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International Immunology, Vol. 13, No. 3, 305-312, March 2001
© 2001 Japanese Society for Immunology

A potential role for protein tyrosine kinase p56lck in rheumatoid arthritis synovial fluid T lymphocyte hyporesponsiveness

Paola Romagnoli1, Donna Strahan1, Michele Pelosi2, Alain Cantagrel1,3 and Joost P. M. van Meerwijk1,4

1 Tolerance and Autoimmunity Section, INSERM U395, IFR 30, CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France
2 Molecular Immunology Unit, Institut Pasteur, 75724 Paris Cedex 15, France
3 Department of Rheumatology, Rangueil Hospital, 31403 Toulouse Cedex 4, France
4 Faculty of Life Sciences (UFR-SVT), University Toulouse III, 31062 Toulouse Cedex 4, France

Correspondence to: P. Romagnoli


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) synovial fluid (SF)-T lymphocytes appear relatively inactive in situ and respond only weakly to diverse stimuli ex vivo. To characterize the molecular defects underlying this hyporesponsiveness we analyzed the expression level of several proteins involved in TCR-proximal signal transduction. As compared to peripheral blood (PB)-T lymphocytes, SF-T cells from some (but not all) of the patients analyzed expressed lower levels of TCR{alpha}ß, CD3{varepsilon}, TCR{zeta}, p56lck and LAT, while p59fyn, phospholipase C-{gamma}1 and ZAP-70 expression was unaltered. Semi-quantitative analysis of T cells from several patients revealed that the degree of TCR{zeta} chain and p56lck modulation correlated statistically significantly with the level of SF-T cell hyporesponsiveness. The differential reactivity of p56lck specific monoclonal and polyclonal antibodies in SF-T but not PB-T lymphocytes indicated that p56lck modulation consists of a conformational change rather than loss of expression. Our results indicate that multiple signaling molecules can be modulated in RA SF-T cells and show for the first time a direct quantitative correlation between T cell hyporesponsiveness and modulation of TCR{zeta} and of p56lck, a critical protein tyrosine kinase required for T cell activation.

Keywords: TCR{zeta}, LAT


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A direct role of T lymphocytes in rheumatoid arthritis (RA) pathogenesis is suggested by the relative predisposition of individuals expressing certain MHC class II haplotypes (in particular DR1 and DR4) to develop this disease (1), and by the synovial invasion of T cells displaying an activated phenotype (2,3). Paradoxically, RA synovial T cells appear to be hyporesponsive. Only low levels of T cell-derived cytokines such as IL-2 are detected in RA inflamed joints (4). In addition, RA patient-derived synovial fluid (SF)-T cells show low proliferative responses upon stimulation in vitro, while responses of their peripheral blood (PB)-T lymphocytes are less affected (5,6). These data suggest that the mechanism leading to T cell hyporesponsiveness is active at the site of inflammation. Experimental evidence suggests that oxidative stress may play a role in the induction of SF-T cell hyporesponsiveness. The SF of RA patients is an oxidative environment (7) and SF-T cells have been shown to have decreased intracellular levels of antioxidant glutathione (GSH) (8). Replenishment of GSH levels partially restores SF-T cell responses (8).

It has recently been shown that TCR-proximal signaling events are affected in RA SF-T cells (9). The TCR is a multisubunit complex composed of at least six different proteins. The clonotypic TCR{alpha}ß heterodimer responsible for antigen recognition is non-covalently associated with the signal-transducing subunits CD3{varepsilon}, CD3{gamma}, CD3{delta} and TCR{zeta}. These subunits contain multiple immunoreceptor tyrosine-based activation motifs (ITAM) that upon TCR engagement are phosphorylated on tyrosine residues by two protein tyrosine kinases (PTK) of the Src family, p56lck and p59fyn (10,11). The phosphorylation of TCR{zeta} is followed by the recruitment and tyrosine phosphorylation-mediated activation of ZAP-70, a PTK that phosphorylates LAT, a critical adaptor molecule linking the activated TCR with its associated PTK to several other adaptors and enzymes such as phospholipase C (PLC)-{gamma}1, SOS, Grb2, Cbl and Vav (12). The combined output of these signaling pathways ultimately results in T cell proliferation, differentiation and cytokine release (13). Interestingly, it has recently been shown that plasma membrane localization and compartmentalization of signaling molecules are essential in the initial tyrosine phosphorylation events (14,15). p56lck, p59fyn and LAT preferentially localize in glycolipid-, sphingolipid- and cholesterol-enriched microdomains (16–18), and TCR ligation triggers the accumulation of tyrosine-phosphorylated proteins in these glycolipid-enriched membrane domains (GEM).

It has been observed that RA SF-T cells express lower levels of TCR{zeta} than PB-T cells (10,19) and because the TCR{zeta} chain is thought to play a central role in TCR signal transduction, it was proposed that the decreased TCR{zeta} expression contributes to SF-T cell hyporesponsiveness (20). The hypothesis that TCR{zeta} plays a central role in TCR signaling was based on two functional and structural properties of the TCR{zeta} chain not shared with other components of the TCR complex: its cytoplasmic tail constituted of three concatenated ITAM motifs that can generate discrete phospho-species (21–23) and the constitutive phosphorylation of some of these ITAM (20). However, it has recently been reported that in P14 TCR transgenic mice uniquely expressing ITAM-less TCR{zeta}, mature CD8+ T cells normally develop. Moreover, these cells have a normal spectrum of activation events and effector functions, suggesting that lack of TCR{zeta} ITAM can be compensated for by other signaling molecules (21).

To investigate whether modulation of signaling molecules other than TCR{zeta} contribute to SF-T cell hyporesponsiveness, we analyzed the expression level of several components of TCR signaling pathways. While little if any change in the expression of p59fyn, PLC-{gamma}1 and ZAP-70 was observed, TCR{alpha}ß, CD3{varepsilon}, TCR{zeta}, p56lck and LAT expression was altered in SF-T cells. Semi-quantitative analysis of T cells from RA patients revealed that the degree of TCR{zeta} chain and p56lck modulation correlated statistically significantly with the level of SF-T cell hyporesponsiveness. Such correlation was not observed for TCR{alpha}ß, CD3{varepsilon} and LAT. We found that the p56lck modulation consists of an as-yet unidentified conformational change of the molecule rather than loss of expression. It therefore appears unlikely that partial TCR{zeta} loss alone can explain SF-T cell unresponsiveness. Together with the recently reported change in LAT localization (22) these data suggest that multiple signaling molecules are affected in SF-T cells of RA patients contributing to their hyporesponsiveness. The statistical significant correlation of TCR{zeta} and p56lck modulation with RA SF-T hyporesponsiveness suggests their important role in this process, and implicate p56lck (which is critical for T cell activation) as a key mediator.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Patients
All patients had active disease with at least one knee joint arthritis from which the SF samples were collected. Clinical findings are listed in Table 1Go. A first group included 19 patients with RA as defined by the criteria of the American College of Rheumatology (23). A second group included six patients with spondyloarthropathies according to the criteria of the European spondyloarthropathy group (24): three with reactive arthritis, two with psoriatic arthritis and one with ankylosing spondylitis. Blood and SF samples were collected the same day for each patient.


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  		Table 1. Clinical and demographic data of arthritis patients
 
mAb and antisera
The following antibodies were used for cell preparation, FACS analysis and immunoblotting: anti-CD4 mAb (SFCl12T4D11, FITC conjugate), anti-CD8 mAb (SFCl21Thy2D3, FITC conjugate), anti-CD3{varepsilon} mAb (UCHT1, FITC conjugate), anti-TCR{alpha}ß mAb (BMA031) and anti-TCR{zeta} mAb (2H2D9, phycoerythrin conjugate) (Immunotech, Marseille, France), anti-CD45 and anti-PLC-{gamma}1 antisera (Santa Cruz Biotechnology, Santa Cruz, NM), anti-CD21 (HB-135) and anti-CD3{varepsilon} mAb (OKT3; ATCC, Rockville, MD), anti-p59fyn rabbit serum (kindly provided by M. F. White, Harvard Medical School, Boston, MA), anti-p56lck (476–509) rabbit serum (Upstate Biotechnology, Lake Placid, NY), anti-ZAP-70 rabbit serum and anti-LAT rabbit serum (kindly provided by O. Acuto, Institute Pasteur, Paris, France), anti-p56lck (22–36) mAb (LCK-01, kindly provided by V. Horejsi, Institute of Molecular Genetics, Prague, Czech Republic), anti-M6PR rabbit serum (kindly provided by R. April, NIH, Bethesda, MD), and anti-caspase 3 rabbit serum (kindly provided by A. Alaim, U395 INSERM, Toulouse, France). Horseradish peroxidase-conjugated goat anti-rabbit antibodies were purchased from Sigma (Sigma-Aldrich, St-Quentin Fallavier, France), mouse IgG was obtained from Pierce (Rockford, IL). Phycoerythrin-conjugated anti-mouse IgG1 mAb was purchased from Southern Biotechnology Associates (Birmingham, AL), FITC-conjugated donkey anti-mouse IgG from Jackson ImmunoResearch (West Grove, PA). Magnetic beads coated with anti-mouse IgG antibodies were obtained from Immunotech (Marseille, France).

Cell isolation
PB and SF mononuclear cells (MC) were purified on Ficoll gradients, washed, and resuspended in RPMI supplemented with 10% FCS, 1 mM non-essential amino acids, 1 mM sodium pyruvate and antibiotics. Part of the cells was directly used for FACS analysis, and part was further purified for proliferation assay and biochemical analysis. Macrophages were depleted by adherence on plastic after 1 h incubation at 37°C. An enriched preparation of T cells (90–94% purity) was routinely obtained after depletion of B cells with anti-CD21 mAb-coated magnetic beads.

Proliferation assays
Round-bottom microplates were coated with 10 µg/ml anti-CD3{varepsilon} mAb (OKT3). SF-T or PB-T cells were added at 30x103 cells/well. Cells were stimulated for 2 days at 37°C, pulsed with 1 µCi of [3H]thymidine and harvested 16 h later.

Surface and intracellular staining
For surface staining, PBMC and SFMC were washed once in PBS containing 2.5% FCS and 0.02% NaN3, and incubated on ice for 20 min with the indicated antibodies. After two washes with PBS containing 2.5% FCS and 0.02% NaN3, cells were incubated with the appropriate secondary reagent for 20 min on ice. The stained cells were analyzed using a Coulter Epics XL cytometer (Coulter, Marseille, France) and data analyzed using CellQuest software (Becton Dickinson, San Jose, CA). For intracellular staining, PBMC and SFMC were washed twice in PBS and fixed for 4 min with 2% paraformaldehide. After two washes with PBS/2.5% FCS/0.02% NaN3, cells were permeabilized in 1% saponin for 7 min at room temperature. Cells were then incubated for 30 min with the indicated antibodies and washed 3 times with PBS/2.5% FCS/0.02% NaN3/0.1% saponin. Cells were subsequently incubated with the appropriate secondary reagent for 30 min, washed and analyzed as above. Live lymphocytes were appropriately gated on forward and side scatter.

Cell lysis and immunoblot analysis
SF-T and PB-T lymphocytes were lysed on ice for 10 min in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin and 2 mM PMSF) containing 1% Triton X-100 (Sigma-Aldrich). Lysates were centrifuged at 14,000 r.p.m. for 15 min at 4°C. Protein concentration in the recovered supernatants was measured using the BioRad (Hercules, CA) protein assay kit. Supernatants containing equal amount of proteins were then resuspended in sample buffer and boiled for 3 min. The samples were resolved on SDS–PAGE under reducing conditions, transferred to a PVDF membrane (Millipore, St-Quentin-Yvelines, France) and immunoblotted with the indicated antibodies. The blots were revealed using ECL (Amersham Pharmacia Biotech, Rainham, UK). Densitometry analysis was performed on scanned films using the program SigmaGel (Sigma-Aldrich). To control for loading, the quantification of the bands was normalized to the expression level of M6PR, caspase 3.

Statistical analysis
The significance of the difference in the expression level (MFI) of signaling molecules between SF-T and PB-T lymphocytes was calculated using an unilateral paired Student's t-test. To analyze whether there was a correlation between SF-T cell hyporesponsiveness and decreased expression level of signaling molecules, Fisher's r to z-test was used.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
TCR{zeta} loss in SF-T cells
It has been suggested that the decreased TCR{zeta} expression by T cells isolated from RA SF and synovial membrane may be responsible for their hyporesponsiveness (20). We therefore analyzed its expression by SF-T and PB-T lymphocytes from arthritis patients (Fig. 1Go). As compared to PB-T, SF-T cells from all (19 RA and six non-RA) patients showed decreased expression of TCR {zeta} chain (Fig. 1Go, patient 7 and Fig. 5aGo), with the exception of two patients, one of which showed the first clinical RA signs 6 months before the SF was taken and analyzed (Fig. 1Go, patient 8 and Fig. 5aGo). To determine if expression of other components of the TCR complex were affected as well, we analyzed the surface expression of CD3{varepsilon} and TCR{alpha}ß in the same cohort of patients. A slight down-modulation of CD3{varepsilon} and TCR{alpha}ß expression by SF-T (as compared to PB-T) cells was observed in 20 of 24 and in 11 of 14 patents respectively (Figs 1 and 5aGoGo), consistent with a previous report (20). As shown in Fig. 5Go(a), the decreased expression level of TCR{alpha}ß, CD3{varepsilon} and TCR{zeta} is statistically significant (TCR{alpha}ß, P < 0.005; CD3{varepsilon}, P < 0.0001; TCR{zeta}, P < 0.0001).



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  		Fig. 1. Expression of TCR complex components. Flow cytometric analysis of TCR{zeta} expression by SF-T and PB-T cells from arthritis patients. SFMC and PBMC were fixed, permeabilized and doubly stained with anti-CD3{varepsilon}–FITC and anti-TCR{zeta}–phycoerythrin. Live T lymphocytes were electronically gated on forward/side scatter and CD3{varepsilon}+. Thick line, isotype-matched control; thin line, PB-T; dotted line, SF-T. CD3{varepsilon} and TCR{alpha}ß expression was analyzed on fresh cells stained with anti-CD3{varepsilon}–FITC and anti-TCR{alpha}ß–phycoerythrin. Lymphocytes were electronically gated on forward/side scatter.

 



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  		Fig. 5. Statistical analysis of the decreased expression level of signaling molecules in SF-T lymphocytes of arthritis patients. Expression levels of SF-T signaling molecules are depicted as percentage of PB-T levels. Statistical analysis were performed using a unilateral paired Student's t-test on: (a) flow cytometry data for TCR{alpha}ß, CD3{varepsilon}, TCR{zeta}, CD8, CD4, CD45 and p56lck, and c.p.m. of the proliferation assays (mean value of triplicate culture, background subtracted), and (b) normalized densitometry counts for p56lck, p59fyn, ZAP-70, LAT and PLC-{gamma}1. Bars indicate means ± SD; **P < 0.01, ***P < 0.001.

 
Normal expression of CD4, CD8 and CD45 in SF-T cells
We next analyzed whether the expression level of other molecules involved in TCR signaling was altered in SF-T cells. Surface expression levels of CD4 and CD8, co-receptors thought to recruit the Src tyrosine kinase p56lck to the engaged TCR complex and thus modulating T cell activation (25,26), was analyzed by flow cytometry (Fig. 2Go). Minimal if any difference in CD4 or CD8 expression levels on SF-T and PB-T cells was observed (Figs 2 and 5aGoGo). The PB-T and SF-T surface expression level of CD45, a tyrosine phosphatase that mediates p56lck activation (27), was also found to be comparable in all patients analyzed (Figs 2 and 5aGoGo).



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  		Fig. 2. Surface expression of CD8, CD4 and CD45 in SF-T and PB-T cells from arthritis patients. Freshly isolated PBMC and SFMC were stained with anti-CD8–FITC, anti-CD4–FITC or anti-CD45–phycoerythrin/anti-CD3{varepsilon}–FITC and analyzed by flow cytometry. Lymphocytes were gated on forward/side scatter. CD45 expression level was evaluated on electronically gated CD3{varepsilon}+ lymphocytes. A representative example (RA patient no. 14) is shown.

 
Altered expression of tyrosine kinase p56lck (but not p59fyn or ZAP70) in SF-T cells
It has recently been reported that TCR engagement-induced tyrosine phosphorylation is generally decreased in RA SF-T as compared to PB-T cells (9). Upon TCR ligation initial tyrosine phosphorylation is mainly mediated by two PTK of the Src family, p56lck and p59fyn, and by a member of the Syk family of PTK, ZAP-70. We therefore analyzed whether the expression level of p56lck, p59fyn and/or ZAP-70 was altered in SF-T cells.

Expression of p56lck was analyzed by flow cytometry using mAb LCK-01 recognizing an epitope within residues 22–36. Interestingly, a statistically significant decreased signal was observed in SF-T (as compared to PB-T) cells from RA and non-RA patients (Figs 3 and 5aGoGo). This decreased staining intensity could be due either to a decreased expression level of the molecule or to masking of the epitope recognized by mAb LCK-01. To distinguish between these two possibilities, Western blot analysis of SF-T and PB-T was performed using a p56lck-specific rabbit polyclonal antiserum recognizing epitopes within residues 476–509. No significant difference in the intensity of SF-T and PB-T p56lck signals was observed in the 13 patients analyzed (Figs 4 and 5bGoGo). These data indicate that in SF-T cells p56lck expression levels are normal but that a conformational change or interactions with other proteins mask the epitope recognized by the mAb LCK-01.



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  		Fig. 3. Expression of p56lck in SF-T cells from patients affected with arthritis. Tyrosine kinase p56lck expression in SF-T and PB-T was evaluated by flow cytometry. PBMC and SFMC from arthritis patients were fixed, permeabilized and doubly-stained with anti-p56lck mAb LCK-01 (followed by Fab' rabbit anti-mouse IgG1–phycoerythrin) and anti-CD3{varepsilon}–FITC. p56lck expression by electronically gated CD3{varepsilon}+ cells is shown. Thick line, isotype matched control; thin line, PB-T; dotted line, SF-T.

 


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  		Fig. 4. Expression of LAT, p59fyn, ZAP-70 and PLC-{gamma}1, in SF-T cells from arthritis patients. Expression of p56lck, p59fyn, ZAP-70, LAT and PLC-{gamma}1 by paired SF-T and PB-T from arthritis patients was analyzed by Western blotting using rabbit polyclonal sera specific for p56lck, p59fyn, ZAP-70, LAT and PLC-{gamma}1.

 
Expression levels of two other TCR proximal tyrosine kinases, p59fyn and ZAP-70 (as measured by Western blot analysis using rabbit polyclonal antibodies), were not altered in SF-T as compared to PB-T cells (Figs 4 and 5bGoGo).

Reduced expression of LAT, but not PLC-{gamma}1, in SF-T cells
It has been reported that TCR ligation-induced Ca2+ mobilization is reduced in RA SF-T as compared to PB-T cells (28,29) and we have confirmed these results (data not shown). Although this may be due to the modulation of p56lck described in this report, expression of LAT and PLC-{gamma}1 [which act upstream of Ca2+ mobilization (12,30,31)] may also be involved. To test this possibility we analyzed SF-T and PB-T expression levels of LAT and PLC-{gamma}1 by Western blot.

LAT is a 36–38 kDa protein highly phosphorylated upon TCR engagement. The heterogeneity of its mol. wt is probably related to palmitoylation, a post-translational modification that mediates LAT localization in GEM microdomains (18). A large heterogeneity in expression levels of LAT in SF-T was observed, ranging from strongly reduced to approximately normal values (Figs 4 and 5bGoGo). In contrast, no significant difference in the expression level of PLC-{gamma}1 has been observed (Figs 4Go and Fig. 5bGo).

Direct correlation of TCR{zeta} and p56lck modulation with ex vivo hyporesponsiveness
Consistent with previous reports, immobilized anti-CD3{varepsilon} mAb OKT-3-induced in vitro proliferation of RA SF-T cells was significantly lower than that of PB-T cells, with the exception of T cells derived from two RA patients (Figs 5a and 6GoGo). Interestingly SF-T cells from two out of four patients affected with other types of arthritis were also hyporesponsive (Figs 5a and 6GoGo).



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  		Fig. 6. Correlation of p56lck and TCR{zeta} expression with SF-T proliferation. Purified SF-T and PB-T cells from arthritis patients were stimulated in vitro with immobilized OKT-3. Proliferation of SF-T (mean value of triplicate culture, background subtracted) is depicted as percentage of PB-T proliferation (mean value of triplicate culture, background subtracted). SF-T TCR{zeta} and p56lck expression levels on CD3{varepsilon}+ cells are depicted as percentage of PB-T levels. Diamond symbols represent spondyloarthropathy patients, while square symbols represent RA patients. Statistical significance of the depicted curves was determined using Fisher's test. A significant direct correlation was found between proliferation and p56lck expression levels of arthritis patients (P < 0.001 black line) and of the cohort of RA patients (P < 0.04 dashed line). No significant direct correlation was found between proliferation and TCR{zeta} expression level of arthritis patients (P < 0.2). When the two cohorts of patients were analyzed separately, a direct correlation was found between proliferation and TCR{zeta} expression level in RA patients (P < 0.05, dashed line), but not in spondyloarthropathy patients (P < 1).

 
To study if the alterations in TCR{alpha}ß, CD3{varepsilon}, TCR{zeta}, p56lck and LAT expression could be responsible for RA SF-T hyporesponsiveness (rather than merely an indirect consequence) we compared their expression levels to anti-CD3 mAb OKT-3-induced proliferative responses. To compensate for patient-to-patient (caused by treatment or other factors) and experimental variations we normalized TCR{alpha}ß, CD3{varepsilon}, TCR{zeta}, p56lck and LAT expression of SF-T to those of PB-T. When considering only patients affected with RA we observed a direct correlation between remaining TCR{zeta} expression and SF-T proliferative response (Fig. 6, PGo < 0.05, n = 10). A statistically significant direct correlation was also observed between SF-T proliferative response and p56lck expression (as analyzed by flow cytometry using mAb LCK-01) (Fig. 6, PGo < 0.001, RA patients n = 9 + non-RA patients n = 4). No significant correlation was found between SF-T hyporesponsiveness and decreased expression of TCR{alpha}ß, CD3{varepsilon} and LAT.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this report we described that in hyporesponsive SF-T lymphocytes from patients affected with inflammatory joint disease expression of TCR{zeta}, p56lck and LAT, but not p59fyn, ZAP-70 and PLC-{gamma}1, is modified (as compared to PB-T lymphocytes from the same patient). TCR{zeta} down-modulation was observed in 24 out of 25 patients analyzed. The degree of TCR{zeta} down-modulation correlated with the level of SF-T lymphocyte hyporesponsiveness. Interestingly, expression levels of tyrosine kinase p56lck appeared normal and its modified expression reflected a yet to be identified conformational change. The degree of p56lck's conformational change correlated directly with the level of T lymphocyte hyporesponsiveness. LAT down-modulation was observed in only nine out of 13 patients analyzed and did not directly correlate with SF-T cell hyporesponsiveness. These data suggest a role for TCR{zeta} in SF-T cell hyporesponsiveness in inflammatory joint diseases. Moreover, the facts that p56lck is critically involved in initial tyrosine phosphorylation upon TCR engagement and that its modification correlated with the level of hyporesponsiveness indicate a key role for this enzyme in SF-T hyporesponsiveness.

An abnormal structure of the TCR complex, in which the TCR{zeta} chain appears to be specifically lost, has been suggested to be responsible of T cell anergy in cancer (32). It has been proposed that TCR{zeta}, due to its particular and unique structure in the TCR complex, may perform a specific function in TCR signal transduction that cannot be fulfilled by other components of the CD3 complex. In our study a significant TCR{zeta} loss was observed in all patients analyzed, with the exception of one patient who showed the first clinical RA signs 6 months before the SF was taken and analyzed. In RA patients we have observed a direct correlation between levels of TCR{zeta} loss and T cell hyporesponsiveness. This direct correlation would suggest an important role for TCR{zeta} down-modulation in the hyporesponsive state. However, strong in vitro T cell stimulation with antibody or saturating densities of MHC–peptide ligand are known to result in T cell activation in the absence of TCR{zeta}–ITAM motifs (21). The apparent in vivo hyporesponsiveness of SF-T cells can be reproduced in vitro using anti-CD3{varepsilon} antibody. Unless TCR{zeta} a has a role other than ZAP-70 recruitment via its phosphorylated ITAM motifs and in stabilization of the TCR complex, it appears therefore unlikely that its down-modulation can explain SF-T cell hyporesponsiveness, at least in vitro. Possibly `activation' signals leading to hyporesponsiveness could in parallel induce TCR{zeta} down-modulation. Analysis of initial events in the process to leading to hyporesponsiveness would be required to resolve this issue.

It has been previously shown that TCR{zeta} is a limiting component for surface expression of the TCR (33). It is therefore rather surprising to find in different pathologies T cells that express strongly reduced levels of TCR{zeta}, but close to normal levels of CD3{varepsilon} and TCR{alpha}ß. Decreased expression of TCR{zeta} has been observed both at the plasma membrane and intracellularly (34). Whether TCR{zeta} disappearance reflects protein loss or a conformational change in the epitope recognized by the specific antibody is still an open issue. It has been recently shown in an in vitro system that caspase 3 can cleave the TCR {zeta} chain (35). Although these results would favor protein degradation as a mechanism for TCR{zeta} disappearance we could not find any evidence of caspase 3 activation in RA SF-T cells (P. Romagnoli et al., unpublished observation), suggesting that in vivo probably other mechanisms are involved.

Our data show that the expression of two intracellular signaling molecules, adaptor molecule LAT and protein tyrosine kinase p56lck, is altered in SF-T cells. In contrast, no modification of the expression of PLC-{gamma}1 and tyrosine kinases p59fyn and ZAP-70 was observed. LAT is a recently cloned adapter molecule of 36–38 kDa essential for the activation of the Ras and PLC-{gamma}1 signaling pathways upon TCR engagement (12). Its dislocation from the plasma membrane has recently been implicated in SF-T cell anergy of RA patients (22). In nine out of 13 patients LAT expression in SF-T cells was reduced, but a direct correlation with reduced proliferation was not observed. The possibility that in the latter case LAT function is altered in an alternative way, e.g. by dislocation from the plasma membrane, cannot be excluded and would require further analysis. Whether LAT loss represents protein loss or a [e.g. phosphorylation-induced (12,36)] conformational change of the adaptor interfering with antibody recognition remains to be determined. In any case, in the patients analyzed we did not find a direct correlation between loss of LAT expression levels and SF-T cell hyporesponsiveness.

While total protein levels of p56lck are unchanged in SF-T cells (as shown by reactivity to polyclonal C-terminal-specific antiserum), detection by a mAb specific for the N-terminus of the protein is hindered. Therefore p56lck's conformation appears to be altered in SF-T cells, as previously reported for AIDS patient-derived T lymphocytes (37). Interestingly, the degree of p56lck modulation strongly correlated with the level of SF-T cell hyporesponsiveness. p56lck's critical role in TCR signaling is underlined by experiments with p56lck-deficient T cell lines and mice. In T cell lines lacking this tyrosine kinase TCR ligation fails to induce any tyrosine phosphorylation and consequently all downstream signaling pathways are blocked (38,39), and in p56lck-deficient mice T cell development is severely impaired (40). Alteration of the N-terminal conformation of p56lck has been shown to correlate with its decreased kinase activity in T cells from AIDS patients (37). Therefore, our data offer an explanation for the previously described decreased TCR engagement induced protein tyrosine phosphorylation in RA SF-T cells (9) and indicate that modification of p56lck plays a key role in SF-T cell hyporesponsiveness.

In addition to the decreased p56lck kinase activity documented in T cells from AIDS patients (37), loss of expression of this protein kinase has been reported in T cells isolated from renal carcinomas (41) and leprosy patients (42). Two common features of T cells in these diseases as well as in RA are hyporesponsiveness upon TCR stimulation and an altered redox balance (8,43,44). These and our observations suggest that p56lck is a key enzyme involved in hyporesponsiveness of T cells exposed to oxidative stress in human pathology. The mechanisms responsible for modulation of p56lck in T cells exposed to oxidative stress are unknown. p56lck could be a direct or indirect target of reactive oxygen species. It has been described that p56lck phosphorylation and its catalytic activity is increased after exposure of T cells to high doses of hydrogen peroxide known to induce apoptosis (45). However, at more physiological doses of hydrogen peroxide such effects could not be detected.

Our results, together with the recently reported change in LAT localization (22), show that multiple signaling molecules are affected in SF-T cells of RA patients and contribute to their hyporesponsiveness. It will be of interest to determine the exact sequence of events leading to these alterations and their contribution to hyporesponsiveness. The use of in vitro models will be required for this endeavor (46).


   Acknowledgments
 
The authors would like to thank Sylvain Fleury for helpful suggestions during the course of this study, Didier Concordet for statistical analysis, Denis Hudrisier and Salvatore Valitutti for helpful reading of the manuscript, Francine Anglade for technical help, and Vaclav Horejsi for the generous gift of antibodies. This work was supported by grants from MENSR (PARMIFR9611), ARP, ARC (7287), Région Midi Pyrénées (RECH/97001940), FRM (10000121-10), and by institutional funds from the INSERM and University Toulouse III. P. R. was supported by Marie Curie training grant BMH4-CT 98-5090 from the European Community. D. S. was an exchange student enrolled in the Erasmus programme (European Community).


   Abbreviations
 
GEM glycolipid-enriched membrane domain
GSH glutathione
ITAM immunoreceptor tyrosine-based activation motif
PBMC peripheral blood mononuclear cell
PB-T peripheral blood T lymphocytes
PLC phospholipase C
PTK protein tyrosine kinase
RA rheumatoid arthritis
SFMC synovial fluid mononuclear cell
SF-T synovial fluid T lymphocytes

   Notes
 
Transmitting editor: T. Saito

Received 7 September 2000, accepted 29 November 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lanchbury, J. S. and Panayi, G. S. 1991. Genetics of RA: the HLA shared epitope hypothesis and its implications. Br. J. Rheumatol. 30:6.
  2. van Boxel, J. A. and Paget, S. A. 1975. Predominantly T-cell infiltrate in rheumatoid synovial membrane. N. Engl. J. Med. 293:517.[Abstract]
  3. Poulter, L. W., Duke, O., Panayi, G. S., Hobbs, S., Raftery, M. J. and Janossy, G. 1985. Activated T lymphocytes of the synovial membrane in rheumatoid arthritis and other arthropaties. Scand. J. Immunol. 22:683.[Web of Science][Medline]
  4. Firestein, G. S., Xu, W. D., Townsend, K., Broide, D., Alvaro-Gracia, J., Glasebrook, A. and Zvaifler, N. J. 1988. Cytokines in chronic inflammatory arthritis. I. Failure to detect T cell lymphokines (interleukin 2 and interleukin 3) and presence of macrophage colony-stimulating factor (CSF-1) and a novel mast cell growth factor in rheumatoid synovitis. J. Exp. Med. 168:1573.[Abstract/Free Full Text]
  5. Salmon, M. and Bacon, P. A. 1988. A cellular deficiency in the rheumatoid one-way mixed lymphocyte reaction. Clin. Exp. Immunol. 71:79.[Web of Science][Medline]
  6. Verwilghen, J., Vertessen, S., Stevens, E. A., Dequeker, J. and Ceuppens, J. L. 1990. Depressed T-cell reactivity to recall antigens in rheumatoid arthritis. J. Clin. Immunol. 10:90.[Web of Science][Medline]
  7. McCord, J. M. 1974. Free radicals and inflammation: protection of synovial fluid by superoxide dismutase. Science 185:529.[Abstract/Free Full Text]
  8. Maurice, M. M., Nakamura, H., van der Voort, E. A., van Vliet, A. I., Staal, F. J., Tak, P. P., Breedveld, F. C. and Verweij, C. L. 1997. Evidence for the role of an altered redox state in hyporesponsiveness of synovial T cells in rheumatoid arthritis. J. Immunol. 158:1458.[Abstract]
  9. Maurice, M. M., Lankester, A. C., Bezemer, A. C., Geertsma, M. F., Tak, P. P., Breedveld, F. C., van Lier, R. A. and Verweij, C. L. 1997. Defective TCR mediated signaling in synovial T cells in rheumatoid arthritis. J. Immunol. 159:2973.[Abstract]
  10. Chan, A. C., Desai, D. M. and Weiss, A. 1994. The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction. Annu. Rev. Immunol. 12:555.[Web of Science][Medline]
  11. Wange, R. L. and Samelson, L. E. 1996. Complex complexes: signaling at the TCR. Immunity 5:197.[Web of Science][Medline]
  12. Zhang, W., Sloan-Lancaster, J., Kitchen, J., Trible, R. P. and Samelson, L. E. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83.[Web of Science][Medline]
  13. Cantrell, D. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:259.[Web of Science][Medline]
  14. Xavier, R., Brennan, T., Li, Q., McCormack, C. and Seed, B. 1998. Membrane compartmentation is required for efficient T cell activation. Immunity 8:723.[Web of Science][Medline]
  15. Montixi, C., Langlet, C., Bernard, A. M., Thimonier, J., Dubois, C., Wurbel, M. A., Chauvin, J. P., Pierres, M. and He, H. T. 1998. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J. 17:5334.[Web of Science][Medline]
  16. Cinek, T. and Horejsi, V. 1992. The nature of large noncovalent complexes containing glycosylphosphatidylinositol-anchored membrane glycoproteins and protein tyrosine kinases. J. Immunol. 149:2262.[Abstract]
  17. Kabouridis, P. S., Magee, A. I. and Ley, S. C. 1997. S-acylation of Lck protein tyrosine kinase is essential for its signalling function in T lymphocytes. EMBO J. 16:4983.[Web of Science][Medline]
  18. Zhang, W., Trible, R. P. and Samelson, L. E. 1998. LAT palmitoylation: its essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation. Immunity 9:239.[Web of Science][Medline]
  19. van Oers, N. S. C., Killeen, N. and Weiss, A. 1994. ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR{zeta} in murine thymocytes and lymph node T cells. Immunity 1:675.[Web of Science][Medline]
  20. Matsuda, M., Ulfgren, A. K., Lenkei, R., Petersson, M., Ochoa, A. C., Lindblad, S., Andersson, P., Klareskog, L. and Kiessling, R. 1998. Decreased expression of signal-transducing CD3{zeta} chains in T cells from the joints and peripheral blood of rheumatoid arthritis patients. Scand. J. Immunol. 47:254.[Web of Science][Medline]
  21. Ardouin, L., Boyer, C., Gillet, A., Trucy, J., Bernard, A. M., Nunes, J., Delon, J., Trautmann, A., He, H. T., Malissen, B. and Malissen, M. 1999. Crippling of CD3-{zeta} ITAMs does not impair T cell receptor signaling. Immunity 10:409.[Web of Science][Medline]
  22. Gringhuis, S. I., Leow, A., Papendrecht-Van Der Voort, E. A., Remans, P. H., Breedveld, F. C. and Verweij, C. L. 2000. Displacement of linker for activation of T cells from the plasma membrane due to redox balance alterations results in hyporesponsiveness of synovial fluid T lymphocytes in rheumatoid arthritis [In process citation]. J. Immunol. 164:2170.[Abstract/Free Full Text]
  23. Arnett, F. C., Edworthy, S. M., Bloch, D. A., McShane, D. J., Fries, J. F., Cooper, N. S., Healey, L. A., Kaplan, S. R., Liang, M. H., Luthra, H. S., et al. 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31:315.[Web of Science][Medline]
  24. Group, E. S. 1991. Preliminary criteria for the classification of spondylarthropathy. Arthritis Rheum. 34:1218.[Web of Science][Medline]
  25. Thome, M., Duplay, P., Guttinger, M. and Acuto, O. 1995. Syc and ZAP-70 mediate recruitment of p56lck/CD4 to the activated T cell receptor/CD3/{zeta} complex. J. Exp. Med. 181:1997.[Abstract/Free Full Text]
  26. Thome, M., Germain, V., Di Santo, J. P. and Acuto, O. 1996. The p56lck SH2 domain mediates recruitment of CD8/p56lck to the activated T cell receptor/CD3/{zeta} complex. Eur. J. Immunol. 26:2093.[Web of Science][Medline]
  27. Sieh, M., Bolen, J. B. and Weiss, A. 1993. CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck. EMBO J. 12:315.[Web of Science][Medline]
  28. Mirza, N. M., Relias, V., Yunis, E. J., Pachas, W. N. and Dasgupta, J. D. 1993. Defective signal transduction via T-cell receptor–CD3 structure in T cells from rheumatoid arthritis patients. Hum. Immunol. 36:91.[Web of Science][Medline]
  29. Allen, M. E., Young, S. P., Michell, R. H. and Bacon, P. A. 1995. Altered T lymphocyte signaling in rheumatoid arthritis. Eur. J. Immunol. 25:1547.[Web of Science][Medline]
  30. Noh, D. Y., Shin, S. H. and Rhee, S. G. 1995. Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim. Biophys. Acta 1242:99.[Medline]
  31. Berridge, M. J. 1993. Inositol trisphosphate and calcium signalling. Nature 361:315.[Medline]
  32. Matsuda, M., Petersson, M., Lenkei, R., Taupin, J. L., Magnusson, I., Mellstedt, H., Anderson, P. and Kiessling, R. 1995. Alteration in the signal-transducing molecules of T cells and NK cells in colorectal tumor-infiltrating, gut mucosal and peripheral lymphocytes: correlation with the stage of the disease. Int. J. Cancer 61:765.[Web of Science][Medline]
  33. Sussman, J. J., Bonifacino, J. S., Lippincott, S. J., Weissman, A. M., Saito, T., Klausner, R. D. and Ashwell, J. D. 1988. Failure to synthesize the T cell CD3-{zeta} chain: structure and function of a partial T cell receptor complex. Cell 52:85.[Web of Science][Medline]
  34. Otsuji, M., Kimura, Y., Aoe, T., Okamoto, Y. and Saito, T. 1996. Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 {zeta} chain of T-cell receptor complex and antigen-specific T-cell responses. Proc. Natl Acad. Sci. USA 93:13119.[Abstract/Free Full Text]
  35. Gastman, B. R., Johnson, D. E., Whiteside, T. L. and Rabinowich, H. 1999. Caspase-mediated degradation of T-cell receptor {zeta}-chain. Cancer Res. 59:1422.[Abstract/Free Full Text]
  36. Brdicka, T., Cerny, J. and Horejsi, V. 1998. T cell receptor signalling results in rapid tyrosine phosphorylation of the linker protein LAT present in detergent-resistant membrane microdomains. Biochem. Biophys. Res. Commun. 248:356.[Web of Science][Medline]
  37. Stefanova, I., Saville, M. W., Peters, C., Cleghorn, F. R., Schwartz, D., Venzon, D. J., Weinhold, K. J., Jack, N., Bartholomew, C., Blattner, W. A., Yarchoan, R., Bolen, J. B. and Horak, I. D. 1996. HIV infection-induced posttranslational modification of T cell signaling molecules associated with disease progression. J. Clin. Invest. 98:1290.[Web of Science][Medline]
  38. Karnitz, L., Sutor, S. L., Torigoe, T., Reed, J. C., Bell, M. P., McKean, D. J., Leibson, P. J. and Abraham, R. T. 1992. Effects of p56lck on the growth and cytolytic effector function of an interleukin-2-dependent cytotoxic T-cell line. Mol. Cell Biol. 12:4521.[Abstract/Free Full Text]
  39. Straus, D. B. and Weiss, A. 1992. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell 70:585.[Web of Science][Medline]
  40. Molina, T. J., Kishihara, K., Siderovski, D. P., Vanewijk, W., Narendran, A., Timms, E., Wakeham, A., Paige, C. J., Hartmann, K. U., Veillette, A., Davidson, D. and Mak, T. W. 1992. Profound block in thymocyte development in mice lacking p56(lck). Nature 357:161.[Medline]
  41. Finke, J. H., Zea, A. H., Stanley, J., Longo, D. L., Mizoguchi, H., Tubbs, R. R., Wiltrout, R. H., O'Shea, J. J., Kudoh, S., Klei, E., Bukowski, R. M. and Ochoa, A. C. 1993. Loss of T-cell receptor {zeta} chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res. 53:5613.[Abstract/Free Full Text]
  42. Zea, A. H., Ochoa, M. T., Ghosh, P., Longo, D. L., Alvord, W. G., Valderrama, L., Falabella, R., Harvey, L. K., Saravia, N., Moreno, L. H. and Ochoa, A. C. 1998. Changes in expression of signal transduction proteins in T lymphocytes of patients with leprosy. Infect. Immun. 66:499.[Abstract/Free Full Text]
  43. Roederer, M., Staal, F. J., Anderson, M., Rabin, R., Raju, P. A. and Herzenberg, L. A. 1993. Disregulation of leukocyte glutathione in AIDS. Ann. NY Acad. Sci. 677:113.[Web of Science][Medline]
  44. Nakamura, H., Nakamura, K. and Yodoi, J. 1997. Redox regulation of cellular activation. Annu. Rev. Immunol. 15:351.[Web of Science][Medline]
  45. Hardwick, J. S. and Sefton, B. M. 1995. Activation of the Lck tyrosine protein kinase by hydrogen peroxide requires the phosphorylation of Tyr-394. Proc. Natl Acad. Sci. USA 92:4527.[Abstract/Free Full Text]
  46. Aoe, T., Okamoto, Y. and Saito, T. 1995. Activated macrophages induce structural abnormalities of the T cell receptor-CD3 complex. J. Exp. Med. 181:1881.[Abstract/Free Full Text]

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