By
From the * Department of Pathology and Laboratory Medicine, Molecular Biology Institute, and § Departments of Pediatrics, Microbiology and Immunology and the Jonsson Comprehensive Cancer
Center, University of California Los Angeles School of Medicine, Los Angeles, California 90095
Galectin-1, a -galactoside binding protein, is produced by thymic epithelial cells and binds to
human thymocytes. We have previously reported that galectin-1 induces the apoptosis of activated T lymphocytes. Because the majority of thymocytes die via apoptosis while still within
the thymus, we tested whether galectin-1 could induce the apoptosis of these cells. We now
report that in vitro exposure to galectin-1 induced apoptosis of two subsets of CD4lo CD8lo
thymocytes. The phenotypes of susceptible thymocytes were consistent with that of both negatively selected and nonselected cells. Galectin-1-induced apoptosis was enhanced by preexposure of thymocytes to antibody to CD3, suggesting that galectin-1 may be a participant in T-cell- receptor mediated apoptosis. In contrast, pretreatment of thymocytes with dexamethasone had
no effect on galectin-1 susceptibility. We noted that 71% of the cells undergoing apoptosis after
galectin-1 treatment had a DNA content greater than 2N, indicating that proliferating thymocytes were most sensitive to galectin-1. We propose that galectin-1 plays a role in the apoptosis of both negatively selected and nonselected thymocytes, and that the susceptibility of thymocytes to galectin-1 is regulated, in part, by entry or exit from the cell cycle.
During T cell maturation in the thymus, only a fraction
of thymocytes are selected to survive and ultimately
migrate out of the thymus as naïve mature T lymphocytes.
The maturation pathway that results in thymocyte survival
is termed positive selection (1). Thymic epithelial cells,
which express MHC molecules, mediate positive selection.
Thymocytes will undergo positive selection when they express a TCR, which can bind with low avidity to self-class I or class II (MHC) (1). During positive selection, thymocytes convert from a CD4+CD8+, double positive (DP)1, phenotype to a CD4+ or a CD8+ single positive (SP), phenotype. In addition thymocytes become CD3hi, CD5hi, and
CD69+ during positive selection (2, 3).
The majority of thymocytes will not undergo positive
selection, but will die by apoptosis within the thymus (4).
Thymocytes that express a functional TCR but are potentially autoreactive will die as a result of negative selection.
After negative selection, thymocytes express intermediate
levels of CD3 (CD3int) and low levels of CD4 and CD8
(CD4lo, CD8lo) immediately before undergoing apoptosis
(5, 6, 7). Thymocytes that do not express a functional
TCR cannot undergo positive or negative selection. This
nonselected population also dies by apoptosis (4).
Thymocyte maturation requires the participation of thymic epithelial cells and extracellular matrix components (8,
9, 10). We reported the expression of galectin-1, a Galectin-1 is a member of a family of animal lectins that
share structural similarities within the carbohydrate binding
domain (13, 14). Galectins are also referred to as S-type
lectins because these molecules require reduced thiol groups
to maintain carbohydrate binding activity. Galectin-1 exists
as a homodimer of 14-kD subunits, is evolutionarily conserved, and developmentally regulated. The preferred carbohydrate ligand for galectin-1 is lactosamine (Gal- Because galectin-1 is abundant within normal thymus
and can mediate the apoptosis of activated T lymphocytes,
we undertook this study to determine whether galectin-1
could mediate the apoptosis of human thymocytes. We report that galectin-1 induced the apoptosis of two distinct
populations of immature thymocytes. Our data is consistent
with a role for galectin-1 in the induction of apoptosis of
negatively selected and nonselected thymocytes.
Preparation of Recombinant Human Galectin-1.
Galectin-1 was prepared by the method of Couraud et al. (23) in Escherichia coli strain
BL21 (DE3) transformed with the expression vector pT7IML-1
(gift of Incyte Pharmaceuticals, Inc., Palo Alto, CA).
Cell Culture and Galectin-1 Treatment.
Human thymocytes were
isolated from surgical specimens and passaged over nylon wool as
previously described (24). Nylon wool passaged thymocytes were
cultured overnight at 37°C in serum-free IMDM with 1% dialyzed BSA, and 85 µg/ml human transferrin (AT-IMDM). The
following day, 106 cells in 0.2 ml of AT-IMDM, 1.1 mM dithiothreitol (DTT) were incubated for 5 h at 37°C with 20 µM recombinant, human galectin-1. DTT was added to maintain the
carbohydrate binding activity of galectin-1. Samples were adjusted to 0.1 M Dexamethasone and CD3 Antibody Pretreatment.
Thymocytes
(2.5 × 106 cells/ml) were incubated with 2 µg/ml of soluble antibody to CD3 TUNEL Labeling and Assessment of DNA Content.
DNA content and apoptosis were assessed by TUNEL using a modification
of the method of Gorczyca (25). Paraformaldehyde-fixed cells
were washed in PBS, resuspended in cold ( We incubated nylon wool passaged thymocytes with and without
galectin-1. Galectin-1 induced apoptosis of a significant
fraction of thymocytes (Fig. 1). In galectin-1-treated samples, 37 ± 5.9% (SEM) of the cells were apoptotic with a
range of 21-41%. In control samples, treated with ATIMDM, 1.1 mM DTT, an average of 22.2 ± 5.9% of the
cells (range, 11-33.5%) were apoptotic. Thus, galectin-1 treatment consistently induced apoptosis of ~15% of the
total thymocyte population.
To determine the stage of differentiation at which thymocytes were most susceptible to galectin-1, we examined
the viable cells in galectin-1-treated and control samples
for expression of a number of cell surface markers. We observed that galectin-1 treatment eliminated a fraction of the
CD3
The apoptotic effects of CD3 stimulation and steroids antagonize each other when the treatments are given simultaneously (26, 27). To determine
whether galectin-1 induces apoptosis via a pathway that interacts or overlaps with that involved in apoptosis induced
by steroids or CD3 antibody, we pretreated thymocytes for
16 h with dexamethasone, or with CD3 antibody before
exposure to galectin-1 (Fig. 3). As expected, the level of
apoptosis in dexamethasone pretreated samples was higher
than in the nontreated controls (19% and 11%, respectively). However, after correcting for background apoptosis, dexamethasone pretreatment did not alter the fraction
of the total population that was susceptible to galectin-1 (10% as compared with 10.9% in controls). These data indicate that galectin-1-induced apoptosis of thymocytes was
independent of steroid pretreatment.
In contrast, pretreatment of the thymocytes with soluble
antibody to CD3 did not alter the background level of apoptosis, but did result in a fourfold increase in the fraction
of cells susceptible to galectin-1-induced apoptosis (40%
above control levels as compared with 10% without CD3 antibody pretreatment). These results demonstrate that engagement of the TCR with CD3 antibodies before galectin-1 exposure increased the sensitivity of thymocytes to
galectin-1.
Previous results from our laboratory showed that galectin-1 induces apoptosis of peripheral T cells that are activated and proliferating, whereas resting peripheral T cells are not susceptible to galectin-1-induced apoptosis (12). Others have
reported cell cycle-dependent differences in the susceptibility of cells to apoptosis in a number of systems (25, 28,
29, 30). To determine at which stage(s) of the cell cycle
thymocytes were undergoing apoptosis, we examined the
DNA content of galectin-1-treated and -untreated thymocytes. By selective gating of TUNEL labeled thymocytes, we found that in untreated samples, the majority of apoptotic thymocytes (74%) had a DNA content greater
than 2N, characteristic of S, G2, or M phase cells (data not
shown). When a greater fraction of the total population was
induced to undergo apoptosis by galectin-1, a comparable
percentage (71%, Fig. 4) of the TUNEL-positive (apoptotic) cells had a DNA content greater than 2N. In contrast, 29% of the total sample and 16% of the TUNEL-negative cells had a DNA content greater than 2N (Fig. 4).
DNA content was not altered by the TUNEL labeling procedure (data not shown). This observation strongly suggests
that thymocytes that are progressing through the cell cycle
are most sensitive to apoptosis and most susceptible to galectin-1.
We have investigated the effects of galectin-1 on thymocyte survival and have examined the surface phenotype
of thymocyte populations before and after galectin-1 treatment. Our results indicate that, after correction for background apoptosis, exogenous galectin-1 induced apoptosis
of 15% of human thymocytes (Fig. 1). This is a significant
fraction when one considers that the thymocytes were exposed to galectin-1 in vivo less than 48 h before use in this
study. Additionally, we found that the susceptible thymocytes were all of an immature phenotype.
Susceptibility to galectin-1 correlated with a number of
events that occur during thymocyte differentiation. The
phenotype of galectin-1-sensitive cells and how galectin-1
susceptibility relates to thymocyte differentiation is shown
schematically in Fig. 5. Examination of the surface phenotype of thymocytes before and after galectin-1 treatment
suggests galectin-1 may be involved in apoptosis of both
nonselected and negatively selected thymocytes.
There were primarily two galectin-1-susceptible populations as determined by flow cytometry. One of the two
populations was CD3 The second susceptible population was CD3intCD4lo
CD8loCD69 Treatment of thymocytes in vitro with antibody specific
for the CD3 If galectin-1 induces apoptosis after negative selection,
then thymic epithelial (TE) cells may participate in negative selection to a greater extent than has previously been
proposed. It is known that TE cells synthesize galectin-1
(11), can induce apoptosis of T cells (7, 38) and can mediate negative selection (39, 40). In our laboratory, we observed that 53% of MOLT-4 cells that bound to TE cells
on glass cover slips were apoptotic after 4 h at 37°C,
whereas only 7% of CEM cells bound to TE cells were apoptotic (data not shown). This is intriguing in light of our previous observation that MOLT-4 cells are sensitive to galectin-1-induced apoptosis, whereas CEM cells are not (12).
Although TE cells can mediate negative selection, dendritic
cells have been reported to do this more efficiently (2). If
galectin-1 is required for negative selection, one would predict that dendritic cells may also synthesize galectin-1, or else
acquire it from the extracellular milieu. This remains to be
investigated.
Three distinct pathways leading to apoptosis of thymocytes have been identified (41). The first requires the
expression of the tumor suppressor protein p53 and can be
induced by DNA damage. The other two pathways, one
induced by steroid treatment, the other by TCR engagement, are p53 independent (41). It has been reported
that DP thymocytes do not express p53 (31). Our data
showing that only DP thymocytes are susceptible to galectin-1 suggest that galectin-1 also induces apoptosis by a p53
independent pathway.
Galectin-1 may be a component of TCR-mediated apoptosis. Ligation of CD3 primed thymocytes for galectin1-induced apoptosis and resulted in an increased fraction of
cells that were susceptible to galectin-1 (Fig. 3). This data is
similar to that found using murine thymocytes treated with
CD3 antibody and galectin-1 (Vespa, G.N.R., and M.C.
Miceli, manuscript in preparation). This synergism between
CD3 antibody and galectin-1 suggests that galectin-1 may be a normal component in the process by which TCR engagement induces apoptosis. Engagement of the TCR, as
occurs during negative selection, may induce intracellular
expression of apoptotic machinery, which is then activated
by galectin-1. This hypothesis is supported by the kinetics
of CD3-mediated apoptosis, which is slow (10-18 h) when
compared with galectin-1-induced apoptosis, which occurs rapidly (1-4 h).
Galectin-1-induced apoptosis was independent of that
induced by steroids. In contrast with the effects of CD3 antibody pretreatment, we observed no effect of dexamethasone pretreatment on galectin-1 susceptibility (Fig. 3). Our
observation that dexamethasone neither antagonizes, nor
augments galectin-1-mediated apoptosis indicates that the
mechanisms leading to steroid and galectin-1-mediated apoptosis can operate independently.
Galectin-1 susceptibility may be regulated by entry of a
vulnerable cell into the cell cycle. We observed that galectin-1 preferentially kills proliferating cells. This observation
is consistent with a previous proposal that the susceptibility
of thymocytes to apoptosis is controlled by entry or exit
from the cell cycle (44). We found that only two subsets of
the DP thymocytes were susceptible to galectin-1 (Fig. 2).
Whereas the majority of DP thymocytes are not dividing,
10-15% of the total thymocyte population is composed of
DP cells that are undergoing cell division (45, 46). This
number corresponds to the average fraction of thymocytes
that we found were susceptible to apoptosis by galectin-1 (15%). Thus, the previously reported size of the population
of proliferating DP thymocytes is sufficient to account for
the fraction of galectin-1-sensitive thymocytes that we observed.
Entry into the cell cycle is unlikely to be the only factor
that determines galectin-1 susceptibility. Thymocytes, which
are in direct contact with TE cells during maturation, may
be in continuous contact with galectin-1 in vivo (11).
However, proliferating thymocytes do not all undergo apoptosis simultaneously in vivo or in vitro. A number of additional factors potentially influence the response of thymocytes to galectin-1. These factors include (a) the expression
of specific cell surface glycoprotein counter receptors recognized by galectin-1; (b) the glycosylation of those specific
counter receptors; (c) the expression of Bcl-2 family members; and (d) the presence of intracellular apoptotic machinery. The latter may be influenced by TCR engagement, which
occurs during negative and positive selection. All of these
factors would affect galectin-1 binding and signal transduction, and most likely combine to determine the susceptibility
of specific thymocyte populations to galectin-1. It is important to note that lactosamine, the carbohydrate ligand
for galectin-1, can be found in varying amounts on more than one cell surface glycoprotein counter receptor. In addition, the dimeric nature of galectin-1 would permit intermolecular and intramolecular cross-linking of cell surface
glycoproteins. Thus, a response to galectin-1 may require
the expression of more than a single glycoprotein species
on the cell surface.
Based on the data presented above and the constitutive
expression of galectin-1 in normal thymus, we hypothesize
that galectin-1 is a participant in the elimination of nonselected and possibly negatively selected cells during normal
thymocyte maturation. A galectin-1 knock out mouse has
been generated (47). This model may be useful for further
investigation of the role of galectin-1 in thymocyte development.
-galactoside binding protein, by human thymic epithelial (TE)
cells in vivo and in vitro. We further demonstrated that galectin-1 bound to thymocytes and mediated the adhesion
of thymocytes to TE cells (11). Recently, we have found
that galectin-1 can induce apoptosis of activated T lymphocytes (12).
1,
4GlcNAc), which can be present on many glycoprotein counterreceptors (14). A number of glycoproteins have
been identified that bind to galectin-1, including laminin
(15), fibronectin (16), lysosome-associated membrane proteins (LAMPS) (17), and the hematopoietic cell surface
membrane proteins CD45 and CD43 (18). Several laboratories have ascribed growth regulatory (19, 20) and/or immunomodulatory activities (21, 22) to galectin-1.
-lactose to dissociate aggregated thymocytes,
washed in 10 mM phosphate buffer, pH 7.4, 140 mM NaCl
(PBS), then either surface stained or fixed with 1% paraformaldehyde for terminal deoxynucleotidyl transferase-mediated dUTP
biotin nick end-labeling (TUNEL) to detect apoptotic cells. Samples were surface stained as previously described (11). PE-conjugated CD3 antibody was used in addition to fluorescein-conjugated antibody to discriminate more easily between cells lacking
CD3 expression (CD3
) and cells expressing low levels of CD3
(CD3lo) (10). The following mouse anti-human antibodies, directly conjugated to fluorochromes were used: CD3-FITC,
CD4-PE, and CD8-peridinin chlorophyll protein (PerCp; Becton Dickinson, San Jose, CA), CD3-PE, CD8-FITC, and CD69-PE
(Caltag, South San Francisco, CA). Isotype-matched controls
were included for all reagents. After surface staining and washing,
cells were either immediately subjected to flow cytometry, or
fixed in 1% paraformaldehyde and stored at 4°C for flow cytometry, which was performed within 24 h. All flow cytometry was
performed using a Becton Dickinson FACScan® with analysis using Lysis II\xa9 or CELLQuest\xa9 software.
(UCHT-1, Dako Corp., Carpinteria, CA), with
20 µM dexamethasone (Calbiochem, La Jolla, CA), or with medium alone at 37°C. After 16 h, galectin-1 or buffer was added. The samples were then incubated, stained, and fixed as described above.
20°C) 80% ethanol and kept on ice for 30 min. Cells were washed in PBS, then incubated at 37°C for 1 h with terminal deoxytransferase (TdT, 120 U/ml; Promega Corporation, Madison, WI), 1× TdT reaction
buffer (provided with enzyme), and biotin-16-2
-deoxyuridine5
-triphosphate (biotin-dUTP, 10 nmol/ml; Boehringer Mannheim, Indianapolis, IN) in a total volume of 50 µl. After washing,
incorporated biotin-dUTP was stained with streptavidin-FITC
(10 µg/ml, Boehringer Mannheim) in PBS with 1% BSA, 0.05%
Triton X-100, and 0.01% NaN3 for 1 h at room temperature.
Samples were washed in the same buffer, then in PBS, 1% BSA,
and stained for DNA content with 1 µg/ml 7-aminoactinomycin D (7-AAD; Molecular Probes, Eugene, OR), or propidium iodide (PI, Calbiochem, Inc., San Diego, CA) with 0.01% RNAase
A, 30 min before flow cytometry was performed. Control samples were treated as above, except the TdT enzyme was omitted.
Galectin-1-induced Apoptosis of Human Thymocytes.
Fig. 1.
Galectin-1 induces apoptosis of thymocytes. Thymocytes
were treated with 1.1 mM DTT (open bars), or with 20 µM galectin-1, 1.1 mM DTT (closed bars) in AT-IMDM medium for 5 h at 37° C. The percentage of apoptotic cells in each sample was determined by flow cytometric analysis after TUNEL labeling. The results shown are duplicate
determinations from three experiments (E1-E3) ± SD.
[View Larger Version of this Image (12K GIF file)]
population, as indicated by comparing absolute
numbers of cells within the region defined by M1 in Fig. 2
A. We also consistently saw a decrease in the population
expressing intermediate levels of CD3 (CD3int, Fig. 2 A;
M3). The same samples were stained by three-color staining using CD3-FITC, CD4-PE, and CD8-PerCP. The
results shown in Fig. 2 B indicate that galectin-1 selectively
eliminated a subset of the CD4+ CD8+ double-positive
(DP) cells that were dim for both markers. CD3 staining in
these samples (data not shown) was consistent with that
shown in Fig. 2, A and C. By comparing absolute numbers
of cells in control and galectin-1-treated samples, we determined that none of the CD4
CD8
double-negative (DN)
cells were lost (data not shown). This indicates that the
CD3
cells that were susceptible to galectin-1 were expressing CD4 and CD8, and were not in the triple negative
fraction. Galectin-1 also eliminated thymocytes from the
CD5lo (data not shown) and CD69
(Fig. 2 C) populations. In summary, the thymocyte populations most susceptible to galectin-1-induced apoptosis were CD3int or
CD3
and CD4loCD8loCD5loCD69
.
Fig. 2.
Distinct subsets of immature thymocytes were susceptible to
galectin-1. Cells treated with galectin-1 or DTT only were surface stained
for CD3-PE (A), CD4-PE and CD8-PerCP (B), or CD3-FITC and
CD69-PE (C). The population within the region circled in B indicates
the CD4loCD8lo population, which comprised 41.2% of the DP cells in
the control sample (left) and 17.3% of the DP cells after galectin-1 treatment (right). In A and C absolute numbers of cells and the percentage in
each marker region or quadrant are indicated in the tables to the right of
the figures. 10,000 events (live plus dead cells) were acquired for each
sample in A, and C, and 5,000 events for each sample in B. Nonviable
cells were excluded from the data shown, by selective gating based on
light scatter profiles. In B and C, appropriate isotype control antibodies
were used to set the cursors.
[View Larger Version of this Image (29K GIF file)]
Fig. 3.
Galectin-1-induced apoptosis of thymocytes was enhanced by
preincubation with CD3 antibody, but not by preincubation with dexamethasone. Thymocytes were preincubated with medium (A), 20 µM
dexamethasone (B), or with 1 µg/ml CD3 antibodies (C) for 16 h before
a 5-h exposure to DTT alone, or to galectin-1 plus DTT. The percentage
of apoptotic cells was determined by flow cytometry after TUNEL staining. The results shown are representative of duplicate determinations.
[View Larger Version of this Image (32K GIF file)]
Fig. 4.
Cells in S, G2, or M (with a DNA content greater than 2N)
were most susceptible to apoptosis mediated by galectin-1. Thymocytes were treated with galectin-1, then stained for apoptosis by nick end-labeling (TUNEL) and for DNA content with propidium iodide. Apoptotic
cells are TUNEL positive. The numbers given indicate the percentage of
each population with a DNA content greater than 2N and were determined using the marker as indicated.
[View Larger Version of this Image (24K GIF file)]
Fig. 5.
Model indicating
galectin-1-sensitive populations
within the human thymus. Asterisk indicates a galectin-1-sensitive phenotype.
[View Larger Version of this Image (11K GIF file)]
CD4loCD8loCD69
(Fig. 2). The
expression of CD4 and CD8 normally occurs during thymocyte ontogeny subsequent to the expression of the TCR
chain (31, 32). Therefore, those DP-susceptible thymocytes that were detected as CD3
by flow cytometry
had already attempted TCR rearrangement. The population that carries this surface phenotype has been shown to
express the intracellular regulator of apoptosis, Bcl-xL, but not Bcl-2 (33, 34). Within this group may be cells that express the pre-T
chain but have unsuccessfully rearranged
a TCR
chain (35). Because this population did not express detectable levels of surface CD3, these cells could not
undergo negative selection. It is reasonable to propose that
these cells represent the nonselected population, i.e., those
thymocytes that fail to express a functional TCR and die
because they are not positively selected. Our observation that DP, CD3
thymocytes are sensitive to galectin-1 suggests a role for galectin-1 in apoptosis due to nonselection.
. This same phenotype precedes apoptosis after negative selection (5). This population may also include
thymocytes that are nonselected because they express a
nonfunctional TCR. Although the viable CD69+ population was not reduced by galectin-1 treatment, 58% of the apoptotic cells were found to express low levels of CD69
(CD69lo, data not shown). This fraction of CD69lo apoptotic thymocytes is similar to that observed by Kersh and Hedrick (5) for murine thymocytes undergoing negative
selection. Our data suggest that a fraction of the CD69
thymocytes became CD69lo upon treatment with galectin-1.
This issue warrants further investigation.
chain mimics antigen stimulation and is used
as a model for negative selection (36). CD3 antibody treatment of thymocytes increases the CD3intCD4loCD8lo population, as does antigen engagement during negative selection (5, 6). Using in vitro models, specific peptide and
MHC binding or CD3 antibody stimulation alone was not
sufficient to induce apoptosis, and a second, unknown, signal was required as well (6, 7). This second signal can be
delivered by some antigen-presenting cell lines, thymic epithelial cells, or dendritic cells (6, 7). Previous attempts to
identify a molecule that mediates this second signal have
yielded conflicting results (6, 36, 37). Galectin-1 is potentially a candidate molecule as a provider of a second (apoptotic) signal needed for negative selection. Our observations
that CD3intCD4loCD8lo cells were eliminated by galectin-1
treatment and that galectin-1 susceptibility was enhanced
by CD3 engagement (Fig. 3) support this hypothesis, and
suggest a role for galectin-1 in negative selection.
Address correspondence to Linda G. Baum, Department of Pathology and Laboratory Medicine, UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095.
Received for publication 11 December 1996 and in revised form 18 February 1997.
1 Abbreviations used in this paper: 7-AAD, 7-amino actinomycin D; DP, double positive; DN, double negative; DTT, dithiothreitol; LAMPS, lysosome associated membrane proteins; PI, propidium iodide; SP, single positive; TdT, terminal deoxytransferase; TE, thymic epithelial; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.We wish to thank Drs. H. Spits, M.C. Miceli, S.M. Hedrick, and L. Goodglick for critical review of the manuscript. We also wish to thank Drs. M.C. Miceli and G.N.R. Vespa for very helpful discussions. We are grateful to the staff of the Jonsson Cancer Center Flow Cytometry core laboratory for technical assistance.
This work was supported by training grant no. CA09056 from USPHS to N.L. Perillo, by National Institutes of Health grant no. AG104015 through the Claude D. Pepper Older American Independence Center, and grants from the Concern Foundation and the University of California Cancer Research Coordinating Committee to L.G. Baum, and by National Institutes of Health grant no. HD29341 to C.H. Uittenbogaart.
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