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Protein Tyrosine Kinase Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus
Article No. au980230
Journal of Autoimmunity (1998) 11, 387–393
Protein Tyrosine Kinase Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus Ana M. Blasini, Veronika Brundula, Magdalena Paris, Liliana Rivas, Susana Salazar, Ivan L. Stekman and Martin A. Rodriguez Centro Nacional de Enfermedades Reuma´ticas, Hospital Universitario de Caracas, Caracas, Venezuela
Key words: PTK, SLE, T cells
We have recently observed an abnormal pattern of protein tyrosine phosphorylation in resting T lymphocytes obtained from peripheral blood of patients with systemic lupus erythematosus (SLE). To examine whether these findings may be related to dysregulated protein tyrosine kinase (PTK) function, we tested the relative amount and enzyme activity of the main PTKs involved in the earliest signalling steps triggered via the CD3 pathway. Cell lysates from peripheral blood T cells in SLE patients showed lower amounts of p59fyn and p56lck as shown by immunoblot. In contrast, the amount of ZAP-70, a PTK of the syk family, was comparable in both groups. However, p59fyn immunoprecipitates obtained from unstimulated peripheral blood SLE T cells showed enhanced PTK activity as compared to controls, whereas the PTK activity of p56lck and ZAP-70 molecules was comparable in both groups. The unchecked activity of the TCR/CD3-associated src kinase p59fyn may alter the balance needed for regulated T cell responses in SLE patients. © 1998 Academic Press
Introduction
augmented TCR/CD3-mediated responses [7, 8]. Similarly, defective protein tyrosine phosphatase (PTP) activity can lead to unchecked cytokine gene activation in T lymphocytes [9]. It is possible that abnormalities in the regulation of tyrosine phosphorylation and dephosphorylation may alter downstream biochemical steps needed for regulated gene responses in T lymphocytes from SLE patients. Our recent experiments seem to suggest that the pattern of tyrosine-phosphorylated proteins is altered in non-stimulated peripheral blood T lymphocytes from patients with SLE [10]. To examine the possible basis for this response pattern we have, in this study, examined the relative amount and enzyme activity of the main kinases involved in early transduction triggered via the CD3 pathway, namely src kinases p56lck, p59fyn and syk ZAP-70, in unstimulated and CD3-activated PB T cells from a group of patients with early and clinically active SLE.
Previous studies have shown a number of abnormalities in the response of peripheral blood (PB) T lymphocytes in patients with SLE upon activation via the TCR/CD3 pathway [1, 2]. We have previously shown that T lymphocytes from SLE patients can have augmented responses upon stimulation via the TCR/CD3 pathway [3]. Also, we have shown that lupus T cells become more sensitive to interleukin-2 (IL-2) after preactivation via the CD3 pathway [4]. More recently, Vassilopoulos et al. reported accelerated and more sustained calcium responses triggered via the CD3 complex in T cells from lupus patients [5]. The biochemical abnormalities underlying this response pattern are unknown, and their examination may unravel important information concerning the mechanisms leading to loss of tolerance in this disease. The sequence of biochemical events triggered by TCR/ CD3 ligation may differ in lupus T lymphocytes, resulting in an altered pattern of downstream signals that may lead to the aforementioned responses. Tyrosine phosphorylation of several substrates is a critical step in signalling via the TCR/CD3 complex [6]. Dysregulation of relevant protein tyrosine kinases (PTK) of the src family, as shown in the cases of transfectant cells with dysregulated p56lck and transgenic mice over-expressing p59fyn kinase activity, can lead to
Materials and Methods Study population Fifteen Hispanic SLE patients, 11 female, four male, mean age 31 (range 22–43 years), were classified according to the 1982 revised ARA criteria [11]. They had early (35±15 months disease duration) and active disease (SLE disease activity index, SLEDAI, score 11±7) [12]. Some patients were receiving treatment
Correspondence to: Ana M. Blasini, PhD, Centro Nacional de Enfermedades Reuma´ticas, Apartado 5495, Caracas 1010, Venezuela. Fax: 58-2-9853883. E-mail: [email protected] 387 0896-8411/98/050387+07 $30.00/0
© 1998 Academic Press
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A
P = 0.0002 12
8
4
0
Resting T cells (2×106) were pelleted and lysed for 15 min in 1% Nonidet P-40 (NP-40) lysis buffer containing 10 mM Tris pH 8, 150 mM NaCl, 3 ìM pepstatin-A, 2 ìM leupeptin, 0.3 ìM aprotinin, 10 mM sodium fluoride, 0.4 mM sodium orthovanadate, and 0.4 mM EDTA. Lysates were electrophoresed on a sodium dodecyl sulphate (SDS)/10% polyacrylamide gel and transferred to nitrocellulose paper. The efficiency of transfer was assessed in each experiment by the Ponceau-S stain method and the relative intensity of the transferred bands measured by scan densitometry (see below). Nitrocellulose filters were blocked by incubation in 3% non-fat milk and immunoblotted with anti-p59fyn (kindly donated by
SLE [p56Ick] P = 0.007
OD
12
8
4
0
C
SLE
C [ZAP-70] 20
NS
16
OD
Western blot
C
B
Cells T cells were isolated from heparinized peripheral blood after Ficoll–Hypaque gradients and plastic adherence, followed by subsequent steps on nylon wool columns and discontinuous density Percoll gradients. The purity of this cell population was 93±3 CD3 + , as tested by flow cytometry with OKT3 MAb. The degree of enrichment for CD3 + cells did not differ in T lymphocyte preparations of individuals from the three comparison groups; therefore, the starting number of T cells for in vitro activation, immunoblot and immunoprecipitation analysis was comparable. Cells were placed in plates precoated with goat antimouse Ig (Tago, Burlingame, CA, USA), and stimulated with 10 ìg of OKT3 MAb alone or OKT3 plus 10 ìg OKT4 MAb (American Type Culture Collection, Rockville, MD, USA) for 3 min at 37°C. Preliminary experiments showed that this was the optimal stimulation time for induction of tyrosine phosphorylation in T cells from SLE patients and controls (data not shown).
[p59fyn]
16
OD
with oral prednisone (range 2.5–50 mg qd, n=7), chloroquine (range 250–400 md qd, n=3) or cyclophosphamide (1 gr iv, monthly pulses, n=3) therapy. A group of 10 non-SLE patients, two male, eight female, mean age 44 (range 20–60 years) consisted of patients with various chronic inflammatory conditions including rheumatoid arthritis (n=3), trombocytopenic purpura (n=1), osteoarthritis (n=3) and primary Sjo¨gren’s syndrome (n=1). They were receiving varying doses of oral prednisone (dose range 5–12.5 mg qd, n=2), non-steroidal antiinflammatory drugs (n=3) and oral metotrexate (dose 7.5 mg weekly, n=4) at the time of study. Two additional patients, recently kidney grafted, receiving oral prednisone and azathriopine were also studied. Healthy controls (n=15) of the same ethnic background, mean age 32 (range 23–32 years), 11 female, four male, were obtained from the blood bank and laboratory personnel. This study received approval from the Ethics Committee of the hospital (Hospital Universitario de Caracas).
12
8
4
0
C
SLE
Figure 1. Distribution of optical density (OD) values obtained by scan densitometry of immunoblot bands corresponding to p56lck, p59fyn and ZAP-70 molecules in unstimulated T lymphocytes from 15 SLE patients and 15 healthy controls. Statistical analysis was by the two-tailed unpaired Student‘s t-test. SLE=SLE patients, C=healthy controls.
PTK activity in SLE T cells
389
Table 1. Phenotypic membrane markers of peripheral blood T lymphocytes in SLE patients and healthy controlsa CD3
SLE (n=15) C (n=15)
(%)
MCF
CD4 (%)
CD8 (%)
CD25 (%)
CD69 (%)
83±7 82±7
5.48±1.5 6.60±3.5
56.0±16 60.9±17
39.0±13 35.1±13
18.2±9 23.0±10
11.9±5 9.0±8
a
1×106 nylon T cells were examined for expression of membrane markers by flow cytometry with MAb OKT3 (anti-CD3), OKT4 (anti-CD4), OKT8 (anti-CD8), anti-Tac (anti-CD25) or LeuTM-23 (anti-CD69). CD3 expression is presented as mean±SD of percentage of positive cells in T lymphocyte preparations (%) or as mean channel fluorescence (MCF). Differences in T cell membrane markers between SLE patients and healthy controls (C) were not statistically significant.
fyn
p59
the enhanced chemiluminescence method, according to the manufacturer’s directions (ECL, Amersham). Optical densities of specific bands were determined using an Imaging Densitometer (BIORAD, Hercules, CA, USA).
eno
Immunoprecipitation and measurement of PTK activity
p56Ick eno
ZAP-70
eno
C
SLE
Figure 2. Representative experiment showing enhanced p59fyn kinase activity, normal p56lck and ZAP-70 kinase activity in one SLE patient and one healthy control (C). T cell extracts were prepared from 107 resting T cells, immunoprecipitated with MAb recognizing p59fyn, p56lck and ZAP-70, and incubated with [ã-32P]ATP in a protein autophosphorylation and phosphorylation of enolase kinase assay. The radiolabelled proteins were identified by autoradiography after electrophoresis in a 10% SDS–polyacrylamide gel.
Dr Kawakami, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA), anti-p56lck, or antiZAP-70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with peroxidaseconjugated anti-mouse IgG antibody (Amersham, Buckinghamshire, UK) or anti-rabbit IgG antibody (Santa Cruz Biotechnology), blots were developed by
10×106 cells were lysed on ice with a 1% NP-40 lysis buffer. PTK molecules were immunoprecipitated by incubating the cell lysates for 1 h with 10 ìg of antip59fyn, anti-p56lck, or anti-ZAP-70 antibody (Santa Cruz Biotechnology) and protein-A/G agarose beads (Santa Cruz Biotechnology). After washing four times in NP-40 washing buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% NP-40) and once with PTK buffer (20 mM MOPS pH 7, 5 mM MnCl2, 5 mM MgCl2), immunoprecipitates were incubated with 15 ìCi of [ã-32P]ATP (6000 Ci/mmol), 10 ìM ATP and 1 ìg of enolase, at room temperature for 18 min. Samples were boiled and resolved in 10% SDS–PAGE. Densitometric analysis of gel autoradiographies was performed by scan densitometry. Background kinase activity, present in cell lysates immunoprecipitated with mouse immunoglobulins of the corresponding isotypes to the anti-PTK Ab used in the study, was subtracted in each experiment. Specific kinase activity could be removed in cell lysates after preclearing by incubation with the corresponding anti-PTK MAb. Similarly, PTK immunoprecipitates obtained from T-cells pretreated overnight with 3 ìM Herbimycin-A (Sigma, St Louis, MO, USA) showed a significant decrease in tyrosine kinase activity. PTK autophosphorylation activity showed a positive correlation with the number of cells in cell lysates and a negative correlation with the enolase concentration on the assay.
Flow cytometry studies Cells (1×106) were incubated with an optimal dilution of the corresponding MAb for 30 min at 4°C (OKT3, OKT4, OKT8 from Ortho Diagnostics, Raritan, NJ, USA; LeuTM-23 from Becton Dickinson, San Jose, CA, USA; anti-CD25 MAb was a kind donation of Dr T.
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Table 2. PTK activity in p56lck, p59fyn and ZAP-70 immunoprecipitates of cell lysates from peripheral blood T lymphocytesa SLE (n=10) p59fyn autophosph. enolase p56lck autophosph. enolase ZAP-70 autophosph. enolase
7.54±0.73* 5.33±0.67** 3.90±1.02 1.85±0.51 3.23±1.56 2.48±0.56
non-SLE (n=10)
C (n=10)
5.38±0.60 3.39±0.75 3.78±1.23 2.60±0.48 3.98±1.71 3.52±1.23
5.84±0.55 3.79±0.28 3.85±0.85 2.49±0.54 4.19±2.18 3.48±1.70
a
T cell extracts were prepared from 107 resting T cells, immunoprecipitated with MAb recognizing p59fyn, p56lck and ZAP-70 molecules, and incubated with [ã-32P]ATP in protein autophosphorylation and phosphorylation of enolase kinase assays. The radiolabelled proteins were identified by autoradiography after electrophoresis in a 10% SDS-polyacrylamide gel. *P=0.012 comparing SLE with non-SLE patients and P=0.015 comparing SLE patients and controls. **P=0.035 comparing SLE with non-SLE patients and P=0.047 comparing SLE patients and controls. There were no statistical differences between p59fyn activity of non-SLE and healthy controls.
Statistical analysis
10
The two-tailed Student’s t-test was used to compare continuous variables.
P = 0.015
Phosphorylation Intensity (OD)
8 P = 0.047
Diminished expression of p56lck and p59fyn src kinases in peripheral blood T lymphocytes from SLE patients
6
4
2
0
Results
Fyn auto-phosp.
Fyn kinase activity on enolase
Figure 3. Enhanced p59fyn kinase activity in unstimulated SLE T cells. Kinase activity was measured by fyn autophosphorylation and phosphorylation on enolase in immunoprecipitates from 107 resting T cells. Mean±SD of OD of fyn autophosphorylation and enolase phosphorylated in immunoprecipitates from 107 resting T cells from SLE patients (") and healthy controls (h) (n=10).
Unstimulated T lymphocytes from SLE patients showed lower amounts of p59fyn and p56lck src kinases as compared to healthy controls (Figure 1). However, the amount of ZAP-70 was similar in both groups. There was a comparable transference of cell lysate proteins to immunoblot filters in patients and controls, as assessed by the Ponceau-S stain method (data not shown). The amount of CD3 å as tested by immunoblot was also similar in both groups, indicating comparable loading of T cell lysates (6.09±2.92 in SLE patients vs. 7.28±2.78 in controls, mean±SD OD). The phenotypic profile of peripheral blood T cells was also comparable in both groups, as assessed by flow cytometry using anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 (OKT8) MAb. The proportion of peripheral blood T lymphocytes expressing CD25 and CD69 did not differ in lupus patients and controls (Table 1).
PTK activity in unstimulated peripheral blood T lymphocytes Waldmann, National Institute of Health, MD, USA). After three washes in 0.01% sodium azide/1% bovine serum albumin PBS, cells were incubated with FITC conjugated goat anti-mouse antiserum (Becton Dickinson) at 4°C, for 30 min in the dark. After three washes in the same buffer, cells were analysed in a Coulter Epics 753 (Coulter Electronics, Hieleah, FL, USA). Controls using non-reactive immunoglobulins of the same isotype as the reactive antibodies were set up in parallel.
As shown in Figure 2, the enzymatic activity of p59fyn immunoprecipitates was enhanced in unstimulated T cells from SLE patients as compared to non-SLE patients and controls. Both the autophosphorylation of p59fyn molecules and their kinase activity on enolase, an exogenous substrate, were augmented in T cells from lupus patients. In contrast, the autophosphorylation and kinase activity on enolase of p56lck and ZAP-70 immunoprecipitates showed no differ-
PTK activity in SLE T cells
391
Table 3. p59fyn kinase activity in unstimulated and CD3-activated T cells from SLE patients and healthy controlsa
CD3 activation − +
p59fyn autophosphorylation C (n=10) SLE (n=10)
p59fyn kinase activity on enolase C (n=10) SLE (n=10)
5.84±0.55 5.69±1.17
7.54±0.73 6.23±0.90
3.79±0.28 4.10±0.92
5.33±0.67 4.32±0.77
(NS)
(P=0.0487)
(NS)
(P=0.034)
a
p59fyn autophosphorylation and kinase activity on enolase were measured in immunoprecipitates from unstimulated and 107 CD3-activated T cells. Cells were stimulated during 3 min with 10 ìg of OKT3 MAb in plates precoated with goat anti-mouse Igs as described in Materials and Methods. Statistical analysis was by Student’s t-test.
ences in SLE patients as compared to non-SLE patients and healthy controls (Table 2). Figure 3 depicts a representative experiment showing the enzymatic activity of the three PTKs examined in SLE patients and healthy controls. The increased activity of p59fyn PTK in lupus peripheral blood T lymphocytes was not related to clinical disease activity or chronic inflammation, nor to effects related to anti-inflammatory or immunosuppressive medication, as assessed in a group of treated non-SLE patients with diverse chronic inflammatory conditions.
Activation via the TCR/CD3 pathway diminishes p59fyn enzymatic activity in SLE but not control T cells In order to examine the role of TCR/CD3 ligation in the regulation of PTK activity, we next stimulated T cells in vitro with anti-CD3 MAb and examined PTK activity. As shown in Table 3, ligation of TCR/CD3 complexes with OKT3 MAb for 3 min induced a significant decrease of p59fyn activity in T lymphocytes from patients but not from controls. In contrast, ZAP-70 and p56lck kinase activities in immunoprecipitates from cell lysates derived from patients and controls remained unaltered after CD3 or CD3/ CD4 co-stimulation (data not shown). Additionally, the kinetics of tyrosine phosphorylation induction was similar in SLE, non-SLE patients and controls (data not shown).
Discussion In the present study we have examined the expression and enzymatic activity of the three main PTKs known to participate in the earliest biochemical events occurring after stimulation via the CD3 pathway, namely the tyrosine phosphorylation of diverse protein substrates. Unstimulated PB T lymphocytes from lupus patients showed diminished amounts in cell lysates of the two main src kinases involved in TCR/CD3 signalling, i.e. p59fyn and p56lck, whereas the expression of ZAP-70, a syk tyrosine kinase, was comparable in
patients and controls. Despite their relative decreased amount in cell lysates, p59fyn molecules displayed an increased kinase activity in lupus as compared to control T cells. Activation via the CD3 pathway in cell cultures in vitro induced a decrease in p59fyn PTK activity only in T lymphocytes from SLE patients. In contrast, activation of cell cultures in vitro induced no change in p56lck and ZAP-70 kinase activity in the three comparison groups. The increased p59fyn activity of unstimulated lupus T cells was not due to in vivo activation of T cells from SLE patients, since expression of CD69 and CD25, markers of early and late T cell activation respectively, was similar to healthy controls. Differences in fyn kinase activity in activated cultures were not due to the response capabilities of SLE vs. non-SLE T cells since the percentage of CD3 + cells, and the mean channel fluorescence (MCF) of CD3 reactivity, were comparable in patients and controls. Furthermore, under these experimental conditions, we have previously observed full proliferative responses by lupus and control T cells [3, 4]. Abnormalities in post-receptor signal transduction may underlie the enhanced CD3-mediated responses seen previously in our lupus patients [3]. There is scant information on the state of the proximal biochemical events occurring after TCR/CD3 stimulation in human SLE. A defect in the breakdown of membrane phosphoinositides and in the mobilization of intracellular calcium has been reported in MRL/lpr-lpr lupus mice [13]. Recently, Vassilopoulos et al. have shown accelerated and more sustained intracytoplasmic calcium mobilization in human lupus T cells after activation via the TCR/CD3 complex [5]. One of the earliest signals triggered by TCR/CD3 ligation is the phosphorylation of several proteins on tyrosine residues, a metabolic event mainly mediated by PTK of the src family [14]. It is possible that dysregulated PTK activity may be responsible for the altered pattern of tyrosinephosphorylated bands in unstimulated T lymphocytes seen in our SLE patients [10]. Katagiri et al. showed enhanced tyrosine-phosphorylation of T cell src kinase p59fyn in the murine lpr lupus model [15]. No such information is available in lupus patients. The potential role of PTK enzyme activity in dys-
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regulated lymphocyte responses is supported by data from transgenic murine models overexpressing p59fyn molecules [8] and in cells transfected with a constitutively activated form of p56lck [7, 16]. Thymic and post-thymic T cells from these mice show enhanced responses upon stimulation via the CD3 pathway. In contrast, expression of a catalytically inactive form of p59fyn blocked the proliferative response that normally follows TCR-mediated activation [8], providing strong support for the conjecture that TCR signalling might proceed through a pathway in which p59fyn plays an obligatory role. Our results suggest in vivo abnormal regulation of the expression and enzyme activity of p59fyn molecules in T cells from SLE patients. Biochemical abnormalities potentially present in lupus p59fyn molecules, either by defective gene transcription or by posttranslational modifications, may simultaneously lead to unchecked enzyme activity and decreased biological half-life. The latter may explain the lower amounts of fyn protein in cell lysates from patients. Alternatively, the metabolic fate and kinase activity may depend on the regulatory control of molecules that are additionally altered in lupus T cells. For example, we have recently observed diminished amounts of TCR æ chains in lupus PB T-cells [17, manuscript submitted]. It is possible that the constitutive coupling of p59fyn and æ chains occurring in normal T cells [18] may play a role in down-regulation of p59fyn activity in the T cell resting state, and at the same time prevent the metabolic degradation of this kinase. It is possible that ligation of TCR/CD3 molecules corrected the enhanced fyn activity in T cells from our lupus patients by restoring coupling of this PTK with down-regulatory molecules such as specific tyrosine phosphatases. It is well known that defects in the tyrosine dephosphorylation of activated substrates by specific phosphatases may contribute to enhanced distal responses in T lymphocytes. For example, inhibition of phosphatases PP1 and PP2A enhanced mitogenic responses and dysregulated IL-2 and IL-2R gene transcription in T lymphocytes [19]. The diminished coupling of tyrosine phosphorylation by scarce amounts of TCR æ in lupus T cells may interfere with this down-regulatory mechanism in TCR/CD3activated cells. The relatively delayed tyrosine phosphorylation of TCR æ chains [20] argues for a potential role in shutting down responses by approximating tyrosine phosphatases to TCR/CD3-coupled signalling molecules such as ZAP-70 and p59fyn. In summary, unstimulated peripheral blood T lymphocytes from lupus patients showed enhanced p59fyn, and normal p56lck and ZAP-70 kinase activities. Given the known role of p59fyn in post-receptor signalling in T cells, it is conceivable that the dysregulation of this critical src kinase may alter the threshold of responses to autoantigens or affect relevant outcomes such as apoptosis and Th1/Th2 functional responses, mechanisms that can potentially lead to a breakdown of the tolerant state of autoreactive clones in SLE patients.
Acknowledgements We are very grateful to Mrs Mayra Mayora for her dedicated and efficient secretarial assistance. This study was supported by grants G-97000808, S1-95000480 from Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICIT) and grant 94-494 RG/BIO/LA from The Third World Academy of Sciences (TWAS).
References 1. Dayal A.K., Kammer G.M. 1991. The T cell enigma in lupus. Arthritis Rheum. 39: 23–332 2. Tsokos G.C., Kovacs B., Liossis S.N.C. 1997. Lymphocytes, cytokines, inflammation and immune trafficking. Curr. Opin. Rheumatol. 9: 380–386 3. Stekman I.L., Blasini A.M., Leon-Ponte M., Baroja M.L., Abadi I., Rodriguez M.A. 1991. Enhanced CD3 mediated T lymphocyte proliferation in patients with systemic lupus erythematosus. Arthritis Rheum. 34: 459–467 4. Blasini A.M., Stekman I.L., Gonzalez F., Tositti M.L., Rodriguez M.A. 1994. T lymphocytes from patients with systemic lupus erythematosus show increased response to interleukin-2 after costimulation with OKT3 monoclonal antibody and phorbol esters. Clin. Immunol. Immunopathol. 70: 66–72 5. Vassilopoulos D., Kovacs B., Tsokos G.C. 1995. TCR/CD3 complex-mediated signal transduction in T cells and T cell lines from patients with systemic lupus erythematosus. J. Immunol. 155: 2269–2281 6. Chan A.C., Desai D.M., Weiss A. 1994. The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction. Ann. Rev. Immunol. 12: 555–592 7. Abraham N., Miceli M.C., Parnes J.R., Veillete A. 1991. Enhancement of T-cell responsiveness by lymphocyte-specific tyrosine protein kinase p56lck. Nature 358: 62–66 8. Cooke M.P., Abraham K.M., Forbush K.A., Perlmutter R.M. 1991. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn). Cell 65: 281–291 9. Lorenz U., Ravichandran K.S., Burakoff S.J., Neel B.G. 1996. Lack of SHPTP1 results in src-kinase hyperactivation and thymocyte hyperresponsiveness. Proc. Natl. Acad. Sci. USA 93: 9624–9629 10. Blasini A.M., Chacon R., Stekman I.L., Alonzo E., Rodriguez M.A. 1995. Diminished tyrosine phosphatase activity of CD45 molecules in non-stimulated peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum. 38: S212 11. Tan E.M., Cohen A.S., Fries J.F., Masi A.T., McShane D.J., Rothfield N.F., Schaller J.G., Talal N., Winchester R.J. 1982. The revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25: 1271–1277 12. Hawker G., Gabriel S., Bombardiere C., Goldsmith C., Caron D., Gladman D. 1993. A reliability study of SLEDAI: A disease activity index for systemic lupus erythematosus. J. Rheumatol. 20: 657–660 13. Scholz W., Isakov N., Mally M.I., Theofilopoulos A., Altman A. 1987. Lpr cell hyporesponsiveness to
PTK activity in SLE T cells
14.
15.
18.
17.
mitogens linked to deficient receptor-stimulated phosphoinositide hydrolysis. J. Biol. Chem. 263: 3626–3631 De Franco A.L. 1994. Signaling pathways activated by protein tyrosine phosphorylation in lymphocytes. Curr. Op. Immunol. 6: 364–371 Katagiri T., Ting J.P., Dy R., Prokop C., Cohen P., Earp H.S. 1989. Tyrosine phosphorylation of a c-src-like protein is increased in membranes of CD4− CD8− T lymphocytes from lpr/lpr mice. Mol. Cell. Biol. 9: 4914–4922 Davidson D., Chow L.M.C., Forunell M., Veillete A. 1992. Enhancement of T cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. J. Exp. Med. 175: 1483–1492 Brundula V., Rivas L., Blasini A.M., Paris M., Stekman I.L., Salazar S., Riera R., Rodriguez M.A. 1997.
393
Diminished levels of TCR æ chains in peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum. 40: S60 18. Samelson L.E., Phillips A.F., Wong E.T., Klausner R.D. 1990. Association of the Fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87: 4358–4362 19. Richards F.M., Milner J., Metcalf S. 1992. Inhibition of the serine-threonine protein phosphatases PP1 and PP2 in lymphocytes: Effect on mRNA levels for interleukin-2, IL-2Rá, krox-24, p53, hsc70 and cyclophilin. Immunol. 76: 642–647 20. Hsi E.D., Siegel J.N., Minami Y., Luong E.T., Klausner R.D., Samelson L.E. 1989. T cell activation induces rapid tyrosine phosphorylation of a limited number of substrates. J Biol. Chem. 264: 10836–10842
Journal of Autoimmunity (1998) 11, 387–393
Protein Tyrosine Kinase Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus Ana M. Blasini, Veronika Brundula, Magdalena Paris, Liliana Rivas, Susana Salazar, Ivan L. Stekman and Martin A. Rodriguez Centro Nacional de Enfermedades Reuma´ticas, Hospital Universitario de Caracas, Caracas, Venezuela
Key words: PTK, SLE, T cells
We have recently observed an abnormal pattern of protein tyrosine phosphorylation in resting T lymphocytes obtained from peripheral blood of patients with systemic lupus erythematosus (SLE). To examine whether these findings may be related to dysregulated protein tyrosine kinase (PTK) function, we tested the relative amount and enzyme activity of the main PTKs involved in the earliest signalling steps triggered via the CD3 pathway. Cell lysates from peripheral blood T cells in SLE patients showed lower amounts of p59fyn and p56lck as shown by immunoblot. In contrast, the amount of ZAP-70, a PTK of the syk family, was comparable in both groups. However, p59fyn immunoprecipitates obtained from unstimulated peripheral blood SLE T cells showed enhanced PTK activity as compared to controls, whereas the PTK activity of p56lck and ZAP-70 molecules was comparable in both groups. The unchecked activity of the TCR/CD3-associated src kinase p59fyn may alter the balance needed for regulated T cell responses in SLE patients. © 1998 Academic Press
Introduction
augmented TCR/CD3-mediated responses [7, 8]. Similarly, defective protein tyrosine phosphatase (PTP) activity can lead to unchecked cytokine gene activation in T lymphocytes [9]. It is possible that abnormalities in the regulation of tyrosine phosphorylation and dephosphorylation may alter downstream biochemical steps needed for regulated gene responses in T lymphocytes from SLE patients. Our recent experiments seem to suggest that the pattern of tyrosine-phosphorylated proteins is altered in non-stimulated peripheral blood T lymphocytes from patients with SLE [10]. To examine the possible basis for this response pattern we have, in this study, examined the relative amount and enzyme activity of the main kinases involved in early transduction triggered via the CD3 pathway, namely src kinases p56lck, p59fyn and syk ZAP-70, in unstimulated and CD3-activated PB T cells from a group of patients with early and clinically active SLE.
Previous studies have shown a number of abnormalities in the response of peripheral blood (PB) T lymphocytes in patients with SLE upon activation via the TCR/CD3 pathway [1, 2]. We have previously shown that T lymphocytes from SLE patients can have augmented responses upon stimulation via the TCR/CD3 pathway [3]. Also, we have shown that lupus T cells become more sensitive to interleukin-2 (IL-2) after preactivation via the CD3 pathway [4]. More recently, Vassilopoulos et al. reported accelerated and more sustained calcium responses triggered via the CD3 complex in T cells from lupus patients [5]. The biochemical abnormalities underlying this response pattern are unknown, and their examination may unravel important information concerning the mechanisms leading to loss of tolerance in this disease. The sequence of biochemical events triggered by TCR/ CD3 ligation may differ in lupus T lymphocytes, resulting in an altered pattern of downstream signals that may lead to the aforementioned responses. Tyrosine phosphorylation of several substrates is a critical step in signalling via the TCR/CD3 complex [6]. Dysregulation of relevant protein tyrosine kinases (PTK) of the src family, as shown in the cases of transfectant cells with dysregulated p56lck and transgenic mice over-expressing p59fyn kinase activity, can lead to
Materials and Methods Study population Fifteen Hispanic SLE patients, 11 female, four male, mean age 31 (range 22–43 years), were classified according to the 1982 revised ARA criteria [11]. They had early (35±15 months disease duration) and active disease (SLE disease activity index, SLEDAI, score 11±7) [12]. Some patients were receiving treatment
Correspondence to: Ana M. Blasini, PhD, Centro Nacional de Enfermedades Reuma´ticas, Apartado 5495, Caracas 1010, Venezuela. Fax: 58-2-9853883. E-mail: [email protected] 387 0896-8411/98/050387+07 $30.00/0
© 1998 Academic Press
A. M. Blasini et al.
388
A
P = 0.0002 12
8
4
0
Resting T cells (2×106) were pelleted and lysed for 15 min in 1% Nonidet P-40 (NP-40) lysis buffer containing 10 mM Tris pH 8, 150 mM NaCl, 3 ìM pepstatin-A, 2 ìM leupeptin, 0.3 ìM aprotinin, 10 mM sodium fluoride, 0.4 mM sodium orthovanadate, and 0.4 mM EDTA. Lysates were electrophoresed on a sodium dodecyl sulphate (SDS)/10% polyacrylamide gel and transferred to nitrocellulose paper. The efficiency of transfer was assessed in each experiment by the Ponceau-S stain method and the relative intensity of the transferred bands measured by scan densitometry (see below). Nitrocellulose filters were blocked by incubation in 3% non-fat milk and immunoblotted with anti-p59fyn (kindly donated by
SLE [p56Ick] P = 0.007
OD
12
8
4
0
C
SLE
C [ZAP-70] 20
NS
16
OD
Western blot
C
B
Cells T cells were isolated from heparinized peripheral blood after Ficoll–Hypaque gradients and plastic adherence, followed by subsequent steps on nylon wool columns and discontinuous density Percoll gradients. The purity of this cell population was 93±3 CD3 + , as tested by flow cytometry with OKT3 MAb. The degree of enrichment for CD3 + cells did not differ in T lymphocyte preparations of individuals from the three comparison groups; therefore, the starting number of T cells for in vitro activation, immunoblot and immunoprecipitation analysis was comparable. Cells were placed in plates precoated with goat antimouse Ig (Tago, Burlingame, CA, USA), and stimulated with 10 ìg of OKT3 MAb alone or OKT3 plus 10 ìg OKT4 MAb (American Type Culture Collection, Rockville, MD, USA) for 3 min at 37°C. Preliminary experiments showed that this was the optimal stimulation time for induction of tyrosine phosphorylation in T cells from SLE patients and controls (data not shown).
[p59fyn]
16
OD
with oral prednisone (range 2.5–50 mg qd, n=7), chloroquine (range 250–400 md qd, n=3) or cyclophosphamide (1 gr iv, monthly pulses, n=3) therapy. A group of 10 non-SLE patients, two male, eight female, mean age 44 (range 20–60 years) consisted of patients with various chronic inflammatory conditions including rheumatoid arthritis (n=3), trombocytopenic purpura (n=1), osteoarthritis (n=3) and primary Sjo¨gren’s syndrome (n=1). They were receiving varying doses of oral prednisone (dose range 5–12.5 mg qd, n=2), non-steroidal antiinflammatory drugs (n=3) and oral metotrexate (dose 7.5 mg weekly, n=4) at the time of study. Two additional patients, recently kidney grafted, receiving oral prednisone and azathriopine were also studied. Healthy controls (n=15) of the same ethnic background, mean age 32 (range 23–32 years), 11 female, four male, were obtained from the blood bank and laboratory personnel. This study received approval from the Ethics Committee of the hospital (Hospital Universitario de Caracas).
12
8
4
0
C
SLE
Figure 1. Distribution of optical density (OD) values obtained by scan densitometry of immunoblot bands corresponding to p56lck, p59fyn and ZAP-70 molecules in unstimulated T lymphocytes from 15 SLE patients and 15 healthy controls. Statistical analysis was by the two-tailed unpaired Student‘s t-test. SLE=SLE patients, C=healthy controls.
PTK activity in SLE T cells
389
Table 1. Phenotypic membrane markers of peripheral blood T lymphocytes in SLE patients and healthy controlsa CD3
SLE (n=15) C (n=15)
(%)
MCF
CD4 (%)
CD8 (%)
CD25 (%)
CD69 (%)
83±7 82±7
5.48±1.5 6.60±3.5
56.0±16 60.9±17
39.0±13 35.1±13
18.2±9 23.0±10
11.9±5 9.0±8
a
1×106 nylon T cells were examined for expression of membrane markers by flow cytometry with MAb OKT3 (anti-CD3), OKT4 (anti-CD4), OKT8 (anti-CD8), anti-Tac (anti-CD25) or LeuTM-23 (anti-CD69). CD3 expression is presented as mean±SD of percentage of positive cells in T lymphocyte preparations (%) or as mean channel fluorescence (MCF). Differences in T cell membrane markers between SLE patients and healthy controls (C) were not statistically significant.
fyn
p59
the enhanced chemiluminescence method, according to the manufacturer’s directions (ECL, Amersham). Optical densities of specific bands were determined using an Imaging Densitometer (BIORAD, Hercules, CA, USA).
eno
Immunoprecipitation and measurement of PTK activity
p56Ick eno
ZAP-70
eno
C
SLE
Figure 2. Representative experiment showing enhanced p59fyn kinase activity, normal p56lck and ZAP-70 kinase activity in one SLE patient and one healthy control (C). T cell extracts were prepared from 107 resting T cells, immunoprecipitated with MAb recognizing p59fyn, p56lck and ZAP-70, and incubated with [ã-32P]ATP in a protein autophosphorylation and phosphorylation of enolase kinase assay. The radiolabelled proteins were identified by autoradiography after electrophoresis in a 10% SDS–polyacrylamide gel.
Dr Kawakami, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA), anti-p56lck, or antiZAP-70 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with peroxidaseconjugated anti-mouse IgG antibody (Amersham, Buckinghamshire, UK) or anti-rabbit IgG antibody (Santa Cruz Biotechnology), blots were developed by
10×106 cells were lysed on ice with a 1% NP-40 lysis buffer. PTK molecules were immunoprecipitated by incubating the cell lysates for 1 h with 10 ìg of antip59fyn, anti-p56lck, or anti-ZAP-70 antibody (Santa Cruz Biotechnology) and protein-A/G agarose beads (Santa Cruz Biotechnology). After washing four times in NP-40 washing buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% NP-40) and once with PTK buffer (20 mM MOPS pH 7, 5 mM MnCl2, 5 mM MgCl2), immunoprecipitates were incubated with 15 ìCi of [ã-32P]ATP (6000 Ci/mmol), 10 ìM ATP and 1 ìg of enolase, at room temperature for 18 min. Samples were boiled and resolved in 10% SDS–PAGE. Densitometric analysis of gel autoradiographies was performed by scan densitometry. Background kinase activity, present in cell lysates immunoprecipitated with mouse immunoglobulins of the corresponding isotypes to the anti-PTK Ab used in the study, was subtracted in each experiment. Specific kinase activity could be removed in cell lysates after preclearing by incubation with the corresponding anti-PTK MAb. Similarly, PTK immunoprecipitates obtained from T-cells pretreated overnight with 3 ìM Herbimycin-A (Sigma, St Louis, MO, USA) showed a significant decrease in tyrosine kinase activity. PTK autophosphorylation activity showed a positive correlation with the number of cells in cell lysates and a negative correlation with the enolase concentration on the assay.
Flow cytometry studies Cells (1×106) were incubated with an optimal dilution of the corresponding MAb for 30 min at 4°C (OKT3, OKT4, OKT8 from Ortho Diagnostics, Raritan, NJ, USA; LeuTM-23 from Becton Dickinson, San Jose, CA, USA; anti-CD25 MAb was a kind donation of Dr T.
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Table 2. PTK activity in p56lck, p59fyn and ZAP-70 immunoprecipitates of cell lysates from peripheral blood T lymphocytesa SLE (n=10) p59fyn autophosph. enolase p56lck autophosph. enolase ZAP-70 autophosph. enolase
7.54±0.73* 5.33±0.67** 3.90±1.02 1.85±0.51 3.23±1.56 2.48±0.56
non-SLE (n=10)
C (n=10)
5.38±0.60 3.39±0.75 3.78±1.23 2.60±0.48 3.98±1.71 3.52±1.23
5.84±0.55 3.79±0.28 3.85±0.85 2.49±0.54 4.19±2.18 3.48±1.70
a
T cell extracts were prepared from 107 resting T cells, immunoprecipitated with MAb recognizing p59fyn, p56lck and ZAP-70 molecules, and incubated with [ã-32P]ATP in protein autophosphorylation and phosphorylation of enolase kinase assays. The radiolabelled proteins were identified by autoradiography after electrophoresis in a 10% SDS-polyacrylamide gel. *P=0.012 comparing SLE with non-SLE patients and P=0.015 comparing SLE patients and controls. **P=0.035 comparing SLE with non-SLE patients and P=0.047 comparing SLE patients and controls. There were no statistical differences between p59fyn activity of non-SLE and healthy controls.
Statistical analysis
10
The two-tailed Student’s t-test was used to compare continuous variables.
P = 0.015
Phosphorylation Intensity (OD)
8 P = 0.047
Diminished expression of p56lck and p59fyn src kinases in peripheral blood T lymphocytes from SLE patients
6
4
2
0
Results
Fyn auto-phosp.
Fyn kinase activity on enolase
Figure 3. Enhanced p59fyn kinase activity in unstimulated SLE T cells. Kinase activity was measured by fyn autophosphorylation and phosphorylation on enolase in immunoprecipitates from 107 resting T cells. Mean±SD of OD of fyn autophosphorylation and enolase phosphorylated in immunoprecipitates from 107 resting T cells from SLE patients (") and healthy controls (h) (n=10).
Unstimulated T lymphocytes from SLE patients showed lower amounts of p59fyn and p56lck src kinases as compared to healthy controls (Figure 1). However, the amount of ZAP-70 was similar in both groups. There was a comparable transference of cell lysate proteins to immunoblot filters in patients and controls, as assessed by the Ponceau-S stain method (data not shown). The amount of CD3 å as tested by immunoblot was also similar in both groups, indicating comparable loading of T cell lysates (6.09±2.92 in SLE patients vs. 7.28±2.78 in controls, mean±SD OD). The phenotypic profile of peripheral blood T cells was also comparable in both groups, as assessed by flow cytometry using anti-CD3 (OKT3), anti-CD4 (OKT4), and anti-CD8 (OKT8) MAb. The proportion of peripheral blood T lymphocytes expressing CD25 and CD69 did not differ in lupus patients and controls (Table 1).
PTK activity in unstimulated peripheral blood T lymphocytes Waldmann, National Institute of Health, MD, USA). After three washes in 0.01% sodium azide/1% bovine serum albumin PBS, cells were incubated with FITC conjugated goat anti-mouse antiserum (Becton Dickinson) at 4°C, for 30 min in the dark. After three washes in the same buffer, cells were analysed in a Coulter Epics 753 (Coulter Electronics, Hieleah, FL, USA). Controls using non-reactive immunoglobulins of the same isotype as the reactive antibodies were set up in parallel.
As shown in Figure 2, the enzymatic activity of p59fyn immunoprecipitates was enhanced in unstimulated T cells from SLE patients as compared to non-SLE patients and controls. Both the autophosphorylation of p59fyn molecules and their kinase activity on enolase, an exogenous substrate, were augmented in T cells from lupus patients. In contrast, the autophosphorylation and kinase activity on enolase of p56lck and ZAP-70 immunoprecipitates showed no differ-
PTK activity in SLE T cells
391
Table 3. p59fyn kinase activity in unstimulated and CD3-activated T cells from SLE patients and healthy controlsa
CD3 activation − +
p59fyn autophosphorylation C (n=10) SLE (n=10)
p59fyn kinase activity on enolase C (n=10) SLE (n=10)
5.84±0.55 5.69±1.17
7.54±0.73 6.23±0.90
3.79±0.28 4.10±0.92
5.33±0.67 4.32±0.77
(NS)
(P=0.0487)
(NS)
(P=0.034)
a
p59fyn autophosphorylation and kinase activity on enolase were measured in immunoprecipitates from unstimulated and 107 CD3-activated T cells. Cells were stimulated during 3 min with 10 ìg of OKT3 MAb in plates precoated with goat anti-mouse Igs as described in Materials and Methods. Statistical analysis was by Student’s t-test.
ences in SLE patients as compared to non-SLE patients and healthy controls (Table 2). Figure 3 depicts a representative experiment showing the enzymatic activity of the three PTKs examined in SLE patients and healthy controls. The increased activity of p59fyn PTK in lupus peripheral blood T lymphocytes was not related to clinical disease activity or chronic inflammation, nor to effects related to anti-inflammatory or immunosuppressive medication, as assessed in a group of treated non-SLE patients with diverse chronic inflammatory conditions.
Activation via the TCR/CD3 pathway diminishes p59fyn enzymatic activity in SLE but not control T cells In order to examine the role of TCR/CD3 ligation in the regulation of PTK activity, we next stimulated T cells in vitro with anti-CD3 MAb and examined PTK activity. As shown in Table 3, ligation of TCR/CD3 complexes with OKT3 MAb for 3 min induced a significant decrease of p59fyn activity in T lymphocytes from patients but not from controls. In contrast, ZAP-70 and p56lck kinase activities in immunoprecipitates from cell lysates derived from patients and controls remained unaltered after CD3 or CD3/ CD4 co-stimulation (data not shown). Additionally, the kinetics of tyrosine phosphorylation induction was similar in SLE, non-SLE patients and controls (data not shown).
Discussion In the present study we have examined the expression and enzymatic activity of the three main PTKs known to participate in the earliest biochemical events occurring after stimulation via the CD3 pathway, namely the tyrosine phosphorylation of diverse protein substrates. Unstimulated PB T lymphocytes from lupus patients showed diminished amounts in cell lysates of the two main src kinases involved in TCR/CD3 signalling, i.e. p59fyn and p56lck, whereas the expression of ZAP-70, a syk tyrosine kinase, was comparable in
patients and controls. Despite their relative decreased amount in cell lysates, p59fyn molecules displayed an increased kinase activity in lupus as compared to control T cells. Activation via the CD3 pathway in cell cultures in vitro induced a decrease in p59fyn PTK activity only in T lymphocytes from SLE patients. In contrast, activation of cell cultures in vitro induced no change in p56lck and ZAP-70 kinase activity in the three comparison groups. The increased p59fyn activity of unstimulated lupus T cells was not due to in vivo activation of T cells from SLE patients, since expression of CD69 and CD25, markers of early and late T cell activation respectively, was similar to healthy controls. Differences in fyn kinase activity in activated cultures were not due to the response capabilities of SLE vs. non-SLE T cells since the percentage of CD3 + cells, and the mean channel fluorescence (MCF) of CD3 reactivity, were comparable in patients and controls. Furthermore, under these experimental conditions, we have previously observed full proliferative responses by lupus and control T cells [3, 4]. Abnormalities in post-receptor signal transduction may underlie the enhanced CD3-mediated responses seen previously in our lupus patients [3]. There is scant information on the state of the proximal biochemical events occurring after TCR/CD3 stimulation in human SLE. A defect in the breakdown of membrane phosphoinositides and in the mobilization of intracellular calcium has been reported in MRL/lpr-lpr lupus mice [13]. Recently, Vassilopoulos et al. have shown accelerated and more sustained intracytoplasmic calcium mobilization in human lupus T cells after activation via the TCR/CD3 complex [5]. One of the earliest signals triggered by TCR/CD3 ligation is the phosphorylation of several proteins on tyrosine residues, a metabolic event mainly mediated by PTK of the src family [14]. It is possible that dysregulated PTK activity may be responsible for the altered pattern of tyrosinephosphorylated bands in unstimulated T lymphocytes seen in our SLE patients [10]. Katagiri et al. showed enhanced tyrosine-phosphorylation of T cell src kinase p59fyn in the murine lpr lupus model [15]. No such information is available in lupus patients. The potential role of PTK enzyme activity in dys-
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regulated lymphocyte responses is supported by data from transgenic murine models overexpressing p59fyn molecules [8] and in cells transfected with a constitutively activated form of p56lck [7, 16]. Thymic and post-thymic T cells from these mice show enhanced responses upon stimulation via the CD3 pathway. In contrast, expression of a catalytically inactive form of p59fyn blocked the proliferative response that normally follows TCR-mediated activation [8], providing strong support for the conjecture that TCR signalling might proceed through a pathway in which p59fyn plays an obligatory role. Our results suggest in vivo abnormal regulation of the expression and enzyme activity of p59fyn molecules in T cells from SLE patients. Biochemical abnormalities potentially present in lupus p59fyn molecules, either by defective gene transcription or by posttranslational modifications, may simultaneously lead to unchecked enzyme activity and decreased biological half-life. The latter may explain the lower amounts of fyn protein in cell lysates from patients. Alternatively, the metabolic fate and kinase activity may depend on the regulatory control of molecules that are additionally altered in lupus T cells. For example, we have recently observed diminished amounts of TCR æ chains in lupus PB T-cells [17, manuscript submitted]. It is possible that the constitutive coupling of p59fyn and æ chains occurring in normal T cells [18] may play a role in down-regulation of p59fyn activity in the T cell resting state, and at the same time prevent the metabolic degradation of this kinase. It is possible that ligation of TCR/CD3 molecules corrected the enhanced fyn activity in T cells from our lupus patients by restoring coupling of this PTK with down-regulatory molecules such as specific tyrosine phosphatases. It is well known that defects in the tyrosine dephosphorylation of activated substrates by specific phosphatases may contribute to enhanced distal responses in T lymphocytes. For example, inhibition of phosphatases PP1 and PP2A enhanced mitogenic responses and dysregulated IL-2 and IL-2R gene transcription in T lymphocytes [19]. The diminished coupling of tyrosine phosphorylation by scarce amounts of TCR æ in lupus T cells may interfere with this down-regulatory mechanism in TCR/CD3activated cells. The relatively delayed tyrosine phosphorylation of TCR æ chains [20] argues for a potential role in shutting down responses by approximating tyrosine phosphatases to TCR/CD3-coupled signalling molecules such as ZAP-70 and p59fyn. In summary, unstimulated peripheral blood T lymphocytes from lupus patients showed enhanced p59fyn, and normal p56lck and ZAP-70 kinase activities. Given the known role of p59fyn in post-receptor signalling in T cells, it is conceivable that the dysregulation of this critical src kinase may alter the threshold of responses to autoantigens or affect relevant outcomes such as apoptosis and Th1/Th2 functional responses, mechanisms that can potentially lead to a breakdown of the tolerant state of autoreactive clones in SLE patients.
Acknowledgements We are very grateful to Mrs Mayra Mayora for her dedicated and efficient secretarial assistance. This study was supported by grants G-97000808, S1-95000480 from Consejo Nacional de Investigaciones Cientı´ficas y Tecnolo´gicas (CONICIT) and grant 94-494 RG/BIO/LA from The Third World Academy of Sciences (TWAS).
References 1. Dayal A.K., Kammer G.M. 1991. The T cell enigma in lupus. Arthritis Rheum. 39: 23–332 2. Tsokos G.C., Kovacs B., Liossis S.N.C. 1997. Lymphocytes, cytokines, inflammation and immune trafficking. Curr. Opin. Rheumatol. 9: 380–386 3. Stekman I.L., Blasini A.M., Leon-Ponte M., Baroja M.L., Abadi I., Rodriguez M.A. 1991. Enhanced CD3 mediated T lymphocyte proliferation in patients with systemic lupus erythematosus. Arthritis Rheum. 34: 459–467 4. Blasini A.M., Stekman I.L., Gonzalez F., Tositti M.L., Rodriguez M.A. 1994. T lymphocytes from patients with systemic lupus erythematosus show increased response to interleukin-2 after costimulation with OKT3 monoclonal antibody and phorbol esters. Clin. Immunol. Immunopathol. 70: 66–72 5. Vassilopoulos D., Kovacs B., Tsokos G.C. 1995. TCR/CD3 complex-mediated signal transduction in T cells and T cell lines from patients with systemic lupus erythematosus. J. Immunol. 155: 2269–2281 6. Chan A.C., Desai D.M., Weiss A. 1994. The role of protein tyrosine kinases and protein tyrosine phosphatases in T cell antigen receptor signal transduction. Ann. Rev. Immunol. 12: 555–592 7. Abraham N., Miceli M.C., Parnes J.R., Veillete A. 1991. Enhancement of T-cell responsiveness by lymphocyte-specific tyrosine protein kinase p56lck. Nature 358: 62–66 8. Cooke M.P., Abraham K.M., Forbush K.A., Perlmutter R.M. 1991. Regulation of T cell receptor signaling by a src family protein-tyrosine kinase (p59fyn). Cell 65: 281–291 9. Lorenz U., Ravichandran K.S., Burakoff S.J., Neel B.G. 1996. Lack of SHPTP1 results in src-kinase hyperactivation and thymocyte hyperresponsiveness. Proc. Natl. Acad. Sci. USA 93: 9624–9629 10. Blasini A.M., Chacon R., Stekman I.L., Alonzo E., Rodriguez M.A. 1995. Diminished tyrosine phosphatase activity of CD45 molecules in non-stimulated peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum. 38: S212 11. Tan E.M., Cohen A.S., Fries J.F., Masi A.T., McShane D.J., Rothfield N.F., Schaller J.G., Talal N., Winchester R.J. 1982. The revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25: 1271–1277 12. Hawker G., Gabriel S., Bombardiere C., Goldsmith C., Caron D., Gladman D. 1993. A reliability study of SLEDAI: A disease activity index for systemic lupus erythematosus. J. Rheumatol. 20: 657–660 13. Scholz W., Isakov N., Mally M.I., Theofilopoulos A., Altman A. 1987. Lpr cell hyporesponsiveness to
PTK activity in SLE T cells
14.
15.
18.
17.
mitogens linked to deficient receptor-stimulated phosphoinositide hydrolysis. J. Biol. Chem. 263: 3626–3631 De Franco A.L. 1994. Signaling pathways activated by protein tyrosine phosphorylation in lymphocytes. Curr. Op. Immunol. 6: 364–371 Katagiri T., Ting J.P., Dy R., Prokop C., Cohen P., Earp H.S. 1989. Tyrosine phosphorylation of a c-src-like protein is increased in membranes of CD4− CD8− T lymphocytes from lpr/lpr mice. Mol. Cell. Biol. 9: 4914–4922 Davidson D., Chow L.M.C., Forunell M., Veillete A. 1992. Enhancement of T cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. J. Exp. Med. 175: 1483–1492 Brundula V., Rivas L., Blasini A.M., Paris M., Stekman I.L., Salazar S., Riera R., Rodriguez M.A. 1997.
393
Diminished levels of TCR æ chains in peripheral blood T lymphocytes from patients with systemic lupus erythematosus. Arthritis Rheum. 40: S60 18. Samelson L.E., Phillips A.F., Wong E.T., Klausner R.D. 1990. Association of the Fyn protein-tyrosine kinase with the T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87: 4358–4362 19. Richards F.M., Milner J., Metcalf S. 1992. Inhibition of the serine-threonine protein phosphatases PP1 and PP2 in lymphocytes: Effect on mRNA levels for interleukin-2, IL-2Rá, krox-24, p53, hsc70 and cyclophilin. Immunol. 76: 642–647 20. Hsi E.D., Siegel J.N., Minami Y., Luong E.T., Klausner R.D., Samelson L.E. 1989. T cell activation induces rapid tyrosine phosphorylation of a limited number of substrates. J Biol. Chem. 264: 10836–10842