| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Microbiology and Immunology and
Pathology and Laboratory Medicine, and
Molecular Biology Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Galectin-1 is a highly evolutionarily conserved ß-galactoside binding protein demonstrated to have immunomodulatory and growth regulatory activities (1). Galectin-1 is expressed as a noncovalently linked homodimer with two ligand binding sites capable of mediating cell/cell interactions by binding to saccharide ligands on apposing cell surfaces. It has no transmembrane domain but remains cell associated by binding to glycoproteins on the surface of the cell producing it. It is expressed in a number of tissues including epithelial cells and APCs in the lymph node, spleen, and thymus (1, 2, 3). Galectin-1 can bind T cells and thymocytes and has been demonstrated to mediate interactions between T cells and galectin-1 expressing endothelial cells (3). Lactosamine is the minimal oligosaccharide structure recognized by galectin-1, though glycoproteins with multiple and complex sugar structures are likely the preferred counterreceptors for galectin-1. Indeed, CD43, CD45, CD4, CD3, and CD7 have been reported to be primary T cell glycoproteins bound by galectin-1 (1, 3). Furthermore, Abs directed against galectin-1 as well as CD45 and CD43 glycoproteins can disrupt thymic epithelial cell/T cell interactions (3). Although initial reports indicated that CD45/galectin-1 interactions may modulate galectin-1-induced apoptosis (9), subsequent studies indicate that this may not always be the case (J.T.N., M.C.M., L.G.B., unpublished observations).
In addition to mediating cell/cell interactions, galectin-1, binding to oligosaccharides containing multiple lactosamine units on T cell surface proteins, may facilitate multimerization or crosslinking and therefore induce signal transduction by these proteins (1). Recent models of TCR activation suggest that CD45 and CD43 must be excluded from the TCR/APC contact site at the same time CD4 and other accessory molecules are recruited in order for efficient T cell activation events to occur (10). Galectin-1 binding may additionally modify this T cell surface-molecular reorganization and thus influence the degree of TCR multimerization (11), inclusion of coreceptor or costimulatory molecules within TCR aggregates, and potentially the functional outcome of antigenic stimulation (10).
Because galectin-1 is expressed in the thymus (3) and induces apoptosis in thymocytes (12), galectin-1 binding may contribute to cell death during thymic selection. Galectin-1 is also expressed in peripheral immune organs including the lymph node and spleen, as well as at sites of immune privilege and on tumor cells, and has been demonstrated to induce apoptosis in activated peripheral T cells (1). Therefore, galectin-1 binding may also contribute to apoptosis of mature T cells in the periphery (9). Because of its abundant expression at sites of self and antigenic presentation, it is likely that a T cell might simultaneously encounter galectin-1 and TCR ligand. However, the consequences of galectin-1 stimulation in the context of coordinate TCR engagement have not yet been examined. Furthermore, most experiments examining galectin-1 T cell immunomodulatory activity have been performed using human T cells, T cell lines, or thymocytes. Here we determine the signal transduction and biological consequences of stimulating a mouse T cell hybridoma or freshly isolated mouse thymocytes with galectin-1 alone or coordinately with anti-TCR Abs. We demonstrate that TCR stimuli that otherwise lead to IL-2 production or proliferation in a T hybridoma and freshly isolated thymocytes, respectively, are converted into efficient stimuli for apoptosis in the context of galectin-1 stimulation. Examination of the TCR signals affected reveal that galectin-1 does not uniformly intensify or dampen all TCR signals. However, a subset of TCR signal transduction events are intensified when TCR is engaged in the presence of galectin-1.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human galectin-1 was purified from Escherichia coli transformed with the expression vector pT7IML-1 as previously described (13). Galectin-1 was stored in 8 mM DTT at -70°C and used in all procedures in medium containing 1.0–1.2 mM of DTT.
Cells, Abs, and Annexin V
BI-141 is a CD4-CD8- MHC class
II-restricted murine T cell hybridoma that recognizes beef insulin in
the context of IA
bßk (14). The BI-141
transfectants expressing F505Lck or R273F505Lck have been described
(6). Thymocytes were obtained from C57Bl/6 females (4–8 wk) and single
cell suspensions were made using standard procedures. Abs against the
TCR ß-chain (H57-597) (15), and CD3
(145-2C11) (16) were purified
from hybridomas. Antisera against extracellular signal-regulated
kinase-2 (ERK-2)3 (C-14) and
4G10 antiphosphotyrosine Ab were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA) and Upstate Biotechnology (Lake Placid, NY),
respectively.
For analysis of thymic subpopulations, 4 x 105
thymocytes were incubated at 37°C with media containing 0.3, 0.6, or
1.2 mM DTT alone, together with 5, 10, or 20 µM galectin-1 and/or
1.25 µg/ml plate-bound anti-CD3 for 5 h. Cells were
resuspended in 0.1 M ß-lactose for 1 min at room temperature, washed
twice with PBS, and incubated with phycoerythrin conjugated
anti-CD4 and biotinylated anti-CD8 (PharMingen, San Diego, CA)
for 30 min at 4°C. Cells were then washed and incubated with
streptavidin tricolor conjugate (Caltag, Burlingame, CA) for 30 min at
4°C, washed, and stained with 2.5 µl Annexin V-FITC for 15 min on
ice as per the manufacturer’s recommendations (PharMingen). Annexin V
positive cells were electronically gated out using Cellquest software
(Becton Dickinson, San Jose, CA), and cell death was calculated
based on the number of live cells in the treated sample relative to the
number in the media alone control containing a comparable concentration
of DTT using the following equation: 100 x [1 - (no. of
Annexin V negative galectin-1 and/or anti-CD3 treated cells)/(no.
of Annexin V negative control media treated cells)]. There were 50,000
total events analyzed in each sample.
Measurement of cell death by propidium iodide (PI) staining
BI-141 or thymocytes at concentration of 2 x 106 or 0.5 x 106 cells/ml, respectively, were incubated at 37°C in medium with 1.2 mM DTT and 20 µM of galectin-1 in the presence or absence of 1 µg/ml of plate-bound or soluble anti-TCR mAbs. Samples were adjusted to 0.1 M lactose to dissociate galectin-1-agglutinated cell clumps and washed 12 h poststimulation. Cell death was measured by staining cells with 0.2 ml of 2 µg/ml PI. PI staining was detected using a FACScan (Becton Dickinson), and data were analyzed using Cellquest software. The percentage of induced death was calculated relative to the percentage of live cells obtained in the unstimulated culture using the following equation: induced death = 100 x [1 - (% treated live cells/% untreated live cell)].
Measurement of IL-2 production
BI-141 (0.5 x 106) cells were stimulated as above. Supernatants were harvested 12 h poststimulation and assayed for IL-2 production by ELISA as per the manufacturer’s recommendations (Endogen, Cambridge, MA).
Measurement of cell proliferation
Thymocytes at concentration of 1 x 106 cells/ml were cultured at 37°C in media containing 1.2 mM DTT and 20 µM of galectin-1 and/or 1 µg of 2C11. Cultures were pulsed for the last 16–18 h of a 30-h incubation with 1 µCi of [3H]thymidine/well, DNA were harvested on glass filters, and counts incorporated were determined using a scintillation counter.
Antiphosphotyrosine Western blot analysis
T cells (2.5 x 106/100 µl) were incubated
with medium alone or with 5 µg anti-CD3 for 30 min on ice and
then incubated for 5 min at 37°C with different stimuli (20 µM
galectin-1, 1.2 mM DTT, or rabbit anti-hamster IgG) (Cappel,
Durham, NC). Cells were washed in RPMI 1640 and lysed in 50 µl of TNE
(50 mM Tris (pH 8.0), 1% Nonidet P-40, and 2 mM EDTA), supplemented
with protease and phosphatase inhibitors for 30 min on ice. Lysates
were boiled in Laemmli buffer containing 2-ME, resolved on 10%
SDS-PAGE, and transferred to nitrocellulose. Immunoblot analysis was
performed using antiphosphotyrosine mAb 4G10 at 1 µg/ml and detected
with 10 µCi [125I] Protein A (ICN, Irvine, CA) and
proteins were visualized by autoradiography.
Measurement of mitogen-activated protein (MAP) kinase/ERK activity
T cells (5 x 106/100 µl) were
incubated with complete medium alone or with 5 µg anti-CD3 for
30 min at 4°C and then incubated for 5 min at 37°C with various
stimuli (20 µM galectin-1, 1.2 mM DTT, or rabbit anti-hamster
IgG). Cells were pelleted and lysed with ERK lysis buffer (25 mM HEPES
(pH 7.6), 0.1% Triton X-100, 20 mM ß-glycerolphosphate, 10 mM
p-nitrophenylphosphate, 150 mM NaCl, and 1 mM
Na2VO3) supplemented with protease inhibitors
for 30 min at 4°C. ERK-2 was immunoprecipitated and in vitro ERK
kinase activity measured using myelin basic protein (MBP) as an
exogenously added substrate essentially as described (17).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Because APCs and stromal cells at sites of T cell antigenic
stimulation express galectin-1 (1), it is likely that an Ag-specific T
cell might encounter both Ag and galectin-1. Therefore, we were
interested in determining the effects of galectin-1 stimulation in the
context of TCR engagement. BI-141 T hybridoma cells were stimulated
with a suboptimal concentration of anti-TCR ß-chain, H57-597, or
with a more optimal concentration of 145-2C11 anti-CD3 alone or
together with purified recombinant galectin-1. We have previously
reported that the BI-141 T hybridoma both secretes IL-2 and undergoes
apoptosis in response to TCR stimulation (6). Here we demonstrate that
this murine T hybridoma also undergoes apoptosis in response to
galectin-1 (Fig. 1
A).
Furthermore, suboptimal stimulation of plate-bound H57-597, unable to
induce apoptosis by itself, enhances the ability of galectin-1 to
induced apoptosis in the BI-141 T cell hybridoma (Fig. 1A).
The ability of galectin-1 and TCR engagement to cooperate in signaling
apoptosis is also evident when optimal concentrations of plate-bound
anti-CD3 (145-2C11) are used in combination with galectin-1
(Fig. 1A). Differences between results obtained with 2C11
and H57-597 relate to the efficacy of the concentration of Ab used
rather than the TCR/CD3 epitope against which they are directed (data
not shown). Similar results are seen when cells are incubated with
plate-bound anti-TCR Ab 12 h before the addition of galectin-1
(results not shown).
|
alone or in combination with galectin-1. As
shown in Fig. 1
B, simultaneous addition of galectin-1
impairs the ability of anti-CD3 to produce IL-2. These results are
not the effect of galectin-1 interfering with IL-2 detection, because
similar inhibition of detection was neither observed when rIL-2 was
directly diluted in an equivalent concentration of galectin-1 nor was
IL-2 activity recovered by preabsorbing galectin-1 from supernatants
using a lactosyl Sepharose column before IL-2 assay (data not shown).
Inhibition of IL-2 production is not likely the result of killing
IL-2-producing cells before they are able to produce sufficient
quantities of IL-2, because maximal levels of IL-2 can be produced by
BI-141 under conditions in which death due to 2C11 alone is as high as
80% (results not shown). Therefore, significant levels of IL-2 can be
produced under conditions leading to death of most of the cells. Galectin-1 cooperates with anti-TCR Abs to induce apoptosis but antagonizes the ability of anti-TCR Abs to induce proliferation in freshly isolated thymocytes
The thymus is a primary site of T cell apoptosis. During positive
and negative selection, TCR engagement cues the apoptotic elimination
of autoreactive and nonfunctional thymocytes and the rescue and
development of T cells bearing TCRs likely to be useful in the
peripheral immune system. Because galectin-1 is expressed by thymic
epithelial and some APCs (1, 2), and has been demonstrated to mediate
thymic epithelial/T cell interactions (9), we were interested in
determining the effects of stimulating freshly isolated mouse
thymocytes with galectin-1 alone or in coordinately with
anti-CD3 Ab. Mouse thymocytes were isolated from 6 wk C57Bl/6
mice and stimulated with galectin-1 and anti-CD3 individually or
in combination. The effects of stimulation with both plate-bound and
soluble anti-CD3 were assessed. Cells were stained with PI and
induced cell death was calculated relative to the percentage of live
cells in unstimulated controls 9 h after stimulation (Fig. 2A). To assess the affects of
galectin-1 on anti-CD3-induced proliferation, tritiated
thymidine uptake was used to measure proliferation of cultures within
30 h of stimulation (Fig. 2B). Fig. 2A
demonstrates that galectin-1 induces cell death in freshly isolated
mouse thymocytes. Furthermore, whereas stimulation of thymocytes with
anti-CD3 alone results in proliferation, coordinate stimulation
with anti-CD3 and galectin-1 inhibits proliferation and induces
even greater apoptosis than treatment with galectin-1 alone (Fig. 2, A and B).
|
To determine which mouse thymic subpopulations undergo apoptosis
in response to anti-CD3/galectin-1 treatment, thymocytes were
coordinately stimulated with varying concentrations of galectin-1 and
anti-CD3 for 5 h. Cells were stained for expression of CD4 and
CD8 and for apoptotic cells using Annexin V and subjected to FACS
analysis. To avoid potential complications due to the nonspecific
uptake of Abs by dead cells, Annexin V positive apoptotic and
preapoptotic cells were electronically gated out and CD4/CD8 expression
on the galectin-1/CD3 insensitive subpopulation examined. Annexin V
negative thymocytes treated with control media show typical relative
proportions of CD4+, CD8+ single positive, and
CD4+CD8+ double positive thymocyte
subpopulations (data not shown). Consistent with data reporting that
human CD4+CD8+ double positive thymocytes
undergo apoptosis in response to galectin-1 alone (12), we observed
that galectin-1 treatment also induced apoptosis in
CD4+CD8+ mouse thymocytes (Fig. 3). However, unlike findings with human
thymocytes in which galectin-1 only induced apoptosis in 
5–15% of
thymocytes (12), we found that the majority of double positive mouse
thymocytes were susceptible to galectin-1 treatment, particularly at
the higher concentrations of galectin-1 tested. Coordinate engagement
of CD3 enhanced the ability of galectin-1 to induce apoptosis in double
positive thymocytes, especially at suboptimal concentrations of
galectin-1 (Fig. 3A). Furthermore, concomitant CD3
engagement and galectin-1 stimulation resulted in apoptosis of a subset
of the CD4-CD8-,
CD4-CD8+, and
CD4+CD8- subpopulations not susceptible to
galectin-1 treatment alone (Fig. 3A).
|
and 3, cell death was determined by the ability of T
cells to exclude PI or bind Annexin V because these assays
allowed for the most accurate quantitation of the total number of cells
dying in response to various stimuli. However, in experiments in which
Annexin V was used in combination with PI staining, an Annexin
V+/PI negative population appeared before the appearance of
th PI positive population (results not shown). These data demonstrate
that galectin-1/TCR stimulation results in the initiation of an
apoptotic program rather than necrosis and are consistent with
previously published reports demonstrating that galectin-1 stimulation
of T cells and TCR stimulation of the BI-141 hybridoma result in
apoptotic-specific events including altered morphology and DNA
fragmentation (6, 11, 12). Similarly, we have detected DNA
fragmentation in response to galectin-1 and galectin-1/anti-CD3
treatment using a TUNEL-based assay (data not shown). Galectin-1 does not uniformly amplify all TCR signals
The TCR initiates signal transduction through the rapid activation of intracellular tyrosine kinases including Lck, Fyn, ZAP70, and the subsequent tyrosine phosphorylation of signal transduction proteins (18). Because T cell surface galectin-1 counterreceptors CD45, CD43, CD3, and CD4 associate with Lck and/or Fyn (19, 20), we examined whether stimulation with galectin-1 directly modifies protein tyrosine phosphorylation or affects TCR-induced protein tyrosine phosphorylation.
As shown in Fig. 4A,
galectin-1 stimulation of BI-141 T cells resulted in the rapid tyrosine
phosphorylation of proteins migrating within at least two bands in the
120- to 130-kDa range. Stimulation with anti-CD3 resulted in less
efficient protein tyrosine phosphorylation of proteins within the 120-
to 130-kDa range and in significant phosphorylation of a 105-kDa
protein (Fig. 4). TCR-induced protein tyrosine phosphorylation of each
of these bands is intensified when goat anti-hamster (GAH) Ig is
included as a crosslinking reagent. Furthermore, phosphorylation of
p115, p70, p42, and p23 kDa (phosphorylated 
-chain) proteins is
additionally detected upon CD3 crosslinking. Coordinate stimulation
with anti-CD3 and galectin-1 results in the tyrosine
phosphorylation of the 120- to 130-kDa proteins to levels comparable
with those seen with galectin-1 alone in addition to inducing the
tyrosine phosphorylation of proteins phosphorylated by anti-CD3
alone (i.e., p105) (Fig. 4). However, the phosphorylation pattern
observed with coordinate galectin-1 and anti-CD3 stimulation is
qualitatively different from that seen with anti-CD3 and
crosslinking GAH, indicating that galectin-1 is not simply acting to
crosslink the TCR or uniformly amplify all TCR signals. Of note the
anti-CD3/GAH-induced p70, p36, and p23 (corresponding to the m.w.
of ZAP70, LAT, and , respectively) are not detected in lysates from
cells coordinately stimulated with galectin-1 and CD3 stimulation,
whereas the 120- to 130-kDa galectin-1-induced bands as well as the
p105 band seen with anti-CD3/GAH crosslinking are readily
detectable (Fig. 4).
|
Because Lck has been reported to associate with the galectin-1
counterligands CD45 (18, 21), CD43 (20, 22), and CD4 (19), and is known
to be pivotal in initiating TCR-induced signals for IL-2 production, we
investigated the role of Lck kinase activity in mediating galectin-1
signals. To this end, we assessed the ability of galectin-1 to
stimulate apoptosis in BI-141 transfectants expressing constitutively
active (F505Lck) and kinase inactive (R273F505Lck) mutant forms of Lck.
We have previously demonstrated that these transfectants are
dramatically modified in their ability to mediate Lck-dependent protein
tyrosine phosphorylation and IL-2 production (6, 23). Although the
expression of F505 enhances Lck-mediated TCR activation events, the
expression of R273F505 leads to the inhibition of Lck-mediated TCR
activation events. We have also reported that TCR-mediated apoptosis in
BI-141 is relatively Lck kinase activity independent in so far as it is
unaffected by the expression of either F505Lck or R273F505Lck (6). As
shown in Fig. 5, the parental BI-141
cells and both F505Lck and R273F505 transfectants respond similarly to
galectin-1 or coordinate galectin-1/TCR stimulation. These data
demonstrate that unlike T cell activation events known to require Lck
kinase activity, the ability of galectin-1 to induce apoptosis or
cooperate with the TCR to induce apoptosis is not dramatically affected
by the manipulation of Lck kinase activity and thus indicate that
galectin-1 is not highly dependent on Lck kinase activity to mediate
these functions.
|
MAP kinases are evolutionarilly conserved mediators of a wide
variety of signal transduction pathways contributing to biological
processes including induction of differentiation, apoptosis, and
proliferation (24). TCR-mediated tyrosine phosphorylation events are
known to result in the activation of the ERK/MAP kinase cascade (18).
To determine whether galectin-1 stimulation induces ERK activation by
itself or whether galectin-1 influences the ability of the TCR to
induce ERK activity, BI-141 and thymocytes were stimulated with
galectin-1 alone or in combination with anti-CD3. ERK was
immunoprecipitated using anti-ERK-2 mAb and immunoprecipitates were
subjected to in vitro kinase assay with MBP as an exogenous substrate.
As shown in Fig. 6, A and
B, galectin-1 and anti-CD3 signals synergize to
dramatically up-regulate ERK activity in both the BI-141 T cell
hybridoma (Fig. 6, A and B) and thymocytes (Fig. 6, C and D). Although galectin-1 and TCR each
stimulate low levels of ERK activity, coordinate stimulation with both
galectin-1 and anti-CD3 induces significantly more ERK activity
than either reagent alone. ERK activation in response to
anti-CD3/galectin-1 is quite rapid, occurring within 1 min of
stimulation, peaking at five minutes, and significantly diminishing by
20 min poststimulation (data not shown). Control stimulations with
media containing DTT alone (Fig. 6, C and D) or
in combination with anti-CD3 (Fig. 6, A–D) demonstrate that the effects observed are
due to galectin-1 rather than DTT present in the stimulating media.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We further demonstrate that coordinate galectin-1 stimulation and TCR engagement result in enhanced apoptosis in the CD4+CD8+ population, especially at suboptimal concentrations of galectin-1. Because galectin-1 is expressed by APCs and thymic epithelial cells, our finding that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis in CD4+CD8+ thymocytes may implicate a role for galectin-1 in TCR-directed apoptosis during negative selection. The electronic gates defining CD4+CD8+ double positive cells in our FACS analysis include transitional subpopulations reported to be sensitive to negative selection (5). Indeed, it is possible that galectin-1 is the thymic costimulatory ligand reportedly required for efficient TCR-induced negative selection (26).
Furthermore, we demonstrate that engagement of the TCR during galectin-1 stimulation induces apoptosis in CD4-CD8-, CD4-CD8+, and to a lesser extent CD4+CD8- subpopulations that are insensitive to galectin-1 simulation alone. The fact that the CD4-CD8- subpopulation is affected by CD3 stimulation at some concentrations of galectin-1 may implicate a role for galectin-1 in modulating pre-TCR signals. Moreover, that CD4+CD8- and CD4-CD8+ subpopulations can be induced to undergo apoptosis in response to galectin-1 treatment in the context of TCR engagement indicate that galectin-1 can modulate TCR-mediated responses in mature T cells and may be relevant to the ability of galectin-1 to modulate TCR-mediated responses in the periphery. In keeping with this idea, we demonstrate that TCR engagement and galectin-1 stimulation cooperate to induce apoptosis in the BI-141 T cell hybridoma.
A previous study reports that 16 h of pretreatment with anti-CD3 increases human thymocyte susceptibility to subsequent 5 h of galectin-1-induced apoptosis (12). However, whether increased susceptibility correlated with the number of cycling cells and which thymic subpopulation was effected were not determined. These data were interpreted to suggest that anti-CD3 treatment may induce intracellular transcription of apoptotic machinery, which is subsequently activated by galectin-1 (12). Assuming that similar phenomena occur in mouse and human thymocytes, our data, demonstrating that enhanced apoptosis is observed within 5 h of CD3/galectin-1 costimulation, indicate that extensive pretreatment with anti-CD3 is not necessary. If anti-CD3 does prime thymocytes for subsequent galectin-1-induced death, such a priming event would have to occur within 1–2 h of TCR engagement. Alternatively, signals resulting from CD3 and galectin-1 coordinate engagement may cooperate to enhance apoptosis.
In this manuscript, we additionally provide evidence that galectin-1 modulates TCR-mediated IL-2 production and proliferation. Although galectin-1 cooperates with TCR engagement to induce apoptosis, it antagonizes TCR-induced proliferation and IL-2 production in thymocytes and T hybridoma cells, respectively. Additional experiments are required to determine which T cell subpopulation is susceptible to the inhibition of TCR-induced proliferation and to determine whether this effect is secondary to the ability of galectin-1 to inhibit TCR-induced IL-2 production. However, the fact that individual TCR-mediated functions are differentially affected by galectin-1 costimulation in the BI-141 T cell hybridoma indicates that galectin-1 independently modulates TCR-mediated signals and functions.
To determine whether galectin-1 stimulation modifies early TCR signal transduction events, we examined the abilities of galectin-1, anti-CD3, and galectin-1/anti-CD3 to affect protein tyrosine phosphorylation and ERK activity. We demonstrate the galectin-1 induces protein tyrosine phosphorylation but does not uniformly amplify all TCR-induced tyrosine phosphorylation. Furthermore, galectin-1/TCR-mediated cell death is resistant to the dominant negative expression of impaired forms of Lck that are capable of modulating TCR-induced IL-2 production. We also demonstrate that galectin-1 synergizes with anti-CD3 to activate ERK. These data indicate that galectin-1 differentially influences particular TCR-induced signals. The fact that galectin-1 differentially modulates CD3-induced protein tyrosine phosphorylation, ERK-2 activity, and downstream functions may account for galectin-1 immunomodulatory properties.
The fact that galectin-1 and TCR cooperate to induce both apoptosis and ERK activation in T cells implicates a potential role for ERK in affecting apoptosis. Experiments are underway to determine whether ERK activity is required for galectin-1/TCR-induced apoptosis or inhibition of IL-2 production and which thymic subpopulation is responsible for the synergistic TCR/galectin-1 ERK activation observed in lysates from total thymocytes. The role of ERK activity in positive and negative selection has been indirectly assessed through the expression of dominant negative upstream regulators of ERK activity, dominant negative Ras, and dominant negative MAP kinase kinase-1 (MEK-1) (27). Interestingly, mice expressing dominant negative forms of Ras and MEK-1 are severely impaired in TCR-mediated positive selection, though negative selection remains unaffected. Interpretation of the effects of disrupting Ras- and MEK-1-mediated ERK activation with regard to the role for galectin-1-induced ERK activity in negative and/or positive selection requires additional characterization of the signaling mechanism through which galectin-1 induces ERK activation. Furthermore, studies comparing thymocyte development in TCR transgenics on wild-type and galectin-1 null (28) backgrounds should lend additional insight as to whether galectin-1 contributes to thymocyte development and which TCR signal transduction pathways are affected by galectin-1 stimulation.
The ability of galectin-1 stimulation to cooperate with TCR signals to mediate apoptosis and inhibit IL-2 production may also be relevant to TCR-directed apoptosis involved in peripheral tolerance induction, immune privilege, or tumor escape from immune surveillance (1). Indeed, galectin-1 is expressed in a number of immune-privileged sites including testes, cornea, brain, placenta, and prostate and by malignancies including ovarian and colon carcinomas (1). If peripheral T cells, like single positive thymocytes and hybridoma cells, respond to coordinate TCR and galectin-1 signals by inducing apoptosis and/or inhibiting IL-2 production and proliferation, cells expressing TCRs with specificities for Ag expressed in immune privileged tissues or tumors would be particularly susceptible to galectin-1 immunomodulatory activity. The ability of galectin-1 to enhance TCR-induced cell death and inhibit IL-2 production may provide a molecular basis for the finding that galectin-1 can prevent disease induction in rat experimental autoimmune encephalomyelitis (EAE) model (29). Indeed, galectin-1 modulation of EAE and down-regulation of lymphokine production is reminiscent of the immunomodulatory capacity of some antagonist peptides that have been demonstrated to interfere with the onset of EAE by shifting a Th1 response to a Th2 response (30). That galectin-1 is an endogenous protein with the potential to independently modulate particular antigenic responses without requiring prior knowledge of the Ag peptide recognized or homogeneity of the specificity of the T cell response makes galectin-1 an intriguing candidate for modulating TCR-mediated T cell responses in vivo. Our demonstration that galectin-1 affects apoptosis and differentially affects TCR signals and functions in mouse T cells and thymocytes lays the groundwork for studies that capitalize on the availability of genetic variants and the ability to manipulate immune responses in the murine system to begin to address these issues.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. M. Carrie Miceli, Molecular Biology Institute, University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095. E-mail address:
3 Abbreviations used in this paper: ERK-2, extracellular signal-regulated kinase-2; PI, propidium iodide; MBP, myelin basic protein; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling; GAH, goat anti-hamster; MAP, mitogen-activated protein.
Received for publication February 11, 1998. Accepted for publication October 7, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
ß T cell receptors. J. Immunol. 142:2736.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. Bi, L. A. Earl, L. Jacobs, and L. G. Baum Structural Features of Galectin-9 and Galectin-1 That Determine Distinct T Cell Death Pathways J. Biol. Chem., May 2, 2008; 283(18): 12248 - 12258. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Pacienza, R. G. Pozner, G. A. Bianco, L. P. D'Atri, D. O. Croci, S. Negrotto, E. Malaver, R. M. Gomez, G. A. Rabinovich, and M. Schattner The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation FASEB J, April 1, 2008; 22(4): 1113 - 1123. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. V. Tribulatti, J. Mucci, V. Cattaneo, F. Aguero, T. Gilmartin, S. R. Head, and O. Campetella Galectin-8 Induces Apoptosis in the CD4highCD8high Thymocyte Subpopulation Glycobiology, December 1, 2007; 17(12): 1404 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kubach, P. Lutter, T. Bopp, S. Stoll, C. Becker, E. Huter, C. Richter, P. Weingarten, T. Warger, J. Knop, et al. Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function Blood, September 1, 2007; 110(5): 1550 - 1558. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Walzel, A. A. Fahmi, M. A. Eldesouky, E. F. Abou-Eladab, G. Waitz, J. Brock, and M. Tiedge Effects of N-glycan processing inhibitors on signaling events and induction of apoptosis in galectin-1-stimulated Jurkat T lymphocytes Glycobiology, December 1, 2006; 16(12): 1262 - 1271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Perone, S. Bertera, Z. S. Tawadrous, W. J. Shufesky, J. D. Piganelli, L. G. Baum, M. Trucco, and A. E. Morelli Dendritic Cells Expressing Transgenic Galectin-1 Delay Onset of Autoimmune Diabetes in Mice J. Immunol., October 15, 2006; 177(8): 5278 - 5289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Hernandez, J. T. Nguyen, J. He, W. Wang, B. Ardman, J. M. Green, M. Fukuda, and L. G. Baum Galectin-1 Binds Different CD43 Glycoforms to Cluster CD43 and Regulate T Cell Death J. Immunol., October 15, 2006; 177(8): 5328 - 5336. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. V. Cabrera, M. Amano, J. Mitoma, J. Chan, J. Said, M. Fukuda, and L. G. Baum Haploinsufficiency of C2GnT-I glycosyltransferase renders T lymphoma cells resistant to cell death Blood, October 1, 2006; 108(7): 2399 - 2406. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Perone, A. T. Larregina, W. J. Shufesky, G. D. Papworth, M. L. G. Sullivan, A. F. Zahorchak, D. B. Stolz, L. G. Baum, S. C. Watkins, A. W. Thomson, et al. Transgenic galectin-1 induces maturation of dendritic cells that elicit contrasting responses in naive and activated T cells. J. Immunol., June 15, 2006; 176(12): 7207 - 7220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Patnaik, B. Potvin, S. Carlsson, D. Sturm, H. Leffler, and P. Stanley Complex N-glycans are the major ligands for galectin-1, -3, and -8 on Chinese hamster ovary cells Glycobiology, April 1, 2006; 16(4): 305 - 317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Fischer, H. Sanchez-Ruderisch, M. Welzel, B. Wiedenmann, T. Sakai, S. Andre, H.-J. Gabius, L. Khachigian, K. M. Detjen, and S. Rosewicz Galectin-1 Interacts with the {alpha}5{beta}1 Fibronectin Receptor to Restrict Carcinoma Cell Growth via Induction of p21 and p27 J. Biol. Chem., November 4, 2005; 280(44): 37266 - 37277. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J M Ilarregui, G A Bianco, M A Toscano, and G A Rabinovich The coming of age of galectins as immunomodulatory agents: impact of these carbohydrate binding proteins in T cell physiology and chronic inflammatory disorders Ann Rheum Dis, November 1, 2005; 64(suppl_4): iv96 - iv103. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Levroney, H. C. Aguilar, J. A. Fulcher, L. Kohatsu, K. E. Pace, M. Pang, K. B. Gurney, L. G. Baum, and B. Lee Novel Innate Immune Functions for Galectin-1: Galectin-1 Inhibits Cell Fusion by Nipah Virus Envelope Glycoproteins and Augments Dendritic Cell Secretion of Proinflammatory Cytokines J. Immunol., July 1, 2005; 175(1): 413 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. K. Dam, S. Oscarson, R. Roy, S. K. Das, D. Page, F. Macaluso, and C. F. Brewer Thermodynamic, Kinetic, and Electron Microscopy Studies of Concanavalin A and Dioclea grandiflora Lectin Cross-linked with Synthetic Divalent Carbohydrates J. Biol. Chem., March 11, 2005; 280(10): 8640 - 8646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sturm, M. Lensch, S. Andre, H. Kaltner, B. Wiedenmann, S. Rosewicz, A. U. Dignass, and H.-J. Gabius Human Galectin-2: Novel Inducer of T Cell Apoptosis with Distinct Profile of Caspase Activation J. Immunol., September 15, 2004; 173(6): 3825 - 3837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
|
