Biphasic Effect of Recombinant Galectin-1 on the Growth and Death of Early Hemat - 《stemcells》2004-2005文献干细胞之家



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发表于 2009-3-5 10:52 |只看该作者 |倒序浏览 |打印

a Stem Cell Biology, National Medical Center, Budapest, Hungary;- N. L$ J% W2 n9 b& E# Q
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b Lymphocyte Signal Transduction Laboratory, Institute of Genetics, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary;  M+ j; Z! r1 T; C" f, k: K$ f* R
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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$ H- A: f, |# ~+ X0 Z

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

ABSTRACT4 K. X7 H+ D* B
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Galectin-1 (gal-1) is a member of the evolutionarily conserved lectin family, galectins with affinity to ?-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 . 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 , and has prophylactic and therapeutic effects on experimental autoimmune myasthenia gravis in rabbits . 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 . 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 . 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 , 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 . 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 . Importantly, a recent paper by Baum et al.  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.. V: K9 R+ T5 ]
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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
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Binding and Intracellular Expression of Gal-1 by Murine Bone Marrow Cells* p0 Z% N* b! o% B
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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 1, gal-1 bound to 70%–80% of nucleated bone marrow cells (Fig. 1A) and to >98% of bone marrow–derived lin– cells (Fig. 1B) obtained from normal adult mice. The gal-1 nonbinding fraction (20%–30%) of the bone marrow cells (Fig. 1A) was comprised of a mixture of cells positive for CD3, 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 1, 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. 2A), whereas lin– cells did not show a detectable amount of intracellular gal-1 protein (Fig. 2B). 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.) [. u0 ^$ f* j2 b4 w! U1 K

Biphasic Modulation of Committed Hematopoietic Progenitor Cell Growth by Gal-1
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The growth regulation of murine and human bone marrow cells by different concentrations of gal-1 was analyzed in semisolid medium (Table 1). 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).' W8 e$ o) D7 X# \" {  I

Table 1. Galectin-1 affects the colony formation of the bone marrow cells in a biphasic fashion0 x7 N9 f, a/ H5 i+ z

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 3A, 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. 3B). 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).

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., U# }# S) S& D; I  ?

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 ?-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 ?-galactoside binding ability of the protein (Table 2).

Table 2. The growth stimulatory activity of galectin-1 is dependent on the carbohydrate binding ability of the lectin
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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) . 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, 4D, respectively), whereas day-21 CAFC frequencies were slightly but reproducibly decreased (Figs 4B, 4E). The 10 ng/ml gal-1 concentration that was the most effective to enhance colony formation (Table 1) 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., G2 b1 W. t( B# e; S9 b. Y4 G( O

Neither mouse nor human day-35 CAFC frequencies were affected by the lectin at this concentration (Figs. 4C, 4F). 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 5, day-35 CAFCs did not respond to gal-1 treatment.

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.
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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 3, 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. 3) induced with high amount of gal-1.. }* d( w- W9 u

Table 3. CAFCs become more sensitive for high amounts of galectin-1–induced growth inhibition during maturation/ V; K) f2 G% B% }

DISCUSSION6 f* k) c, _. e/ J! k# B% x& X0 F

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).! n) i, G4 W6 j6 {& _0 E
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