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Stem Cells 2005;23:279-287 www.StemCells.com
© 2005 AlphaMed Press

Biphasic Effect of Recombinant Galectin-1 on the Growth and Death of Early Hematopoietic Cells

Virág Vasa, Roberta Fajka-Bojab, Gabriela Ionb, Valéria Dudicsc, Éva Monostorib, Ferenc Uhera

a Stem Cell Biology, National Medical Center, Budapest, Hungary;
b Lymphocyte Signal Transduction Laboratory, Institute of Genetics, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary;
c Polyclinic of Hospitaller Brothers of St. John of God, Budapest, Hungary

Key Words. Apoptosis • Cobblestone area–forming cells • Galectin-1 Hematopoietic stem and progenitor cells • Human • Mouse

Correspondence: Ferenc Uher, Ph.D., National Medical Center, Stem Cell Biology, Diószegi ut 64., Budapest, Hungary, H-1113. Telephone: 36-1-372-4334; Fax: 36-1-372-4352; e-mail: uher{at}ohvi.hu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Galectin-1 is a member of the family of ß-galactoside binding animal lectins, galectins. Its presence in the bone marrow has been detected; however, its role in the regulation of hematopoiesis is unknown. In the present study, we have evaluated the effect of recombinant human galectin-1 on the proliferation and survival of murine and human hematopoietic stem and progenitor cells. We show that low amount of galectin-1 (10 ng/ml) increases the formation of granulocyte-macrophage and erythroid colonies and the frequencies of day-7 cobblestone area–forming cells on a lactose-inhibitable fashion. In contrast, high amount of galectin-1 (10 µg/ml) dramatically reduces the growth of the committed blood-forming progenitor cells as well as the much younger, lineage-negative hematopoietic cells (day-28 to -35 cobblestone area–forming cells). This inhibition is not blocked by lactose and, therefore, is largely independent of the ß-galactoside–binding site of the lectin. Furthermore, assays to detect apoptosis render it likely that the high amount of galectin-1 acts as a classical proapoptotic factor for the premature hematopoietic cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Galectin-1 (gal-1) is a member of the evolutionarily conserved lectin family, galectins with affinity to ß-galactoside containing glycoconjugates. It is involved in a variety of normal and pathological processes, including cell adhesion, cell growth regulation, immunomodulation, inflammation, apoptosis, embryogenesis, and cancer progression [1–5]. As an effective regulator of the immunological processes, it prevents the clinical and histopathological signs of experimental encephalomyelitis, a T cell–mediated autoimmune disease in susceptible Lewis rats [6], and has prophylactic and therapeutic effects on experimental autoimmune myasthenia gravis in rabbits [7]. Moreover, it has also been demonstrated that recombinant gal-1 and its genetic delivery suppress the inflammatory response in collagen-induced arthritis, an experimental model of rheumatoid arthritis [3]. Although the precise mechanism involved in these properties in vivo still remains to be elucidated, it has been proposed that gal-1 may shift the T-cell response from Th1 to Th2 and contributes to the deletion of antigen-specific activated T cells [8]. Experimental data have been accumulated in the last few years concerning the implication of gal-1 in apoptosis of activated peripheral T cells, tumor T-cell lines, and immature cortical thymocytes [9–12], which also contribute to immunomodulatory effect. In addition to the inhibitory role of gal-1 on T-cell proliferation and survival, this protein promotes proliferation of vascular endothelial and bone marrow stromal cells [13,14]. Gal-1 produced by bone marrow stromal cells has been implicated in the synapse formation between pre-B and marrow stromal cells, resulting in pre-B cell activation [15]. Importantly, a recent paper by Baum et al. [16] has provided evidence that Gal-1 efficiently prevents the development of the graft-versus-host disease (GVHD), the major complication of bone marrow transplantation. However, because of the implication of the lectin as a therapeutical agent in GVHD, the role of gal-1 in early hematopoiesis should be analyzed.

In the present study, we have evaluated the effect of recombinant human gal-1 on the growth and death of murine and human hematopoietic stem and progenitor cells. We show that low amount (10–20 ng/ml) of gal-1 increases the formation of the colony-forming units granulocyte-macrophage (CFU-GM) and erythroid colonies (BFU-E) and the frequencies of the day-7 cobblestone area–forming cells (CAFCs). In contrast, high amount (5–10 µg/ml) of gal-1 inhibits the growth of the committed blood-forming progenitor as well as the much younger hematopoietic (stem) cells (day-28 to -35 CAFCs) in a fashion that is independent of the lectin property. It is also shown that high amount of gal-1 acts as a classical proapoptotic factor on hematopoietic cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of Bone Marrow Cells
Murine bone marrow was extracted from the femurs and tibias of 6- to 8-week-old (C57Bl/6 x DBA/2) F1 (BDF1) mice (National Institute of Oncology, Budapest, Hungary). After standard erythrocyte lysis, nucleated cells were placed into 25-cm2 culture flasks (Costar, Cambridge, MA) and incubated overnight in {alpha}-minimal essential medium (MEM) (Gibco, Grand Island, NY) supplemented with 20% fetal calf serum (FCS) and antibiotics (Gibco). Subsequently, nonadherent cells were harvested and stained with a panel of biotinylated lineage-specific antibodies (mouse lineage panel, containing anti-CD3{varepsilon}, anti-CD45R/B220, anti-CD11b, anti-Ly-6G, and anti-TER-119; BD PharMingen, San Diego) for 30 minutes at 4°C. Cells were washed twice and incubated with streptavidin-coated magnetic particles (BioSource, Camarillo, CA) for an additional 30 minutes at 4°C. The particle-free cells were separated and collected as the lin– (lineage-negative) fraction of bone marrow cells. This noncommitted cell population represented 0.2%–0.6% of original unfractionated bone marrow nucleated cells. The efficiency of the purification was verified by flow cytometry (see below).

Normal human bone marrow cells were obtained as leftover cells from allogeneic transplants according to procedures approved by the ethics committee at the National Medical Center. Mononuclear cells were isolated using Ficoll Hypaque (1.077 g/ml, Lymphoprep; Nycomed, Oslo, Norway) density centrifugation.

Recombinant Gal-1
Human recombinant gal-1 was cloned and purified as previously described [9]. Briefly, gal-1 cDNA, kindly provided by Dr. Jun Hirabayashi and Ken-Ichi Kasai (Teikyo University, Japan) [17], was cloned into pQE-60 prokaryotic expression vector (Qiagen, Valencia, CA) as described [9]. The vector was transformed into Escherichia coli strain, BL21, and transformants were selected in LB medium containing ampicillin (100 µg/ml). The bacteria producing gal-1 were grown up and lysed. The bacterial lysate containing the recombinant protein was loaded onto lactosyl agarose column, and gal-1 was isolated in one step with affinity chromatography. The homogeneity of the purified gal-1 was verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC in a Vydac C4 column. In addition, gal-1 was analyzed by mass spectrometry, and the measured molecular mass was in good agreement with the calculated one (14.925 kDa) (data not shown).

Flow Cytometry
Unfractionated or lin– cells were resuspended in phosphate-buffered saline (PBS). Cells were stained with phycoerythrin (PE)–conjugated monoclonal anti-Sca-1 (clone D7), fluorescein isothiocyanate (FITC)–conjugated anti-CD34 (clone RAM34), or biotinylated anti–c-kit (clone 2B8) antibodies (BD PharMingen). C-kit+ were detected with streptavidin-PE (Sigma, St. Louis).

For gal-1 binding assay, cells were incubated with the recombinant lectin in the first step and then with a polyclonal rabbit anti–gal-1 antibody in the second step and with the FITC-conjugated goat anti-rabbit immunoglobulin G (IgG) reagent (BD PharMingen) in the third step. Control samples without gal-1 were included for each sample.

In all cases, the stained cells were washed with PBS containing 12.5 µg/ml propidium iodide to gate for dead cells and analyzed immediately on FACS can flow cytometer using CellQuest software (Becton, Dickinson, Mountain View, CA).

Intracellular staining for gal-1 was performed using the DAKO IntraStain Fixation and Permeabilisation Kit (Dako A/S, Glostrup, Denmark), polyclonal rabbit anti–gal-1 antibody, and FITC-conjugated goat anti-rabbit IgG.

Annexin-V Labeling
To detect phosphatidyl serine exposure, the cells were treated as indicated and then washed twice with PBS and resuspended in binding buffer (0.01 M HEPES, 0.14 M NaCl, and 2.5 mM CaCl2). Annexin V-FITC and propidium iodide (10 µg/ml) were added to the cells for 15 minutes in the dark at room temperature. After washing, the cells were analyzed on FACS can cytofluorimeter.

Hypodiploid, Sub-G1 Cell Population
Cells were treated as indicated and subjected to DNA content analysis. Briefly, the cells were harvested and washed two times with PBS containing 0.1% glucose and then permeabilized and stained in the following solution: PBS supplemented with 0.1% Triton X-100, 0.1% Na3-citrate, 10 µg/ml RNase, and 10 µg/ml propidium iodide. After incubation in dark for 30 minutes at room temperature, the cells were analyzed on FACS can. The sub-G1 (hypodiploid) population was determined with cell cycle analysis using CellQuest software programs and was considered as apoptotic cells.

Progenitor (Colony-Forming Cell) Assays
Quantification of the number of CFU-GM and BFU-E was performed using a semisolid colony-forming cell assay. Mouse bone marrow cells were plated in Iscove’s modified Dulbecco’s medium supplemented with 1% methylcellulose, 30% horse serum (Gibco), 10%WEHI-3Bconditioned medium as a source of growth factors, 4 x 10–3 M/l L-glutamine, 2.5 x 10–4 M/l a-thioglycerol (Gibco), 1% deionized bovine serum albumin (Sigma), and antibiotics (Gibco). Cells were cultured in 35-mm Petri dishes (Costar) at 37°C in 5% CO2. CFU-GM and BFU-E were counted on day 7 and 9 of culture in the same dish.

For human bone marrow mononuclear cells, the medium was supplemented with FCS (Gibco) and 5637 (HTB-9) human bladder carcinoma-conditioned medium (10% vol/vol) as a source of growth factors. CFU-GM and BFU-E were counted on day 9 and 14 of culture, respectively.

CAFC Assay
In vitro determination of hematopoietic stem and progenitor cell frequencies was performed by limiting dilution analysis of CAFCs in microcultures according to methods previously described [18], with some modification [19]. Bone marrow cells were extracted and purified as described above. The murine lin– cells or whole bone marrow cells (unfractionated cells) or the mononuclear fraction of human bone marrow cells were washed twice with CAFC medium before the CAFC assay. The CAFC medium was comprised of {alpha}MEM (Gibco) supplemented with 12.5% FCS, 12.5% horse serum, HEPES (3.5 mM), L-glutamine (2 mM) (all from Gibco), ß-mercaptoethanol (10–4 M), and hydrocortisone hemisuccinate (10–5 M) (Sigma). For each sample, six to eight serial twofold dilutions of cells were prepared and plated in flat-bottom 96-well plates (Costar) over pre-established confluent layers of GY30 stromal cells [19]. Twenty-four to 36 wells per dilution were plated for each group of cells and maintained at 33°C and 5% CO2. Once a week, half of the medium (100 µl) was carefully removed from each well, and an equal volume of fresh medium was added. The wells were evaluated for cobblestone areas weekly from day 7 until day 35. The proportion of negative wells at each dilution was used in a Poisson-based limiting dilution analysis calculation to determine the CAFCs using the L-Calc software (Stem Cell Technologies, Vancouver, Canada).

Statistical Analysis
Results are presented as the mean ± standard error. Significance was determined using the two-tailed paired Student’s t-test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Binding and Intracellular Expression of Gal-1 by Murine Bone Marrow Cells
To confer any regulatory activity, it was essential to show that ligand sites for the human recombinant gal-1 are present on the surface of bone marrow cells. As shown in Figure 1Go, gal-1 bound to 70%–80% of nucleated bone marrow cells (Fig. 1AGo) and to >98% of bone marrow–derived lin– cells (Fig. 1BGo) obtained from normal adult mice. The gal-1 nonbinding fraction (20%–30%) of the bone marrow cells (Fig. 1AGo) was comprised of a mixture of cells positive for CD3{varepsilon}, CD45R/B220, CD11b, Ly-6G, and TER-119 (data not shown), indicating that these cells belonged to the more matured cell population. As also shown in Figure 1Go, high amount (100 mM) of lactose strongly, but not completely, inhibits gal-1 binding to marrow cells.



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  		Figure 1. Flow cytometric analysis of galectin-1 binding by bone marrow cells. Unfractionated (A) or lineage-negative (B) murine bone marrow cells were incubated with recombinant galectin-1 or bovine serum albumin in the presence or absence of 100 mM lactose before staining with rabbit anti–galectin-1 F(ab')2 and fluorescein isothiocyanate–labeled goat anti-rabbit immunoglobulin G antibodies. For negative controls, the recombinant lectin or the anti–galectin-1 antibody was omitted. Data of one representative experiment out of three.

 
Next, we examined the endogenous expression of gal-1 in the bone marrow cells by flow cytometric analysis of permeabilized cells using polyclonal rabbit anti-gal-1 F(ab')2 and FITC-labeled goat anti-rabbit IgG. A small shift was detected between the peaks of the control and the anti-gal-1–stained unfractionated bone marrow cells, indicating a low expression of intracellular gal-1 in these cells (Fig. 2AGo), whereas lin– cells did not show a detectable amount of intracellular gal-1 protein (Fig. 2BGo). Notably, the gal-1–expressing bone marrow cells were mainly plastic adherent cells (data not shown), indicating a stromal origin of these cells.



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  		Figure 2. Flow cytometric analysis of galectin-1 expression by bone marrow cells. Unfractionated (A) or lineage-negative (B) murine bone marrow cells were permeabilized and stained with rabbit anti–galectin-1 F(ab')2 and fluorescein isothiocyanate–labeled goat anti-rabbit immunoglobulin G antibodies. For negative controls, the anti–galectin-1 antibody was omitted. Data of one representative experiment out of three.

 
Biphasic Modulation of Committed Hematopoietic Progenitor Cell Growth by Gal-1
The growth regulation of murine and human bone marrow cells by different concentrations of gal-1 was analyzed in semisolid medium (Table 1Go). Formation of CFU-GM and BFU-E marrow cells was significantly increased in the presence of low amount (10 ng/ml) of gal-1, whereas the growth of these committed cell populations was entirely blocked in the presence of high amount (10 µg/ml) of gal-1. Thus, gal-1 had a dose-dependent biphasic effect on the plating efficiency of colony-forming cells. When exogenous hematopoietic growth factors were not added to the cultures, no colony formation was observed in the presence of gal-1 (data not shown).


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  		Table 1. Galectin-1 affects the colony formation of the bone marrow cells in a biphasic fashion
 
To infer the nature of the inhibitory process on the cultured cells, we performed routine assays to detect apoptotic cells. We recorded DNA cytograms by fluorescence-activated cell sorter analysis, in which apoptotic cells were expected to show up in the clearly defined sub-G0/G1 peak. Murine bone marrow cells were monitored at various time points between 1 and 4 hours after addition of gal-1. As shown in Figure 3AGo, all nonadherent bone marrow cells were dead after 4 hours of incubation in the presence of 10 µg/ml lectin. Moreover, non-adherent marrow cells showed signs of early apoptosis, i.e., annexin V binding, very rapidly after exposure to gal-1 (Fig. 3BGo). Thus, high amount of gal-1 did not simply inhibit hematopoietic cell proliferation; rather, it induced apoptotic death of these cells in vitro. In contrast, adherent marrow (stromal) cells were completely resistant for gal-1–induced death (data not shown).



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  		Figure 3. Galectin-1–induced apoptosis of bone marrow cells. (A): DNA-content histograms of nonadherent murine bone marrow cells were incubated in the presence of 10 µg/ml galectin-1 for 4 hours and labeled by propidium iodide. (B): Annexin V–fluorescein isothiocyanate binding by marrow cells incubated with galectin-1 for 10, 20, 30, 40, and 60 minutes, respectively. Data of one representative experiment out of three.

 
To determine whether the growth regulatory effect of gal-1 was dependent on lectin-cell surface glycoconjugate interaction, we performed experiments in the presence of high concentration of lactose. Higher amounts (50–100 mM) of lactose were toxic for the marrow cells and thus could not be used in these experiments. The inhibition of colony formation in the presence of 10 µg/ml (720 nM) gal-1 was not affected by 30 mM lactose (representing a 4 x 103-fold molar excess); thus, it was largely independent of the ß-galactoside binding site. In contrast, the growth-stimulatory activity of the mitogenic concentration (10 ng/ml) of gal-1 was susceptible to inhibition by lactose but not by sucrose, and thus it was attributable to the ß-galactoside binding ability of the protein (Table 2Go).


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  		Table 2. The growth stimulatory activity of galectin-1 is dependent on the carbohydrate binding ability of the lectin
 
Effect of Gal-1 on the Development of Cobblestone Area–Forming Cells
Unfractionated murine bone marrow cells or human mononuclear bone marrow cells were deposited in a limiting dilution setup in 96-well plates containing a pre-established confluent stromal cell layer, as described in Materials and Methods. It has been shown repeatedly that the time of the appearance of various cobblestone area–like colonies in long-term bone marrow cultures strongly correlates with the maturity of the hematopoietic cells. In mouse system, committed progenitors form colonies after 7–14 days (hence referred as day-7 to -14 CAFCs), whereas the more primitive stem cells start proliferating only after 4–5 weeks (day-28 to -35 CAFCs) [18]. The presence of low concentration (20 ng/ml) of gal-1 had different consequence to the cells at various maturation stages, because both mouse and human day-7 CAFC frequencies were increased (Figs. 4A, 4DGo, respectively), whereas day-21 CAFC frequencies were slightly but reproducibly decreased (Figs 4B, 4EGo). The 10 ng/ml gal-1 concentration that was the most effective to enhance colony formation (Table 1Go) did not increase the day-7 CAFC formation. Therefore, we determined the optimal concentration of gal-1 used in CAFC assay (data not shown), and hence the 20 ng/ml concentration was applied. A conceivable explanation for the difference in the optimal gal-1 concentrations in colony formation and CAFC assays is that in CAFCs, additionally to the bone marrow cells, there are stroma cells used as feeder layer. Because stroma cells also contain gal-1 binding cell-surface structures, they may partially compete for gal-1 and hence increase the effective gal-1 concentration from 10 to 20 ng/ml.



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  		Figure 4. CAFC frequencies of bone marrow cells in the presence or absence of low amount of recombinant galectin-1. Murine (A, B, C) and human (D, E, F) bone marrow cells were isolated as described in Materials and Methods and overlaid on 96-well plates in multiple dilutions for limiting dilution analysis of CAFCs in the presence of 20 ng/ml galectin-1. Wells were evaluated for cobblestone areas weekly from day 7 through day 35. Frequencies of day-7 (A, D), day-21 (B, E), and day-35 (C, F) CAFCs were calculated by the L-Calc software (Stem Cell Technologies). Data of one representative experiment out of three. Abbreviation: CAFC, cobblestone area–forming cell.

 
Neither mouse nor human day-35 CAFC frequencies were affected by the lectin at this concentration (Figs. 4C, 4FGo). To confirm that the failure of the response by this cell population to gal-1 treatment was not due to the very low frequency of the most immature cells, we carried out the following experiment: The lin– murine marrow cells were highly enriched (from 1:80,000–120,000 to 1:400–500) and subjected to day-35 CAFC assay. As shown in Figure 5Go, day-35 CAFCs did not respond to gal-1 treatment.



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  		Figure 5. Day-35 CAFC frequencies of lin– bone marrow cells in the presence or absence of low amounts of recombinant galectin-1. Murine lin– marrow cells were isolated as described in the Materials and Methods section and overlaid on 96-well plates in multiple dilutions for limiting dilution analysis of CAFCs in the presence of 20 ng/ml galectin-1. Wells were evaluated for cobblestone areas on day 35. The L-Calc software (Stem Cell Technologies) was used to calculate frequencies of day-35 CAFCs. Data of one representative experiment out of three. Abbreviation: CAFC, cobblestone area–forming cell.

 
The presence of high amount (10 µg/ml) of gal-1 completely inhibited CAFC formation from day 7 to day 35 (data not shown). However, some CAFC formation was observed when the unfractionated or lin– bone marrow cells were seeded after preincubation with gal-1, which was then removed from the cultures. As shown in Table 3Go, the more mature CAFC precursors were more sensitive whereas immature cells were much less sensitive for gal-1–mediated growth inhibition, in the following order of sensitivity: day-7 > day-14 > day-21 > day-28 > day-35 CAFCs. This suggests that during maturation and differentiation, the hematopoietic cells become progressively more and more sensitive for growth inhibition by apoptosis (Fig. 3Go) induced with high amount of gal-1.


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  		Table 3. CAFCs become more sensitive for high amounts of galectin-1–induced growth inhibition during maturation
 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The role of gal-1 in the regulation of the hematopoiesis has not been studied in detail so far. Therefore, in the present study, we have measured the effect of recombinant human gal-1 on murine and human hematopoietic stem and progenitor cell proliferation and survival. We have found that gal-1 acts in a biphasic manner on the bone marrow cells depending on its concentration. Low amount of gal-1 (10–20 ng/ml) promotes the differentiation of the bone marrow cells into CFU-GM–committed and BFU-E–committed progenitors and, accordingly,day-7 CAFC frequencies in cellculture. In contrast, high amount of gal-1 (10 µg/ml) dramatically inhibits the growth of the committed blood-forming progenitor cells as well as the much younger hematopoietic (stem/early progenitor) cells (day-28 to -35 CAFCs). The growth inhibition correlates with the apoptotic death of these hematopoietic cells, rendering gal-1 as a classical proapoptotic factor on the blood-forming cells. The apoptotic effect of gal-1, however, depends on the maturation/differentiation states of the cells, because the most matured (7-day CAFCs) cells are the most sensitive and the youngest (35-day CAFCs) cells are the less sensitive for the growth inhibitory activity of the lectin.

The biphasic growth stimulatory and inhibitory effect of gal-1 observed on bone marrow cells is not unique. Several studies have suggested that the seemingly paradoxical positive and negative effects of gal-1 on cell growth are highly dependent on the type and activation state of the cells and might also be influenced by the relative distribution of monomeric versus dimeric forms of the lectin [20]. First, Wells and Mallucci [21] showed that gal-1 acts as an autocrine/paracrine negative growth factor and inhibits the proliferation of mouse embryonic fibroblasts in a carbohydrate-independent way. Adams et al. [22] found a biphasic modulation of cell growth by the recombinant form of the lectin. Furthermore, it is well known that gal-1 induces the death of activated and effector T cells but is not cytotoxic to naive or memory T cells [2,8]. Therefore, our data are essentially consistent with previous works done with other cells isolated from different tissues.

Of note, it is well known that gal-1 is expressed by bone marrow stromal cells [23,24], and we also detected intracellular lectin in the adherent fraction of marrow cells. Nevertheless, in this work, exogenous recombinant gal-1 has been added to hematopoietic cell cultures, which do not give information on how the exogenous gal-1 contributes to the regulation of hematopoiesis. Thus, the fine expression pattern and secretion of gal-1 by bone marrow cells in situ remain to be elucidated in the future. Experiments are already in progress to clarify this point.

Finally, the fact that gal-1 is a stimulator for committed hematopoietic progenitor cell growth is in good agreement with recent data from Baum et al. [16]. They found that gal-1 treatment increases bone marrow cellularity and, at the same time, reduces severity of GVHD in an animal model. The gal-1–mediated death pathway is distinct from that induced by corticosteroids or by Fas [25,26], indicating that gal-1 may synergize with other immunosuppressive agents to optimize therapeutic benefit in both inflammatory and autoimmune diseases. Therefore, additional study on the mechanisms involved in gal-1 expressions and functions in bone marrow may open new avenues not only in basic biomedical research but also in clinical therapy.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are grateful to Dr. Jun Hirabayashi and Ken-Ichi Kasai (Teikyo University) for the generous gift of gal-1 cDNA. We thank Dr. Susan R. Hollan for reading the manuscript and for helpful suggestions. This work was supported by grants from the Hungarian Scientific Research Found (OTKA T037579), Hungarian Ministry of Welfare (ETT 203/2001 and ETT 369/2003), and National Office for Research and Technology (OMFB-00541/2004).


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Danguy A, Camby I, Kiss R. Galectins and cancer. Biochim Biophys Acta 2002;1572:285–262.[Medline]

  2. Rabinovich GA, Baum LG, Tinari N et al. Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response? Trends Immunol 2002;23:313–320.[CrossRef][Medline]

  3. Rabinovich GA, Sotomayor CE, Riera CM et al. Evidence of a role for galectin-1 in acute inflammation. Eur J Immunol 2000;30:1331–1339.[CrossRef][Medline]

  4. Rabinovich GA. Galectins: an evolutionarily conserved family of animal lectins with multifunctional properties; a trip from the gene to clinical therapy. Cell Death Differ 1999;6:711–721.[CrossRef][Medline]

  5. Rabinovich GA, Rubinstein N, Fainboim L. Unlocking the secrets of galectins: a challenge at the frontier of glyco-immunology. J Leukoc Biol 2002;71:741–752.[Abstract/Free Full Text]

  6. Offner H, Celnik B, Bringman TS et al. Recombinant human beta-galactoside binding lectin suppresses clinical and histological signs of experimental autoimmune encephalomyelitis. J Neuroimmunol 1990;28:177–184.[CrossRef][Medline]

  7. Levi G, Tarrab-Hazdai R, Teichberg VI. Prevention and therapy with electrolectin of experimental autoimmune myasthenia gravis. Eur J Immunol 1983;13:500–507.[Medline]

  8. Rabinovich GA, Toscano MA, Ilarregui JM et al. Shedding light on the immunomodulatory properties of galectins: novel regulators of innate and adaptive immune responses. Glycoconj J 2004;19:565–573.[CrossRef][Medline]

  9. Fajka-Boja R, Szemes M, Ion G et al. Receptor tyrosine phosphatase, CD45 binds galectin-1 but does not mediate its apoptotic signal in T cell lines. Immun Lett 2002;82:149–154.[CrossRef][Medline]

  10. Pace KE, Hahn HP, Pang M et al. CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death. J Immunol 2000;165:2331–2334.[Abstract/Free Full Text]

  11. Perillo NL, Pace KE, Seilhamer JJ et al. Apoptosis of T cells mediated by galectin-1. Nature 1995;378:736–739.[CrossRef][Medline]

  12. Perillo NL, Uittenbogaart CH, Nguyen JT et al. Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes. J Exp Med 1997;185:1851–1858.[Abstract/Free Full Text]

  13. Moiseeva EP, Javed Q, Spring EL et al. Galectin 1 is involved in vascular smooth muscle cell proliferation. Cardiovasc Res 2000;45:493–502.[Abstract/Free Full Text]

  14. Andersen H, Jensen ON, Moiseeva EP et al. A proteome study of secreted prostatic factors affecting osteoblastic activity: galectin-1 is involved in differentiation of human bone marrow stromal cells. J Bone Miner Res 2003;2:195–203.

  15. Gauthier L, Rossi B, Roux F et al. Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicate in synapse formation between pre-B and stromal cells and in pre-B triggering. Proc Natl Acad Sci USA 2002; 99: 13014–13019.

  16. Baum LG, Blackall DP, Arias-Magallano S et al. Amelioration of graft versus host disease by galectin-1. Clin Immunol 2003;109:295–307.[CrossRef][Medline]

  17. Hirabayashi J, Ayaki H, Soma G et al. Production and purification of a recombinant human 14 kDa ß-galactoside-binding lectin. FEBS Lett 1989;250:161–165.[CrossRef][Medline]

  18. Ploemacher RE, van der Sluijs JP, Voerman JS et al. An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood 1989;74:2755–2763.[Abstract/Free Full Text]

  19. Vas V, Szilagyi L, Paloczi K et al. Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay. J Leukoc Biol 2004;75:714–720.[Abstract/Free Full Text]

  20. Sacchettini JC, Baum LG, Brewer CF. Multivalent protein-carbohydrate interactions: a new paradigm for supermolecular assembly and signal transduction. Biochemistry 2001;40:3009–3015.[CrossRef][Medline]

  21. Wells V, Mallucci L. Identification of an autocrine negative growth factor: mouse ß-galactoside-binding protein is a cytostatic factor and cell growth regulator. Cell 1991;64:91–97.[CrossRef][Medline]

  22. Adams L, Scott GK, Weinberg CS. Biphasic modulation of cell growth by recombinant human galectin-1. Biochim Biophys Acta 1996;1312:137–144.[Medline]

  23. Abedin MJ, Kashio Y, Seki M et al. Potential roles of galectins in myeloid differentiation into three different lineages. J Leukoc Biol 2003;73:650–656.[Abstract/Free Full Text]

  24. Harrison FL, Catt JW. Intra- and extracellular distribution of an endogeneous lectin during erythropoiesis. J Cell Sci 1986;84:201–212.[Abstract]

  25. Bryder D, Ramsfjell V, Dybedal I et al. Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation. J Exp Med 2001;194:941–952.[Abstract/Free Full Text]

  26. Maguer-Satta V, Oostendorp R, Reid D et al. Evidence that ceramide mediates the ability of tumor necrosis factor to modulate primitive human hematopoietic cell fates. Blood 2000;96:4118–4123.[Abstract/Free Full Text]

Received April 13, 2004; accepted for publication September 10, 2004.



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