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Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-associated protein of 70 kDa (ZAP-70) upon TCR
engagement. The molecular basis underlying the deficient
phosphorylation of LAT and consequently the hyporesponsiveness of the
synovial fluid T lymphocytes lies in the membrane displacement of LAT.
We demonstrate that the subcellular localization of LAT is sensitive to
changes in the intracellular levels of the antioxidant glutathione. The
membrane anchorage of LAT, and consequently the phosphorylation of LAT
and the cellular activation of the synovial fluid T lymphocytes upon
TCR engagement, is restored in synovial fluid T lymphocytes after
supplementation of the intracellular glutathione levels with
N-acetyl-L-cysteine. These data suggest a
role for the membrane displacement of LAT in the hyporesponsiveness of
the synovial fluid T lymphocytes as a consequence of oxidative
stress. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to
TCR stimulation (4, 5, 6, 7). Although unable to respond to the
Ag-dependent triggering of the TCR/CD3 complex, evidence exists that
the T lymphocytes in the inflamed joint induce damage of the joint
cartilage through Ag-independent cell-cell contact with macrophages
(8, 9, 10, 11). Furthermore, Salojin and coworkers recently
suggested that the hyporesponsiveness of regulatory T lymphocytes that
normally confer protection from autoimmune disease due to altered TCR
signaling might contribute to the breakdown of tolerance and increase
susceptibility to autoimmune disease (12). The prolonged
residence of the T lymphocytes in the synovium thus seems to contribute
to and perpetuate the disease. The hyporesponsive state of the SF T lymphocytes in RA correlates with markers of oxidative stress: the intracellular levels of the antioxidant glutathione (GSH) are significantly decreased in SF T lymphocytes. Moreover, the extracellular levels of another important redox regulator, thioredoxin are significantly increased in the SF (13, 14). GSH is an important regulator of the cellular redox balance and plays a major role in the protection against oxidative stress both by reacting directly with reactive oxygen radicals and acting as a substrate in the detoxification of H2O2 and organic peroxides. In addition, GSH is involved in maintaining the sulfhydryl groups of intracellular proteins in a reduced state, which is required for their functional conformation (15, 16). It has previously been shown that the depletion of the intracellular GSH levels in T lymphocytes through treatment with DL-buthionine (S,R)-sulfoximine (BSO) results in hyporesponsiveness due to the abrogation of the proximal TCR-mediated signaling events (17).
The proximal signaling pathways induced by the engagement of the
TCR/CD3 complex by ligand include the activation of two classes of
protein tyrosine kinases, the Src and Syk
families. The recruitment of the Syk kinase -associated
protein of 70 kDa (ZAP-70) to the TCR -chain plays a central role as
it serves to bring the kinase in close proximity of its substrates
(18, 19, 20, 21). Two substrates of ZAP-70 have been identified:
the adaptor proteins Src homology 2 (SH2) domain-containing leukocyte
protein of 76 kDa (SLP-76) and linker for activation of T cells (LAT)
(22, 23, 24). LAT has recently been cloned and identified as
the 36- to 38-kDa protein that had previously been shown to play an
important role in the TCR-mediated signaling pathways (22, 25, 26, 27). LAT is selectively expressed by T lymphocytes and NK
cells, where it is localized in the plasma membrane through its
N-terminal 
-helical structure (22, 28). Several
signaling proteins, including Grb2, the p85 subunit of
phosphatidylinositol 3-kinase, Grb2-like accessory protein (Grap), and
phospholipase C1 (PLC1) associate with phosphorylated LAT through
their SH2 domains to generate multiprotein complexes that amplify the
TCR-induced signals (22, 26, 27, 28, 29).
The molecular basis underlying the hyporesponsiveness of the SF T
lymphocytes in RA remains elusive, although some leads have evolved.
First, it has been observed that the expression of the signaling chain
of the TCR/CD3 complex, the TCR -chain, is down-regulated in SF T
lymphocytes (30, 31). Second, the tyrosine phosphorylation
of substrates upon TCR stimulation shows an aberrant pattern in SF T
lymphocytes. Specifically, the phosphorylation of a protein of 36–38
kDa is absent in SF but not peripheral blood (PB) T lymphocytes from RA
patients (31). Third, a more distal event in the
TCR-mediated signaling pathways, the influx of
Ca2+ from the endoplasmic reticulum, is reduced
in PB T lymphocytes from RA patients, which also display some degree of
hyporesponsiveness (32, 33, 34). These observations seem to be
consistent with a defect in the proximal TCR-mediated signaling events
in SF T lymphocytes in RA.
In the present study, we set out to elucidate the molecular mechanisms
underlying the hyporesponsiveness of the T lymphocytes in the synovium
of RA patients and establish the role oxidative stress plays in this
process. We present evidence that the 36- to 38-kDa phosphoprotein that
does not become phosphorylated in TCR-stimulated SF T lymphocytes is
the adaptor protein LAT. We also show that LAT is displaced from the
membrane in SF T lymphocytes due to the severely reduced intracellular
GSH levels. The displacement of LAT is responsible for the abrogation
of its phosphorylation upon TCR stimulation, as well as the subsequent
association of PLC1 with LAT, hence blocking the TCR-induced
signaling pathways leading to the influx of Ca2+
and eventually to the expression of IL-2. The treatment of SF T
lymphocytes with N-acetyl-L-cysteine
(NAC) elevates the intracellular GSH levels and consequently restores
the membrane localization and phosphorylation of LAT and ultimately the
cellular activation of the SF T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes from heparine-collected PB or SF of RA patients were isolated through a negative selection procedure. Mononuclear cell suspensions were prepared by Ficoll-Hypaque density-gradient centrifugation. Monocytes, B lymphocytes, and NK cells were depleted by incubating with mAbs against CD14, CD16, and CD19 (10 µg of each mAb per 20 x 106 of mononuclear cells; Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB), Amsterdam, The Netherlands) and sheep anti-mouse IgG coated-dynabeads (Dynal, Oslo, Norway) for 1.5 h, after which cells rosetted with immunomagnetic beads were removed with a Dynal magnetic particle concentrator. The remaining cell preparations contained >95% T lymphocytes as assessed by flow cytometric analysis after staining with a peridinin chlorophyl protein (PerCP)-conjugated anti-CD3 mAb (Becton Dickinson, San Jose, CA). Control human PB T lymphocytes were obtained from healthy volunteer platelet donors, and mononuclear cell suspensions were prepared by Ficoll-Hypaque density-gradient centrifugation. T lymphocytes were isolated by 2-aminoethylisothiouronium bromide-treated SRBC rosetting. The SRBC were lysed with 155 mM NH4Cl, 10 mM KHCO3, and 0.1 mM EDTA according to standard procedures. The remaining cell preparations contained >92% T lymphocytes as assessed by FACS analysis after staining with a PerCP-conjugated anti-CD3 mAb (Becton Dickinson). After isolation, T lymphocytes were kept at 37°C, 5% CO2 in IMDM (Life Technologies, Gaithersburg, MD) containing 10% FCS (Life Technologies) supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin (Boehringer Mannheim, Mannheim, Germany).
Stimulation
T lymphocytes (5 x 106/ml) were incubated for various time periods with 1 µg/ml anti-CD3 mAb (1XE; CLB), in combination with 1 µg/ml anti-CD28 mAb (15E8; CLB) as indicated. NAC (Sigma, St. Louis, MO) was added at a final concentration of 5 mM, while BSO (Sigma) was added at a final concentration of 200 µM.
Immunoprecipitation, Western blotting, and immunodetection
Whole-cell lysates were prepared from 5 x 106 T lymphocytes, either unstimulated or stimulated with anti-CD3 for 3 min, when indicated after incubation with either NAC or BSO. Cells were harvested, washed twice with PBS, and lysed in 300 µl lysis buffer (10 mM triethanolamine, pH 7.8, 150 mM NaCl, 5 mM EDTA, 10 mM Na3VO4, 1% Nonidet P-40) supplemented with protease inhibitors (10 µg/ml leupeptin (Sigma), 0.4 mM PMSF (Sigma)) during 45 min on ice. Insoluble debris was spun down in a microcentrifuge for 15 min at 4°C.
To detect tyrosine phosphorylation of LAT or association of PLC1
with LAT, LAT was immunoprecipitated from 300 µl of cell lysate
(equivalent to 5 x 106 cells) by incubating
with 3 µg rabbit anti-LAT pAb (06-807; Upstate Biotechnology,
Lake Placid, NY) during 16 h at 4°C while rotating, and an
additional 2 h after the addition of 25 µl of protein A-Agarose
beads (50% slurry; Santa Cruz Biotechnology, Santa Cruz, CA). The
immunoprecipitates were spun down in a microcentrifuge for 30 s at
4°C, washed twice with lysis buffer, and then boiled in 1x SDS-PAGE
sample buffer. The proteins were separated by SDS-PAGE on a 5%
(PLC1 detection) or 10% (LAT detection) gel, using Rainbow-colored
protein m.w. markers (Amersham, Little Chalfont, U.K.) as a reference,
and transferred onto a polyvinylidene difluoride membrane (Millipore,
Bedford, MA). The membrane was blocked in PBS containing 5% skin milk
and 0.01% Tween 20 during 1 h. Detection of tyrosine
phosphorylated proteins was performed by incubating the membranes with
a mouse mAb against phospho-Tyr (PY99) (1:1000; sc-7020;
Santa Cruz Biotechnology), while LAT and PLC1 were detected by
incubating the membranes with the rabbit polyclonal Abs (pAbs) against
LAT (1:1000) and PLC1 (1:250) for 16 h. The membranes were
subsequently incubated with the appropriate secondary Abs
(HRP-conjugated rabbit anti-mouse Ig (1:5000; Dako, Glostrup,
Denmark) or swine anti-rabbit Ig-HRP (1:5000; Dako)) for 3 h,
and then assayed using the enhanced chemiluminescence (ECL) detection
system (Amersham). Membranes were stripped of bound Abs by incubating
the membranes for 30 min at 50°C in stripping buffer (100 mM 2-ME,
2% SDS, 62.5 mM Tris-HCl, pH 6.7) to allow for a second round of
detection.
Flow cytometric analysis
A total of 2.5 x 105 T lymphocytes
were fixed for 10 min in 1% para-formaldehyde in PBS at
room temperature (RT), washed twice with PBS, and stored overnight at
4°C. The expression of LAT, ZAP-70, or PLC1 was measured via a
two-step intracellular staining procedure. Cells were incubated for 10
min at RT in PBS containing 1 mg/ml BSA (Sigma), 10% FCS, and 100
µg/ml saponin (Sigma) as a permeabilizing agent. Cells were washed
three times with PBS/BSA/saponin and incubated for 1 h at 4°C
with appropriate dilutions of the primary Abs (LAT, 1:250; ZAP-70 pAb
(sc-574; Santa Cruz Biotechnology), 1:35; PLC1 pAb
(sc-81; Santa Cruz Biotechnology), 1:35). After three washes
(PBS/BSA/saponin), cells were incubated for 30 min at 4°C with PE
(R-PE)-conjugated goat anti-rabbit IgG (Harlan Sera-Lab, Crawley
Down, U.K.). A negative control was incubated with the secondary Ab
only. After three washes (PBS/BSA/saponin), the free
F(ab')2 sites of cell-bound goat
anti-rabbit-PE were blocked using 10% normal rabbit serum,
whereafter cells were stained with a PerCP-conjugated anti-CD3 mAb
(Becton Dickinson) (1:25) for 1 h at 4°C. CD3-positive cells
were analyzed on a FACScan using the CellQuest software (Becton
Dickinson). The levels of LAT, ZAP-70, and PLC1 are expressed as the
ratio of the mean fluorescence intensity (MFI), i.e., the MFI of LAT,
ZAP-70, or PLC1 divided by the MFI of the negative control.
ZAP-70 kinase activity assay
Total cell lysates were prepared from 5 x 106 T lymphocytes, either unstimulated or stimulated with anti-CD3 for 3 min. Cells were harvested and washed once with PBS, resuspended in 500 µl lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 1 mM DTT, 1% Triton X-100, 10% glycerol) supplemented with protease inhibitors (10 µg/ml leupeptin, 0.4 mM PMSF) and phosphatase inhibitors (50 mM ß-glycerophosphate, 1 mM Na3VO4), and incubated on ice for 30 min. Insoluble debris was spun down in a microcentrifuge for 15 min at 4°C.
ZAP-70 was immunoprecipitated from 500 µl of cell lysate (equivalent
to 5 x 106 cells) by incubating with 800 ng
rabbit anti-ZAP-70 pAb (Santa Cruz Biotechnology) during 1 h
at 4°C while rotating, and an additional 16 h after the addition
of 25 µl of protein A-Agarose beads (50% slurry; Santa Cruz
Biotechnology). The immunoprecipitates were spun down in a
microcentrifuge for 30 s at 4°C, washed twice with lysis buffer,
twice with LiCl buffer (500 mM LiCl, 100 mM Tris-HCl, pH 7.6, 1 mM DTT,
0.1% Triton X-100), and finally twice with assay buffer (25 mM
Tris-HCl, pH 7.0, 150 mM NaCl, 10 mM MnCl2, 1 mM
DTT, 0.1 mM Na3VO4, 0.1%
Triton X-100). The immunoprecipitates were assayed for kinase activity
in 30 µl assay mix containing 25 mM Tris-HCl, pH 7.0, 150 mM NaCl, 10
mM MnCl2, 1 mM DTT, 0.1 mM
Na3VO4, 0.1% Triton X-100,
250 µM ATP, and 10 µCi [-32P]ATP (3000
Ci/mmol; Amersham) during 20 min at 30°C, using 10 µg tubulin
(Sigma) as substrate. The reactions were terminated by the addition of
5x SDS-PAGE sample buffer and boiling. Proteins were separated by
SDS-PAGE on a 10% gel, using Rainbow-colored protein m.w. markers
(Amersham) as a reference. Quantification of phosphorylated substrates
was performed using a PhosphorImaging system and the ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Immunofluorescence staining and microscopy
A total of 1 x 105 T lymphocytes were mounted onto adhesive microscope slides, air dried, and kept frozen until staining. Before staining, the cells were fixed in 4% para-formaldehyde in PBS for 15 min at RT. After three washes in PBS containing 5% BSA, cells were permeabilized using 0.1% Triton X-100 in PBS for 4 min at RT, washed three times (PBS/BSA), and preblocked for 45 min at RT in PBS containing 10% FCS. Cells were then incubated with a rabbit pAb against LAT (1:250) in PBS/FCS for 45 min at RT, washed three times (PBS/BSA), and incubated with FITC-conjugated swine anti-rabbit Ig (1:100; Dako) in PBS/FCS for 45 min at RT. A negative control was incubated with the secondary Ab only. After three final washes (PBS), cells were imbedded in 1 mg/ml p-phenyleendiamine (Sigma) in 90% glycerol, 10% PBS and covered with a coverslip. To determine whether the cell membrane was intact after BSO treatment, cells were stained immediately after fixation with a mouse mAb against CD3 (1:25; Becton Dickinson) and detected using tetramethylrhodamine isothiocyanate (TRITC)-conjugated rabbit anti-mouse Ig (1:200; Dako). Cells were viewed using a Leitz Aristoplan microscope (Leica, Wetzlar, Germany) equipped with a 100x objective and optics for FITC and TRITC. Images were taken with a Sony 3 CCD Color Video Camera, model DXC-9508 (Sony, Tokyo, Japan).
Preparation of membrane and cytoplasmic cell fractions
A total of 10 x 106 T lymphocytes were disrupted in 500 µl extraction buffer (50 mM Tris-HCl, pH 7.0, 10 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na3VO4) supplemented with protease inhibitors (10 µg/ml leupeptin, 0.4 mM PMSF) by shearing through a 25-gauge needle. After centrifugation at 1600 x g for 10 min at 4°C, this extraction step was repeated on the pellet. The membrane and cytoplasmic fractions were separated from the pooled supernatants at 100,000 x g for 60 min at 4°C. The supernatant after ultracentrifugation contains the cytoplasmic proteins (Triton X-100 was added to 1%). The pellet containing the membrane proteins was resuspended in 500 µl buffer (10 mM triethanolamine, pH 7.8, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1% Nonidet P-40) supplemented with protease inhibitors (10 µg/ml leupeptin, 0.4 mM PMSF). LAT was immunoprecipitated from the extracts and detected with ECL Western blotting as described above.
Measurement of secreted IL-2 protein
Human T lymphocytes (3 x 106/ml) were left unstimulated or stimulated with anti-CD3 and anti-CD28 for 24 h when indicated after incubation with either NAC or BSO. Secreted IL-2 protein was quantified in cell-free supernatants using a human IL-2 ELISA kit (Genzyme, Cambridge, MA) as recommended by the manufacturer.
Statistical analysis
Statistical analyses were performed on the data using the Fisher’s exact test for paired observations. Statistical significance of the data was set at p < 0.05.
Patient identification
Patients enrolled in this study fulfilled the 1987 revised
criteria of the American College of Rheumatology for rheumatoid
arthritis, differed between 27 and 72 years of age (mean age 57 years),
with a female:male ratio of 9:8 (see Table I
).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tyrosine phosphorylation pattern upon TCR stimulation of SF T
lymphocytes from RA patients is aberrant, with, most particular, the
absence of the phosphorylation of a 36- to 38-kDa protein
(31). To determine whether this phosphoprotein of 36–38
kDa is identical with the adaptor protein LAT, we immunoprecipitated
LAT from whole-cell extracts of SF and PB T lymphocytes from RA
patients that were either left unstimulated or stimulated for 3 min
with anti-CD3 and assessed the phosphorylation status of LAT
through Western blotting analysis. We observed no phosphorylated LAT in
unstimulated T lymphocytes. In PB T lymphocytes from RA patients, LAT
becomes extensively phosphorylated on tyrosine residues upon TCR
stimulation, as occurs in T lymphocytes from healthy controls (Fig. 1). However, after stimulation of the
TCR/CD3 complex in SF T lymphocytes from RA patients, we did not detect
any phosphorylated LAT, indicating that the missing 36- to 38-kDa
phosphoprotein in SF T lymphocytes is indeed LAT (Fig. 1). Western
blotting analysis revealed that the impaired phosphorylation of LAT is
not due to a lack of expression of LAT, because we could detect LAT in
extracts from SF as well as PB T lymphocytes from RA patients, although
the level of LAT expression seemed slightly lower in both SF and PB T
lymphocytes compared with the expression of LAT in T lymphocytes from
healthy controls (Fig. 1). To quantitatively determine the expression
of LAT at the individual cell level, we measured the MFI after the
intracellular immunofluorescence staining of LAT by flow cytometric
analysis. The MFI ratio for LAT in both PB and SF T lymphocytes was
2-fold lower compared with the LAT MFI ratio in T lymphocytes from
healthy controls: 104.4 ± 5.1 (mean ± SEM;
n = 5) for healthy controls vs 52.2 ± 2.2
(mean ± SEM; n = 4; p = 0.003)
for PB T lymphocytes and 48.9 ± 1.7 (mean ± SEM;
n = 4; p < 0.001) for SF T lymphocytes
(Fig. 2). These data support the
conclusion that the significantly decreased expression of LAT in SF T
lymphocytes does not account for the lack of phosphorylation of LAT,
because the phosphorylation of LAT remains intact in PB T lymphocytes
from RA patients that contain similar levels of LAT.
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The phosphorylation of LAT upon TCR ligation is regulated by the
syk family tyrosine kinase ZAP-70 (22). One
explanation for the lack of phosphorylation of LAT in SF T lymphocytes
could be that either the expression of ZAP-70 or the activation of the
kinase activity of ZAP-70 is affected by the severely reduced
intracellular GSH levels. The expression of ZAP-70 was similar in T
lymphocytes from healthy controls and RA patients (MFI ratios:
24.3 ± 0.9 (mean ± SEM; n = 6) for healthy
controls, 26.5 ± 1.6 (mean ± SEM; n = 4)
for PB T lymphocytes, 25.1 ± 1.5 (mean ± SEM;
n = 5) for SF T lymphocytes; Fig. 2
). To determine the
kinase activity of ZAP-70, we performed a kinase assay specific for
ZAP-70. ZAP-70 was immunoprecipitated from whole-cell extracts of SF
and PB T lymphocytes from RA patients that were either left
unstimulated or stimulated for 3 min with anti-CD3, and the
specific ZAP-70 kinase activity was determined by measuring the
phosphorylation of its substrate, tubulin. The basal kinase activity of
ZAP-70 present in unstimulated T lymphocytes from healthy controls was
enhanced 2.2-fold ± 0.23 (mean ± SEM; n
= 3) in anti-CD3-stimulated T lymphocytes (Fig. 3). The basal ZAP-70 kinase activity in
both unstimulated PB and unstimulated SF T lymphocytes from RA patients
is up-regulated almost 2.1-fold (2.1-fold ± 0.16 (mean ±
SEM; n = 3; p < 0.001) for PB T
lymphocytes, 2.1-fold ± 0.07 (mean ± SEM; n
= 3; p = 0.003) for SF T lymphocytes) upon TCR/CD3
stimulation to reach a level of kinase activity that is only slightly
lower than the kinase activity observed in stimulated T lymphocytes
from healthy controls (p = 0.37 for PB T
lymphocytes, p = 0.17 for SF T lymphocytes; Fig. 3).
These results demonstrate that the defective phosphorylation of LAT in
SF T lymphocytes is not due to a defect in the kinase activity of
ZAP-70.
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The recruitment of signaling proteins to the membrane is a common
theme in the proximal events of many signaling pathways (20, 22, 35). To explore whether LAT is absent from the membrane in SF T
lymphocytes and therefore the recruitment of ZAP-70 to the membrane
upon TCR engagement does not result in the phosphorylation of LAT, we
analyzed the subcellular localization of LAT by immunofluorescence
staining of T lymphocytes on cytospins. In healthy controls, LAT is
clearly expressed in the plasma membrane of the T lymphocytes (Fig. 4B). In PB T lymphocytes from
RA patients, LAT is also localized in the cellular membrane, although
the level of expression is clearly diminished compared with T
lymphocytes from healthy controls (Fig. 4C). In contrast, in
SF T lymphocytes from RA patients, we detected LAT in the cytoplasm of
the cell instead of the membrane (Fig. 4D). Western blot
detection of LAT in separated membrane and cytoplasm fractions
confirmed the membrane and cytoplasm localization of LAT in,
respectively, PB and SF T lymphocytes from RA patients (Fig. 4E). These results signify that the displacement of LAT from
the membrane in SF T lymphocytes is responsible for its deficient
phosphorylation.
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The intracellular GSH levels are significantly reduced in SF but
not PB T lymphocytes from RA patients (13). To examine
whether the decreased intracellular GSH levels play a role in the
membrane displacement of LAT, we treated T lymphocytes from healthy
controls with BSO to decrease the intracellular GSH reserves (13, 17) and determined the subcellular localization of LAT at
successive time points. Immunofluorescence staining of LAT revealed
that LAT still resided in the cellular membrane of the T lymphocytes
after 16 h of treatment with BSO (Fig. 5B). However, after 48 h
of BSO treatment, LAT started to accumulate in the cytoplasm, although
we also could detect membrane-associated LAT (Fig. 5C).
After 72 h of BSO treatment, we did not observe any expression of
LAT in the plasma membrane, while the cytoplasm showed massive
expression of LAT (Fig. 5D). Immunofluorescence staining for
the membrane receptor CD3 ascertained that the cellular
mem- brane of the T lymphocytes was still intact after 72 h
of BSO treatment (Fig. 5E). Moreover, the cellular
activation of the T lymphocytes treated for 72 h with BSO was
completely impaired because the cells were unable to produce any IL-2
while untreated T lymphocytes produced high levels of IL-2 after
24 h of stimulation with anti-CD3 and anti-CD28 (data not
shown). T lymphocytes cultured for 72 h in medium showed normal
LAT membrane localization (Fig. 5F). These results suggest
that the membrane displacement of LAT in SF T lymphocytes from RA
patients is due to the severely decreased intracellular levels of
GSH.
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B). In SF T lymphocytes that
were left untreated for 48 h, LAT was still present in the
cytoplasm of the cells, excluding the possibility that LAT returns
spontaneously to the membrane after 48 h of culture (Fig. 6A). These data denote that the subcellular localization of
LAT is highly sensitive to the intracellular concentration of
GSH.
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1 with
LAT, and the secretion of IL-2 upon TCR stimulation
Because the elevation of the intracellular GSH levels restored the
membrane localization of LAT in SF T lymphocytes from RA patients, we
also examined whether the phosphorylation of LAT and downstream
signaling events in the TCR-mediated signaling pathways were restored.
We immunoprecipitated LAT from whole-cell extracts of either resting or
activated SF T lymphocytes both before and after a 48-h treatment with
NAC and determined the tyrosine phosphorylation of LAT through Western
blotting analysis. As shown before, LAT remained unphosphorylated in
untreated SF T lymphocytes from RA patients after TCR stimulation (Fig. 7A). However, in SF T
lymphocytes that had been treated with NAC for 48 h, LAT becomes
extensively phosphorylated on tyrosine residues upon TCR ligation (Fig. 7A).
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1 with LAT upon TCR engagement, necessary to
transmit the TCR-induced signal from LAT to the inositol
1,4,5-triphosphate-sensitive Ca2+ channels, is
dependent on the phosphorylation of LAT because the N-terminal SH2
domain of PLC1 interacts with the multiple phosphorylated tyrosine
residues of LAT (22, 25, 28, 36). FACS analysis showed
that the expression of PLC1 was slightly but not significantly
reduced in both PB and SF T lymphocytes from RA patients compared with
T lymphocytes from healthy controls: 31.2 ± 1.8 (mean ±
SEM; n = 5) for healthy controls vs 27.2 ± 1.6
(mean ± SEM; n = 4) for PB T lymphocytes and
26.5 ± 2.9 (mean ± SEM; n = 4) for SF T
lymphocytes (Fig. 2). To determine whether PLC1 associates with LAT,
we subjected whole-cell extracts of either resting or stimulated T
lymphocytes to immunoprecipitation with anti-LAT Abs followed by
Western blotting detection of PLC1. PLC1 coimmunoprecipitated
with LAT only in stimulated T lymphocytes from healthy controls and
also from PB T lymphocytes of RA patients, but not in stimulated
untreated SF T lymphocytes from RA patients due to the lack of
phosphorylation of LAT (Fig. 7B). To determine whether the
modulation of the intracellular GSH levels would restore the
association of PLC1 with LAT, we evaluated the ability of PLC1 to
coimmunoprecipitate with LAT from whole-cell extracts of NAC-treated SF
T lymphocytes. Indeed, after treatment of the SF T lymphocytes with NAC
for 48 h, not only the phosphorylation of LAT but also the
association of PLC1 with LAT upon TCR stimulation was restored (Fig. 7B).
To establish whether the restoration of the membrane localization of
LAT, and hence the phosphorylation of LAT and the association of
PLC1 with LAT upon TCR ligation, through increasing the
intracellular GSH levels, also results in a reversal of the
hyporesponsiveness of the SF T lymphocytes from RA patients, we
determined the secretion of IL-2 by the T lymphocytes in response to
stimulation with anti-CD3 and anti-CD28 for 24 h.
Unstimulated T lymphocytes secreted only very low levels of IL-2.
Stimulation of T lymphocytes from healthy controls with
anti-CD3/anti-CD28 for 24 h resulted in a secretion of
2381 ± 319 pg/ml IL-2 (mean ± SEM; n = 7;
Fig. 7C). The IL-2 secretion of anti-CD3 plus
anti-CD28-stimulated PB T lymphocytes from RA patients was
2-fold lower: 1380 ± 58 pg/ml IL-2 (mean ± SEM;
n = 4; Fig. 7C). The stimulation of
untreated SF T lymphocytes induced an IL-2 production of only 293
± 58 pg/ml IL-2 (mean ± SEM; n = 4; Fig. 7C). The treatment of the SF T lymphocytes with NAC for
48 h elevated the IL-2 secretion upon anti-CD3/anti-CD28
stimulation significantly to a level of expression comparable to the
expression by PB T lymphocytes: 1316 ± 137 pg/ml IL-2 (mean
± SEM; n = 3; p = 0.023; Fig. 7C). These results indicate that the reduced GSH levels play
a critical role in the abrogation of the cellular activation of the SF
T lymphocytes in RA through the membrane displacement of LAT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several observations have suggested that the T lymphocytes in the
inflamed joints of RA patients exhibit defects in the TCR-induced
signal transduction routes rendering them unresponsive upon mitogenic
or antigenic stimulation. These observations include a decrease in the
expression levels of the TCR -chain, an aberrant pattern of protein
tyrosine phosphorylation, and a block in the Ca2+
influx upon TCR activation, any of which could contribute to the
hyporesponsiveness of the T lymphocytes (30, 31, 32, 33, 34).
The reduced expression of the TCR -chain has been proposed as
underlying mechanism responsible for the negative regulation of the TCR
signaling pathways. The decreased expression of the TCR -chain is
not only observed in the hyporesponsive SF T lymphocytes from RA
patients but also in tumor-infiltrating T lymphocytes from human cancer
patients and in the hyporesponsive T lymphocytes from HIV-infected
individuals. In all cases, only the expression of the TCR -chain is
reduced, while the expression of other components of the TCR/CD3
complex remains constant (30, 31, 37, 38, 39). Although the
correlation between the hyporesponsive state of the T lymphocytes and
the reduced TCR expression suggests a causal relationship, formal
evidence for this hypothesis is not available. Our data show that the
kinase activity of ZAP-70 is not affected in SF T lymphocytes from RA
patients when compared with the ZAP-70 kinase activity in T lymphocytes
from healthy controls and PB T lymphocytes from RA patients, suggesting
that the signaling pathway from the TCR -chain leading to the
activation of ZAP-70 is intact and functional.
Previously, we have reported the absence of a 36-to 38-kDa
phosphoprotein in SF T lymphocytes upon TCR stimulation
(31). A similar observation has been described for
autoimmune nonobese diabetic mice, where it was shown that a decrease
in the phosphorylation of a 36-kDa protein upon TCR engagement in
hyporesponsive T lymphocytes correlates with a decreased membrane
targeting of PLC1 and the Grb2/Sos complex, and an impaired
activation of Ras (40, 41). The current data indicate that
in SF T lymphocytes from RA patients this phosphoprotein represents the
membrane-anchored 36-kDa adaptor protein LAT. The expression of LAT is
reduced by 50% in SF T lymphocytes from RA patients compared with T
lymphocytes from healthy controls. However, the reduced expression is
not likely to account for the deficient phosphorylation of LAT, because
the level of LAT expression is SF T lymphocytes is approximately
similar to the level of expression in PB T lymphocytes from RA
patients, which demonstrate efficient phosphorylation of LAT upon TCR
engagement. Furthermore, the deficient phosphorylation of LAT is not
due to a defect in the kinase activity of ZAP-70, because this activity
is intact in SF T lymphocytes. Intracellular immunofluorescence
staining of LAT reveals that the impaired phosphorylation of LAT in SF
T lymphocytes correlates with a cytoplasmic localization of LAT,
whereas in T lymphocytes from healthy controls and PB T lymphocytes
from RA patients, which exert intact LAT phosphorylation, LAT is
exclusively expressed in the membrane. These data indicate that, due to
the displacement of LAT from the cellular membrane, the membrane
recruitment of ZAP-70 in SF T lymphocytes upon TCR engagement is
ineffective in evoking the phosphorylation of LAT. Furthermore, the
membrane displacement and the impaired phosphorylation of LAT in SF T
lymphocytes also correlate with an impaired recruitment of PLC1 to
the membrane through association with phosphorylated LAT. These data
indicate that the disturbed subcellular localization of LAT accounts
for the hyporesponsiveness displayed by the SF T lymphocytes through
the abrogation of the TCR-induced signaling pathways.
The functioning of T lymphocytes is markedly influenced by alterations
in the intracellular redox balance. Exposure to reactive oxygen
radicals has been demonstrated to down-regulate the activity of T
lymphocytes (42, 43, 44). In SF T lymphocytes from RA
patients, a significant decrease of the intracellular GSH levels has
been shown to correlate with the hyporesponsive state of these cells
(13). A similar decrease in the intracellular GSH levels
has been reported for T lymphocytes from HIV-infected individuals
(45). Moreover, the hyporesponsiveness displayed by the SF
T lymphocytes from RA patients clearly resembles the hyporesponsiveness
observed in aging T lymphocytes. Aging T lymphocytes contain severely
reduced intracellular levels of antioxidants including GSH, which
inhibits the phosphorylation of a 35- to 36-kDa protein, which is most
likely LAT, the phosphorylation of PLC1, the generation of inositol
1,4,5-triphosphate, and the influx of Ca2+ upon
TCR engagement. Increasing the intracellular GSH levels in aged T
lymphocytes by either GSH or NAC treatment restores the generation of
inositol 1,4,5-triphosphate and the Ca2+ influx
in response to TCR ligation (46, 47). The data presented
here indicate that redox balance alterations play a critical role in
the abrogation of the cellular activation of the SF T lymphocytes in RA
through the membrane displacement of LAT. The treatment of SF T
lymphocytes with NAC not only restores the membrane localization
of LAT, but also the phosphorylation of LAT upon TCR engagement and the
subsequent association of PLC1 with phosphorylated LAT. By mimicking
the situation of the SF T lymphocytes through the treatment of T
lymphocytes from healthy controls with BSO, which depletes the
intracellular GSH levels, we could induce the membrane displacement of
LAT. Similar results were reported upon the treatment of T lymphocytes
with N-ethylmaleimide, which induces the sulfhydryl
oxidation of intracellular proteins. In
N-ethylmaleimide-treated T lymphocytes, the phosphorylation
of a 35-kDa protein, which most likely represents LAT, upon TCR
stimulation is inhibited. Also, the tyrosine phosphorylation of PLC1
is abrogated, probably as a result of the impaired membrane recruitment
of PLC1 because the phosphorylation of LAT is also abrogated
(48).
Preliminary data indicate that the membrane displacement of LAT due to decreased GSH levels is relatively specific for RA as compared with other arthritic diseases. Only in patients with psoriatic arthritis have we so far observed a partial displacement of LAT from the membrane, but not in patients with spondylarthropathy or juvenile RA (our unpublished observations). Further studies are in progress to establish the disease-specificity of the membrane displacement of LAT.
Several hypotheses can be proposed to explain the displacement of LAT
from the membrane. First, a posttranslational modification of LAT
resulting in a conformational change could account for the membrane
displacement of LAT. The primary functions of GSH include conserving
the reduced state of the sulfhydryl groups of intracellular proteins to
maintain the correct conformation of the proteins (16, 49). LAT contains four cysteine residues, of which two are
within the N-terminal -helix, which mediates the membrane
localization of LAT, one is just proximal of the -helix, and the
fourth is present in the cytoplasmic part of the protein
(22). It seems probable that in SF T lymphocytes from RA
patients that contain decreased levels of the main antioxidant GSH, the
sulfhydryl groups of LAT become oxidized and form disulfide bonds
either intramolecular or intermolecular with small cytoplasmic
sulfhydryl-containing proteins and hence interfere with the placement
of the N-terminal -helix within the cellular membrane. Second, a
change in the cellular architecture could result in the membrane
displacement of LAT. Oxidative stress has been reported to induce
severe changes in the cytoskeletal structure (50, 51, 52).
Several proteins involved in the TCR-mediated signaling pathways
including the TCR -chain, the guanine nucleotide exchange factor
Vav, and SLP-76 are or upon TCR ligation become physically linked with
components of the cytoskeleton (15, 53, 54, 55). It seems
possible that LAT is also connected either directly or indirectly with
the cytoskeleton, and any changes in the cytoskeletal structure due to
oxidative stress therefore might somehow result in the displacement of
LAT from the cellular membrane.
An important point of consideration is the role of functionally
defective T lymphocytes in chronic inflammation. It is generally
accepted that T lymphocytes contribute to the perpetuation of the
disease in RA. Because SF T lymphocytes exert impaired responsiveness
to TCR-mediated activation, it is clearly important to know what
mechanism might be involved. In this respect it is worth note that
despite suppressed cytokine expression and proliferation, SF T
lymphocytes display an activated phenotype (1, 2, 3), which
may be important for the activation of macrophages and fibroblasts via
direct Ag-independent cell-cell contact. Chronically activated
macrophages and fibroblasts produce matrix metalloproteinases and
proinflammatory cytokines like TNF- and IL-1, which may aggravate
the disease (8, 10, 11). The induction of oxidative stress
through the stimulation of neutrophils by TNF- thus creates a
vicious circle, responsible for the chronicity of the disease. Evidence
exists that the persistence of T lymphocytes in the chronically
inflamed joints is a consequence of the active inhibition of T cell
apoptosis by environmental factors (56). Recently, it has
been shown that the expression of Fas ligand (FasL) is deficient in SF
T lymphocytes from RA patients that express high levels of Fas
(57). Because the intact TCR-signaling pathways, and
especially the ZAP-70-mediated Ca2+ influx, are
required to control the expression of FasL and the homeostasis of T
lymphocytes through Fas/FasL-mediated apoptosis (58, 59, 60),
the membrane displacement of LAT is likely to underlie this defect.
Therefore, we propose that the treatment of RA patients with an
antioxidant like NAC might contribute to the increased apoptosis of the
SF T lymphocytes and eventually ameliorate the disease in RA.
In summary, our present findings indicate that the hyporesponsive state of the T lymphocytes in the SF of the inflamed joints of patients with RA results from the displacement of LAT from the plasma membrane due to the reduced intracellular GSH levels. The localization of LAT in the cytoplasm of the SF T lymphocytes interferes with its phosphorylation and subsequent association with other signaling proteins, and hence abrogates the TCR-mediated signaling pathways.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: RA, rheumatoid arthritis; SF, synovial fluid; GSH, glutathione; BSO, DL-buthionine (S,R)-sulfoximine; ZAP-70, -associated protein of 70 kDa; SH2, Src homology 2; SLP-76, SH2 domain-containing leukocyte protein of 76 kDa; LAT, linker for activation of T cells; PLC1, phospholipase C1; PB, peripheral blood; NAC, N-acetyl-L-cysteine; pAb, polyclonal Ab; RT, room temperature; ECL, enhanced chemiluminescence; MFI, mean fluorescence intensity; FasL, Fas ligand; PerCP, peridinin chlorophyl protein.
Received for publication April 28, 1999. Accepted for publication December 3, 1999.
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