(Received for publication, March 7, 1997, and in revised form, April 7, 1997)
From the Superantigens (SAgs) activate T-cells in a manner
specific to the V Staphylococcal enterotoxins are prototypic superantigens
(SAgs)1 that stimulate a large population
of T-cells in a manner specific to the V Previous studies demonstrated that Lck, a member of the Src family
protein-tyrosine kinases, plays an essential role in the TCR signal
transduction pathway (4, 5). The T-cell line that lacks functional Lck
is defective in TCR activation (6). Thymocyte differentiation is
severely disturbed in mice lacking Lck expression and in mice
expressing inactive forms of Lck (7-9). Biochemical analysis has shown
that Lck plays at least two roles in the TCR signaling pathway, namely
phosphorylation of the immunoreceptor tyrosine-based activation motifs
(ITAMs) to recruit ZAP-70 and activation of membrane-recruited ZAP-70
(4, 5, 10-15). Another Src family protein-tyrosine kinase, Fyn, has
also been implicated in playing a role in TCR signal transduction (4,
5, 16, 17). However, the phenotype of T-cells from Fyn knockout mice is
much less dramatic than that of Lck knockout mice (18, 19).
CD45, a receptor-type protein-tyrosine phosphatase, also plays a
critical role in TCR signal transduction (20-22). Loss of CD45
expression results in abrogation of the proximal TCR signaling process
in both mature and immature T-cells (20-22). CD45 has been shown
in vitro to up-regulate Src family protein-tyrosine kinases by dephosphorylating C terminus-negative regulatory tyrosine (23, 24).
Impairment of the TCR signaling pathway in CD45-deficient cell lines
appears to be due to the loss of function of certain populations of Src
family protein-tyrosine kinases (25).
To understand the unique characteristics of SAg-induced T-cell
activation, we analyzed the signal transduction pathway triggered by
staphylococcal enterotoxins using the leukemic T-cell line Jurkat and
its mutant derivatives. The results demonstrate that Lck is not
required for T-cell activation by SAgs. In the absence of Lck, Fyn was
clearly activated by SAg stimulation. In addition, loss of CD45
severely impaired SAg-induced activation. This impairment was partly
reconstituted by the expression of the active form of Fyn. Thus, in
place of Lck, SAgs appear to utilize Fyn for initial events in the
signal transduction pathway.
Jurkat and J.CaM1.6 (26),
J.RT3.T3.5 (27), and J.45.01 (28) (gifts from Dr. Arthur Weiss,
University of California, San Francisco) cells were maintained in RPMI
1640 medium supplemented with 5% fetal calf serum, 100 µg/ml
penicillin, and 100 µg/ml streptomycin.
The NF-AT
(nuclear factor of activated
T-cells)/luciferase reporter construct (29) was a gift from
Dr. Gerald Crabtree (Stanford University, Stanford, CA). The expression
construct for the kinase-inactive form of Fyn, Fyn.K299M (mutated at
Lys-299 to Met) (17) was a gift from Dr. Noemi Fusaki (Science
University of Tokyo, Chiba, Japan). For the active form of Fyn,
wild-type Fyn (thymus form) cDNA (a gift from Dr. Roger Perlmutter,
University of Washington, Seattle) was mutated at Tyr-525 to Phe using
polymerase chain reaction-based mutagenesis as described (12). cDNA
from the active form of Fyn was cloned into the pREP3 expression
vector (Invitrogen, Carlsbad, CA).
The
Jurkat cell line and its mutant derivatives were transfected with the
NF-AT/luciferase reporter construct as described (13). After
transfection, each cell line was stimulated for 10 h with Raji
cells pretreated with staphylococcal enterotoxin E or D (Toxin
Technology, Sarasota, FL). Cells were harvested, and the activity of
NF-AT from each transfectant was determined as described (13). The
maximum response of each transfectant was determined by stimulation
with 1.0 µM ionomycin (Calbiochem) and 10 ng/ml phorbol
12-myristate 13-acetate (Calbiochem). For experiments with Fyn.K394M
and Fyn.Y525F, J.CaM1.6 and J.45.01 cells were transfected with the
expression constructs (40 µg) 10 h prior to stimulation with
staphylococcal enterotoxin E (SEE)-treated Raji cells. Normalization of
the transfection efficiency was performed using the cytomegalovirus
promotor-based LacZ expression vector (pCR3/LacZ, Invitrogen) and the
Cells (5 × 105) were loaded with 5 µM Fura-2/AM (Dojindo, Kumamoto, Japan) in Hanks'
balanced saline solution for 30 min at 37 °C and plated on a
glass-bottom microwell dish (Mattek, Ashland, MA) coated with Cell-Tack
(Collaborative Research, Bedford, MA). To cells attached to the plate
were added 1 × 105 Raji cells incubated with 10 ng/ml
SEE (30 min at 37 °C in Hanks' balanced saline solution). The ratio
of emitted fluorescence signals at 340 and 360 nm excitation
(F340/F360) was monitored
and analyzed as described (30) using a SIT camera (C2400-08, Hamamatsu
Photonics, Hamamatsu, Japan) and the Argus 50/CA system (Hamamatsu
Photonics).
J.CaM1.6 cells (1 × 108)
were combined with Raji cells (1 × 107) pretreated
with 100 ng/ml SEE and incubated at 37 °C for 20 min. Cells were
harvested, and cell lysates were immunoprecipitated as described (31)
with 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), anti- J.CaM1.6 and Jurkat cells (1 × 106) were stimulated with 2 µg/ml phytohemagglutinin
(PHA)-P (Honen, Tokyo) or 100 ng/ml SEE and Raji cells (5 × 104) for 16 h. After wash with staining buffer
(Hanks' balanced saline solution containing 5% calf serum and 0.02%
sodium azide), cells were stained with fluorescein
isothiocyanate-conjugated anti-human CD69 (Pharmingen, San Diego, CA)
in staining buffer. Samples were analyzed by flow cytometry using
FACScan (Becton-Dickinson, San Jose, CA).
Jurkat cells, which express V
Superantigen stimulation, as well as other activation through TCR,
results in several changes in the cell phenotype such as expression of
surface antigens (33). PHA is a strong T-cell mitogen and activates
T-cells in a TCR-dependent manner (26). Treatment of Jurkat
cells with PHA induced a high level of CD69 expression (Fig.
2, upper left panel). In contrast, PHA
stimulation of J.CaM1.6 cells resulted in minimum, if any, induction of
CD69 expression. Treatment with C305 also resulted in induction of CD69
in Jurkat cells, but not in J.CaM1.6 cells (data not shown). On the
contrary, SEE induced CD69 to the same level in Jurkat and J.CaM1.6
cells (Fig. 2, right panels). CD25 was also induced in both
Jurkat and J.CaM1.6 cells by SEE (data not shown). In addition,
programmed cell death was observed when J.CaM1.6 cells were cultured
with SEE and Raji cells.2
To further characterize the mechanism of SEE-induced T-cell activation,
we investigated early signaling events that followed TCR engagement (4,
5). First, the increase in intracellular Ca2+ levels in
Jurkat and J.CaM1.6 cells was determined. It was previously shown that
anti-TCR antibody (C305) treatment results in no increase in
Ca2+ in J.CaM1.6 cells (6). To measure the increase in
Ca2+ induced by SEE presented by Raji cells, microscopic
fluorometric analysis was applied. Jurkat and J.CaM1.6 cells were
compared at the single cell level with regard to the change in
intracellular free Ca2+. As shown in Fig.
3A, both cell lines responded to SEE-treated Raji cells in an identical manner. An increase in Ca2+ was
observed 10-15 min after the addition of Raji cells. Equivalent increases in Ca2+ levels were observed in both Jurkat and
J.CaM1.6 cells. In addition, F340/F360 ratios were
maintained at the maximum level over a 10-min period in both Jurkat and
J.CaM1.6 cells. This demonstrates that SEE induces an increase in
intracellular free Ca2+ in a manner distinctive from
antibody-mediated stimulation. A comparison between the pseudo-color
F340/F360 ratio image and the bright-field image confirmed that cells that show an increase in
Ca2+ colocalize with the cells forming de novo
cell-cell complexes, presumably composed of SEE-presenting Raji cells
(identified as cells not showing any fluorescence) and J.CaM1.6 cells
(Fig. 3B).
Protein tyrosine phosphorylation is an essential step in TCR signal
transduction. Lck plays a major role in phosphorylating two tyrosines
in ITAMs, recruiting ZAP-70, and initiating downstream events (4, 5,
10-15). Recruitment of ZAP-70 to the
The functional role of Fyn was further tested by expressing the
kinase-inactive form, Fyn.K299M (17), in J.CaM1.6 cells. As shown in
Fig. 5A, the expression of Fyn.K299M in
J.CaM1.6 cells significantly suppressed NF-AT activation induced by
differing doses of SEE. This shows that Fyn.K299M functions in a
dominant-negative manner in the signal transduction pathway activated
by SEE. The role of Fyn was also tested using J.45.01 cells.
Previously, it was shown that the loss of CD45 expression leads to
greatly impaired reactivity of T-cells to anti-TCR antibody stimulation
(20-22). Loss of CD45 expression in T-cells was associated with the
increased level of tyrosine phosphorylation of Tyr-505 in Lck and Fyn
(35-37). As shown in Fig. 1A, the reactivity of J.45.01
cells against SEE is also severely impaired (Fig. 1A). When
the active form of Fyn (Tyr-528 to Phe mutation in Fyn) was expressed
in J.45.01 cells, NF-AT activation by SEE was enhanced to a level
equivalent to that in Jurkat cells (Fig. 4B; also see Fig.
1). In contrast, no enhancement was observed when wild-type Fyn was
expressed, indicating that Tyr-528 of Fyn plays a critical function in
relation to the function of CD45.
Our results presented here demonstrate that SAgs activate T-cells
in an Lck-independent manner and that Fyn may play a critical role in
the signaling process. Two other reports support this. Migita et
al. (38) demonstrated that staphylococcal enterotoxin B activates
Fyn, but not Lck, in mouse splenic T-cells in vivo. In Fyn
knockout mice, the proliferative response of splenocytes to
staphylococcal enterotoxin B was substantially impaired (17). There are
at least two roles that Lck plays in the TCR signal transduction
pathway (4, 5, 10-15). One is tyrosine phosphorylation of ITAMs in the
TCR complex to create binding sites for ZAP-70, and the other is
activation of ZAP-70 by tyrosine phosphorylation. It was previously
demonstrated in a heterologous system using COS cells that Fyn and Lck
induce tyrosine phosphorylation of ITAMs and activation of ZAP-70 at an
equivalent efficiency (10). Fyn was found in association with the It is still unclear, however, why SAgs, but not other TCR stimulators,
activate T-cells independently of Lck. This is not due to the strength
of SAg-induced activation since limited amounts of SEE activated
J.CaM1.6 cells in an identical manner to Jurkat cells, whereas anti-TCR
antibody and PHA completely failed to activate J.CaM1.6 cells. Thus,
the differences between SAg stimulation and other stimulation derive
from the qualitative differences in the manner in which TCR is
triggered. One possibility is that the activation by SAgs involves a
T-cell surface molecule other than TCR. Such a coreceptor could
function in activating Fyn only in case of SAg stimulation. Another
possibility is that SAgs induce a unique conformational change in the
TCR complex so that Fyn can be activated. Recently, the crystal
structure of a complex between the Finally, the biological responses that are unique to SAg activation may
be contributed by the involvement of Fyn in its signal transduction
process. A difference in the substrate specificity of Lck and Fyn is a
factor that could contribute to the biological consequences. In
addition, we have recently shown that the SH2 and SH3 domains of Lck
play critical roles in T-cell activation initiated by
membrane-localized ZAP-70 (13). It is likely that the SH2 and SH3
domains of Fyn also play roles in the signaling process initiated by
SAg stimulation. These domains of Lck and Fyn may have different
repertoires of targets in vivo (44). This implies that there
can be differing molecules that are involved in the distal events of
the signal transduction pathway when Fyn instead of Lck is involved. In
such cases, the molecules associated specifically with the SH2 and SH3
domains of Fyn could contribute to the unique biological consequences
provoked by SAg stimulation.
We are very grateful to Gerald Crabtree,
Noemi Fusaki, Roger Perlmutter, and Arthur Weiss for reagents; to
Masako Takamatsu and Yi-Ying Huang for excellent technical assistance;
to Hideyoshi Higashi for operation of the Argus system; to Fumie Sahira
for secretarial assistance; and to Makoto Iwata and Julie Hambleton for
critical reading of the manuscript. We also are grateful to Ko Okumura,
Minoru Muramatsu, and Hiroto Hara for support.
Division of Cell and Information,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
region of the T-cell antigen receptor.
Stimulations by SAgs provoke drastic T-cell activation that leads to
programmed cell death or the anergic state of responding cells. To
characterize the signal transduction pathway initiated by SAgs, mutant
lines derived from the human leukemic T-cell line Jurkat were tested for their reactivities against prototypic SAgs, staphylococcal enterotoxins. The J.CaM1.6 cell line, which lacks Lck expression and
lost reactivity against T-cell antigen receptor-mediated stimulation, was activated by staphylococcal enterotoxins in a manner
indistinguishable from the Jurkat cell line. In contrast, the J.45.01
cell line, which lacks expression of functional CD45, showed severely
impaired reactivity. The role of Lck appears to be replaced by another Src family protein-tyrosine kinase, Fyn. In J.CaM1.6 cells, Fyn was
rapidly phosphorylated and activated after staphylococcal enterotoxin
treatment. The kinase-inactive mutant of Fyn significantly suppressed
the reactivity against staphylococcal enterotoxin E in J.CaM1.6 cells,
and the expression of the active form of Fyn reconstituted reactivity
against staphylococcal enterotoxin E in J.45.01 cells. These results
demonstrate that SAgs activate T-cells in an Lck-independent pathway
and that Fyn plays a critical role in the process.
region of the T-cell
antigen receptor (TCR) (1, 2). SAgs have been implicated as a causative
factor in a number of human diseases, such as toxic shock syndrome,
rheumatoid arthritis, and diabetes mellitus (2, 3). Activation by
staphylococcal enterotoxins induces rapid proliferation followed by the
anergic state and programmed cell death of mature T-cells and
thymocytes (1, 2). Although T-cell activation by SAgs is
distinguishably unique, the precise signal transduction mechanism has
not been well characterized.
Cell Lines and Culture Conditions
-galactosidase-based luminescence system (Tropix Inc., Bedford, MA).
Each experiment was performed more than once with either duplicate or
triplicate samples, and the representative results are given.
(Transduction Laboratories, Lexington, KY), and anti-Fyn (Transduction
Laboratories). Each sample was analyzed by Western blotting using 4G10
or anti-Fyn, horseradish peroxidase-conjugated anti-mouse IgG (Zymed,
Laboratories, Inc., South San Francisco, CA), and the ECL system
(Amersham International, Buckinghamshire, United Kingdom). For in
vitro kinase assay, J.CaM1.6 cells (1 × 107)
were mixed with Raji cells (5 × 106) pretreated with
or without 100 ng/ml SEE. After 20 min of incubation at 37 °C, cell
lysates were prepared, and Fyn was immunoprecipitated. In
vitro kinase assay was performed as described previously (31).
8, were previously shown to
respond strongly to staphylococcal enterotoxins A, D, and E presented by Raji cells, a leukemic B-cell line (32). To analyze the detailed mechanism of T-cell activation by staphylococcal enterotoxins, NF-AT-dependent transcriptional activity was measured after
stimulation. As shown in Fig. 1A, treatment
with SEE plus Raji cells strongly induced the NF-AT activity of Jurkat
cells. Surprisingly, J.CaM1.6 cells, which are Lck-deficient (6),
showed equivalent reactivity against SEE compared with Jurkat cells.
This activation by SEE is TCR-dependent since the
TCR-negative Jurkat mutant, J.RT3.T3.5 (27), did not show any response.
SED also activated J.CaM1.6 and Jurkat cells equally well
(Fig. 1B). There was no difference between Jurkat and
J.CaM1.6 cells in reactivity against limited amounts of SEE (Fig.
1C). On the other hand, the anti-idiotypic antibody against
Jurkat TCR, C305 (27), completely failed to activate J.CaM1.6 cells
(Fig. 1D). It should be noted that the CD45-deficient
mutant, J.45.01 (28), showed a greatly reduced response to SEE (Fig.
1A).
Fig. 1.
Lck-independent activation of NF-AT by SEE
and SED. A, NF-AT activation by SEE in mutant lines derived
from Jurkat cells. Jurkat cells and mutant derivatives were stimulated
with Raji cells pretreated with SEE (100 ng/ml). Each cell line, except J.RT3.T3.5, expresses an equivalent level of TCR (not shown). SEE-induced NF-AT activity is presented as the percent activity against
maximum responses induced by stimulation with phorbol 12-myristate
13-acetate plus ionomycin. The J.CaM1.6 cell line is the Lck
kinase-deficient derivative of the Jurkat cell line. J.RT3.T3.5 cells
lost expression of TCR due to the mutation of the TCR chain.
J.45.01 cells are deficient in expressing CD45 protein-tyrosine
phosphatase. B, comparison of stimulatory ability of SEE and
SED in J.CaM1.6 and Jurkat cells. Jurkat (open bars) and
J.CaM1.6 (closed bars) cells were stimulated with Raji cells pretreated with 100 ng/ml SEE or SED. NF-AT activity in cells stimulated for 10 h was determined and is presented as the -fold increase over unstimulated Jurkat cells. C, reactivity of
Jurkat and J.CaM1.6 cells against limited amounts of SEE. The
sensitivity of Jurkat and J.CaM1.6 cells to SEE was tested by serially
diluting SEE. NF-AT activity under each condition was determined and is presented as the percent activity of the maximum response of Jurkat cells. D, anti-receptor antibody-induced NF-AT activity in
Jurkat cells, but not in J.CaM1.6 cells. Jurkat and J.CaM1.6 cells were stimulated for 10 h with serially diluted ascites of anti-TCR monoclonal antibody C305. NF-AT activity was determined and is presented as described for C.
[View Larger Version of this Image (25K GIF file)]
Fig. 2.
Cell-surface expression of CD69 in Jurkat and
J.CaM1.6 cells stimulated with SEE or PHA. J.CaM1.6 and Jurkat
cells were stimulated with PHA-P (left panels) or SEE and
Raji cells (right panels) for 16 h. Stimulated
(thick lines) and unstimulated (thin lines) cells
were analyzed with fluorescein isothiocyanate-conjugated anti-CD69.
[View Larger Version of this Image (20K GIF file)]
Fig. 3.
Microscopic fluorometric analysis of
cytoplasmic Ca2+ increase induced by SEE. A,
changes in the cytoplasmic free Ca2+ concentration induced
by SEE in individual Jurkat and J.CaM1.6 cells. Jurkat and J.CaM1.6
cells were loaded with Fura-2/AM and stimulated with Raji cells
pretreated with SEE. Changes in the F340/F360 ratio were
monitored by microscopic fluorometry. Data from five representative
cells for each cell line are presented. B, de
novo formation of the cell-cell complex between J.CaM1.6 and Raji
cells. The bright-field images (left panels) and the pseudo-color images of the
F340/F360 ratio
(right panels) are from the same view. The upper
panels are images taken at the beginning of measurement (0 min),
and lower panels are images taken at the end of measurement
(30 min). Added Raji cells are indicated by the green
arrows.
[View Larger Version of this Image (33K GIF file)]
chain by SED stimulation was
previously described (34). Thus, it was likely that staphylococcal
enterotoxins activate T-cells through a pathway similar to that of
antigenic stimulation via ITAM tyrosine phosphorylation and that a
kinase other than Lck plays a major role in the process. As shown in
Fig. 4A (left panel), SEE
stimulation induced tyrosine phosphorylation in various cellular
proteins. Fyn, an Src family protein-tyrosine kinase that associates
with the TCR complex, was previously indicated as playing a role in the
TCR signal transduction pathway (4, 5, 16-19). An increase in tyrosine
phosphorylation on Fyn and the
chain was confirmed (Fig.
4A, upper and middle right panels). An
equivalent amount of the
chain (data not shown) and Fyn was
detected in the immunoprecipitates from unstimulated and stimulated
J.CaM1.6 cells (Fig. 4A, lower right panel).
Moreover, the in vitro kinase assay of anti-Fyn immunoprecipitates showed strongly induced kinase activity in SEE-activated J.CaM1.6 cells (Fig. 4B).
Fig. 4.
Induction of tyrosine phosphorylation and Fyn
kinase activity in J.CaM1.6 cells following stimulation with SEE.
A, Western blot analysis of protein tyrosine phosphorylation
in J.CaM1.6 cells treated with SEE and Raji cells. Immunoprecipitates
(IP; antibodies used were anti-phosphotyrosine
(anti-pY; left panel), anti- (upper
right panel), and anti-Fyn (middle right panel)) from
unstimulated (
) and SEE-stimulated (+) samples were analyzed by
Western blotting using anti-phosphotyrosine monoclonal antibody 4G10.
Equivalent amounts of the
chain (not shown) and Fyn (lower right panel) were detected for each immunoprecipitate.
B, increase in the in vitro kinase activity of
Fyn immunoprecipitated from SEE-stimulated J.CaM1.6 cells. Fyn was
immunoprecipitated from unstimulated (
) or SEE-stimulated (+)
J.CaM1.6 cells, and the in vitro kinase activity of
autophosphorylation was determined.
[View Larger Version of this Image (32K GIF file)]
Fig. 5.
NF-AT activation by SEE in cells expressing
mutant forms of Fyn. A, SEE-induced NF-AT activation in
J.CaM1.6 cells transfected with the expression construct for
kinase-inactive Fyn. J.CaM1.6 cells was transiently transfected with
the expression construct for the kinase-inactive form of Fyn and
stimulated with various amounts of SEE. NF-AT activity was determined
and is presented as the -fold induction over the unstimulated mock
transfectant. B, NF-AT activation in J.45.01 cells
expressing the active form of Fyn. J.45.01 cells were transfected with
the expression construct for no insert (mock), wild-type Fyn, and the
active form of Fyn, Fyn.Y528F, along with the NF-AT/luciferase reporter
construct. Transfectants were stimulated with SEE (100 ng/ml) with Raji
cells as describe for A, and NF-AT activity was
determined.
[View Larger Version of this Image (20K GIF file)]
chain (16), and the overexpression of Fyn induces hyperactivity of
antigen-specific T-cells (17). Thus, it is likely that SAg stimulation
leads to activation of Fyn in the absence of Lck and initiates the
signaling process. Recent reports also indicate the functional
redundancy of Fyn and Lck in T-cell development (39, 40).
chain of TCR and staphylococcal
enterotoxin B was reported (41). The complex formed by SAg with TCR is
distinctive in structure from the complex formed by TCR with the
MHC+ peptides (42). In addition, antibody treatment of the
CD3
chain induced Syk activation and a Ca2+ increase in
J.CaM1.6 cells (26, 43). Thus, Fyn may be activated by the structural
alteration within the TCR complex by SAg stimulation.
*
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.
To whom correspondence should be addressed: Mitsubishi Kasei
Inst. of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan. Tel./Fax: 81-427-25-8191; E-mail: makio{at}libra.ls.m-kagaku.co.jp.
1
The abbreviations used are: SAgs, superantigens;
TCR, T-cell antigen receptor; ITAM, immunoreceptor tyrosine-based
activation motif; SEE, staphylococcal enterotoxin E; SED,
staphylococcal enterotoxin D; PHA, phytohemagglutinin.
2
M. Tachibana and M. Iwashima, unpublished
observation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.