| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of Medicine (Rheumatology Division) and Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
calcineurin-dependent NF-AT pathway. When exposed
to the anergy protocol, lupus T cells, in marked contrast to normal T
cells, did not phosphorylate Cbl/Cbl-b but continued to express
strongly phosphorylated extracellular signal-regulated kinase (ERK);
U0126, a specific inhibitor of mitogen-activated protein kinase kinase
ERK, could block both the early and the prolonged hyperexpression
of CD40L. Thus, pathways regulating the activities of Cbl and one
particular mitogen-activated protein kinase, ERK, are involved in the
prolonged hyperexpression of CD40L in lupus T
cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The cognate interaction between autoimmune Th and B cells of lupus in the production of pathogenic anti-DNA autoantibodies (14, 15) is dependent on costimulatory signals (4, 16). Blocking the interaction between CD40 ligand or CD154 (CD40L)3 on the autoimmune Th cells and CD40 on autoimmune B cells, even briefly with anti-CD40L Ab therapy in lupus-prone mice, produces unexpected long-term benefits (4, 5). But, even more interesting is the prolonged hyperexpression of CD40L in lupus T cells that occurs irrespective of disease status. In normal T cells, CD40L expression is tightly regulated (17, 18). Persistent and increased expression of CD40L could disrupt this regulatory checkpoint and allow autoimmune cells to expand. T cells in lupus-prone SNF1 mice (4) and not only T cells (5, 6), but also B cells (5, 19), of lupus patients express abnormally high levels of CD40L without any deliberate stimulation (5). Moreover, upon stimulation with suboptimal doses of mitogens, the up-regulation of CD40L in T cells (5) and in B cells (5) from lupus patients in long-term remission, and in T cells from preautoimmune lupus-prone mice (4), is markedly greater than normal, suggesting an intrinsic regulatory defect associated with a lowered threshold of activation of T cells as well as B cells of lupus (5, 20, 21, 22). The possession of promiscuous Th cell receptors (10) and abnormally prolonged expression of CD40L by lupus T cells probably lower the threshold for recognition of apoptotic cell Ags, resulting in a pathogenic autoimmune response (9).
Herein, we have begun to study the mechanisms underlying the prolonged expression of CD40L in lupus T cells. T cells from patients with active lupus might be exhausted by continuous stimulation in vivo and thus become refractory to further stimulation in vitro. However, these T cells recover after resting in vitro (23). Therefore, to reveal intrinsic T cell defects we have studied short-term T cell lines that were expanded by one-time stimulation and then fully rested (5, 24, 25). Moreover, half of these T cell lines were obtained from patients in long-term remission of their disease (5, 25). We have previously shown that the one-time expansion to make these short-term lines by anti-CD3, anti-CD28, and IL-2 stimulation yields a polyclonal population that retains the original fraction of autoimmune T cells with their autoantibody-inducing ability and specificity for nucleosomal histone peptides (5, 10, 25, 26). We found herein that in contrast to T cell lines from normal subjects, T cell lines from lupus patients, even those from patients with inactive disease, do not down-regulate CD40L expression in the face of anergy-inducing conditions.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T cells from eight patients with active lupus (all females, aged 18–50 years), eight patients in long-term remission, and 15 normal (healthy) subjects (10 females and 5 males, aged 22–40 years) were studied. Disease activity by System Lupus Activity Measure ranged between 7 and 20 for the active patients. None of the patients in remission had detectable proteinuria or serum anti-DNA autoantibodies at the time of testing, and their System Lupus Activity Measure ranged between 0 and 4. The patients in remission had never received any cytotoxic drugs, and they were not receiving any steroids at the time their blood samples were drawn for the assays. Steroid had been discontinued for several years in the remission patients, as reported previously (5, 25).
Abs and reagents
Anti-CD3 (OKT3) mAb-secreting hybridomas were obtained from American Type Culture Collection (Manassas, VA). The hybridoma supernatants were concentrated by 47% saturated ammonium sulfate precipitation and dialysis before use. Anti-CD28 Ab (mAb 9.3) containing ascites was provided by Kathy Cabrian (Bristol-Myers Squibb, Seattle, WA). Anti-CD3 (clone UCTH1) and PE-conjugated anti-CTLA-4 (clone BNI3) mAbs were obtained from Immunotech (Marseille, France). Purified anti-human CD40L (CD154, clone 24-31) FITC, anti-human CD80 (B7-1, clone BB1), anti-CD152 (CTLA-4, clone ANC152.2/8H5), as well as isotype-matched control IgG1 Ab were obtained from Ancell (Bayport, MN). Anti-CD86 (B7.2, clone IT2.2, clone FUN-1), anti-CTLA4 (clone BNI3), anti-IL2 neutralization Ab (clone MQ1-17H12), and isotype control rat IgG2a (clone R35-95) were obtained from PharMingen (San Diego, CA). Purified anti-human CD28 (clone L293), PE-conjugated mAb to CD69 and anti-CD45RO-PE were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Mitogen-activated protein kinase (MAPK) kinase (MKK (MEK)) Inhibitor U0126 and anti-active MAPK polyclonal Ab were obtained from Promega (Madison, WI). SB202190, an inhibitor of P38 MAPK, was purchased from Calbiochem (San Diego, CA). All mAbs were used at optimal saturating concentrations as recommended by the manufacturers. c-Jun (N-terminal amino acid positions: 1–79) recombinant proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell preparations and short-term T cell lines
PBMC were obtained from lupus patients and healthy donors by centrifugation of heparinized blood over Ficoll-Hypaque (Pharmacia LKB Biotechnology, Piscataway, NJ) gradient. CD4 cells were selected from PBMC using anti-CD4 mAb-coupled magnetic beads (Dynal, Oslo, Norway). CD4 T cell lines were made within 10–14 days, after one round of stimulation of the CD4 T cells with plate-bound anti-CD3 and anti-CD28 mAb and rIL-2 (20 U/ml), as described (5, 25).
Flow cytometry analysis
Viable T cells were harvested and stained with various FITC-conjugated or PE-conjugated mAbs as described (5, 25). The cells were analyzed by flow cytometry using FACScan and CellQuest software (Becton Dickinson). For intracellular CTLA-4 staining, the T cells were first treated with IntraPrep permeabilization reagent obtained from Immunotech as recommended by the manufacturer, and then were incubated with PE-conjugated anti-CTLA-4 Ab.
Induction of anergy
CD4+ short-term T cell lines that were previously expanded by OKT3 and anti-CD28 were fully rested for 7–10 days (first rest). These rested T cells (1 x 106/ml) were then stimulated in complete RPMI 1640 medium with plate-bound anti-CD3 (OKT3; coated at 1 µg/ml) for 16–18 h (first stimulation). After this anergy induction, cells were transferred into fresh uncoated plates and incubated for 2 days (second rest), and then were rechallenged with the same concentrations of plate-bound anti-CD3 (second stimulation). At different time points, the T cells were harvested to determine expression of the CD40L and other markers by flow cytometry.
Cytokine assays
Concentrations of IL-2 were measured by ELISA using
anti-human IL-2 (clone B33-2; PharMingen) as a detection Ab and
anti-human IL-2 (clone 5344.11; PharMingen) as a capture Ab
(25). Concentrations of IL-4 and IFN-
in T cell culture
supernatants were determined by Quantikine per manufacturer
instructions (R&D Systems, Minneapolis, MN).
Western blot and immunoprecipitation analysis
T cells were stimulated for the indicated times by anti-CD3 Ab at 37°C. Stimulation was stopped with cold wash buffer containing PBS, 5 mM EDTA, and 0.5 mM vanadate, then cells were lysed in a 1% Nonidet P-40 (Sigma, St. Louis, MO) lysis buffer containing Tris-HCl, pH 7.6; 150 mM NaCl; 1 mM EGTA; 5% glycerol; 10 mM tetrasodium pyrophosphate; 0.5% sodium deoxycholate; 10 mM NaF; aprotinin (5 µg/ml); pepstatin (1 µg/ml); soybean trypsin inhibitor (2 µg/ml); and 1 mM PMSF for 20 min on ice. Protein content of the lysate was determined with the BCA protein assay kit (Pierce, Rockford, IL), and 30 µg was subjected to SDS-PAGE. Immunoblotting, stripping, and reprobing of the immunoblots were performed with various Abs, such as anti-phosphotyrosine mAb (PY99), mAb anti-Fyn, mAb anti-extracellular signal-regulated kinase 2 (ERK2), and rabbit polyclonal Ab against Cbl/Cbl-b, which were all obtained from Santa Cruz Biotechnology, and HRP-conjugated anti-phosphotyrosine mAb (clone 4G10), rabbit anti-human Fyn polyclonal Ab, rabbit anti-human Vav polyclonal Ab, mAb anti-Vav, and mAb anti-Cbl, which were obtained from Upstate Biotechnology (Lake Placid, NY). All blots were developed with enhanced chemiluminescence substrate (Amersham, Arlington Heights, IL). Immunoprecipitations of lysates from 5 x 106 cells were performed with anti-Cbl, anti-Fyn, or anti-Vav antiserum (5 µg/sample), respectively, followed by protein A/G-conjugated agarose (Santa Cruz Biotechnology), and analyzed by SDS-PAGE and immunoblotting, as above.
Jun kinase assay
Solid state c-Jun N-terminal kinase assay was performed by incubating cell lysates with GST-c-Jun 1–79(1–79) agarose beads as described (27).
Bead preparation to test CTLA-4 function
Anti-CD3, anti-CD28, and one of the anti-human CTLA-4 mAbs were covalently attached to polyurethan-coated tosyl-activated Dynabeads (Dynal, Lake Success, NY) according to the manufacturer’s instruction (bead/cell ratio, 1:1). Beads were prepared with a constant amount of anti-CD3 mAb that represented 5% of the total protein bound and a titration of anti-CD28 and anti-CTLA-4 or control mAb to make up the remaining 95%. Ab-coated beads were tested at anti-CD28 and anti-CTLA-4 ratios of 10:0, 9:1, 5:5, 3:7, 1:9, and 0:10, as described (28, 29). CD4+ T cells from rested cell lines were cultured in 96-well plates at a density of 5 x 105 cells with the beads coated with a constant amount anti-CD3 and various concentrations of anti-CD28 and anti-CTLA4 mAbs. T cell proliferation was determined in 72-h culture assays that were pulsed with 1 µCi of [3H]TdR during the last 18 h. Staining of T cells for CD40L in duplicate cultures with the beads were performed after 5 h.
Blocking with anti-B7 mAbs
CD4+ T cells were cultured in the presence of anti-B7 (B7.1 and B7.2) mAbs or control Ab (20 µg/ml) during the entire anti-CD3 stimulation and anergy induction period. CD40L expression levels were measured by flow cytometry.
Blocking with anti-IL2 mAb
Rested CD4+ T cells were stimulated by plate-coated anti-CD3 and anti-CD28 in the presence of soluble anti-IL2 neutralizing mAb or rat IgG2a control Ab (20 µg/ml) for 48 h. CD40L expression levels were measured by flow cytometry.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Short-term CD4 T cell lines from 16 lupus patients and 15 normal
subjects were analyzed. Kinetics from a representative experiment are
shown in Fig. 1
. The CD4 T cell lines
were fully rested for 7–10 days in uncoated wells and then stimulated
in vitro by plate-bound anti-CD3 Ab for 5, 18, or 48 h (first
stimulation). The T cells from either normal or lupus subjects had
similar resting levels of CD40L-positive T cells (2 and 1%,
respectively). After stimulation, both types of cell lines showed
almost identical early response at 5 h with a 23- to 46-fold
increase in the percentage of CD40L-positive T cells. However, in the
case of normal T cells, the percentage of CD40L-positive cells dropped
quickly from 46 to 6% by 18 h and went down further to background
level (3%) at 48 h. By contrast, in the case of lupus T cells,
the expression of CD40L was prolonged, as 22% of the T cells were
CD40L positive even at 48 h. Next, the same T cells that were
stimulated by plate-bound anti-CD3 for 48 h were rested for 2
days in fresh uncoated wells (second rest) when surface CD40L came down
to background levels also in the lupus T cells in the absence of
continued anti-CD3 stimulation. At this stage, viability was
comparable between the normal and lupus T cell cultures with
20–30% dead cells. Only viable T cells were harvested by
Ficoll-Hypaque centrifugation and then restimulated by plated-bound
anti-CD3 (second stimulation) (Fig. 1B). The normal T
cell lines showed weak response to the second anti-CD3 stimulation
with a peak level of CD40L-positive T cells reaching 10% at 5 h,
which declined to background levels thereafter (Fig. 1B).
However, when these anergic normal T cells were treated with either
IL-2 (50 U/ml) or PMA (10 ng/ml) plus ionomycin (100 ng/ml), the
hyporesponsiveness to anti-CD3 could be reversed completely,
demonstrating that the cells were still viable (Fig. 1C). In
contrast to normal T cells, lupus T cells behaved differently after
exposure to the anergy induction protocol; they responded to the second
anti-CD3 stimulation with prolonged hyperexpression of CD40L with
kinetics similar to the first anti-CD3 stimulation.
|
, A and B, at two
time points; one is at 5 h of the first anti-CD3 stimulation
when optimal expression occurs before anergy sets in, and the other is
at 18 h of second stimulation when anergy is well established.
Consistent with the kinetics in Fig. 1
, the normal T cells failed to
up-regulate CD40L after exposure to the anergy induction protocol, in
marked contrast to the lupus T cells (p <
0.01). We also measured another T cell activation marker, CD69, in
these experiments (Fig. 2, C and D). In marked
contrast to the pattern of CD40L expression, both types of cell lines
had similar increases in CD69 expression (p >
0.1), either on first stimulation with anti-CD3 after being fully
rested or after second stimulation following the anergy induction
protocol.
|
Because anergy is associated with a block in IL-2 production, we
measured IL-2 production by the lupus and normal T cells at different
time points (Fig. 3). In general, normal
and lupus T cell lines produced comparable level IL-2 on the first
anti-CD3 stimulation, but after exposure to the anergy protocol,
normal T cell lines produced IL-2 at background levels after second
stimulation, in marked contrast to lupus T cell lines
(p < 0.01). Indeed, addition of IL-2 could
restore CD40L expression by the anergized, normal T cells (Fig. 1C). This data suggested that resistance to anergy induction
was associated with prolonged CD40L expression in lupus T cells. When T
cells are optimally stimulated with both anti-CD3 and anti-CD28
mAbs, prolonged expression of CD40L occurs even in normal T cells
(30). However, although the prolonged CD40L expression by
the optimally stimulated, normal T cells could be significantly
inhibited by anti-IL2 Ab (Ref. 30 ; Fig. 3B), that was not the case in lupus T cells (Fig. 3B). There were no particular deviations to either Th1 or
Th2 type among the lupus or normal T cell lines. All of the short-term
T cell lines produced both IFN and IL-4 on optimal stimulation with
anti-CD3 (data not shown), which was consistent with previous
observations (25). Moreover, there were no differences in
memory markers between the lupus and normal T cell lines (Fig. 3C).
|
Because it is well known that Ca2+
calcineurin-dependent NF-AT activation is responsible for inducing
CD40L expression (17, 31, 32), we tested lupus T cells for
abnormalities in this pathway. Cyclosporin A (CsA) is a specific
inhibitor that blocks calcineurin-dependent NF-AT activation.
Therefore, CsA was added along with plate-bound anti-CD3
stimulation to fully rested T cell lines, and CD40L expression was
measured. At earlier time points, up to18 h, both lupus and normal T
cells were very sensitive to inhibition by CsA, and titration of CsA
failed to show differences between normal and lupus T cell lines in
dose response (data not shown). Representative results from a lupus T
cell line is shown in Fig. 4. When lupus
T cell lines were treated with CsA (200 ng/ml) at early stages of
activation (added at time 0 or 6 h of stimulation by anti-CD3)
the degree of inhibition was very high (between 81 and 97%). However,
when CsA was added at later stages (18 h or later during continued
anti-CD3 stimulation), it was much less effective; inhibition was
only 11% when CsA was present in the cultures during the last 18- to
26-h period of anti-CD3 stimulation, or 35% when CsA was present
during the last 18–42 h of anti-CD3 stimulation. Normal T cells
were not tested with CsA at these later stages of anti-CD3
stimulation because they had spontaneously down-regulated CD40L
expression (Fig. 1A). The data so far suggested that other
factors might determine the prolonged expression of CD40L in lupus T
cells besides Ca2+ calcineurin-dependent
NF-AT pathway.
|
We searched for possible mechanisms in TCR signal transduction
pathways that could be responsible for persistent CD40L expression in
lupus T cells following exposure to the anergy induction protocol.
Representative results are shown in Fig. 5. In Fig. 5A, preanergy
samples from fully rested T cells before first stimulation and
postanergy samples from T cells that were rested for 2 days after 18-h
exposure to anergy induction protocol are shown side by side. The
phosphotyrosine immunoblots were obtained from the respective T cells
either before stimulation (-) or 10 min after stimulation (+) with
anti-CD3. There were no obvious differences in the preanergy
samples from normal (lanes 1 and 2) and
lupus T cells (lanes 5 and 6) up to 10 min
of anti-CD3 stimulation (first stimulation). However, among
postanergy samples, when the normal T cells were stimulated for the
second time with anti-CD3 (lanes 4 and
8), they showed increased tyrosine phosphorylation of bands,
including those at higher molecular mass regions (110–140 kDa), in
marked contrast to lupus T cells under this condition. Total protein
loadings in the last two lanes of Fig. 5A were comparable to
the other lanes (as shown in Fig. 6).
Similar results were obtained with a 5- or 30-min stimulation of the T
cells by anti-CD3; the differences in phosphorylation of proteins
on second stimulation, particularly at the higher molecular mass
regions were evident also at these time points (data not shown).
Because the high molecular mass bands could include phosphorylated Cbl,
which is important for anergy maintenance (33), we
performed Cbl-specific immunoprecipitation and immunoblotting, which
showed that in marked contrast to normal T cells, Cbl/Cbl-b was not
phosphorylated in lupus T cells under anergy-inducing conditions (Fig. 5B). Moreover, normal T cells showed an increase in
phosphorylated Fyn as compared with lupus T cells after exposure to
anergy protocol, but no differences in phosphorylation of Vav could be
detected under those conditions (data not shown).
|
|
Inactivation of the ERK pathway is clearly involved in T cell
anergy (34, 35). We measured ERK activity in normal and
lupus T cells under preanergy or postanergy conditions using
anti-active or phospho-ERK Ab. The same membrane from Fig. 5A was reprobed with anti-phospho ERK Ab, as shown in
Fig. 6 (upper panel, labeled as phospho ERK), stripped, and
further reprobed with anti-total ERK protein Ab, as shown in Fig. 6
(lower panel), demonstrating equal loading of total
Erk protein among the different samples. The phosphorylated ERK band
was not detectable in any of the unstimulated T cell lysates. Strongly
phosphorylated ERK bands were found in preanergy samples of both normal
and lupus T cells upon stimulation by anti-CD3 for 10 min. After
anergy induction protocol (postanergy), the active ERK bands in lupus T
cell lysates persisted and were markedly greater in intensity than in
normal T cells after stimulation by anti-CD3 (second stimulation).
Indeed, the same difference between lupus and normal T cells in the
level of active ERK was seen as early as 60 min after the first
stimulation with plate-bound ant-CD3 (data not shown). The
down-regulation of ERK activity in normal T cells could be bypassed by
treating the cells with PMA and ionomycin (P+I) for 1 min, which also
indicated that the T cells were not apoptosed after anergy induction.
These data suggested that there is a possible defect in down-regulation
of the Ras-MAPK pathway in lupus T cells that causes resistance to
induction of anergy, which may also be responsible for abnormally
prolonged expression of CD40L by lupus T cells.
There are at least three known MAPK family members, ERKs, c-Jun N-terminal kinases, and p38 MAPK. We failed to detect any difference in p38 MAPK activity between normal and lupus T cell lysates under the above conditions using anti-phospho p38 Ab (data not shown). Moreover, addition of up to 5 µM SB202190, which specifically inhibits p38 MAPK at a 4 µM range (36), did not block the prolonged hyperexpression of CD40L in lupus T cells (data not shown). Furthermore, using the Jun kinase assay we did not detect any differences in phosphorylation of c-Jun between normal and lupus T cells during the first stimulation (preanergy) or second stimulation (postanergy) (data not shown).
To determine whether persistent ERK activity in lupus T cells was
involved in prolonged expression of CD40L, one of MKK (MEK)
ERK-specific inhibitors, U0126, was used. This inhibitor specifically
inhibits MKK1/2 activation at a range of 10–100 µM (Promega). After
exposure to anergy induction protocol, lupus T cell lines were treated
with either CsA (200 ng/ml) or U0126 (25 µM) either from the
beginning of plate-bound anti-CD3 stimulation or 18 h later
(Fig. 7). When cells were treated with
inhibitors from the beginning of anti-CD3 stimulation, inhibition
of CD40L expression was comparable between CsA (down from 32%
CD40L+ T cells to 6.7%) and U0126 (from 32 to
7.3%), but when the inhibitors were added into cell cultures after
18 h of activation with anti-CD3, U0126 was still a potent
inhibitor (from 32% CD40L+ T cells to 6.5%),
but CsA was less effective at this time (from 32 to 18.7%). Thus,
prolonged CD40L expression in preactivated lupus T cells was only
partially inhibited by CsA but almost completely inhibited by
U0126.
|
Because activated T cells can express B7 molecules as well as
CD28, autocrine or paracrine signaling could make them relatively
resistant to anergy, i.e., independent of exogenous CD28 signals.
Moreover, CTLA-4-mediated negative signaling is important for anergy in
mouse T cells (37). Therefore, we addressed the
correlation between resistance to anergy and expression of
costimulatory molecules (CD28, B7) or CTLA-4 in human lupus T cell
lines before and after anergy induction, as compared with the normal T
cell lines (Fig. 8). The expression of
CD28 in T cells was constitutive and high at all time points we
measured without any major difference between normal and lupus T cells
(consistently around 70–90%). However, B7.1 and B7.2 expression on
normal T cells was markedly lower than that on lupus T cells during the
postanergy period (p < 0.01).
|
10% CTLA-4+ in the
case of normal T cells (p < 0.01). The above
stainings included both surface and intracellular pool of CTLA-4.
Because maximal CTLA-4 surface expression can be achieved by treating
cells with the phosphatase inhibitor pervanadate, we treated both
normal and lupus T cells with pervanadate as described
(38). We found that, after anergy induction, the
percentage of CTLA-4+ T cells in lupus cell lines
was 3-fold higher than that among normal T cell lines upon pervanadate
treatment (data not shown). Although there were differences in expression of B7 and CTLA-4 between lupus T cells and normal T cells, we failed to block the anti-CD3-induced prolonged expression of CD40L on lupus T cells using any of the mAbs available against B7.1 and B7.2 that are known to block costimulatory function (39, 40) (data not shown). We also tested various Abs against CTLA-4 (clone ANC152.2/8H5, Ancell; clone BNI3, PharMingen) using published protocols (28, 29), and none of them showed inhibition of either proliferation or CD40L expression in normal or lupus T cells upon optimal anti-CD3 stimulation (data not shown).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Indeed, we have found that selective inhibition of one of the MAPKs, ERK, blocks the prolonged expression of CD40L in lupus T cells. Our results suggest that the early expression of CD40L requires the CsA-sensitive NF-AT pathway, but prolonged expression of CD40L is more dependent on ERK. Increased and persistent ERK activity in lupus T cells even after exposure to the anergy induction protocol, could contribute to CD40L hyperexpression via activation of AP-1 transcription factors and possibly also by stabilizing CD40L mRNA (27, 47). Indeed, PMA + ionomycin greatly increases the stability and expression of CD40L mRNA, even in normal T cells (6, 47, 48), which could have been due to PKC-mediated Ras-MAPK activation.
Thus, prolonged expression of CD40L by lupus T cells is associated with
defects in anergy induction and maintenance. Ligation of TCR without
costimulation leads to functional unresponsiveness or anergy. Anergic T
cells have a block in Ras activation by TCR signaling, but the pathway
downstream of Ras is intact because PMA restores normal function in
such cells by activating Ras directly (34, 35, 49). In
anergic T cells from normal subjects MAPKs are down-regulated, but the
mechanism is upstream because direct inhibition of the MAPKs does not
lead to anergy (34, 35, 50). One proposed mechanism of
anergy is that mediated by Fyn kinase-phosphorylating Cbl, which then
activates Rap1. Rap-1 antagonizes Ras function by sequestering Raf-1
kinase and thus preventing activation of MAPK cascade and downstream
transcription factors (33, 51, 52). We found that the
Fyn-Cbl-Rap1 pathway, as evidenced by phosphorylation status of Cbl, is
operative in normal T cells after exposure to anergy induction
protocol, but not in the lupus T cells. In anergic T cells,
phosphorylated Cbl may interact with a negative regulatory tyrosine in
ZAP-70 to down-regulate its activity and, consequently, lead to
inactivation of Ras-MAPK pathway (53, 54, 55). Consistent with
this scenario, we found persistently active or phosphorylated ERK in
lupus T cell lines exposed to anergy induction protocol, in marked
contrast to normal T cell lines. However, phosphorylated Cbl might also
mediate ubiquitination and degradation of activating molecules in the T
cell signaling pathway (56, 57). Although decreased
tyrosine phosphorylation of other bands was apparent in the lupus T
cells after exposure to the anergy protocol, we did not examine
phosphorylation status of TCR
chain, as its expression is variable
in lupus T cells and its functional significance is uncertain because
other TCR subunits may take over the job of the chain
(58, 59, 60).
Furthermore, we did not detect any gross differences in cytokine
profile (Th1 or Th2 deviation), memory marker (CD45RO), or rates of
apoptosis between the T cell lines from lupus and normal subjects.
Resistance to anergy could be due to high level expression of B7 (B7.1
and B7.2) molecules by the lupus T cells, which could cause autocrine
and paracrine stimulation via constitutively expressed CD28 as has been
thought to be the case in another autoimmune disease, multiple
sclerosis (MS; Refs. 61, 62, 63). However, the situation in
lupus is different. In the case of MS, anergy resistance was seen only
in T cells from patients with actively progressive disease, whereas in
lupus, we find resistance to anergy in T cells from patients in
long-term remission, suggesting an intrinsic defect rather than some
adjuvant effect during relapse of disease (63, 64).
Interestingly, the resistance to anergy coincides with
hyperinducibility of CD40L expression in lupus T cells from patients
with inactive disease (5), and these phenomena are
probably connected. Moreover, anergy resistance is not just a feature
of any T cell that has been activated before (61, 62),
because we and others (65) do not observe it in
established T cell lines and clones from normal subjects. In conflict
with the possibility proposed for MS T cells (61, 62, 63),
constitutive overexpression of transgenic B7 molecules in T cells of
mice did not precipitate any autoimmune disease or alter susceptibility
to anergy induction (66). Indeed, we found that blocking
of B7-CD28 interaction among the lupus T cells fails to diminish the
prolonged CD40L expression in lupus T cells, indicating that other
mechanisms are at work. Moreover, B7 CTLA-4 mediated negative
signaling, which is thought to be important for anergy induction and
maintenance, and inactivation of MAPK in mouse T cells (29, 37, 67, 68), was not important in down-regulating the prolonged
hyperexpression of CD40L in lupus T cells.
Common underlying mechanisms could determine increased
Ca2+ mobilization on TCR signaling
(3), deficiency of PKA I activity (69),
various effects caused by lupus-inducing drugs (70, 71),
and the lowered activation threshold with prolonged hyperexpression of
CD40L we find in lupus T cells (4, 5, 6). However,
Ca2+ calcineurin-dependent NF-AT pathway
is not solely involved for CD40L hyperexpression as expected. We found
that prolonged expression of CD40L in lupus T cells is associated with
a lack of Cbl/Cbl-b phosphorylation and persistent activation of one
particular MAPK, ERK, under conditions that would induce anergy in
normal T cells. These observations open up the possibility of using
ERK-specific inhibitors for therapy of lupus.
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Syamal K. Datta, Rheumatology Division, Ward 3-315, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611.
3 Abbreviations used in this paper: CD40L, CD40 ligand (CD154); CsA, cyclosporin A; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MKK (MEK), MAPK kinase; MS, multiple sclerosis.
Received for publication July 7, 2000. Accepted for publication September 6, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chain. J. Exp. Med. 187:367./
expressing double negative (CD4-/CD8-) and CD4+ T helper cells in humans augment the production of pathogenic anti-DNA autoantibodies associated with lupus nephritis. J. Immunol. 143:103.[Abstract]
/
T-cell receptor. Proc. Natl. Acad. Sci. USA 87:7020. chain. J. Clin. Invest. 101:1448.[Medline]
phosphorylation is not required for the induction of T cell antagonism by altered peptide ligands. J. Immunol. 163:599. ITAMs does not impair T cell receptor signaling. Immunity 10:409.[Medline]
and ZAP 70. J. Exp. Med. 186:1645.This article has been cited by other articles:
|
|
 |
|
  S Gorjestani, V Rider, B. Kimler, C Greenwell, and N. Abdou Extracellular signal-regulated kinase 1/2 signalling in SLE T cells is influenced by oestrogen and disease activity Lupus, June 1, 2008; 17(6): 548 - 554. [Abstract] [PDF]
|
|
|
|
 |
|
 M. R. Gilbert, D. G. Carnathan, P. C. Cogswell, L. Lin, A. S. Baldwin Jr, and B. J. Vilen Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion J. Immunol., April 15, 2007; 178(8): 4803 - 4810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Dranitzki-Elhalel, J. H. Huang, M. Sasson, J. Rachmilewitz, M. Parnas, and M. L. Tykocinski CD40{middle dot}FasL inhibits human T cells: evidence for an auto-inhibitory loop-back mechanism Int. Immunol., April 1, 2007; 19(4): 355 - 363. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. C. Kyttaris, Y. Wang, Y.-T. Juang, A. Weinstein, and G. C. Tsokos Increased Levels of NF-ATc2 Differentially Regulate CD154 and IL-2 Genes in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., February 1, 2007; 178(3): 1960 - 1966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X Li, V Rider, B F Kimler, and N I Abdou Estrogen does not regulate CD154 mRNA stability in systemic lupus erythematosus T cells Lupus, December 1, 2006; 15(12): 852 - 857. [Abstract] [PDF]
|
|
|
|
|
|
E. C. Jury, D. A. Isenberg, C. Mauri, and M. R. Ehrenstein Atorvastatin Restores Lck Expression and Lipid Raft-Associated Signaling in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., November 15, 2006; 177(10): 7416 - 7422. [Abstract] [Full Text] [PDF]
|
|
|
</ |
) and lupus (
) subjects. Representatives from 15 normal and 16
lupus T cell lines are shown. A, First stimulation of
fully rested T cell lines with plate-bound anti-CD3 for 5, 18, or
48 h. B, After anergy induction and second rest,
the T cells were rechallenged with plate-bound anti-CD3 (second
stimulation) for 5, 18, or 48 h. C, The normal
(open columns) and lupus T cells (filled columns) after their second
rest were restimulated by plate-coated anti-CD3 (second
stimulation) alone for different durations (left), and
also for 5 h with anti-CD3 in the presence of IL2 (50 U/ml) or
PI (10 ng/ml PMA + 100 ng/ml ionomycin) (right). This is
a representative of six experiments.
, CD28; 
,
B7.2), and total CTLA-4 (