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J Biol Chem, Vol. 274, Issue 20, 13744-13747, May 14, 1999
From the Departments of T cell development and function in complex
ganglioside-lacking (GM2/GD2 synthase gene-disrupted) mice were
analyzed. GM1, asialo-GM1, and GD1b were representative gangliosides
expressed on T cells of the wild type mice and completely deleted on
those of the mutant mice. The sizes and cell numbers of the mutant mice spleen and thymus were significantly reduced. Spleen cells from the
mutant mice showed clearly reduced proliferation compared with the wild
type when stimulated by interleukin 2 (IL-2) but not when treated with
concanavalin A or anti-CD3 cross-linking. Expression levels of IL-2
receptor Gangliosides are enriched in nervous systems of vertebrates,
suggesting their importance in the neuronal functions (1). They are
also present in cells of the immune system and have been thought to
contribute in cell to cell recognition or cellular signal modulation
(2). In particular in human T cells,
GD31 was induced by various
stimulations via up-regulation of GD3 synthase gene (3). Moreover,
anti-GD3 monoclonal antibody
(mAb)2 induced T cell
activation, suggesting that GD3 could mediate growth signals by binding
endogenous ligands (4).
In murine T lymphocytes, a number of ganglioside components were
present (5, 6) showing changes depending on the stage of development
(7) and differentiation of the cells (8). These results indicate that
gangliosides play important roles in the immune system. However, many
of these studies were done by the addition of exogenous gangliosides
(9) or anti-ganglioside antibody to cultured cells (10) or into
experimental animals (11). These studies do not address the functions
of endogenously generated gangliosides.
We have established mutant mouse lines that completely lacked GM2/GD2
synthase activity and expression of all complex gangliosides including
those of asialo-series (12). They showed mild defects in the nervous
system, and severe dysfunction in male genital organ (13). In this
study, we analyzed the phenotypic and functional changes in the immune
system of the complex ganglioside-lacking mice. Most significantly, it
was found that mature T cells from the mutant mice respond very weakly
to interleukin 2 (IL-2). We elucidated here that the signaling pathway
for IL-2/IL-2 receptor (IL-2R) was largely disrupted by the altered
ganglioside arrangement on cell surface. This is the first report to
study glycolipid functions by the genetic modification of their
carbohydrate structures and showed critical roles for endogenous
glycolipids in the transduction of proliferation signals introduced by lymphokines.
Flow Cytometry--
For the analysis of IL-2 receptors and
gangliosides, spleen cells and thymocytes from 6-8-week-old mice were
used. Spleen cells resuspended in phosphate-buffered saline/0.5
mM EDTA/5% fetal calf serum were panned, and then unbound
cells were collected and served to flow cytometry. Antibodies used were
as follows: PC61 (IL-2R T Cell Proliferation Assay--
Spleen cells from mice (6-8
weeks old) were dispersed and washed twice in plain RPMI 1640 medium at
4 °C. They were adjusted at 3.75 × 106/ml in RPMI
1640 containing 10% fetal bovine serum (FBS) and plated into 96-well
plates at 100 µl/well. 100 µl of IL-2 was added to make final
concentration of 1 unit/ml and cultured. At days 2, 3, 4, and 5 of
culture, cells were harvested after incorporation of
[3H]thymidine for 18 h (1 µCi/well) by being
trapped with a glass filter, and then the filter was counted using a
liquid scintillation counter (Aloka, Tokyo). To analyze the response to
concanavalin A (ConA) stimulation, ConA was added to the spleen cells
prepared as described above at 1~5 µg/ml, and proliferation was
examined as performed in IL-2 treatment. Proliferation by stimulation
with anti-CD3 antibody was also examined by cultivating spleen cells in
96-well plates coated by mAb145-2C11 at 10 µg/ml, otherwise as done
in IL-2 stimulation.
Preparation of Panning Plates--
Anti-mouse IgG (H and L)
(Cappel) was coated in 6-cm bacteriological plates (Falcon) at 20 µg/ml in 50 mM Tris-HCl (pH 9.5) for 3 h at room
temperature. After washing in saline, the plates were blocked by 0.1%
bovine serum albumin in phosphate-buffered saline and then stocked in a freezer.
Immunoprecipitation--
Spleen cells from mice of 6-8 weeks
old were cultured in 24-well plates at the density of 1 × 107/ml/well in RPMI 1640 containing 10% FBS. The cells
were cultured for 0, 5, 10, and 30 min after addition of IL-2 (1 unit/ml, Takeda Pharmaceutical Co.) (1 Takeda unit = 383 Japan
reference unit). The cells were collected at each time point, and then
the pellets were lyzed by adding 200 µl of lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Northern Blotting--
Spleen cells from mice (6-8 weeks old)
were adjusted at 1 × 107/ml in RPMI 1640 containing
10% FBS and plated in 24-well plates at 2 × 107/well/2 ml. They were cultured for 0, 10, 30, and 60 min
in the presence of IL-2 (1 unit/ml). At each time point, cells were
collected, and then total RNA was extracted by Trizol® (Life
Technologies, Inc.). For Northern blot analysis, total RNA (15 µg/lane) was separated in 1.2% agarose-formaldehyde gel and
transferred onto a nylon membrane (GeneScreen Plus, NEN Life Science
Products). Prehybridization and hybridization was performed with
32P-radiolabeled cDNA probes synthesized from
c-fos or c-myc containing plasmids as described
previously (3). Then the membrane was washed and analyzed by a
BioImaging Analyzer BAS-2000 (Fuji, Tokyo).
Ganglioside Expression on T Lymphocytes in the Mutant
Mice--
GM1, asialo-GM1, and GD1b gangliosides detected in the wild
type spleen T cells were completely absent in the mutant spleen cells,
as expected from the proposed ganglioside synthetic pathway (Fig.
1, A and B).
Instead, GD3 expression was slightly increased in the knock-out mice,
probably as a result of precursor accumulation.
Size of Immune Tissues and Numbers of Total Cells and T
Cells--
Spleens and thymuses from the mutant mice were smaller than
the wild type organs (Fig. 1C). Total cell numbers in the
spleens or thymuses of the knock-out mice were significantly reduced as shown in Fig. 1C. The ratios of CD3 positive T cells in the
whole spleen cells were almost equivalent between the two groups,
i.e. 31.8 ± 1.1% and 31.2 ± 1.6% for the wild
type and the mutant mice, respectively.
Proliferation of Spleen Cells--
To compare their proliferation,
spleen cells were stimulated with ConA, anti-CD3 antibody, or IL-2.
Those from the mutant mice showed clearly decreased incorporation of
[3H]thymidine compared with the wild type when stimulated
by IL-2 (Fig. 2) but not when treated
with ConA or anti-CD3 cross-linking. Because T cell population in the
mutant spleen was equivalent to that of the wild type, the results
described above indicate that T lymphocytes in the mutant have defects
in IL-2/IL-2R-mediated signaling.
Expression of IL-2 Receptors--
Expression levels of IL-2
receptors were analyzed by flow cytometry. All three components of IL-2
receptor, Activation of JAK-STAT System with IL-2--
To analyze the
activation of the JAK/STAT pathway, the phosphorylation patterns of
JAK1, JAK3, and STAT5 proteins that are the representative signal
molecules responsible for transmission of the IL-2 signal were
examined. JAK1 and JAK3 were phosphorylated at 5 min after IL-2
treatment in the wild type mouse T cells, and the activation level was
sustained for 10 and 30 min for JAK1 and JAK3, respectively (Fig.
4). On the other hand, JAK1 was scarcely phospholylated, and JAK3 phosphorylation detected at 5 min quickly returned to the basal line in the mutant mice. STAT5 was also phosphorylated at 5 min after IL-2 stimulation and kept the activated form even after 30 min in the wild type. In contrast, the mutant spleen
cells showed delayed response and already declined at 30 min after IL-2
treatment (Fig. 4C).
c-myc and c-fos Gene Expression during IL-2 Stimulation--
c-fos/c-jun, c-myc, or bcl2
has been reported to be involved in the cell proliferation by IL-2
(14). We analyzed the regulation of c-fos and
c-myc gene transcription when stimulated with IL-2. mRNA
of c-fos in the wild type mice was readily detected by
Northern blotting 10 min after IL-2 stimulation and then gradually
decreased. In the mutant mouse, c-fos mRNA levels
reached the plateau at 30 min and soon fell (Fig.
5A). On the other hand,
c-myc mRNA levels exhibited very similar patterns in the
mutant and wild type mice.
Ganglioside GM2/GD2 synthase gene knock-out mice showed defects in
ganglioside expression on T cells in accord with the proposed pathway
of ganglioside synthesis (15). The comparative study of the immune
system using these mutant mice, as well as the wild type mice, is
expected to unambiguously elucidate the actual roles of gangliosides
in vivo. In fact, results obtained here clearly showed
important roles of gangliosides in the regulation of
IL-2/IL-2R-mediated signals in a straightforward manner.
In many studies, the roles of glycolipids in the immune system have
been analyzed by observing the effects of exogenous gangliosides, and
they exhibited suppressive effects on T cell functions such as CD4
internalization (9) and reduced response to mitogens or allogeneic
antigens (16). Actually, gangliosides were reported to bind to IL-2
(17) and thereby inhibit T cell proliferation (18). These
immunosuppressive effects of gangliosides have been considered to
explain immunosuppression in cancer patients (19). However, results
obtained in these experiments do not seem likely to reflect the real
effects of endogenously generated gangliosides.
Compartmentalization through protein-protein or protein-lipid
interactions has now been recognized as a fundamental mechanism for
efficient and organized cell signaling (20, 21). Some membrane
components are apparently organized into glycolipid-enriched membrane
domains or detergent-insoluble glycolipid-enriched domains (22) known
as rafts. These rafts are enriched in sphingolipids, cholesterol,
glycosylphosphatidyl-inositol-anchored proteins (23), and a variety of
signaling molecules. Recently, dynamic T cell receptor recruitment to
such a microdomain upon T cell activation has been reported in a T cell
line (24, 25) and in thymocytes (26). These data suggest that
gangliosides expressed on T cells associate with receptors and
signaling molecules to transduce the IL-2 signals at such a
microdomain, although it is yet unclear that IL-2 receptors are
enriched in glycolipid-enriched membrane domains.
Defects in the IL-2 signal transduction found in the mutant mice T
cells should take place due to lack of GM1/GD1b/GA1 and may be due to
serious structural defects of IL-2 receptors. In particular, the
configuration of Results presented in this report demonstrate a possibility that cell
surface gangliosides regulate the proliferation signals by modulating
the growth factor receptors. These findings will provide insights into
the roles of gangliosides in the proliferation of activated T
lymphocytes (3) and T cell leukemia cells (31, 32) and also may provide
clues to developing new therapeutic approaches for malignant lymphoid tumors.
We thank Dr. K. O. Lloyd at Memorial
Sloan-Kettering Cancer Center for critically reading the manuscript and
Dr. T. Tabeshita for providing anti-IL-2R mAbs. We also thank the
members of the Laboratory Animal Center for Biomedical Research,
Nagasaki University School of Medicine for the care of the mice.
*
This work was supported by a Grant-in-Aid for Scientific
Research of Priority Areas 10178105 and by a grant from the Core of
Excellence from the Ministry of Education, Science, Sports, and Culture
of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§§
Present address: Dept. of Neuroscience, Johns Hopkins University,
Baltimore, MD 21205.
||
To whom correspondence should be addressed: Dept. of
Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai,
Showa-ku, Nagoya 466-0065 Japan. Tel.: 81-52-744-2070; Fax:
81-52-744-2069; E-mail: koichi{at}med.nagoya-u.ac.jp.
1
Ganglioside nomenclature is based on that of
Svennerholm (33).
The abbreviations used are:
mAb, monoclonal
antibody;
FBS, fetal bovine serum;
IL-2, interleukin 2;
IL-2R, IL-2
receptor;
FITC, fluorescein isothiocyanate, ConA, concanavalin A.
COMMUNICATION
Attenuation of Interleukin 2 Signal in the Spleen Cells of
Complex Ganglioside-lacking Mice*
,
,§§,,, and Oncology and
Pediatrics, Laboratory of Morphogenesis,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 
, and 
were almost equivalent, and up-regulation of
chain after T cell activation was also similar between the mutant
and wild type mice. Activation of JAK1, JAK3, and SAT5 after IL-2
treatment was reduced, and c-fos expression was delayed and
reduced in the mutant spleen cells, suggesting that the IL-2 signal was
attenuated in the mutant mice probably due to the modulation of IL-2
receptors by the lack of complex gangliosides.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 1 µl of ascites), TUm122 (IL-2R , 1 µl of ascites), TUGm2 (IL-2R, 1 µl of ascites) (these three
antibodies were kindly provided by Dr. T. Takeshita at Tohoku
University), R24 (anti-GD3 provided by Dr. L. J. Old, 0.5 µl of
ascites), 220 (anti-GD2, supernatant), 2D4 (anti-GA2 obtained from
ATCC, supernatant), 229-6-22 (anti-GA1, supernatant), M2590 (anti-GM3
purchased from Nihon Biotest Research Institute, 5 µg/ml), 370 (anti-GD1b, 0.5 µl of ascites), 549 (anti-GT1b, 0.5 µl of ascites),
and 9-22 (anti-GD1a, 0.5 µl of ascites). Additional antibodies used
were FITC anti-rat IgG for PC61, TUm122 and TUGm2, FITC anti-mouse IgG
for R24 and 220, and FITC anti-mouse IgM for other mouse IgM mAbs.
Samples without mAbs were used as negative controls. CD3 expression was analyzed by anti-CD3 mAb145-2C11 and FITC anti-hamster IgG with nonpanned spleen cells. To detect GM1, biotin-labeled choleratoxin B
subunit (List Biological Laboratory, Campbell, CA) was used followed by
avidin-FITC (EY Laboratories).
-amidinophenyl-methanesulfonyl-fluoride hydrochloride, aprotinin (10 µg/ml), 1% (v/v) Brij96). After
centrifugation at 15,000 rpm, the supernatants were precleared with
protein A-Sepharose (Amersham Pharmacia Biotech) and then incubated
with 10 µl of antibodies (anti-JAK1 (Q-19), anti-JAK3 (C-21), or
anti-STAT5b (C-17)) (Santa Cruz Biotechnology Inc.) and then with 20 µl of protein A-Sepharose for 2 h at 4 °C. After washing with
lysis buffer three times, the immunoprecipitates were applied to 7.5% gel of SDS-polyacrylamide gel electrophoresis. Proteins were
electrically blotted onto polyvinylidene difluoride membranes, and
immunoblot was performed by PY-20 (ICN Biomedical Inc.) at 1:2000,
anti-JAK1 at 1:4000, anti-JAK3 at 1:4000, and anti-STAT5b at 1:4000
dilution. Second antibodies used were biotinylated anti-mouse IgG for
anti-JAK1 and anti-JAK3 and biotinylated anti-rabbit IgG for
anti-STAT5b. Signals of antibody binding were detected by ECL detection
kit (Amersham Pharmacia Biotech).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (41K):
[in a new window]
Fig. 1.
Expected defects of ganglioside synthesis and
phenotypic changes in GM2/GD2 synthase gene knock-out mice.
A, glycolipids in the box are expected to be
deleted in the mutant mice. B, flow cytometric analysis of
spleen cells with mAbs. Used antibodies and second antibodies are
described under "Experimental Procedures." Solid lines
are controls with second antibody alone. C, morphogenesis
and cell numbers of spleen and thymus. Total cell numbers were counted
and presented as averages of six mice each ± S.D. p
values are 0.01 and 0.02 for spleen and thymus, respectively.
View larger version (17K):
[in a new window]
Fig. 2.
Proliferative reaction of spleen cells to
ConA, IL-2 and anti-CD3 antibody. Spleen cells prepared as
described under "Experimental Procedures" were stimulated by ConA
(2.5 µg/ml), IL-2 (1 unit/ml), or anti-CD3 (10 µg/ml immobilized),
and [3H]thymidine uptake was examined. The numbers are
average counts ± S.D. ConA stimulation was examined at least
three times and showed similar results.
, , and , were equally detected by specific
monoclonal antibodies between the wild type and the mutant mice (Fig.
3). After culture with IL-2, their
expression patterns were also similar. When they were cultured in the
presence of ConA, marked up-regulation of IL-2R chain was observed
in both groups (Fig. 3). Thus, there are no marked differences in the
expression levels of IL-2 receptors and in the up-regulation of chain.
View larger version (24K):
[in a new window]
Fig. 3.
Expression levels of IL-2 receptors.
Expression of three components consisting of IL-2 receptor was analyzed
by flow cytometry. Spleen T cells from the wild type (+/+) and the
mutant mice ( 
/) were analyzed before (left) or after
treatment with IL-2 (middle) or with ConA (right)
for 3 days as described under "Experimental Procedures."
View larger version (72K):
[in a new window]
Fig. 4.
JAK/STAT activation after IL-2 stimulation of
spleen T cells. Spleen cells were cultured in the presence of IL-2
for the times indicated and then served to immunoprecipitation
(IP) and Western immunoblots as described under
"Experimental Procedures" and as indicated in the figure.
Immunoprecipitation was performed by anti-JAK1 (A),
anti-JAK3 (B), or anti-STAT5b (C) and then
blotted by anti-phosphotyrosine mAb (PY20) or individual antibodies
used for immunoprecipitation as controls.
View larger version (30K):
[in a new window]
Fig. 5.
c-fos and c-myc
expression during IL-2 stimulation of spleen cells. Spleen
cells were cultured in the presence of IL-2 for the times indicated and
then served to Northern blotting as described under "Experimental
Procedures." The intensity of bands were measured by
Bas2000 imaging analyzer (Fuji, Tokyo), and both graphs
assign an intensity of 1 to time 0 of the wild type and express
intensity at subsequent time points relative to time 0.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and chains may be affected because it has been
reported that JAK1 and JAK3 are differentially associated with IL-2R
and IL-2R, respectively (27). Among many signaling molecules, JAK3
is reported to mainly act in cell proliferation and to activate STAT5
(28) and induce c-fos expression (29). Reduced activation of
JAK3 and JAK1 followed by reduced activation of STAT5 and reduced
c-fos induction upon IL-2 stimulation may explain the
suppressed proliferation of the mutant T cells. The fact that all
mutant mice deficient for IL-2/IL-2Rs, except for those deficient in
IL-2R, show deregulated T cell activation and autoimmune diseases
(30) suggests that the defects in the immune system of our mutant mice
might come mainly from attenuation in the chain-mediated signal.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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