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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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain signaling
cytokines, IL-2, IL-4, and IL-7, in activated T lymphocytes, whereas
the inflammatory cytokines including TNF-
and IFN- 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 -chain signaling cytokines • proliferation • anti-sense
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 -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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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)-, IL-10, murine rIL-4, IL-7, interferon
(IFN)-, 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
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
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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain signaling
cytokines-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-, TNF-, 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 -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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-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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1.6x105,
P<0.05 at 5x105 cells/ml), but sense
oligonucleotides did not (Fig. 9B) .
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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
-chain signaling cytokines, such as IL-2, IL-4, and IL-7, but not by
various inflammatory cytokines including IFN- and TNF-. This
observation suggests that galectin-3 may be involved in another,
physiological immune response including activated lymphocytes. The
observation that common -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 molecules used as control were detected in nuclear
and membrane fractions (Fig. 4) . The observation that CD3 molecules
are localized in the nucleus is in agreement with previous observations
by Nakano et al. [22
]; CD3 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 -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.
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ACKNOWLEDGEMENTS |
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Received June 28, 2000; revised November 22, 2000; accepted November 22, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bcl-xs) gene expression Eur. J. Immunol. 26,294-299[Medline]
chain of the T cell receptor complex J. Biol. Chem. 271,6483-6489This article has been cited by other articles:
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