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Stem Cells 2004;22:544-555 www.StemCells.com
© 2004 AlphaMed Press

Hyaluronic Acid Facilitates the Recovery of Hematopoiesis following 5-Fluorouracil Administration

Vera Y. Matrosovaa, Irina A. Orlovskayab, Naira Serobyanc, Sophia K. Khaldoyanidic

a National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland;
b Institute for Clinical Immunology, Novosibirsk, Russia;
c Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California, USA

Key Words. Bone marrow aplasia • Chemotheraphy • Cytokines • Hematopoietic progenitor cells Microenvironment • Hyaluronan

Correspondence: Sophia K. Khaldoyanidi, M.D., Ph.D., Division of Vascular Biology, La Jolla Institute for Molecular Medicine, 4570 Executive Drive, Suite 100, San Diego, CA 92121, USA. Telephone: 858-587-8788 ext. 105; Fax: 858-587-6742; e-mail: sophia{at}ljimm.org


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The fate of hematopoietic stem cells (HSCs) is determined by microenvironmental niches, but the molecular structure of these local networks is not yet completely characterized. Our recent observation that glycosaminoglycan hyaluronic acid (HA), a major component of the bone marrow extracellular matrix, is required for in vitro hematopoiesis led us to suggest a role for HA in structuring the hematopoietic niche. Accordingly, HA deprivation induced by various treatments might lead to an imbalance of normal HSC homeostasis. Since 5-fluorouracil (5-FU) administration sharply decreases the amount of cell surface–associated HA in bone marrow, we examined whether the administration of exogenous HA enhances suppressed hematopoiesis in 5-FU–treated mice. HA administered to mice following 5-FU infusion facilitated the recovery of leukocytes and thrombocytes in the peripheral blood. Intravenously infused HA was found in the bone marrow, where it bound endothelial cells and resident macrophages and increased expression of the hematopoiesis-supportive cytokines interleukin-1 and interleukin-6. In agreement with these observations, enhanced hematopoietic activity was detected in the bone marrow, as measured by elevated counts of long-term culture-initiating cells (LTC-ICs), committed progenitors, and the total number of mature bone marrow cells. Overall, our results suggest that HA is required for regulation of the hematopoiesis-supportive function of bone marrow accessory cells and, therefore, participates in hematopoietic niche assembly.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The behavioral choices of hematopoietic stem cells (HSCs) are regulated by multiple signals provided by the hematopoietic microenvironmental niche in response to physiological and pathophysiological demands [1]. The cellular compartment of the niche is rather heterogeneous and is represented by cells of hematopoietic (macrophages, lymphocytes, osteoclasts, etc.) and mesenchymal (stromal cells, osteoblasts, adipocytes, etc.) origin [reviewed in 2, 3]. Over the past decade, our understanding of molecular mechanisms mediating the regulatory signals provided by the cells of the hematopoietic microenvironment has significantly advanced [4]. Soluble and cell surface–associated factors and extracellular matrix (ECM) molecules are produced by the cells that comprise the hematopoietic niche and contribute to its highly complex structure [5 and reviewed in 6–8]. ECM components, such as collagens, fibronectin, laminin, and hemonectin, were shown to participate in the bone marrow regulatory network, whereas the role of numerous other ECM molecules, including hyaluronic acid (HA), is not yet understood.

HA, a member of the glycosaminoglycan (GAG) family, is a major component of bone marrow ECM [9]. Our recently published observation provided the first indication of the involvement of HA in the regulation of hematopoiesis [10]. We demonstrated that HA deprivation induced by hyaluronidase treatment inhibits both myelopoiesis and lymphopoiesis in long-term bone marrow culture (LTBMC), whereas exogenous HA stimulates hematopoiesis, at least in part, via CD44. In line with these findings, CD44-specific antibodies directed against the HA-binding domain inhibit hematopoiesis [11,12]. The involvement of HA in the regulation of multiple cell functions, such as cell proliferation [13,14], migration [15], cytokine production [10, 16–18], and adhesion molecule expression [19], requires strict regulation of HA turnover. Degradation of HA or alteration of its synthesis and accumulation can be induced by various treatments, such as UV irradiation [20,21] or administration of 5-fluorouracil (5-FU) [22], hydrocortisone, or other chemicals [23]. A treatment-induced alteration of the GAG composition in the bone marrow can significantly affect the function of the bone marrow microenvironment and, consequently, lead to an imbalance of hematopoietic homeostasis.

5-FU, a remedy commonly used in oncology, exhibits a high toxicity that leads to severe myelosuppression and various other symptoms [24–27]. We have shown here that administration of HA to mice with 5-FU–induced suppression of bone marrow hematopoiesis leads to a significant acceleration of bone marrow hematopoiesis and recovery of the white blood cell (WBC) and platelet (PLT) numbers in the peripheral blood through stimulation, at least in part, of interleukin-1 (IL-1) and interleukin-6 (IL-6) production. Our findings support the concept that HA is an essential regulatory component of the bone marrow hematopoietic microenvironment.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
5-FU–Induced Bone Marrow Hypoplasia
The experiments were conducted in agreement with institutional policy on animal use and approved by the Institutional Animal Care and Use Committee (IACUC). Eight- to 12-week-old female mice C57Bl/6J x DBA/2J (B6D2F1) were obtained from Harlan Inc. (Indianapolis, IN) or bred in-house. The animals were kept under standard pathogen-free conditions. The mice were treated with an intraperitoneal injection of 150 mg/kg 5-FU (Sigma Chemical Corp., St. Louis, MO). Where indicated, the mice were intravenously administered 100 µg/mouse sodium HA (Sigma Chemical Corp. or Lifecore Biomedical Inc., Chaska, MN), 100 µg/mouse chondroitin sulfate (CS; Sigma Chemical Corp.) or phosphate-buffered saline (PBS; 200 µ1). Lifecore’s medical grade HA (1.5 million Da) was produced by streptococcal fermentation (group A). Sigma’s HA was from rooster comb (0.75–2.0 million Da). The source of CS was shark cartilage. Both HA and CS were proven to be endotoxin free.

Mice were euthanized by an overdose of CO2. Femurs were dissected and cleaned from muscle tissues. Thereafter, epiphyses were cut off with scissors at each end of the femur. The contents were flushed out of the bone with PBS supplemented with 5% fetal calf serum (FCS) using a needle (21G) attached to a 1-ml syringe. To ensure the preparation of single-cell suspension, the cell suspension was aspirated several times through a smaller needle (25G). The cells were kept on ice until used.

Colony-Forming Unit (CFU) Assay
Bone marrow cells were harvested and plated at a concentration of 1 x 104 cells/ml in 24-well plates in semisolid methyl-cellulose containing 30% FCS, 1% bovine serum albumin (BSA), 10–4 M 2-mercaptoethanol, and 2 mM l-glutamine (StemCell Technologies, Vancouver, BC, Canada). A conditioned medium from cell line WEHI-3B was added (15% v/v) as a source of IL-3 [28]. To culture erythroid burst-forming units (BFU-E), 10 U/ml of erythropoietin (Boehringer, Mannheim, Germany) was added. Colonies containing at least 500 cells were counted after 14 days. For growing granulocyte-macrophage colony-forming units (GM-CFU), 10 ng/ml GM-CSF was added. The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. Colonies containing more than 20 cells were counted under the inverted microscope after 7 days of culture. Where indicated, monoclonal antibodies directed against IL-1 and IL-6 (R&D Systems, Minneapolis, MN) were added at a concentration of 100 µg/ml.

Long-Term Culture-Initiating Cell (LTC-IC) Assay
Single-cell suspension obtained from the bone marrow of mice to be examined was plated in Dulbecco’s Modified Eagle Medium (DMEM; Gibco-Invitrogen, Carlsbad, CA) supplemented with 20% horse serum (StemCell Technologies) and 10–6 M hydrocortisone (Sigma Chemical Corp.) in limiting dilution into 96-well plates containing the stromal cell line S17. The cultures were fed weekly, and the number of wells containing LTC-IC colonies was evaluated after 14 days of culture [29].

Morphological Analysis
Smears of bone marrow cells were fixed on glass slides in methanol at –20°C for 20 minutes and dried at room temperature. Fixed cells were stained according to the standard procedure [30]. Briefly, the slides were incubated with Filipson’s dye (25% Giemsa dye in 96% ethanol) for 15 minutes, extensively washed with distilled water (pH 7.0), dried, and covered with a cover slip. The slides were then examined under the microscope.

Northern Blot Analysis
Total RNA isolation from the mouse bone marrow was done using a commercially available kit (Qiagen Inc., Santa Clarita, CA). Northern blot analysis was performed as described elsewhere [31]. Murine IL-6 cDNA was provided by E. Sterneck (National Institutes of Health [NIH], Frederick, MD), IL-1{alpha} cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA using primers 5'-GGCAGGCAGTATCACTCATT and 5'-TCTCTTTGAACAGAATGTGC. Quantification of northern hybridization signals was done with Scion Image PC ("Image 2") software (NIH).

Enzyme-Linked Immunosorbent Assay (ELISA)
Serum samples from both HA-treated and control mice were kept at –80°C until use. The samples were tested by ELISA (Endogen, Woburn, MA) for IL-1 and IL-6, according to the instructions of the manufacturer.

Flow Cytometry
Bone marrow cells were incubated with rat antimouse CD44-specific antibodies directed against CD44 standard (CD44s, clone KM81, American Type Culture Collection [ATCC], Manassas, VA); CD44 splice variant 4 and 6 (CD44v4 and CD44v6, clones 10D1 and 9A4 were provided by Dr. Jonathan Sleeman, Institute of Toxicology and Genetics, Karlsruhe Research Center, Karlsruhe, Germany); and CD44 splice variant 10 (CD44v10, clone K926 was provided by Dr. Margot Zoeller, Department of Tumor Progression and Immune Defense, German Cancer Research Center, Heidelberg, Germany). Rat IgG (Pharmingen, San Diego, CA) was used as the control antibody. Staining was visualized by secondary goat antirat fluorescein isothiocyanate (FITC)-conjugated antibody. Phycoerythrin (PE)-labeled CD31- and CD11b-specific antibodies were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ). FITC-labeled HA was obtained from Calbiochem (San Diego, CA). The receptor for HA-mediated motility (RHAMM)-specific monoclonal antibody (clone 3T3.5) was provided by Dr. Linda Pilarski (University of Alberta, Canada). Cell surface–associated HA was detected by biotin-conjugated HA-binding protein, followed by incubation with FITC-conjugated avidin (both from Sigma Chemical Corp.). Cell-surface staining was determined by fluorescence-activated cell sorter (FACS) analysis. Briefly, stained cells were washed twice with FACS buffer (PBS, 2% FCS, 0.1% BSA, 0.01% NaN3). Fluorescence intensity was analyzed on FACScan (Becton, Dickinson) according to standard procedures.

Statistical Analysis
Statistical analysis was carried out by Student’s t-test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
5-FU Induces Bone Marrow Hypoplasia and Panleukocytopenia
5-FU was injected into mice intraperitoneally at 150 mg/kg. The counts of WBCs, red blood cells (RBCs), PLTs, hemoglobin (HGB), and hematocrit (HCT) were monitored daily for 2 weeks. The treatment of mice with 5-FU induced severe bone marrow hypoplasia and panleukocytopenia. The number of WBCs and PLTs dropped from 8.4 ± 1.5 x 106/ml and 678.4 ± 82 x 106/ml before 5-FU administration to 2.52 ± 0.5 x 106/ml and 388 ± 50 x 106/ml, respectively, 7 days later (Fig. 1A, CGo). The total number of mononuclear cells in the bone marrow decreased from 15.3 ± 2.2 x 106/femur before to 5.00 ± 0.65 x 106/femur 7 days after 5-FU administration (Fig. 2AGo). All parameters recovered to normal 14 days after 5-FU administration.



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  		Figure 1. Hyaluronic acid (HA) facilitates recovery of peripheral blood cells after 5-fluorouracil (5-FU) administration. B6D2F1 hybrid mice (n = 10 per group) were administered 5-FU (150 mg/kg) on day 0. HA (100 µg/mouse) or phosphate buffered-saline (PBS) was injected on day 4, 6, 10, and 13. Blood was harvested daily, and the numbers of (A) leukocytes (white blood cells [WBCs]) and (C) thrombocytes (platelets [PLTs]) were measured and expressed as mean ± standard deviation (SD). Control measurements from untreated mice were taken on day 0, before 5-FU administration. To demonstrate a dose-dependent effect of HA on WBC recovery, mice (n = 5 per group) were administered with various doses of HA (0–1,000 µg/mouse), and the peripheral blood samples were evaluated for the leukocyte number on day 7; cell counts are expressed as mean ± SD (B). A significant difference (*p < .01) in cell counts between 5FU/PBS and 5FU/HA groups was detected for WBCs from day 6 to day 12 and for PLTs from day 6 to day 11. The results of one representative out of three similar experiments are shown.

 


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  		Figure 2. Hyaluronic acid (HA) facilitates bone marrow recovery after 5-fluorouracil (5-FU) treatment. B6D2F1 hybrid mice (a total number of 40) were administered 5-FU (150 mg/kg) on day 0. HA (100 µg/mouse) or phosphate-buffered saline (PBS) was injected on day 4, 6, 10, and 13. (A): The bone marrow of control, untreated mice (n = 5) was analyzed on day 0. On day 7, 14, 21, and 28, bone marrow cells (Bmcs) were harvested from 5FU/PBS and 5FU/HA mice (n = 5 for every group per each time point), counted, and expressed as mean ± standard deviation (SD). The results of one representative out of two similar experiments are shown. (B): Bmcs from 5FU/PBS and 5FU/HA mice were harvested on day 7, assayed for numbers of granulocyte-macrophage colony-forming unit (CFU-GM) and BFU-E in methylcellulose media, and presented as mean ± SD. (C): Smears of bone marrow harvested at day 7 were fixed and stained with Giemsa dye. Numbers of blast cells and megakaryocytes (MKC) were calculated and expressed as the percentage of cells examined. (D): Bmcs were harvested from 5FU/PBS and 5FU/HA mice (n = 3) on day 7 and assayed for number of long-term culture-initiating cells (LTC-ICs) in the limiting dilution assay in two independent experiments. The LTC-IC frequency in the bone marrow from one representative experiment is shown as mean ± SD. A statistically significant difference is indicated by an asterisk.

 
5-FU Decreases the Amount of Cell Surface–Associated HA in the Bone Marrow
The effect of 5-FU on the cell surface–associated HA was investigated next. Bone marrow cells from 5-FU–treated (day 0) mice were harvested on day 4 and analyzed by flow cytometry. The amount of HA on the surface of bone marrow cells from 5-FU–treated mice was detected using biotin-conjugated HA-binding protein and compared with that in non-treated control mice. While cell surface–associated HA was not detectable on populations R3 and R4 due to low number of cells, populations R1 and R2 demonstrated a significant (p < .01) decrease in the amount of cell surface HA in 5-FU–treated mice (Table 1Go). The fluorescent intensity of R1 and R2 populations from 5-FU–treated mice was decreased 5-fold and 2.6-fold, respectively, over that of the control.


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  		Table 1. The effect of 5-fluorouracil (5-FU) on the cell surface–associated hyaluronic acid (HA)
 
5-FU treatment also affected the expression of CD44, a major receptor for HA. The total expression of CD44 proteins on the cell surface of bone marrow cells was significantly decreased on day 2 and day 4 after 5-FU treatment, as detected by a pan-CD44-specific antibody (Fig. 3AGo). Interestingly, the expression of CD44 splice variant 6 (CD44v6), a high-affinity receptor for HA [32] present on large granulated bone marrow cells (R4), was not changed by 5-FU on day 2 after the treatment (Fig. 3BGo). Furthermore, the cell surface expression of RHAMM, a second major HA receptor [33,34], was increased on the R4 subpopulation of bone marrow cells. The fluorescent intensity of the R4 population stained with RHAMM-specific antibodies was increased from 26.6 ± 2.9 in nontreated mice to 44.9 ± 9.8 in 5-FU–treated mice (day 2). The expression of both CD44v6 and RHAMM on the R4 subpopulation of bone marrow cells on day 4 after 5-FU infusion was not measured due to the low number of cells (<5%). These data suggest that while 5-FU interferes with the expression of pan-CD44, both the high-affinity HA receptors CD44v6 and RHAMM are present on the subpopulation of bone marrow cells.



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  		Figure 3. 5-Fluorouracil (5-FU) interferes with the cell surface expression of CD44. B6D2F1 mice were administered 150 mg/kg 5-FU (day 0). Bone marrow cells were harvested on days 2 and 4. (A): The expression of pan-CD44 was detected by a CD44-specific antibody (transparent field) and analyzed by flow cytometry. Isotype-matched immunoglobulin G was used as a negative control (black field). Histograms represent a fluorescent intensity of total nongated bone marrow cells from control, nontreated mice and 5-FU–treated mice (days 2 and 4). (B): The expression of CD44v6 on gated R4 subpopulation of bone marrow cells from control and 5-FU–-treated (day 2) mice is shown.

 
Since HA is required for hematopoietic homeostasis in bone marrow, we further investigated whether the infusions of exogenous HA facilitate the recovery of hematopoiesis following 5-FU administration.

HA Accelerates Recovery of Peripheral Blood Cells
To examine the effect of HA on 5-FU–perturbed hematopoiesis, 5-FU–treated (day 0) mice were administered 100 µg/mouse HA on days 4, 6, 10, and 13. A control group of animals was treated with a 200-µ1 injection of PBS. The peripheral blood from HA- and PBS-treated mice was collected daily and examined for the number of WBCs, RBCs, and PLTs and the amount of HGB and HCT. While RBCs, HGB, and HCT were unchanged (p > .01; Table 2Go), the number of WBCs in HA-treated mice was significantly higher than those in the control PBS-treated group, starting from day 5 (2-fold to 2.5-fold; Fig. 1AGo). The effect of HA on WBC recovery was dose dependent. The optimal concentration of HA was found to be 100 µg/mouse (Fig. 1BGo). Different specimens of HA demonstrated similar biological effects. The number of PLTs in HA-treated mice was increased starting from day 5 and was elevated by a factor of 1.7 on day 8 (Fig. 1CGo). Six days after the first HA treatment, the parameters observed in the HA-treated group corresponded to those in normal mice prior to 5-FU treatment. Thus, the administration of HA rescued mice from 5-FU–induced leukocytopenia and thrombocytopenia. Because mature blood cells are a product of proliferation and differentiation of hematopoietic progenitors, we next examined the effect of HA on bone marrow hematopoiesis in 5-FU–treated mice.


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  		Table 2. Effect of hyaluronic acid (HA) on peripheral blood cell counts after 5-fluorouracil (5-FU) administration
 
HA Facilitates Hematopoiesis in 5-FU–Impaired Bone Marrow
Mice (n = 40) were treated with 5-FU (day 0) followed by administration of PBS (5FU/PBS) or 100 µg/mouse HA (5FU/HA) on day 4, 6, 10, and 13. Control mice (n = 5) that were not treated with 5-FU were sacrificed on day 0 to determine the number of bone marrow cells before treatment. Bone marrow cells were harvested from 5FU/PBS (n = 5 per each time point) and 5FU/HA (n = 5 per each time point) on days 7, 14, 21, and 28. On day 7 the total number of bone marrow cells in the HA-treated mice was 3.5-fold higher than that in the PBS-treated mice (Fig. 2AGo). A morphological analysis of the bone marrow cells revealed that the numbers of mature myeloid and lymphoid elements in the bone marrow were increased in the mice that received HA treatment following 5-FU administration (Table 3Go). At day 7, a ninefold increase in neutrophil numbers in HA-treated mice over that in the controls was found. In addition, a three-fold and 10-fold elevated number of lymphocytes and platelets, respectively, was detected at day 14 in the bone marrow of HA-treated mice. Interestingly, the HA-treated animals also showed an increased number of bone marrow cells in mitosis (1.7 per 100) over that of the control (0.3 per 100). These data suggest a higher number of proliferating progenitor cells in the bone marrow of HA-treated mice.


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  		Table 3. Effect of hyaluronic acid (HA) on bone marrow cell counts after 5-fluorouracil (5-FU) administration
 
To examine this assumption, bone marrow cells were cultured in methylcellulose to evaluate the number of lineage-committed progenitors. The number of myeloid progenitors in HA-treated mice was 2.9-fold higher, and the number of early erythroid progenitors was 21.5-fold higher than in the controls (Fig. 2BGo). The number of megakaryocytes in the bone marrow of HA-treated mice showed a 3.7-fold increase (Fig. 2CGo). Thus, HA promoted the bone marrow hematopoietic activity that had been impaired by 5-FU.

We next investigated whether HA stimulation benefited the pool of committed progenitors at the cost of damaging more primitive progenitors that were measured by using an LTC-IC assay. The number of LTC-ICs in the bone marrow of HA-treated mice was evaluated using a limited-dilution assay. We monitored a trend in the increase in number of LTC-ICs in the bone marrow of 5FU/HA versus 5FU/PBS mice: Although the difference was not statistically significant (p > .1), the number of LTC-ICs was elevated from 14.8 ± 9.6/femur in the controls to 30.6 ± 13.3/femur in HA-treated animals (Fig. 2DGo). These findings suggest that the increase in the number of mature cells and committed progenitors in the bone marrow of HA-treated mice does not result in the exhaustion of the pool of more primitive stem cells as measured by number of LTC-ICs.

Intravenously Administered HA Targets Bone Marrow Cells
To address the question of whether HA targets bone marrow, 5-FU–administered mice were infused with FITC-labeled HA (HA-FITC). After 2 hours, the animals were sacrificed, then bone marrow and peripheral blood cells were isolated and their fluorescent activity was examined using FACScan. While blood cells harvested from HA-FITC–administered mice did not change their fluorescein signal (data not shown), bone marrow cells exhibited a 1.5-fold increase in fluorescein signal over that of the hyaluronidase-treated control (Fig. 4AGo). Double staining with lineage-specific antibodies revealed that at least CD31+ and CD11b+ cells bind HA-FITC (Fig. 4BGo). This finding is in line with our observation that CD44 splice variants, high-affinity receptors for HA [32], are expressed on the bone marrow–derived macrophages (Fig. 4CGo) but not on peripheral blood leukocytes (data not shown). This suggests that intravenously injected HA can be transported into the bone marrow, where it binds its cellular target(s).



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  		Figure 4. Hyaluronic acid (HA) binds bone marrow cells in vivo. B6D2F1 mice were administered 150 mg/kg 5-fluorouracil. (A): On day 4, HA labeled with fluorescein isothiocyanate (HA-FITC) was injected intravenously; 2 hours later bone marrow and peripheral blood cells were harvested, and the cell samples were examined using flow cytometry (transparent field). The fluorescent activity of hyaluronidase-treated cells is shown as a dark field. The fluorescent signal was analyzed with CellQuest-Pro (Becton, Dickinson). One representative histogram out of three is shown. (B): HA-FITC (fluorescein isothiocyanate) binding by CD11b (right) and CD31 (left) positive cells (FL2) was evaluated by fluorescence-activated cell sorter analysis. One representative dot plot out of three is shown. (C): Expression of CD44s, CD44v4, CD44v6, and CD44v10 (FL1) on bone marrow-derived macrophages is shown.

 
We have previously demonstrated that HA does not directly promote the proliferation of hematopoietic progenitors [10]. Therefore, it is likely that HA upregulates the production of hematopoiesis-mediating cytokines by the cells of the bone marrow hematopoietic microenvironment. We next examined whether the HA administered in vivo stimulates cytokine production by bone marrow accessory cells in mice with chemotherapeutically perturbed bone marrow hematopoiesis.

HA Induces IL-6 and IL-1 Expression by Bone Marrow Cells In Vivo
Mice were administered 5-FU followed by PBS (5FU/PBS) or HA (5FU/HA) treatment. CS, a second major constituent of bone marrow ECM, was used as a GAG control (5FU/CS). To control the effect of stress induced by the injection and handling on cytokine expression, the control group of mice was twice administered PBS (PBS/PBS). After 24 hours, bone marrow cells were harvested, mRNA was purified, and the presence of IL-1 and IL-6 transcripts was determined by northern blot. We found that IL-1 and IL-6 gene expression in the bone marrow was significantly higher in the HA-treated group than in the control groups (PBS- or CS-treated mice) (Fig. 5AGo). Relative fold induction of IL-1 and IL-6 genes in the HA-treated mice was, respectively, 2.5 and 3.4 times higher than in the CS- or PBS-treated group (Fig. 4BGo). We further examined the presence of IL-1 and IL-6 gene products in the serum of HA-treated mice using ELISA. Twenty-four hours after HA administration, the concentration of IL-1 and IL-6 was significantly higher in serum obtained from mice treated with HA than in PBS- or CS-administered mice (Fig. 5C, DGo).



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  		Figure 5. Hyaluronic acid (HA) upregulates expression of interleukin-1 (IL-1) and IL-6 in the bone marrow. B6D2F1 mice were administered 150 mg/kg 5-fluorouracil (5-FU). On day 4, the mice were administered 100 µg/mouse HA, chondroitin sulfate (CS), or 200 µ1 phosphate-buffered saline (PBS). Bone marrow cells were harvested, and total RNA was isolated. (A): The number of IL-1{alpha}, IL-6, and ß-actin transcripts were analyzed by northern blot. (B): Quantification of northern hybridization signals was done with Scion Image PC ("Image 2") software (version1.6.1, NIH, Bethedsa, MD). (C, D): Serum levels of IL-1 and IL-6 were examined by enzyme-linked immunosorbent assay and expressed as mean ± standard deviation (SD). (E): The colony-promoting activity of serum samples was examined in colony-forming unit (CFU) assay in the presence of 5% WEHI-3B condition media. Where indicated, IL-1- and IL-6-specific neutralizing antibodies (100 µg/ml) were added. The number of colonies is expressed as mean ± SD. A statistically significant difference is indicated by an asterisk.

 
To prove that increased concentrations of IL-1 and IL-6 detected in the serum obtained from 5FU/HA versus 5FU/PBS mice mediate enhanced hematopoietic activity, we used IL-1 and IL-6 neutralizing antibodies in a methylcellulose clonogenic assay. Freshly harvested bone marrow cells were cultured in methylcellulose media supplemented with the 5FU/PBS or 5FU/HA serum samples in the presence or absence of IL-1 and IL-6 neutralizing antibodies. We found that in cultures supplemented with IL-1- and IL-6-specific neutralizing antibodies, the number of CFUs was significantly decreased (twofold), suggesting that IL-1 and IL-6, at least in part, contribute to the colony-promoting activity of serum obtained from HA-treated mice (Fig. 5EGo).

Thus, we have demonstrated that replacement of HA in vivo stimulates bone marrow accessory cells to produce hematopoiesis-supportive cytokines and provides more favorable conditions for recovery of suppressed hematopoiesis.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The most undesirable consequences of chemotherapy and radiotherapy, used for the treatment and cure of a large variety of malignancies, are severe bone marrow aplasia and pancytopenia. Recovery of the pool of mature blood cells and their committed progenitors depends on HSC, which make up the cellular compartment of the bone marrow. The destiny of HSCs is regulated by a vast multiplicity of factors, produced by cells of the hematopoietic microenvironment, which mediate extrinsic signals [reviewed in 35]. Among various pathways, the CD44/HA has been implicated in the regulation of hematopoiesis (for a recent review, see Ghaffari et al. [38]) [10, 36, 37]. In this study, we demonstrated that HA replacement facilitates bone marrow and peripheral blood recovery after perturbation of bone marrow hematopoiesis by 5-FU in mice.

It has previously been shown that, in addition to exhibiting high levels of myelotoxicity and lymphotoxicity, 5-FU impairs bone marrow megakaryocytopoiesis, resulting in a low platelet number [39,40]. Furthermore, bone marrow transplantation following 5-FU administration delays both platelet recovery and the rebound of thrombocytosis [41]. Recombinant human G-CSF (rhG-CSF), used in cancer chemotherapy to shorten the period of neutropenia, gives rise to consistent, severe thrombocytopenia in mice [39] and in patients [42]. The results of our study demonstrated that the administration of HA following 5-FU injections not only prevented the decrease of WBCs but also rescued mice from the 5-FU–induced thrombocytopenia. These effects were especially remarkable during the second week after 5-FU administration. The fast recovery of WBCs and PLTs in HA-treated mice was due to an accelerated hematopoietic activity in the bone marrow. In addition to preventing the long-term depletion of bone marrow cells induced by 5-FU, HA significantly increased the numbers of megakaryocytes and committed hematopoietic progenitors in the marrow. Furthermore, we observed a higher number of cells in mitosis in 5FU/HA versus 5FU/PBS mice, indicating a higher proliferating rate. Importantly, the expansion of the pool of committed rapidly proliferating progenitors does not occur at the cost of damaging more primitive hematopoietic stem cells, as has been shown by examining the number of LTC-ICs in the bone marrow of 5FU/HA versus 5FU/PBS mice. These findings are in agreement with recently published observations by Nilsson and colleagues, who have demonstrated that the ligation of HA expressed on the cell surface of HSCs prevents them from entering the cell cycle [43]. Hence, it allows us to suggest a dual function for HA in the regulation of hematopoiesis (Fig. 6AGo). It is likely that in steady-state hematopoiesis, HA keeps a pool of the most primitive self-renewing stem cells in a quiescent stage, whereas committed progenitors are provided with growth factors produced by HA-stimulated microenvironmental cells and are driven to proliferation and maturation. Evidently, the degradation of HA that could be induced by various factors in vivo and in vitro leads to an imbalance of hematopoietic homeostasis (Fig. 6BGo). Since HSCs lose one of the "quiescent signals," they might enter the cell cycle, especially under conditions of physiological or pathophysiological demand, and undergo commitment that eventually can lead to stem cell exhaustion. This assumption is in line with our previously published observation that HA’ase-induced HA deprivation leads to severe reduction in the number and size of hematopoietic "loci" in LTBMC [10]. Apart from that, cells composing a microenvironmental niche might not have the essential signaling to produce the sufficient amount of growth factors required for proliferation of the committed progenitors, which then conceivably undergo maturation switches. At this stage, the protective role of HA on primitive stem cells is rather hypothetical and requires further confirmation by performing long-term reconstitution studies. These experiments are currently in progress in our laboratory.



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  		Figure 6. Hypothetical dual function of hyaluronic acid (HA) in regulation of hematopoiesis. (A): Endogenous HA binds its receptors (CD44, RAHMM, HARE, yet unknown) expressed on the surface of cells composing hematopoietic niche. HA-receptor interactions result in activation of microenvironmental cells, followed by the production of positive growth factors that stimulate proliferation of committed hematopoietic progenitor cells (HPCs). Ligation of HA initiated by HA-receptor interactions leads to the arrest of hematopoietic stem cell (HSC) proliferation. (B): Degradation of HA allows proliferation and commitment of HSCs. In the absence of growth factors, the pool of committed progenitors can undergo fast maturation.

 
While HA does not stimulate proliferation of hematopoietic progenitor cells directly [10], it does interact with the cell surface of bone marrow macrophages and triggers their activation. We have previously demonstrated that by activating two independent signal transduction pathways, HA stimulates the production of IL-1 and IL-6 by activated bone marrow macrophages in vitro [10]. Using HA-blocking anti-CD44 antibodies, we have demonstrated that upregulation of IL-1 is mediated by CD44, while IL-6 is mediated by an as-yet-unknown molecule. Among the large variety of factors involved in myelopoiesis, both IL-1 and IL-6 have been shown to produce a direct effect on thrombocytopoietic recovery when administered alone [44]. It was assumed that IL-1 acts on a primitive, 5-FU–resistant multilineage progenitor, while IL-6 might also influence megakaryocyte ploidy and enhance the production of thrombocytes [44,45]. Since IL-1 and IL-6 are both involved in the regulation of thrombocytopoiesis and myelopoiesis, we examined expression of these cytokines in the bone marrow of HA-treated mice. HA stimulated both IL-1 and IL-6 synthesis and production in vivo, as shown by northern blot analysis and ELISA, while levels of G-CSF, GM-CSF, M-CSF, and IL-3 were not changed (data not shown). In contrast, treatment with CS, another major CD44-binding component of bone marrow ECM, did not induce upregulation of IL-1 and IL-6 expression. This finding is in line with our observation that an in vivo administration of CS does not interfere with hematopoietic recovery in 5-FU–treated mice (Khaldoyanidi et al., unpublished observation). Interestingly, using IL-1- and IL-6-specific neutralizing antibodies demonstrated only a partial inhibition of the colony-promoting activity of serum obtained from HA-treated mice. This finding suggests that in addition to IL-1 and IL-6, HA-stimulated bone marrow cells produce other activities that are positive regulators of proliferation of the committed progenitors.

Overall, our findings suggest that replacement of HA has been shown to be effective in the fast recovery of supressed hematopoiesis by providing a more favorable microenvironment in the bone marrow hematopoietic niche.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by grants R21 DK067084 to S.K.K. from NIDDK NIH and grants 10KT-0036 and 11IT-0020 to S.K.K. from the Tobacco-Related Disease Research Program (TRDRP), University of California, Oakland. N.S. was supported by a Cornelius Hopper Diversity Award. V.M. and I.O. contributed equally to this work.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received November 4, 2003; accepted for publication January 2, 2004.



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