| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Pharmacia & Upjohn, Lund Research Center, and
Department of Cell and Molecular Biology, Section for Tumor Immunology, The Wallenberg Laboratory, University of Lund, Lund, Sweden
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Production of IL-2 in T cells is strongly regulated at the
transcriptional level. Activation of the IL-2 promoter requires
cooperative interactions of several transcription factors, including
AP-1, NF-
B, NF-AT, and NF-IL-2 (6, 7, 8, 9). AP-1 proteins seem to play a
central role in IL-2 regulation by binding to the functionally
important AP-1 site in the IL-2 promoter (10) as well as participating
in the formation of transcriptionally active NF-AT and NF-IL-2 (8, 9).
The AP-1 transcription factor is a complex between different members of
the Fos (c-Fos, FosB, Fra-1, and Fra-2) and Jun (c-Jun, JunB, and JunD)
families of proteins (11). These proteins contain a leucine zipper that
permits dimerization with other members of the Fos/Jun family (12).
Homodimers of Jun, but not of Fos, bind to
12-O-tetradecanoylphorbol-13-acetate-responsive elements
(TREs). However, the DNA binding affinity of heterodimers is
significantly higher (13).
AP-1 is regulated at the level of both jun and fos gene transcription and by posttranslational modifications of their gene products. Synthesis of c-Fos and the trans-activating capacity of c-Jun and c-Fos have been shown to be regulated by the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK), Jun N-terminal kinases (JNK), and Fos-regulating kinase (FRK), respectively. ERK activation depends on coupling of the TCR-CD3 complex to p21ras (14), with subsequent activation of the Raf-MEK kinase cascade (15, 16). JNK activation also occurs through p21ras, but requires an additional signal emerging from the CD28 costimulatory receptor (17). This involves activation of small GTP binding proteins such as Rac1 and Cdc42 (18, 19) that induce the kinase cascade MEKK-SEK-JNK (20).
Kang et al. previously showed that AP-1-dependent DNA binding and trans-activation at the IL-2 gene are defective in anergic Th1 cells (5). Recent studies have demonstrated that the TCR-inducible activities of ERK and JNK also are reduced in in vitro anergized T cell clones (21), which correlated with reduced expression of c-Fos, FosB, and JunB proteins (22). However, the physiologic relevance of these findings is unclear, since only limited information is available on in vivo induced anergic T cells. We have previously shown that repeated injections of staphylococcal enterotoxin A (SEA) to mice transduce a state of long-lasting IL-2 hyporesponsiveness in the Vß3+CD4+ T cell compartment (23) associated with reduced DNA binding of AP-1 and Fos/Jun-containing NF-AT (24). In this study we demonstrate lack of AP-1-dependent transcription in anergic CD4+ T cells. The defective AP-1 transcriptional activity seems to involve both reduced c-Fos protein levels and lack of JNK-mediated posttranslational modifications of AP-1 protein. We suggest that the perturbation of the JNK pathway may be a critical factor in anergic CD4+ T cells in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Transgenic C57BL/6 mice expressing a rearranged TCR-Vß3 gene under the influence of an inserted Ig heavy chain enhancer (25) were provided by Dr. M. Davis (Stanford, CA). AP-1-luciferase reporter transgenic mice (AP-1-luc) expressing the firefly luciferase gene driven by four human collagenase TREs (7) were provided by Dr. R. Flavell (Howard Hughes Medical Institute, Yale University, New Haven, CT). c-fos transgenic mice (H2-c-fos) carrying the mouse c-fos gene under the control of the H-2Kb gene promoter (26) were provided by Dr. T. Tokuhisa (Chiba University School of Medicine, Chiba, Japan). The AP-1-luc and the H2-c-fos mice were crossed with TCR-Vß3 transgenic mice and used for experiments. Recombinant SEA was expressed in Escherichia coli and purified to homogeneity as described previously (27). Various doses of SEA in PBS with 1% normal syngeneic serum or PBS alone were injected i.v. at 4-day intervals. The TCR-Vß3 transgenic mice expressed 90–95% of TCR-Vß3 in the CD3+ T cell population before and after SEA treatment.
Reagents
mAbs directed to murine CD3, CD4, B220, CD19, and TCR-Vß3 were purchased from PharMingen (San Diego, CA). Recombinant murine IL-2 and mAbs to murine IL-2 (JES6-1A12 and JES6-5H4) were obtained from PharMingen. PMA and ionomycin were purchased from ICN Pharmaceuticals (Costa Mesa, CA). A GST-c-Jun1–79-expressing plasmid was provided by Dr. M. Karin (University of California-San Diego). Production and purification of GST-c-Jun protein were performed as previously described (28).
Cell separation
Spleens were prepared from mice injected i.v. with SEA or PBS at different times before analysis. Purified CD4+ T cells (>95% CD4+ as determined by FACS analysis) and B220+ B cells (>95% CD19+) were obtained by positive selection using magnetic beads coated with anti-CD4 mAb or anti-B220 mAb (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions.
Luciferase activity analysis
Purified CD4+ T cells were incubated at 1 x 106 cells/well (24 wells/plate) in the presence or the absence of various stimuli. Purified B220+ B cells were used as APCs at 1 x 105 cells/well. After specific periods of time, cells from each independent well were harvested, washed twice in PBS, and lysed in lysis buffer (luciferase assay, Promega, Madison, WI) for 30 min at room temperature. Lysate was spun down for 5 min, and total supernatant was analyzed using the luciferase reagent (Promega) and measured in a luminometer (MicroLumat LB 96 P, Berthold, Nashua, NH). Background measurement was subtracted from each sample, and experimental values are expressed as recorded light units of luciferase activity.
IL-2 protein levels
Blood samples were drawn at various time points after i.v. injections of SEA or PBS and were tested for IL-2 content by a specific ELISA using mAbs JES6-1A12 and JES6-5H4 according to instructions from the manufacturer. The same method was used for IL-2 measurements in culture supernatants.
Preparation of nuclear extracts
Nuclear extracts were made according to the method described by Schreiber et al. (29). Purified CD4+ T cells (5–10 x 106) were used for extraction, and the nuclear proteins were finally dissolved in 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.5 µg/ml leupeptin, and 0.5 µg/ml antipain. The protein concentration of all extracts was measured by the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA), and the extracts were stored in aliquots at -70°C until used.
Western blot analysis
Before analysis by Western blotting, protein extracts were separated on 10% SDS-PAGE and electroblotted onto nitrocellulose membranes (0.2 µM; Bio-Rad). The membranes were probed with specific rabbit antisera. The immune complexes were detected by a 1/3000 dilution of goat anti-rabbit Ig-horseradish peroxidase conjugate (Bio-Rad) and were visualized using enhanced chemiluminescence detection (Amersham, Little Chalfont, U.K.). For analyses of Jun and Fos expression, nuclear protein extracts and the following rabbit polyclonal Abs were used: anti-c-Fos (4), anti-JunB (N-17), and anti-JunD (329) purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse anti-c-Jun mAb and a goat anti-mouse Ig-horseradish peroxidase conjugate for detection were obtained from Transduction Laboratories (Lexington, KY). To examine JNK protein abundance, cellular extracts and JNK-1 (C-17) and JNK-2 (FL) or pan-JNK (FL) rabbit polyclonal Abs (Santa Cruz Biotechnology) were used.
Electrophoretic mobility shift assay (EMSA)
The AP-1 consensus oligonucleotide used contained the following
sequence: 5'-CTAGTGATGAGTCAGCCGGATC-3'. The probe was end labeled with
[
-32P]ATP using T4 polynucleotide kinase (Promega),
according to instructions from the manufacturer and was purified on 5%
polyacrylamide gels in 1x TBE (89 mM Tris, 89 mM boric acid, and 2 mM
EDTA). Binding reactions were performed with the same amount of protein
in each reaction (0.5–1 µg) in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl,
1 mM EDTA, 1 mM DTT, 5% glycerol, and 2 µg of poly(dI-dC)
(Pharmacia, Piscataway, NJ). The reactions were incubated at 37°C for
30 min with 15,000 cpm of double-stranded 32P-labeled
oligonucleotides. The samples were electrophoresed on 5%
polyacrylamide gels in 1x TBE. The gels were dried under vacuum and
exposed to autoradiography at -70°C.
Preparation of cellular extracts
Total cellular extracts for immunocomplex protein kinase assays and Western blots were made from 5–10 x 106 purified CD4+ T cells according to the method of Hibi et al. (28). In individual experiments, the same number of cells from each group was used for extraction. The cells were lysed in 600 µl of cold lysis buffer consisting of 20 mM Tris-HCl (pH 7.7), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, and 0.5% Nonidet P-40; to which protease and phosphatase inhibitors had been freshly added. After a 30-min incubation on ice, the extracts were spun for 10 min in an Eppendorf centrifuge at 4°C to pellet cellular debris. The supernatants were removed and stored at -70°C.
Immunocomplex protein kinase assay
Immunocomplex kinase assays were performed as described by Hibi
et al. (28). Clarified cellular extracts were incubated with protein
A/G-agarose beads (Santa Cruz) precoated with polyclonal Ab to JNK-1
(C-17; Santa Cruz) for 2 h at 4°C with gentle rotation. Immune
complexes were washed and then assayed for enzyme activity as
described, using GST-c-Jun1–79 as a substrate. The beads
were pelleted and resuspended in 30 µl of kinase buffer (20 mM HEPES
(pH 7.6), 2 mM DTT, 10 mM MgCl2, 1 mM EDTA, and 1 mM EGTA,
to which phosphatase inhibitors had been freshly added) containing 1
µg of GST-c-Jun, 1 µM ATP, and 1 µCi of
[-32P]ATP. Incubations were conducted for 20 min at
30°C, then reactions were stopped by addition of 10 µl of 4x SDS
sample buffer. Phosphorylation of the substrates was analyzed by 10%
SDS-PAGE followed by autoradiography of the dried gel.
[32P]ATP incorporation was determined by cutting the gel
and measuring radioactivity in a beta counter.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously shown that repeated injections of SEA to
TCR-Vß3 transgenic mice induce a state of IL-2 hyporesponsiveness in
the CD4+ compartment that correlates with reduced DNA
binding of the AP-1 transcription factor (24). To confirm that AP-1 is
also perturbed at the transcriptional level, we used transgenic mice
expressing a luciferase reporter gene under the control of AP-1 binding
sites (AP-1-luc) (7). Double-transgenic TCR-Vß3xAP-1-luc mice were
treated with one injection of SEA to induce activation and with
repeated injections to induce anergy. CD4+ transgenic
spleen T cells were purified, stimulated in vitro with SEA-coated
syngeneic B220+ B cells, and analyzed for AP-1
transcriptional activity. Activated CD4+ T cells (1x SEA)
exhibited significant AP-1 transcriptional activity in response to SEA
(Fig. 1
A), while no activity
could be detected in the anergic CD4+ T cells (3x SEA)
(Fig. 1A). Addition of PMA resulted in an enhanced response
in activated cells, but did not restore activity in the anergized cells
(Fig. 1A). However, the combination of PMA and ionomycin
resulted in a similar response in both groups (Fig. 1A),
suggesting that both a rise in intracellular calcium and activation of
protein kinase C are required to circumvent the block in the anergic T
cells. Stimulation with anti-CD3 and anti-CD28 Abs induced AP-1
activity in the activated T cells (1x SEA), but failed to elicit a
similar response in the in vivo anergized T cell population (3x SEA)
(data not shown), ruling out the possibility that these cells are
unresponsive because of a lack of specificity to SEA. Parallel analysis
of IL-2 production in cultures showed patterns similar to those for
AP-1 transcriptional activities (Fig. 1B), emphasizing the
importance of AP-1 in IL-2 regulation.
|
AP-1 activity is controlled by synthesis of its subunits Fos and
Jun and by posttranslational protein phosphorylations (30). To
determine whether the defective AP-1 transcriptional activity observed
in anergic T cells was due to reduced expression of Jun and Fos
proteins, we analyzed nuclear Fos and Jun protein content by Western
blotting. We have previously shown that large amounts of AP-1 DNA
binding were induced in CD4+ T cells 1 h after a
single SEA injection (24). The AP-1 complexes detected by EMSA
contained mainly heterodimers between c-Fos and JunB or JunD (24).
Using Western blot analysis of the nuclear extracts, we found that JunD
was constitutively expressed, and the amount did not change following
activation either by a single (1x SEA) or repeated (3x SEA)
injections (Fig. 2A). In
contrast, c-Fos, JunB, and c-Jun were expressed only after stimulation
(Fig. 2, B–D). Interestingly, the induction of c-Fos was
defective in anergic CD4+ T cells (Fig. 2D). On
the contrary, JunB protein was induced in both groups to a similar
extent (Fig. 2B). c-Jun was expressed at low levels, which
were further reduced in the anergic cells (Fig. 2C). Taken
together, these results suggested that the reduced c-Fos expression may
explain the defective AP-1 DNA binding and trans-activation
in the anergic CD4+ T cells.
|
In an attempt to restore the possible c-Fos defect in the anergic
T cells, we used transgenic mice that express c-fos under an
MHC class I promoter (H2-Kb; H2-c-fos) (26). These mice
were crossed with the TCR-Vß3 transgenic mice and used for anergy
experiments. Constitutive expression of c-fos was confirmed
by RT-PCR (data not shown). Double-transgenic mice were injected one or
three times with SEA. Nuclear extracts from purified CD4+ T
cells were analyzed by EMSA for AP-1 DNA binding. Interestingly,
CD4+ T cells from H2-c-fosxTCR-Vß3 transgenic mice
anergized by SEA (3x SEA) contained significantly more AP-1 than
anergic CD4+ T cells from control mice (Fig. 3A). The levels of AP-1 in
H2-c-fos transgenic mice anergized by SEA corresponded to the levels
observed in CD4+ T cells from control mice activated by a
single injection of SEA (1x SEA), suggesting that c-Fos indeed was a
limiting factor for obtaining AP-1 band shifts. Supershift analysis of
the obtained AP-1-binding protein complex in H2-c-fos transgenic mice
demonstrated the same constitution as that previously described for
SEA-treated TCR-Vß3 transgenic mice (24), consisting mainly of
heterodimers between c-Fos and JunB or JunD (data not shown). In
contrast, analysis of IL-2 production in serum from double-transgenic
mice revealed a perturbed IL-2 response similar to that in control mice
(Fig. 3B). This suggests that there is an additional defect
in the AP-1 pathway, possibly due to a perturbation in the
posttranslational modifications of Fos and Jun.
|
To exclude the possibility that AP-1 transcriptional activity was
restored in the H2-c-fos mice and that the absence of IL-2 production
instead represented additional defects in other transcription factors,
we crossed the H2-c-fos mice with the AP-1-luc mice on
TCR-Vß3+ background. Triple-transgenic mice were
activated (1x SEA) or anergized (3x SEA) by SEA in vivo. Purified
CD4+ T cells were stimulated with SEA-coated syngeneic
B220+ B cells and PMA in vitro and analyzed for AP-1
transcriptional activity. Activated H2-c-fos CD4+ T cells
(1x SEA) exhibited significant AP-1 transcription in response to SEA
and PMA (Fig. 4A), while the
anergic H2-c-fos CD4+ T cells failed to respond (Fig. 4A). Addition of ionomycin restored the response in the
anergic cells (Fig. 4A), as shown above (Fig. 1
A). Similarly, the AP-1 transcriptional activity correlated
with the ability to synthesize IL-2 (Fig. 4B). Thus, these
results suggest that the AP-1 complex induced by overexpression of
c-Fos is transcriptionally inactive.
|
Synthesis of Fos and Jun proteins is not sufficient to induce AP-1
activity. In addition, these proteins need to be phosphorylated to
become transcriptionally active (30). Protein kinase activities that
phosphorylate and activate Jun (JNK) and Fos (FRK) have been described
(28, 31). However, the c-Fos kinase FRK is poorly understood. To
investigate whether JNK activity was reduced in the anergic
CD4+ T cells, we used a specific immunocomplex protein
kinase assay. TCR-Vß3 transgenic mice were injected one or three
times with SEA to induce activation or anergy, respectively. Analysis
of JNK activity in purified CD4+ T cells revealed that a
significant response was rapidly induced in activated CD4+
T cells (1x SEA), with peak activity after 0.5 h in vivo (Fig. 5A). In contrast, the anergic
cells exhibited significantly lower basal JNK activity before the last
SEA injection and a minor activation-induced increase (Fig. 5A). Analysis of JNK protein levels by Western blotting
showed that these proteins were constitutively expressed (Fig. 5B) in conformity with earlier studies (21). Thus, the
defective AP-1 activity in anergized CD4+ T cells may be
the result of deficient JNK activation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
AP-1 protein activity is regulated both at the level of gene transcription and by posttranslational modifications, which are largely controlled by protein kinases of the MAPK family (30). c-fos transcription is controlled by ERK-dependent phosphorylation of the ternary complex factor Elk-1 that regulates the c-fos promoter (32). Activation of JNK results in the phosphorylation of c-Jun and JunD, which augments their transcriptional activities (28, 33). In addition, JNK phosphorylates ATF-2, which together with c-Jun bind to a nonconventional TRE site in the c-jun promoter and induce c-jun transcription (34). It was recently demonstrated that c-Fos, FosB, and JunB proteins are reduced in Th1 clones anergized by anti-CD3 treatment in vitro (22), which correlated with impaired activities of the MAPK family members ERK and JNK (21). In this study, we observed that in vivo anergized CD4+ T cells expressed less c-Fos protein than activated CD4+ T cells. In contrast, both JunB and JunD proteins were expressed at similar levels in activated and anergic CD4+ T cells. Thus, a potential explanation for defective AP-1 trans-activation in anergic cells in vivo would be poor activation of MAPK and subsequent suboptimal Fos protein expression. However, overexpression of c-Fos protein in H2-c-fos transgenic mice restored AP-1 DNA binding, but failed to induce IL-2 production and proper AP-1-dependent transcription. Thus, in addition to the observed defect in synthesis of the Fos/Jun family of proteins observed here and by others (22), anergic T cells lack posttranslational modifications of AP-1 proteins required for AP-1-dependent trans-activation.
Protein kinase activities that phosphorylate and activate Jun (JNK) and
Fos (FRK) have been described. Whereas the c-Fos kinase FRK is poorly
understood, the c-Jun kinase JNK has been studied in detail. The JNK
protein kinases bind Jun proteins at a docking site between amino acids
30 and 60 (30) and subsequently phosphorylate the
NH2-terminal activation domain of c-Jun, causing increased
transcriptional activity (28, 33). JunD is also a substrate for JNK,
but contains a weaker docking site for JNK than c-Jun (33).
Interestingly, full activation of JNK1 and JNK2 has been reported to
require two signals in T cells, i.e., ligation of both TCR and CD28
(17). Studies of T cell cultures in vitro have further shown that the
activity of JNK correlates to the ability of T cells to synthesize IL-2
(17). In contrast, MAPK ERK1 and ERK2 were activated by TCR stimulation
alone and were not affected by CD28 (17). Hence, integration of signals
that lead to full T cell activation may occur at the levels of JNK
activation. In this study rapid induction of JNK activity in
CD4+ T cells was observed after SEA priming in vivo. In
contrast, we observed inhibition of JNK activation in SEA-anergized T
cells. This could explain the absence of AP-1 transcriptional activity
in the c-Fos-overexpressing cells, although prominent AP-1 DNA binding
was recorded. Supershift analysis of the induced AP-1 complexes showed
that both the activated and anergic T cells contained mainly
heterodimers between c-Fos and JunB or JunD (24) (data not shown). We
did, however, detect c-Jun protein by Western blotting and found
reduced protein levels in the anergic cells (Fig. 2). Thus, it could be
speculated that c-Jun may be present at other important regulatory
elements of the IL-2 promoter, such as the CD28RE/AP-1 (35) and NF-AT
(9) sites, and that a defect in JNK activation may result in reduced
transcriptional activity of those sites. Indeed, a dominant negative
mutant form of c-Jun was shown to inhibit NF-AT-driven transcription
and to prevent IL-2 gene expression (36).
Inhibition of JNK activation may also explain the observed defective expression of c-Fos protein in the anergic T cells. In addition to its ability to activate Jun synthesis and transcriptional activity, it was recently demonstrated that JNK activates the c-fos promoter through phosphorylation of TCF/Elk-1 (37). Furthermore, expression of the protein kinase MEKK-1 in the JNK cascade induced c-fos transcription and Elk-1 transcriptional activity in the absence of considerable ERK activation (37). Interestingly, induction of MEKK-1 activity also requires two signals in T cells (38), suggesting similar activation requirements for JNK and MEKK-1 proteins. However, the roles of ERK and JNK in the induction of Fos and Jun proteins and in the control of AP-1 transcription remain to be completely established in T cells. The absence of AP-1 transcriptional activity in anergized CD4+ T cells could in addition include a failure to phosphorylate c-Fos. However, little information is available about FRK (31). FRK is a protein kinase of the MAPK family that is stimulated via the p21ras pathway and phosphorylates c-Fos at Thr232 (31). The role of FRK in regulation of AP-1 activity in T cells is not known. Interestingly, when analyzing the induced c-Fos protein using higher resolution electrophoresis, we observed the presence of a slower migrating band in the activated CD4+ T cells that was absent in the anergic cells (data not shown). Whether this represents a phosphorylated form of c-Fos remains to be determined.
In a recent study by Li et al., it was demonstrated that the block in ERK and JNK activation upon restimulation of anergic T cells in vitro could be completely overcome by stimulation with phorbol ester (21). They further showed that triggering of anergic cells resulted in dephosphorylation and translocation of the calcium/calcineurin-dependent transcription factor NF-AT (21). Taken together, these results suggested that the block in anergic T cells was not due to a global defect in TCR/CD3 signal transduction, but occurred specifically at the level of p21ras. In T cells, PMA activates protein kinase C, which, in turn, activates p21ras by inhibiting Ras GTPase-activating protein (39). A defect in Ras activation was directly demonstrated by Fields et al., who showed that conversion of GDP-p21ras to the activated GTP-bound form was prevented in the anergized cells (39). In this study we were unable to restore AP-1 transcription by PMA alone. A rise in intracellular calcium by ionomycin treatment was necessary to induce AP-1 activity in the anergized T cells. Thus, in addition to the defect in Ras activation observed in vitro (21, 40), the in vivo anergized CD4+ T cells seem to mobilize insufficient levels of calcium to trigger AP-1 transcription.
Rincón and Flavell (7) demonstrated the occurrence of DNA
binding, but transcriptionally inactive AP-1 in primary T cells in
response to PMA. Furthermore, they reported that blocking of the
calcium-dependent pathway by cyclosporin A, which inhibits the protein
phosphatase calcineurin (41), had minor effect on AP-1 DNA binding, but
completely inhibited the transcriptional activity (7). In a recent
study on B cells by Goodnow and co-workers, it was demonstrated that
the amplitude and duration of calcium signals contribute to selective
activation of NF-AT, NF-B, and JNK (42). NF-B and JNK required a
large transient Ca2+ rise for activation, whereas NF-AT was
activated by a low sustained Ca2+ plateau (42).
Furthermore, stimulation of B cells tolerized by self Ag was shown to
trigger low calcium increases and activated NF-AT and ERK but not
NF-B and JNK (43). The need for activation of the calcium- and
calcineurin-dependent pathway by ionomycin to restore AP-1
transcription may imply that suboptimal calcium mobilization
contributes to the insufficient response in in vivo anergized
CD4+ T cells. Indeed, studies on T cell clones have
implicated that low amplitude Ca2+ responses are involved
in the induction of T cell anergy by altered peptide ligands (44).
In conclusion, we have demonstrated that in vivo anergized CD4+ T cells contain multiple defects in the AP-1 pathway, which strongly correlated to the inability of these cells to induce JNK kinase activity and to synthesize IL-2. The requirements for both PMA and ionomycin to restore AP-1 and IL-2 responses led us to propose that a block in p21ras activation and suboptimal increase in intracellular Ca2+ may be involved in the induction of anergy in vivo. Identification of the upstream targets responsible for regulation of calcium amplitude and uncoupling of the JNK pathway in anergic T cells will be a major focus for future studies.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: AP-1, activating protein-1; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive element; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, Jun N-terminal kinase; FRK, Fos-regulating kinase; MEK, MAPK/ERK kinase; SEK, Stress-activated protein lignase kinase; SEA, staphylococcal enterotoxin A; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay.
Received for publication March 20, 1998. Accepted for publication July 27, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B transcription factor activation in T cells. Ann. NY Acad. Sci. 766:245.[Medline]
B transcription factors. Proc. Natl. Acad. Sci. USA 93:979.This article has been cited by other articles:
|
|
 |
|
  J. Garaude, R. Farras, G. Bossis, S. Charni, M. Piechaczyk, R. A. Hipskind, and M. Villalba SUMOylation Regulates the Transcriptional Activity of JunB in T Lymphocytes J. Immunol., May 1, 2008; 180(9): 5983 - 5990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Morton, B. McManus, P. Garside, A. McI. Mowat, and M. M. Harnett Inverse Rap1 and Phospho-ERK Expression Discriminate the Maintenance Phase of Tolerance and Priming of Antigen-Specific CD4+ T Cells In Vitro and In Vivo J. Immunol., December 15, 2007; 179(12): 8026 - 8034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Su, N. Lineberry, Y. Huh, L. Soares, and C. G. Fathman A Novel E3 Ubiquitin Ligase Substrate Screen Identifies Rho Guanine Dissociation Inhibitor as a Substrate of Gene Related to Anergy in Lymphocytes J. Immunol., December 1, 2006; 177(11): 7559 - 7566. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Ise, K. Nakamura, N. Shimizu, H. Goto, K. Fujimoto, S. Kaminogawa, and S. Hachimura Orally Tolerized T Cells Can Form Conjugates with APCs but Are Defective in Immunological Synapse Formation J. Immunol., July 15, 2005; 175(2): 829 - 838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Su, R. J. Creusot, E. M. Gallo, S. M. Chan, P. J. Utz, C. G. Fathman, and J. Ermann Murine CD4+CD25+ Regulatory T Cells Fail to Undergo Chromatin Remodeling Across the Proximal Promoter Region of the IL-2 Gene J. Immunol., October 15, 2004; 173(8): 4994 - 5001. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Adams, A. M. Grierson, A. M. Mowat, M. M. Harnett, and P. Garside Differences in the Kinetics, Amplitude, and Localization of ERK Activation in Anergy and Priming Revealed at the Level of Individual Primary T Cells by Laser Scanning Cytometry J. Immunol., August 1, 2004; 173(3): 1579 - 1586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Grundstrom, P. Anderson, P. Scheipers, and A. Sundstedt Bcl-3 and NF{kappa}B p50-p50 Homodimers Act as Transcriptional Repressors in Tolerant CD4+ T Cells J. Biol. Chem., February 27, 2004; 279(9): 8460 - 8468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson CTLA-4 Is Not Required for Induction of CD8+ T Cell Anergy In Vivo J. Immunol., November 1, 2001; 167(9): 4936 - 4941. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Guerder, M. Rincon, and A.-M. Schmitt-Verhulst Regulation of Activator Protein-1 and NF-{{kappa}}B in CD8+ T Cells Exposed To Peripheral Self-Antigens J. Immunol., April 1, 2001; 166(7): 4399 - 4407. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Attinger, H. Acha-Orbea, and H. R. MacDonald Cutting Edge: Cell Autonomous Rather Than Environmental Factors Control Bacterial Superantigen-Induced T Cell Anergy In Vivo J. Immunol., August 1, 2000; 165(3): 1171 - 1174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Utting, S.-J. Teh, and H.-S. Teh A Population of In Vivo Anergized T Cells with a Lower Activation Threshold for the Induction of CD25 Exhibit Differential Requirements in Mobilization of Intracellular Calcium and Mitogen-Activated Protein Kinase Activation J. Immunol., March 15, 2000; 164(6): 2881 - 2889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Frauwirth, M.-L. Alegre, and C. B. Thompson Induction of T Cell Anergy in the Absence of CTLA-4/B7 Interaction J. Immunol., March 15, 2000; 164(6): 2987 - 2993. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Labuda, A. Sundstedt, and M. Dohlsten Selective induction of p38 mitogen-activated protein kinase activity following A6H co-stimulation in primary human CD4+ T cells Int. Immunol., March 1, 2000; 12(3): 253 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Auphan, S. Ghosh, R. A. Flavell, and A.-M. Schmitt-Verhulst Differential Requirements for NF-{kappa}B and AP-1 trans-Activation in Response to Minimal TCR Engagement by a Partial Agonist in Naive CD8 T Cells J. Immunol., November 15, 1999; 163(10): 5219 - 5227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. K. Teague, D. Hildeman, R. M. Kedl, T. Mitchell, W. Rees, B. C. Schaefer, J. Bender, J. Kappler, and P. Marrack Activation changes the spectrum but not the diversity of genes expressed by T cells PNAS, October 26, 1999; 96(22): 12691 - 12696. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
