs in particular, are involved in the regulatory steps through which hBMS inhibits megakaryocytic differentiation. Altering human bone marrow stromal PGs by desulfation or enzymatic cleavage reduced adhesion of HPCs to hBMS, increased release of megakaryocytopoiesisinhibiting cytokines, and decreased megakaryocytic differentiation of HPC. These data indicate that in bone marrow of healthy individuals PGs inhibit megakaryocytic differentiation by colocalizing HPC with megakaryocytopoiesisinhibiting cytokines. Desulfation of hBMS causes an additional decrease in megakaryocytes as not only adherent but also nonadherent cells are exposed to high levels of megakaryocytopoiesis-inhibiting cytokines. We determined levels of well-known inhibitors of megakaryocytopoiesis, that is, IL-8, TGF-h, MIP-1a, and PF4 [23–25]. We propose that IL-8 is the most likely candidate being responsible for the decrease in megakaryocytopoiesis. Firstly, the rise in concentration of IL-8 after desulfation was most pronounced. Secondly, IL-8 levels, similar to what we found after sodium chlorate treatment, have been described to inhibit megakaryocytopoiesis in a lineage-specific way [23]. Furthermore, although the levels of TGF-h in conditioned media were sufficient to maximally inhibit megakaryocytic colony formation [26], the levels hardly increased after desulfation. Therefore, TGF-h probably not explains the additional decrease in megakaryocytes after desulfation. MIP-1a significantly increased after sodium chlorate treatment, confirming MIP-1a being bound to sulfated glycoconjugates [27,28]. The levels of MIP-1a, however, were in the range of 50 pg/ml, whereas levels of at least 5 ng/ml are necessary to inhibit megakaryocytic colony forming [23]. PF4 was below the detection limit in conditioned medium of both sulfated and desulfated hBMS. Desulfation of hBMS also led to release of megakaryocytopoiesis-promoting cytokines. Apparently, IL-6 and TPO are not able to overcome the inhibiting effect of hBMS in our model. Likewise, IL-6 did not restore platelet production in TPO-deficient mice, unless HPC escaped

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from the inhibiting interaction with stromal cells by relocating to the vascular niche [1]. Furthermore, IL-6 did not affect megakaryocytic colony forming after sublethal irradiation of mice [29]. Moreover, IL-6 is not likely to play an essential role in normal steady state megakaryocytopoiesis, since in c-Mpl/IL-6 knock out mice, the number of platelets was comparable with those in mice lacking c-Mpl only [30]. Although the essential regulator of megakaryocyte proliferation, TPO, has been described to be produced by bone marrow stroma [31], the low TPO levels we observed are in accordance with the lack of promotion of megakaryocytopoiesis by either desulfated or untreated hBMS. We showed that HSPG is one and probably the major sulfated glycoconjugate involved in cytokine binding. HSreleased products did not inhibit megakaryocytopoiesis. However, this is likely explained by the low levels of cytokines in the released products (only about 10% of the levels in conditioned medium of hBMS). Therefore, the sensitivity of this particular assay is probably not high enough for detecting an effect on megakaryocytopoiesis. Therefore, we cannot definitely conclude that particularly HSPGs inhibit megakaryocytopoiesis. It would be interesting in future studies to address the effect of HSPG on the ERK/MAPK and PKC-q pathways, as stromal inhibition of megakaryocytopoiesis is correlated with the blocking of these pathways [32,33]. HS has been described to inhibit ERK activation in myocardial cells [34], suggesting that stromal HSPG might also be involved in the regulation of ERK/MAPK signaling. Besides hBMS, CD34-positive cells were found to express HS. Expression of PGs on hematopoietic progenitor cell (HPCs) lines has been described by others [35,36]. Furthermore, HPC were found to express chondroitin sulfate [37]. However, to our knowledge, only Siebertz et al. [38] showed HSPG (glypican-4) expression on primary HPC earlier. Our studies suggest that megakaryocytopoiesisinhibiting cytokines also need to some extent progenitor cell HS to bring about their effects, as desulfation of HS on HPC results in a small decrease in megakaryocytic differentiation. However, we cannot rule out the possibility that desulfation of chemokine receptors also plays a role. Recently, CCR5 and CXCR4 were found to contain sulfated tyrosine residues, which are important in chemokine binding and HIV-1 entry [39,40]. We did not detect differences in CXCR4 expression of CD34-positive cells upon sodium chlorate treatment. It cannot be explained why concomitant addition of sulfate does not completely reverses the increase in IL-8 after sodium chlorate treatment (Fig. 3). One might argue that sodium chlorate treatment increased cytokine levels by mechanisms other than influencing sulfation. However, sodium chlorate is known to abolish sulfation without inhibiting cell growth or protein synthesis [41,42]. In addition, in our model, the viability and composition of stromal cells were not affected by sodium chlorate treatment

(data not shown). Moreover, in most of our experiments, the increase in cytokine levels was reversed by concomitant addition of sodium sulfate, which is known to negate the desulfating effects of sodium chlorate [19]. As the reversal of IL-8 was not complete, it was ruled out that sodium chlorate treatment induced significant expression of IL-8 at the mRNA level by a quantitative PCR. The number of IL-8 copies obtained from mRNA of sodium chlorate-treated compared to untreated hBMS was almost similar (0.46 F 0.31% vs. 0.36 F 0.20% of GAPDH copies, n = 5). An extra argument favoring release of IL-8 from desulfated stromal proteoglycans is the similar increase of IL-8 observed after hepari(ti)nase treatment of hBMS. Finally, the specific action of desulfation is confirmed by our observation that in contrast to the decrease in megakaryocytes, a higher granulocytic output was noticed when CD34-positive cells were cultured in conditioned medium of desulfated stroma after removal of sodium chlorate by PD10 columns (39.6 F 16.0% vs. 24.7 F 9.3%, n = 3). This is in accordance with the observation that stromal HSPG bind IL-3 and granulocyte monocyte-colony stimulating factor, thereby promoting granulocytopoiesis [43]. In conclusion, we show that PGs on hBMS participate in stromal inhibition of megakaryocytopoiesis, by exposing HPC to high levels of megakaryocytopoiesis-negative cytokines produced by hBMS. As stromal inhibition has been proposed as an explanation for the impaired or delayed platelet recovery observed after stem cell transplantation [1,33], altered PG structure might well be involved. Indeed, alkylating agents were described to change extracellular matrix production [44]. The influence of anticancer therapy on the composition and function of stromal PGs is currently under investigation.

Acknowledgment We greatly appreciate the technical assistance of S. Marjolein Klaver.

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