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(Journal of Leukocyte Biology. 2001;69:555-564.)
© 2001 by Society for Leukocyte Biology

Expression and function of galectin-3, a ß-galactoside-binding protein in activated T lymphocytes

Hong-Gu Joo, Peter S Goedegebuure, Noriaki Sadanaga, Makoto Nagoshi, Wolfram von Bernstorff and Timothy J. Eberlein

Laboratory of Biologic Cancer Therapy, Department of Surgery, Washington University School of Medicine, St. Louis, Missouri

Correspondence: Dr. Peter S. Goedegebuure, Department of Surgery, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. E-mail: goedegep{at}msnotes.wustl.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A soluble beta-galactoside-binding lectin, galectin-3 has been shown to be involved in cell adhesion and activation of immune cells. Although galectin-3 is known to be expressed in various types of cells, it has not been shown whether galectin-3 is expressed in T lymphocytes. We present evidence here that galectin-3 is expressed in activated murine T lymphocytes including CD4+ and CD8+ T cells but not in resting T cells. Galectin-3 expression was induced by anti-CD3 mAb or mitogen and enhanced by common {gamma}-chain signaling cytokines, IL-2, IL-4, and IL-7, in activated T lymphocytes, whereas the inflammatory cytokines including TNF-{alpha} and IFN-{gamma} did not. Galectin-3 expression and proliferation were down-regulated by withdrawal of IL-2 and gamma irradiation. Anti-sense but not sense phosphorothioated oligonucleotides for galectin-3 inhibited galectin-3 expression and blocked proliferation of T cells significantly. This study suggests that up-regulation of galectin-3 plays an important role in proliferation of activated T lymphocytes.

Key Words: common {gamma}-chain signaling cytokines • proliferation • anti-sense


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectin-3 was first identified as a cell-surface molecule on thioglycollate-elicited murine peritoneal macrophages [1 ]. Subsequently, intracellular galectin-3 was detected in various cell types including 3T3 fibroblast and colon carcinoma [2 , 3 ]. Galectin-3 is a ß-galactoside-binding protein, which was named Mac-2, CBP-35, L-29, L-34, and epsilon BP previously. It has been demonstrated that this protein has multiple functions including formation of tumor metastases, immunoglobulin (Ig)E-mediated activation of neutrophils, involvement of proliferation, and pre-mRNA splicing. Proliferating 3T3 mouse fibroblasts express higher levels of galectin-3 than quiescent 3T3 cells. The presence of galectin-3 in the nucleus is correlated with the proliferation state of the cells [2 ]. Furthermore, recent results showed that recombinant galectin-3 is a mitogen capable of stimulating fibroblast cell proliferation in a paracrine manner [4 ]. The ability of galectin-3 to bind extracellular matrix, mainly laminin, has been shown to be closely related to the metastatic potential of tumor cells [5 ].

Galectin-3 has several potential roles on immune cells in inflammatory processes. It down-regulates interleukin (IL)-5 gene expression in human eosinophils, the eosinophilic cell line EoL-3, peripheral blood mononuclear cells (PBMC), and in Ag-specific CD4+ T lymphocytes [6 ]. Although the expression and function of galectin-3 in several types of immune cells have been shown, not much is known about galectin-3 in relation to T lymphocytes.

Recently, it was shown that the human leukemia cell line, Jurkat-transfected with the galectin-3 gene, displayed higher growth rates than control transfectants and showed resistance to apoptotic signals [7 ]. However, because Jurkat cells do not express galectin-3, it is not clear what the exact function of galectin-3 is in normal cells under physiological conditions. It has not been shown whether galectin-3 is expressed in T lymphocytes, because immunohistochemistry on lymphoid organs and various lymphoid cell lines including EL4 cells by Northern blot analysis did not detect galectin-3 [8 , 9 ]. Thus, we investigated the expression and function of galectin-3 in primary T lymphocytes.

The data presented here demonstrate that galectin-3 is expressed in activated T lymphocytes but not in resting T cells. The expression of galectin-3 was enhanced by common {gamma}-chain signaling cytokines, such as IL-2, IL-4, and IL-7, and correlated with the proliferation of activated T lymphocytes. Galectin-3 is located predominantly in intracellular compartments but not on the surface and is secreted by activated T lymphocytes. These findings on the presence and secretion of galectin-3 in activated T lymphocytes may describe the roles of galectin-3 in T lymphocytes and explain galectin-3-mediated interactions between T lymphocytes and other immune cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and antibodies
Female 6- to 8-week-old C57BL/6 mice were purchased from Taconic Farms (Germantown, NY). The mice were housed under NIH-approved animal subject conditions. All mice received animal laboratory chow and water ad libitum and were used at the age of 7–9 weeks. Rat monoclonal antibody (mAb) M3/38 against mouse galectin-3 was purified from the culture supernatant of TIB166 hybridoma (American Type Culture Collection, ATCC, Rockville, MD) by using protein G-Sepharose beads (Sigma, St. Louis, MO).

Lymphocyte preparation and culture
Spleen cells, lymph node lymphocytes, or thymocytes from C57BL/6 mice were prepared by mechanical disruption and hypotonic lysis of red blood cells. To remove adherent cells, the cell suspensions were first passed through nylon wool and subsequently incubated in a culture flask for 2 h. The nonadherent cells were centrifuged on Lympholyte®-M (Cedarlane, Ontario, Canada). After washing twice with Hanks’ balanced saline solution (HBSS), the cells were used for surface or intracellular staining and flow cytometry analysis. Typically, 5x107 spleen cells were retrieved from a single mouse. Spleen cells were cultured in 2 ml culture medium (CM) in a 24-well plate at 37°C in a 5% CO2 incubator. Culture medium was RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS), 2 mM fresh L-glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 100 international units (IU)/ml penicillin, 100 µg/ml streptomycin (all from Bio-Whittaker, Walkersville, MD), and 5x10-5 M 2-mercaptoethanol (Sigma).

Activation of T lymphocytes
Spleen cells were activated by adding 5 µg/ml concanavalin A (Con A; Sigma) or by placing the cells onto solid-phase anti-CD3 mAb (hybridoma 145-2C11; ATCC)-coated flasks [10 ]. After 2 days, the viable cells were purified by Lympholyte®-M and then cultured at 1x106 cells/well in 2 ml CM in the absence or presence of human recombinant (hr)IL-2 (a gift from Amgen, Thousand Oaks, CA), granuloctye-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-{alpha}, IL-10, murine rIL-4, IL-7, interferon (IFN)-{gamma}, and ultrapure transforming growth factor (TGF)-ß (all from Genzyme, Cambridge, MA) in 24-well plates. The number and viability of cells were determined by the trypan blue exclusion test.

Flow cytometry analysis
For intracellular staining, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at 4°C, washed twice, resuspended in HBSS, and then permeabilized by incubation in BSS containing 0.1% saponin. The cells were incubated with 2 µg anti-galectin-3 mAb for 30 min at 4°C and washed twice with saponin-containing buffer. Staining was performed with incubation in 0.5 µg anti-rat IgG-fluorescein isothiocyanate (FITC). After intracellular staining, cell-surface staining was performed with 1 µg anti-mouse CD4-phycoerythrin (PE), CD8-PE, or CD19-PE (PharMingen, San Diego, CA). For propidium iodide (PI)-staining analysis, cells were fixed in ice-cold 70% ethanol followed by overnight incubation at -20°C. Cells were stained with PBS containing 50 µg/ml PI, 0.1% Triton X-100, 0.5 mM ethylenediaminetetraacetate (EDTA), and 50 µg/ml RNase A (Sigma) for 60 min at room temperature. The bromodeoxyuridine (BrdU, PharMingen) incorporation assay was performed according to the manufacturer’s instruction. Briefly, cells were incubated with BrdU (10 µM) for 2 h and treated with DNase (300 µg/ml). Incorporated BrdU was labeled by 1 µg anti-BrdU Ab-PE and stained with 2 µg anti-galectin-3 mAb and 1 µg anti-rat IgG-FITC. Flow cytometry analysis of stained lymphocytes was performed on an Epics C cytometer (Coulter, Hialeah, FL).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and Western blot analysis
SDS/PAGE was carried out on 12% or 14% polyacrylamide gels by the method of Laemmli [11 ]. Briefly, cells were lysed in buffer consisting of 1% Triton X-100, 10 mM Tris (pH 7.4), 0.15 M NaCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 25 µg/ml phenylmethylsulfonyl fluoride (PMSF) for 5 min on ice. Cells were centrifuged at 13,000 rpm for 10 min at 4°C, and the supernatant was collected. An aliquot was mixed with equal parts of 2 x sample loading buffer and was denatured at 100°C for 5 min. Protein concentration in the lysates was determined using a protein assay kit (Bio-Rad, Hercules, CA), and each sample was loaded at a concentration of 100 µg/lane in the gel. After electrophoresis, proteins were transferred onto nitrocellulose membranes and probed with 1.3 µg/ml anti-galectin-3 mAb or 1 µg/ml anti-mouse CD3{varepsilon} Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and appropriate secondary antibodies. The blot was developed by chemiluminescence (Amersham, Arlington Heights, IL).

Detection of galectin-3 by using asialofetuin-Sepharose 4B
Asialofetuin was immobilized on CNBr-activated Sepharose 4B according to the manufacturer’s instructions. To investigate the binding affinity of galectin-3 to asialofetuin, 1 x 107-activated T lymphocytes were lysed. After centrifugation at 13,000 rpm for 10 min at 4°C, the supernatant was incubated with 10 µl asialofetuin-Sepharose 4B in the absence or presence of 250 mM lactose or sucrose with bidirectional agitation at 4°C for 1 h. After the beads were washed three times with the lysis buffer, the bound and subsequently eluted protein was analyzed by Western blot using anti-galectin-3 mAb.

Subcellular localization of galectin-3
Activated T lymphocytes were centrifuged, washed in HBSS, and resuspended at a cell density of 5 x 107 cells/ml in lysis buffer containing 10 mM Hepes (pH 7.4), 38 mM NaCl, 25 µg/ml PMSF, 1 µg/ml leupeptin, and 1 µg/ml aprotinin [12 ]. The cell suspension was homogenized using a Dounce homogenizer and centrifuged at 900 g to pellet the nuclei. The postnuclear supernatant was centrifuged at 130,000 g to pellet the membrane compartments. The membrane and nuclear pellet were resuspended in a volume of Triton X-100 containing lysis buffer equal to that of cytosolic supernatant. The relative expression of galectin-3 and CD3{varepsilon} in cytosolic, crude membrane, and nuclear fractions was analyzed by Western blot.

Anti-sense studies
Purified phosphorothioated sense and anti-sense oligonucleotides were designed for the inhibition of galectin-3 expression in activated T lymphocytes. Sense oligonucleotides (5' AGG AAA ATG GCA GAC AGC) specific for mouse galectin-3 gene [9 ] and the complementary anti-sense oligonucleotides (5' GCT GTC TGC CAT TTT CCT) were synthesized and purified by Oligos Etc. (Wilsonville, OR). Activated T lymphocytes pretreated with Con A were prepared by Lympholyte®-M and then cultured with sense or anti-sense oligonucleotides. The efficacy of oligonucleotides was determined by flow cytometry analysis. Thymidine incorporation was assessed after exposure to 1 µCi [3H] thymidine (ICN Pharmaceuticals, Irvine, CA) during the last 18 h of culture. For apoptosis analysis, annexin-V and PI staining were used. Cells were stained using Annexin-V FITC kit (BioSource International, Camarillo, CA) and analyzed by an Epics C cytometer (Coulter).

Statistical analysis
Each experiment was performed at least three times. The statistical significance of the experimental data was evaluated by Student’s t-test. P < 0.05 was accepted as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectin-3 is expressed in activated T lymphocytes but not in resting cells
The expression level of galectin-3 was examined in resting and activated T lymphocytes from the spleen and lymph nodes. Consistent with earlier observations [8 , 9 ], no significant expression of galectin-3 was observed in resting lymphocytes including CD4+, CD8+ T lymphocytes (Fig. 1A and B ) and B cells (unpublished results) by intracellular and surface staining. However, galectin-3 expression was detected in activated T lymphocytes (Fig. 1C and 1D) , which were pretreated with 5 µg/ml Con A and incubated with 100 IU/ml IL-2-containing medium, and LPS-activated B cells (unpublished results). In double-staining results of flow cytometry analysis, CD4+ and CD8+ T lymphocytes expressed galectin-3 molecules after activation. Similar results were observed with resting and activated T lymphocytes of lymph nodes (unpublished results).



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Figure 1. Galectin-3 is expressed in activated T lymphocytes. Murine spleen cells were double-stained for CD4 or CD8 (cell surface) and for galectin-3 (intracellular). Analysis was performed by flow cytometry. Resting splenic CD4+ or CD8+ T lymphocytes do not express intracellular galectin-3 (A and B). CD4+ and CD8+ T lymphocytes activated with Con A (5 µg/ml) for 48 h and then cultured in IL-2-containing media (100 IU/ml) for 96 h express intracellular galectin-3 (C and D).

 
Activation is required for galectin-3 expression in T lymphocytes
We investigated the relationship between activation and galectin-3 expression by using intracellular staining and flow cytometry analysis. Murine spleen cells were incubated with Con A, anti-CD3 mAb, or medium alone for 48 h and then incubated with IL-2 (100 U/ml) or medium alone for another 48 h. It is interesting that the expression level of galectin-3 in T lymphocytes activated with mitogen and IL-2 was significantly higher (P<0.05) than that of T lymphocytes activated with mitogen only (Fig. 2 ). However, IL-2 alone did not enhance galectin-3 expression in T lymphocytes. These data suggest that an activation signal is required for the induction of galectin-3 expression, but this activation signal by itself is insufficient to enhance galectin-3 expression.



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Figure 2. Activation is required for galectin-3 expression in activated T lymphocytes. Spleen cells at 2 x 106 cells/ml were cultured in the absence or presence of plate-bound anti-CD3 mAb (145-2C11) or 5 µg/ml Con A for 48 h and then cultured in the absence or presence of 100 IU/ml IL-2. Cells were stained intracellularly with anti-galectin-3 mAb followed by anti-rat IgG-FITC and assayed by flow cytometry. Mean fluorescence intensity (MFI) and percent-positive of each sample were obtained through subtraction of value obtained using galectin-3 mAb from the value obtained using rat IgG as primary Ab. *, P < 0.05; **, P < 0.01 as compared with the medium alone.

 
Galectin-3 expression is regulated by common {gamma}-chain signaling cytokines
Previously, it was demonstrated that galectin-3 expression in macrophages was induced by inflammatory stimuli [13 ]. Surprisingly, in our study, common {gamma}-chain signaling cytokines, such as IL-4 and IL-7, enhanced galectin-3 expression significantly (P<0.01), whereas other cytokines including inflammatory cytokines, IFN-{gamma}, TNF-{alpha}, GM-CSF, TGF-ß, and IL-10 did not affect the expression level (Table 1 ). Furthermore, IL-2, IL-4, and IL-7 increased the viability of activated T lymphocytes significantly, whereas the other cytokines had no effect. The effects of the common {gamma}-chain signaling cytokines on galectin-3 expression and T cell viability were similar for Con A- and anti-CD3 mAb-activated T lymphocytes. These observations suggest that activated T lymphocytes require specific cytokines, such as IL-2, IL-4, or IL-7, for survival, which is associated with an increased expression of galectin-3.


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Table 1. Effects of Various Cytokines Including IL-2, IL-4, and IL-7 on Intracellular Galectin-3 Expression and Viability of Activated T Lymphocytes

 
Characterization of galectin-3 in activated T lymphocytes
To determine the molecular weight of galectin-3 in activated T lymphocytes, Western blot analysis was performed using an anti-galectin-3 mAb. Galectin-3 expressed in activated T lymphocytes was recognized by the rat anti-galectin-3 mAb (M3/38) as a single band, and the molecular weight was 35 kDa (Fig. 3A ). In addition, galectin-3 analysis in the mouse 3T3 cells was used as a positive control; M3/38 mAb detected a band at the same molecular weight, whereas no band was detected in the human leukemia cell line, Jurkat (Fig. 3A) . These results are consistent with previous data showing that galectin-3 is a 35 kDa molecule in mouse 3T3 cells and is not expressed in Jurkat T cells [7 , 14 ]. We measured the change of galectin-3 expression in a time-dependent manner after incubation in IL-2 (100 IU/ml)-containing medium. Kinetics experiments showed that the expression level of galectin-3 increased from 0–48 h after activation of lymphocytes with Con A (Fig. 3B) . To investigate the binding affinity of galectin-3 in activated T lymphocytes to galactose residues, galectin-3 molecules were harvested from T cell lysates by using asialofetuin beads in the presence of lactose or sucrose (Fig. 3C) . The binding of galectin-3 to asialofetuin beads was inhibited completely by lactose but not by sucrose at a concentration of 250 mM. This binding affinity was consistent with previous data [9 ].



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Figure 3. Galectin-3 is expressed in activated T cells and has binding affinity to asialofetuin with lactose specificity. (A) Western blot analysis of cell lysates using anti-galectin-3 mAb. T cells were stimulated with 5 µg/ml Con A and incubated in IL-2-containing medium. (B) Enhancement of galectin-3 in T cells activated with 5 µg/ml Con A for 48 h. Analysis was started when T cells were transferred into medium plus 100 IU/ml IL-2 (lane 1, t=0). (C) Galectin-3 molecules from activated T lymphocytes were assayed for their specificity for galactoside residues. Total cell lysate from 2.5 x 106 cells was analyzed in lane 1. Lysates from 1 x 107 cells were mixed with asialofetuin-conjugated beads (lanes 2–4) in the absence (lane 2) or presence (lane 3) of lactose or sucrose (lane 4) at 250 mM. The bound and subsequently eluted protein was probed with anti-galectin-3 mAb in Western blot.

 
Activated T lymphocytes express intracellular galectin-3 predominantly
Surface and subcellular localization of galectin-3 was determined in activated T lymphocytes. No significant surface expression of galectin-3 on thymocytes, resting spleen cells, or activated T lymphocytes could be detected by flow cytometry analysis, whereas the pancreatic cancer cell line, CAPAN I, was highly positive (Fig. 4A ). Intracellular galectin-3 molecules were found in the nucleus, cytoplasm, and membrane fractions of activated T lymphocytes (Fig. 4B) .



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Figure 4. Galectin-3 is expressed predominantly in intracellular compartments. The surface expression level of galectin-3 was measured by flow cytometry in thymocytes, resting spleen cells, T lymphocytes activated for 1 week with Con A/IL-2, and pancreatic tumor cells, Capan I, as positive control (A). The activated T lymphocytes were incubated in the presence of 100 U/ml IL-2. Cell lysates were separated into nucleus, cytosolic, and membrane fractions and analyzed by Western blot with anti-galectin-3 mAb or anti-CD3{varepsilon} Ab (B).

 
Calcium ionophores enhance the spontaneous secretion of galectin-3
To investigate whether galectin-3 is secreted from activated T lymphocytes, supernatants from T cell cultures were evaluated for the presence of galectin-3 by Western blot analysis. Activated T lymphocytes were cultured in IL-2 (100 IU/ml)-containing medium for 1 week after Con A stimulation. The viability was 95–98%. After 1 week, the T cells (5x106 cells/ml) were kept in medium with or without calcium ionophore A23187 (2.5 µM or 10 µM) or ionomycin (1 µg/ml or 4 µg/ml) for 3 h. Subsequently, supernatants were harvested by centrifugation and used for evaluation of galectin-3. Soluble galectin-3 was detected in the supernatants of activated T lymphocytes incubated in IL-2-containing medium alone, suggesting a constitutive secretion of galectin-3 (Fig. 5 ). In addition, the amount of secreted galectin-3 was increased after incubation of cells in both calcium ionophores.



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Figure 5. Galectin-3 is secreted by activated T lymphocytes spontaneously, and the secretion can be enhanced by calcium ionophores. Activated T lymphocytes stimulated with Con A/IL-2 for 1 week were incubated in medium alone (lane 1), with calcium ionophore A23187 (lanes 2+3) or with ionomycin (lanes 4+5) for 3 h. Each supernatant was harvested by centrifugation and then assayed for the amount of secreted galectin-3 molecules by Western blot analysis.

 
IL-2 withdrawal decreases galectin-3 expression and cell viability
To further investigate whether galectin-3 expression is correlated with viability of activated T lymphocytes, we examined the effect of IL-2 withdrawal on galectin-3 expression. Spleen cells were activated with 5 µg/ml Con A for 2 days and subsequently incubated in 100 IU/ml IL-2 for more than 5 days. The medium was changed every 2 days. The culture consisted of 93–95% T lymphocytes after 1 week. The activated T lymphocytes were centrifuged on Lympholyte®-M and stained by PI for the evaluation of viability. Galectin-3 expression was decreased from 100% to 69 ± 10% at 8 h after IL-2 withdrawal (Fig. 6A ), whereas the percentage of cells with hypodiploid DNA content only increased marginally from 1–2% (Fig. 6B) . Similarly, at 24 and 48 h after IL-2 withdrawal, the expression of galectin-3 had decreased much more rapidly than the viability. Thus, IL-2 withdrawal decreased galectin-3 expression and cell viability.



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Figure 6. IL-2 withdrawal decreases galectin-3 expression and viability in activated T lymphocytes. Activated T lymphocytes, which were pre-activated with 5 µg/ml Con A and then cultured in IL-2-containing media (100 IU/ml) for more than 5 days, were used. After washing the cells twice with PBS, the cells were incubated in medium alone for 48 h. The viability was determined by trypan blue exclusion, and MFI was calculated after intracellular staining (A). The percentage of cells with hypodiploid DNA content was determined by flow cytometry analysis after staining with 50 µg/ml PI (B).

 
Galectin-3 expression is correlated with cumulative cell number
It is interesting that the common {gamma}-chain signaling cytokines, IL-2, IL-4, and IL-7, showed different effects on cumulative cell number, whereas these cytokines showed a similar effect on T cell viability from days 2–6 after activation of lymphocytes. At the same time, galectin-3 expression correlated with an increase of the cumulative cell number. Although at day 2, the cells treated with IL-4 were similar in cell number and galectin-3 expression as those treated with IL-2 at days 4 and 6, cell number and galectin-3 expression were higher in the IL-2-supplemented culture than in the IL-4- or IL-7-supplemented cultures (Fig. 7 ). Although the viability of IL-7-supplemented cultures was similar to that of IL-2- or IL-4-supplemented cultures (88–95%), the cells treated with IL-7 did not increase in cell number and galectin-3 expression (Fig. 7) .



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Figure 7. Increase of cumulative cell number is associated with galectin-3 expression. Activated T lymphocytes stimulated with Con A (5 µg/ml) for 48 h were cultured in the absence or presence of 100 IU/ml IL-2, 250 U/ml IL-4, or 250 U/ml IL-7 for 6 days. Each cytokine-containing medium was replaced with new medium containing an equal amount of cytokine every 2 days. The cell number was counted by trypan blue exclusion (A), and galectin-3 staining (B) was performed as described in Materials and Methods.

 
Irradiation blocks cell proliferation and decreases galectin-3 expression
After culture of activated T lymphocytes in IL-2-, IL-4-, or IL-7-containing medium for 6 days, we investigated the change of galectin-3 expression induced by irradiation. Indeed, irradiation blocked proliferation of T lymphocytes and also decreased galectin-3 expression (Fig. 8 ). With regard to galectin-3 expression, the activated T lymphocytes treated with IL-2 or IL-4 were affected with irradiation to a lesser extent than those cells treated with IL-7. This observation suggests that galectin-3 expression is correlated to proliferation of activated T lymphocytes.



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Figure 8. Irradiation blocks T cell proliferation and decreases galectin-3 expression. Activated T lymphocytes, which were incubated in 100 IU/ml IL-2, 250 U/ml IL-4, or 250 U/ml IL-7 for 6 days after Con A stimulation, received 1000 rad irradiation. The cells were cultured 24 h after irradiation and measured for intracellular galectin-3 by flow cytometry. The number represents MFI.

 
Inhibition of galectin-3 expression by anti-sense oligonucleotides decreases the proliferation of activated T lymphocytes
To investigate the function of galectin-3 further, activated T lymphocytes were treated with sense or anti-sense oligonucleotides specific for murine galectin-3. Activated T lymphocytes pretreated with Con A were incubated in IL-2-containing media in the presence of sense or anti-sense oligonucleotides. The inhibitory effect of anti-sense oligonucleotides on galectin-3 expression was measured by flow cytometry analysis. Anti-sense oligonucleotides inhibited the galectin-3 expression, but sense oligonucleotides did not (Fig. 9A ). At the tested concentration (10 µM), sense oligonucleotides did not show a decrease in viability or morphological changes (unpublished results). Anti-sense oligonucleotides inhibited the proliferation of activated T lymphocytes significantly at various cell concentrations (P<0.01 at <=1.6x105, P<0.05 at 5x105 cells/ml), but sense oligonucleotides did not (Fig. 9B) .



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Figure 9. Inhibition of galectin-3 expression by anti-sense oligonucleotides decreases the proliferation of activated T lymphocytes. Activated T lymphocytes pretreated with Con A were incubated in the presence of IL-2. The cells were treated with 10 µM sense or anti-sense oligonucleotides for 2 days and then analyzed for galectin-3 expression. The number represents MFI (A). Activated T lymphocytes were cultured in 10 µM concentration of sense or anti-sense oligonucleotides at various cell concentration for 2 days (B). The [3H]-thymidine incorporation was measured after exposure to 1 µCi [3H] thymidine during the last 18 h of culture. Results are normalized to control cells, grown in medium only.

 
Correlation of DNA synthesis and galectin-3 expression
To strengthen the correlation between proliferation and galectin-3 in activated lymphocytes, we performed BrdU labeling and double-stained for BrdU and galectin-3 (Fig. 10 ). Activated lymphocytes were 45–50% BrdU-positive in the presence of IL-2, whereas <5% cells were labeled in the absence of IL-2 (Fig. 10A) . We compared the expression level of galectin-3 in BrdU-positive and -negative cells. The galectin-3 expression level in BrdU-positive cells was higher than in BrdU-negative cells based on mean fluorescence and percentage positive cells (Fig. 10B) .



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Figure 10. Galectin-3 is expressed at higher level in BrdU-positive cells than in BrdU-negative cells. Activated T lymphocytes pretreated with ConA were cultured in the absence or presence of IL-2 (100 U/ml) for 2 days. Cells were incubated with BrdU at a concentration of 10 µM for the last 2 h and labeled with anti-BrdU Ab-PE, anti-galectin-3 mAb and anti-rat IgG-FITC.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although galectin-3 has been studied in tumor cells and other immune cells [1 , 3 , 9 ], the expression and functional studies of galectin-3 in T lymphocytes have not been shown previously. We demonstrated here that galectin-3 is expressed clearly in activated T lymphocytes but not in resting T cells, and activated CD4+ and CD8+ T lymphocytes express galectin-3. An activation signal, provided by anti-CD3 mAb, ConA, or other, is required for induction of galectin-3, and the expression is enhanced by common {gamma}-chain signaling cytokines such as IL-2, IL-4, and IL-7.

To investigate the function of galectin-3 in activated T lymphocytes, we designed anti-sense oligonucleotides specific for murine galectin-3 [9 ]. Anti-sense oligonucleotides inhibited the up-regulation of galectin-3 by IL-2, whereas sense oligonucleotides tested at the same concentration did not affect galectin-3 expression. Previous results demonstrated that galectin-3 transfected into Jurkat cells modulated the growth and apoptosis of Jurkat cells [7 ]. Thus, proliferation and apoptosis analysis were performed by thymidine incorporation, annexin-V, and PI staining in the presence of sense or anti-sense oligonucleotides. Anti-sense oligonucleotides inhibited the proliferation of activated T lymphocytes significantly at various cell concentrations (Fig. 9) . However, we did not detect any evidence of apoptosis in spite of the multiple attempts.

Several possibilities might explain this discrepancy. First, it is possible that the effect of galectin-3 down-regulation by anti-sense oligonucleotides may be overcome by anti-apoptotic molecules, such as bcl-2 and bcl-xL, which are increased sharply by IL-2 in activated T lymphocytes [15 ]. Although binding affinity of galectin-3 for bcl-2 was demonstrated [7 ], it is not clear that bcl-2 binds galectin-3 functionally in activated T lymphocytes. Second, there are significant differences between the two experimental systems. Whereas Jurkat cells do not express galectin-3 naturally, activated T lymphocytes express significant amounts of various anti-apoptotic molecules. Thus, it is possible that anti-apoptotic effects observed in Jurkat cells may not appear in activated T lymphocytes treated with IL-2, because Jurkat cells are well-known to be sensitive to various apoptotic signals. Taken together, our results suggest that galectin-3 is involved in proliferation of activated T lymphocytes mainly, although it is still possible that galectin-3 has anti-apoptotic effects.

Because galectin-3 had been found on the surface of thioglycollate-elicited macrophages, it is known to be related with inflammation [1 , 13 ]. Surprisingly, galectin-3 expression in T cells is increased sharply by common {gamma}-chain signaling cytokines, such as IL-2, IL-4, and IL-7, but not by various inflammatory cytokines including IFN-{gamma} and TNF-{alpha}. This observation suggests that galectin-3 may be involved in another, physiological immune response including activated lymphocytes. The observation that common {gamma}-chain signaling cytokines increase the viability and proliferation of activated T lymphocytes is consistent with previous studies [15 , 16 ].

Galectin-3 is a member of the family of animal lectins with ß-galactoside binding affinity. Although galectin-1 and galectin-3 are included in the same family, the two proteins demonstrated different effects on lymphocytes. Recombinant human galectin-1 induces apoptosis in thymocytes and activated T lymphocytes and inhibits IL-2 production [17 , 18 ].

We demonstrated here that activated T lymphocytes secreted galectin-3 spontaneously, and the secretion could be enhanced by calcium ionophores. To evaluate the effects of secreted galectin-3, we measured the proliferation and viability of activated T lymphocytes in the presence of anti-galectin-3 mAb or control antibody. However, we did not detect significant effects of anti-galectin-3 mAb at a range of 1–10 µg/ml (unpublished results). The expression level of intracellular galectin-3 in viable cells treated with and without ionophores was similar (unpublished results). Therefore, it seems that the rate of synthesis of galectin-3 has increased, but the newly synthesized protein does not accumulate in the cells. The amount of secreted galectin-3 was decreased rapidly from the cells after IL-2 withdrawal by Western blot analysis (unpublished results). Therefore, IL-2 withdrawal-induced apoptosis is unlikely a result of secreted galectin-3.

Galectin-3 has been shown to be an adhesion molecule that binds to the extracellular matrix including laminin [19 ]. Galectin-3 is expressed on the surface of macrophages and tumor cells and closely correlated with the formation of tumor metastasis [5 , 13 , 20 ]. To investigate the presence of surface galectin-3 on T lymphocytes, we measured the expression level on thymocytes, resting spleen cells, and activated T lymphocytes by flow cytometry analysis. In our study, no galectin-3 molecules were detected on the surface of lymphocytes in spite of the multiple attempts. However, the finding that galectin-3 is released from activated T cells spontaneously suggests that secreted galectin-3 may be involved in the adhesion of activated T lymphocytes to other immune cells.

Intracellular galectin-3 in activated T lymphocytes is distributed in the nucleus, cytosol, and membrane compartments. The nuclear and cytosolic localization of galectin-3 was demonstrated previously [2 , 21 ]. In our study, galectin-3 was detected in membrane fractions as well as nuclear and cytosolic fractions. CD3{varepsilon} molecules used as control were detected in nuclear and membrane fractions (Fig. 4) . The observation that CD3{varepsilon} molecules are localized in the nucleus is in agreement with previous observations by Nakano et al. [22 ]; CD3{varepsilon} is present in the nuclear fraction, and the expression is increased upon T cell activation.

This study is the first to show the expression and function of galectin-3 in normal T lymphocytes. In summary, galectin-3 expression is induced by activation signals including TcR-mediated activation and enhanced by common {gamma}-chain signaling cytokines, IL-2, IL-4, and IL-7. Galectin-3 is located predominantly in intracellular compartments not on the cell surface, and intracellular galectin-3 plays an important role in the proliferation of activated T lymphocytes.


    ACKNOWLEDGEMENTS
 
Financial support for this work was received in part from the National Institutes of Health grants CA60662 and CA68500 and the postdoctoral fellowship program of the Korea Research Foundation.

Received June 28, 2000; revised November 22, 2000; accepted November 22, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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