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Rheumatology Advance Access originally published online on July 31, 2006
Rheumatology 2007 46(2):238-245; doi:10.1093/rheumatology/kel180
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The systemic and SmD183–119–autoantigen-specific cytokine memory of Th cells in SLE patients

S. Langer1, D. Langnickel1, P. Enghard1, R. Undeutsch1, G. R. Burmester1, F. Hiepe1,2, A. Radbruch2 and G. Riemekasten1

1Department of Rheumatology and Clinical Immunology, Charité University Hospital, Humboldt University of Berlin and 2German Rheumatism Research Center, D-10117 Berlin, Germany

Correspondence to: G. Riemekasten, MD, Department of Rheumatology and Clinical Immunology, Charité University Hospital, Schumannstr. 20/21, D-10117 Berlin, Germany. E-mail: gabriela.riemekasten{at}charite.de


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. The aim of the study was to analyse the cytokine memory of T-cells derived from systemic lupus erythematosus (SLE) patients and healthy donors enriched for autoantigen-specific T-cells by in vitro stimulation with SmD183–119, a common autoantigen in SLE.

Methods. Autoreactive CD3+ T-cells derived from 37 SLE patients and 14 healthy donors were enriched by repetitive ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with SmD183–119. For control, PBMCs were stimulated only with interleukin-2 (IL-2). After two rounds of antigenic stimulation, cultures were stimulated with PMA/ionomycin to probe the cytokine memory by intracellular cytokine staining. Frequencies of cytokine-expressing T-cells were analysed and, in SLE patients, compared with disease activities and autoantibody levels.

Results. Comparing the cytokine memory in the cultures, SLE patients displayed higher frequencies of tumour necrosis factor-{alpha} (TNF-{alpha})+ T-cells than healthy donors and the frequencies correlated with disease activity. Frequencies of SmD183–119-specific TNF-{alpha}+ T-cells and of memory T-cells expressing interferon-{gamma} (IFN-{gamma}) correlated with serum anti-dsDNA antibody levels. The frequencies of IL-10 expressing SmD183–119-specific T-cells were lower among PBMCs of SLE patients. Relatively higher frequencies of IL-10+ T-cells in SLE patients correlated with low disease activities, and low anti-dsDNA and anti-SmD183–119 antibody concentrations in culture supernatants.

Conclusions. The memory of autoreactive SmD183–119-specific and unspecific stimulated peripheral Th cells for re-expression of cytokines is shifted towards more cells expressing TNF-{alpha} and less IL-10+ cells, when compared SLE patients with normal donors. This shift towards proinflammatory memory effector Th cells correlates with disease severity and humoral autoimmunity.

KEY WORDS: SLE, T-cell cytokines, Cytokine memory, IL-10+ T-cells, Autoantigens


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
In systemic lupus erythematosus (SLE), the increased number of activated and affinity-maturated T-cells as well as the presence of autoreactive T-cells suggests an impaired T-cell effector function that seems to be restricted to a distinct panel of autoantigens [1]. The impaired effector function is usually best exemplified by the pattern of cytokines produced [2]. Therefore, characterization of T-cell cytokine expression, especially of autoantigen-specific T-cells, is central to understand T-cell help in the etiopathogenesis of lupus.

T-cell cytokines are involved in the control of B- and T-cell interaction and, therefore, are target candidates as regulators of pathogenicity in SLE. Interferon-{gamma} (IFN-{gamma}) stimulates B-cells [3, 4], induces B- and plasma-cell migration into the inflamed tissue [5] and plays a pivotal role in directing cell-mediated immune responses. The increased expression of IFN-{gamma} in autoreactive T-cell clones indicates a role for IFN-{gamma}+ T-cells in lupus pathogenesis [4]. Interleukin-4 (IL-4), termed ‘B-cell growth factor’, also induces B-cell migration [6] and class switch to the immunoglobulin (Ig) isotypes G1/G4 over-represented in SLE [7, 8]. IL-10 induces differentiation of B-cell blasts into plasma cells and also participates in the control of isotype switching towards IgG1 and IgG3 [8] isotypes also over-represented in SLE [9]. Furthermore, IL-10 has a regulatory function in lupus, (summarized in [10, 11]) especially when produced by T-cells, and recently, autoantigen-specific IL-10 expressing T-cells were shown to decrease anti-dsDNA antibody generation in vitro [12]. Tumour necrosis factor-{alpha} (TNF-{alpha}) is an important proinflammatory cytokine in lupus and other autoimmune diseases [13]. Furthermore, it is a survival factor for plasma cells [14, 15].

Naive and central memory T-cells do not produce effector cytokines except IL-2. Upon primary activation, naive T-cells become instructed for expression of cytokines. Repetitive stimulations with the corresponding antigen result in a cytokine memory with a faster recall expression of previously instructed cytokines without the requirement of the original instructing signal. This allows a stable effector function [16, 17]. Therefore, T-cells expressing effector cytokines such as IFN-{gamma}, IL-4, IL-10 and TNF-{alpha} exhibit effector/memory cells previously instructed by antigens or, in SLE, by autoantigens.

Studying the T-cell cytokine memory among all freshly isolated memory Th cells, most authors have not found any differences between healthy donors and lupus patients [18–20]. This is probably related to the low frequencies of autoreactive T-cells, which is considered to be <1 per 105 T-cells [21, 22]. Furthermore, the missing differences in the T-cell cytokine memory may reflect the heterogeneity of the patients, or the varying balance between different autoreactive T-cell subsets as defined by their cytokine expression during flares and remissions in SLE. In the present study, we attempted to analyse the cytokine memory by a novel approach. We cultured peripheral blood mononuclear cells (PBMCs) over an extended period of time to enrich T-cells specific for autoantigens present in cell cultures as well as for the SmD183–119 autoantigenic peptide. Cultures were finally stimulated with PMA/ionomycin to circumvent possible unresponsiveness to physiological T Cell Receptor (TCR) triggering due to extended culturing and repeated restimulation. The T-cell cytokine memory was correlated with clinical findings and autoantibody concentration in the culture supernatants.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Acknowledgements
 References
 
Patients
This study was approved by the Ethics Committee of the Charité University Hospital, and is in compliance with the Helsinki Declaration. Blood samples were obtained, after written consent from 37 patients with SLE and 14 healthy donors. Patients fulfilled the American Congress of Rheumatology (ACR) classification criteria for SLE [23]. Most of the patients received immunosuppressive drugs (14 received azathioprine, three cyclophosphamide, four mycophenolate, six hydroxychloroquine, two methotrexate, one leflunomide, one ciclosporin-A, and three patients had a combination of two different immunosuppressive drugs). Two patients received steroids in a dosage >10 mg/day.

The disease activity was measured by SLE Disease Activity Index (SLEDAI) as described elsewhere [24]. Three patients were tested twice at 6-month intervals; one of them with different disease activity, the others remained stable.

Antigen
The SmD183–119 peptide (VEPKVKKREAVAGRGRGRGRGRGRGRG-RGRGGPRR) was synthesized as free peptide according to the protocol described by Atherton [25] and purified by a reverse phase chromatography (C18 Vydac column).

Isolation of PBMCs and culture conditions
Approximately 40 ml of venous blood were collected using heparinized S-monovettes (Sarstedt, Nümbrecht, Germany) from SLE patients and healthy donors.

PBMCs were isolated by Ficoll PaqueTM PLUS (Amersham Pharmacia Biotech AB, Sweden) density centrifugation, washed twice in Phosphate Buffer Saline (PBS)/Bovine Serum Albumine (BSA) and split: One fraction was suspended in growth medium [Roswell Park Memorial Institute (RPMI) 1640 (all supplements 2 mM L-glutamine, PAA Laboratories, Coelbe, Germany)], 5% heat inactivated human AB serum (PAA, Austria), 0.3 mg/ml glutamine, 50 µM 2-mercaptoethanol (GIBCO Brl, Paisley, UK), 100 U/ml penicillin, 0.1 mg/ml streptomycin (glutamine; PAA Laboratories, Coelbe, Germany) and cultured at 37°C and 5% CO2. The other fraction was resuspended in chilled freezing medium (70% RPMI 1640, 20% fetal bovine serum, 10% DiMethyl SulfOxide (DMSO)) at the concentrations of approximately 10 x 106 cells/ml and immediately frozen to –70°C.

At day 3, one portion of the cultured cells received 10 µg/ml of the SmD1-peptide, the other portion did not receive any. At day 7, both cultures received 5 U/ml of recombinant (r)IL-2 (Strathmann Biotec AG, Hamburg, Germany). At day 11, the frozen cells were quickly thawed by adding growth medium at room temperature and washed in a high surplus of growth medium. These cells were {gamma}-irradiated (30 Gy, 10 min) and added as autologous feeder cells to the respective cultures. In addition, 10 µg/ml of the SmD1-peptide was given only to the culture previously stimulated with the SmD183–119 peptide. At day 18, 5 U/ml of rIL-2 were added to the cultures. Some days later (median at day 22), supernatants of the culture pairs (with and without SmD1 stimulation) were harvested and cells were restimulated with PMA (10 ng/ml) and ionomycin (250 ng/ml) for 6 h. After 3 h, 10 µg/ml brefeldin A was added to the cells.

The cells were harvested, washed in PBS and fixed in 2% paraformaldehyde for 20 min at room temperature. After washing with PBS/BSA, the cells were stored in PBS/BSA/acid at 4°C overnight. The variability of the used culture system was controlled using PBMCs from healthy donors, demonstrating reproducible results in parallel cultures of cells from the same donor (no outliner values, S.D. comprising 9–54% of the mean value depending on the produced cytokine).

Intracellular cytokine staining and flowcytometric analysis
The cells were first subjected to anti-CD3-Biotin and then to streptavidin-Peridinin-chlorophyll-protein complex (PerCP) (PharMingen), then divided into two groups. One group was stained with anti-IFN-{gamma}-Fluorescein IsoThioCyanate (FITC), anti-IL-4-Phycoerythrin (PE) and anti-IL-10-Cy5, the other group with anti-TNF-{alpha}-FITC, anti-IL-2-PE and anti-IL-4-Cy5. 16 cultures derived from 5 SLE patients were also stained for Cluster of Differentiation (CD)25 (anti-CD25-FITC, PharMingen, San Diego, CA, USA). For intracellular and surface staining, the following monoclonal antibodies (all from PharMingen) were used according to the manufacturer's instructions: IFN-{gamma}-FITC (clone N748A and clone 4SB3), TNF-{alpha}-FITC (clone 1D12), IL-2-PE (clone N7.4A), IL-4-PE (clone 4D9), CD3-Biotin (clone UCHT1), IL-4-Cy5 (clone 4D9, clone 4D9), IL-10-Cy5 (clone Jes3.19F1).

Intracellular staining was performed in PBS/BSA/acid containing 0.5% saponin. All cells were blocked with 10% polyclonal human IgG (Flebogamma, Grifols, Langen, Germany) in a total of 50–70 µl staining volume. The incubation time for surface staining was 10 min, and 15 min for intracellular staining. Samples were analysed by a 4-colour-analysis using Fluorescence Activated Cell Sorting (FACS). Calibur flow cytometer (Becton Dickinson) and FACS-Express software, version 2.0.

Serum anti-SmD183–119 peptide antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as described [26]. Reactivity of a standard serum at a dilution of 1:100 was defined as 10 000 arbitrary units (AU). Supernatants were diluted 1:2, sera were analysed at dilution 1:100. Serum reactivity above 123 AU was defined as positive as described before [26].

Anti-dsDNA antibodies were detected by ELISA as described previously [27]. Serum were tested at dilution 1:100, supernatants at dilution 1:2. Serum levels above 6 µg/ml were defined as positive.

Statistics
Graphpad Prism 3.0 was used for statistical analysis. The frequencies of T-cells expressing cytokines showed a skewed distribution. Therefore, Mann–Whitney U-test was used to compare cytokine expression in different culture conditions. Linear regression was used to compare disease activity and autoantibody levels with cytokine expression or ratios of cytokine expression. Wilcoxon matched pair test as well as paired t-test was used to compare the frequency of CD25+ T-cells in cultures with and without SmD183–119. P-values <0.05 were defined as significantly independent from the R2 values.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Acknowledgements
 References
 
SLE patients reveal higher frequencies of TNF-{alpha}-expressing memory T-cells and lower frequencies of IL-10-expressing T-cells compared with healthy donors
Comparing the cytokine memory of peripheral T-cells in SLE patients with healthy donors, frequencies of TNF-{alpha}-expressing memory T-cells were higher in cultures from SLE patients (n = 35) than in those from healthy donors (n = 14) in both cultures stimulated either with SmD183–119 or the medium control (P = 0.045 and P = 0.038, respectively, Fig. 1A for the SmD183–119 culture). In contrast, SLE patients showed a decreased frequency of IL-10+ T-cells compared with healthy controls only after stimulation with SmD183–119 (P = 0.0317, Fig. 1B).


Figure 1
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  		FIG. 1. Frequency of cytokine-expressing memory T-cells in SLE patients vs healthy donors by systemic T-cell stimulation. SmD183–119-reactive memory Th cells activated and expanded ex vivo showed a shift towards more cells expressing TNF-{alpha} (A) and less cells expressing IL-10 (B), when SLE patients are compared with healthy donors. After ex vivo restimulation with SmD183–119, ratios of memory Th cells expressing TNF-{alpha} vs IL-10 (C) and of those expressing IFN-{gamma} vs IL-10 (D) were higher in SLE patients compared with the ratios of healthy donors. In 34 SLE cultures, the frequency of TNF-{alpha}+ T-cells was measured, 31 SLE cultures were analysed for IL-10-expressing T-cells and 37 SLE cultures were analysed for IFN-{gamma}.

 
SLE T-cells revealed a higher ratio of memory Th cells expressing TNF-{alpha} vs IL-10 in cultures stimulated with SmD183–119 and with medium control (P = 0.018 and P = 0.023, respectively, Fig. 1C for the SmD183–119 culture) compared with cultures from healthy donors. Furthermore, the ratio of T-cells expressing IFN-{gamma} vs IL-10 in both cultures compared with healthy donors (P = 0.02 for the SmD1-stimulated culture (Fig. 1D) and P = 0.03 for the medium control) further suggesting the lower IL-10 expression in lupus T-cells.

Proinflammatory Th cytokine memory correlates to disease activity in SLE patients
To analyse a possible role of memory T-cells expressing one of the investigated cytokine on disease activity, the frequencies of memory/effector T-cells positive for the different cytokines were compared with the disease activity in the corresponding SLE patient.

In both cultures, with and without SmD183–119, the frequency of TNF-{alpha} expressing T-cells correlated with disease activity classified according to SLEDAI (P = 0.027 for the medium control, P = 0.021 for the SmD183–119 culture). Patients with active disease showed an increased cytokine memory for TNF-{alpha} compared with inactive patients (Fig. 2A). Higher frequencies of IL-10 memory T-cells were associated with lower disease activity in both cultures (P = 0.039 for the medium control, P = 0.032 for the SmD1 culture, Fig. 2B). The frequencies of IFN-{gamma}, IL-2, and IL-4 expressing memory Th cells did not correlate with disease activity. The correlation between disease activity and ratio of cells expressing TNF-{alpha} vs IL-4 was only present in cultures stimulated with SmD183–119 (P = 0.007, R2 = 0.028, Fig. 2C), Furthermore, there were significant correlations between disease activity and increased ratio of IFN-{gamma} vs IL-10-expressing Th cells (P = 0.024 for the SmD183–119 cultures, data not shown) and ratio of Th cells expressing IL-2 vs IL-10 (P = 0.007 for the SmD183–119 culture, Fig. 2D). The increased ratio of cells expressing IFN-{gamma} vs IL-10 and IL-2 vs IL-10 correlated with disease activity also in the cultures stimulated without SmD183–119 peptide (P = 0.042 and P = 0.003, respectively). The increase in the frequencies of memory/effector T-cells expressing proinflammatory cytokines and the decrease in the frequency of IL-10-expressing T-cells during active disease were also supported by a 6-month follow-up investigation of one SLE patient suffering from a lupus flare reflected by an increase in SLEDAI from 4 to 9 (Fig. 3). In this patient, increased frequencies of IFN-{gamma}+CD3+ T-cells (from 2.9 to 4.8%), of TNF{alpha}+ (from 3.4 to 4.5%) and of IL-2+ T-cell (from 3.6 to 12%) were detected as well as increased frequencies of IL-4+ T-cells (from 0.4 to 1.7%). In contrast, the frequency of IL-10 expression slightly decreased when active disease was compared with inactive disease (from 0.55 to 0.34%, Fig. 3). Another lupus patient with long-term clinical remission showed stable, but very low frequencies of cytokine-expressing T-cells.


Figure 2
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  		FIG. 2. Proinflammatory systemic Th cytokine memory correlates to disease activity in SLE patients. Cytokine memory of ex vivo activated and expanded Th cells expressing the proinflammatory cytokine TNF-{alpha} correlated to disease activity classified according to SLEDAI (A). In contrast, higher frequencies of memory Th cells expressing IL-10 were associated with lower disease activity (B). The cytokine memory was very similar in both cultures, but more intensified by enrichment of SmD183–119-specific T-cells. 34 cultures were measured for TNF-{alpha} and 31 for IL-10. There was a correlation between disease activity and ratio of cells expressing TNF-{alpha} vs IL-4 (C) and ratio of Th cells expressing IL-2 vs IL-10 (D) only present in cultures stimulated with SmD183–119.

 

Figure 3
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  		FIG. 3. Follow-up investigation in a lupus patient shows activity-dependent differences in the systemic cytokine memory. Follow-up investigation of a patient with a lupus flare changed the cytokine memory towards proinflammatory cytokines. The figure shows the frequencies of T-cells in cultures of T-cells stimulated with SmD183–119 prior to the PMA/ionomycin stimulation. To measure the disease activity, SLEDAI was used.

 
Activation and expansion of SmD183–119-reactive T-cells revealed different memory T-cells expressing different cytokines
Stimulation with the SmD183–119 peptide and expansion of SmD183–119-reactive T-cells revealed a heterogeneous cytokine response. No differences were detected comparing the frequency of memory T-cells in the SmD183–119 culture with the frequency in the culture stimulated only with IL-2 (Table 1). However, all cultures stimulated with SmD183–119 revealed increased frequencies of CD25+ activated T-cells when compared with the medium control (P = 0.008, Fig. 4).


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  		TABLE 1. Cytokine memory in cultures either stimulated with SmD183–119 or only with IL-2. Median frequencies (%) of cytokine-expressing CD3+ memory Th cells in 22-day cultures stimulated with SmD183–119 (SmD) and without SmD183–119 (control) and detected by intracellular cytokine staining and flow cytometry after PMA/ionomycin stimulation in healthy donors (HD) as well as in SLE patients. In paragraphs, 25th and 75th percentiles are shown. In SLE patients, 37 corresponding cultures were measured for IFN-{gamma}, 34 for TNF-{alpha}, 27 for IL-2, 29 for IL-4 and 31 for IL-10. In HD, 14 corresponding cultures were measured for IFN-{gamma}, TNF-{alpha}, IL-10 and IL-4, and 10 for IL-2.

 

Figure 4
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  		FIG. 4. Increased frequencies of T-cells positive for the activation marker CD25 by culturing T-cells with SmD183–119 compared with the cultures of the same donor stimulated wih medium control. 16 different cultures from five different patients were analysed.

 
Comparing the cytokine memory of CD3+ T-cells from SLE patients and healthy donors, 57% of the ex vivo stimulated cultures from healthy donors reacted to SmD183–119 by IFN-{gamma} expression, whereas only 40% of the cultures from SLE patients did. The T-cell memory of SmD183–119-reactive Th cells was shifted towards IL-4 expressing cells in 59% of the SLE patients, but only in 33% of the healthy donors (data not shown). Four SLE patients had increased frequencies of Th cells expressing any of the cytokines investigated when cultures stimulated with SmD183–119 were compared with medium controls. The results indicate that the balances of cytokine-expressing T-cells might be more important.

The increase in frequencies of SmD183–119-specific T-cells expressing IFN-{gamma}, TNF-{alpha}, and IL-10 correlate with clinical findings in SLE patients
The frequency of CD3+ memory Th cells in cultures with and without SmD183–119 was assessed to analyse cytokine memory of Th cells after activation and expansion of SmD183–119-reactive T-cells. Higher frequencies of T-cells expressing a certain cytokine in the SmD183–119-expanded culture reflect the cytokine memory of SmD183–119-specific T-cells. To analyse different SLE patients with different basal frequencies of SmD183–119-specific T-cells, the relative increase in frequencies of SmD183–119-specific memory T-cells expressing one of the investigated cytokines was compared with the serum autoantibody levels and disease activity in the corresponding lupus donors.

The increase in frequencies of IFN-{gamma} expressing Th cells in the cultures after expansion of SmD183–119-specific T-cells (in%) correlated with serum anti-Sm and anti-dsDNA antibody levels (P = 0.003 and P = 0.016, respectively, Fig. 5A and B). The higher the increase in SmD183–119-specific T-cells expressing IFN-{gamma}, the higher were the corresponding serum antibody concentrations. The increase in frequencies of memory Th cells expressing TNF-{alpha} upon SmD183–119 stimulation also correlated with serum anti-dsDNA antibody concentrations (P = 0.038, Fig. 5C). The increase in frequencies of SmD183–119-specific T-cells expressing IL-10 correlated with disease activity (P = 0.006, Fig. 5D), but not with serum antibody levels.


Figure 5
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  		FIG. 5. Cytokine memory of SmD183–119-specific T-cells correlates with clinical findings in SLE patients. The percentage increase in frequencies of SmD183–119-specific memory Th cells expressing IFN-{gamma} correlated with the levels of serum anti-SmD183–119 (A, n = 9) and anti-dsDNA antibodies (B, n = 12) as detected by ELISA. The increase in frequencies of TNF-{alpha} expressing Th cells upon expansion and activation of SmD183–119-specific memory Th cells was also associated with serum anti-dsDNA antibody levels in the corresponding donor (C, n = 15). The increase in frequencies of Th cells expressing IL-10 correlated with disease activity (D, n = 9), but not with serum antibody levels.

 
Increase in frequencies of SmD183–119-specific T-cells expressing IL-10 is associated with low antibody levels in culture supernatants
Comparing the cytokine memory in cultures stimulated with SmD183–119 with medium controls, differences in frequencies of IL-10 expressing memory T-cells upon stimulation by the SmD183–119 peptide ranged from +53% (in the case of increased frequencies of IL-10 expressing T-cells upon SmD183–119 stimulation compared with the medium control) to –58% (in the case of a decrease in frequencies of IL-10 expressing T-cells upon SmD183–119 restimulation). An enhanced frequency of SmD183–119-specific T-cells expressing IL-10 was associated with low anti-SmD183–119 and anti-dsDNA antibody concentrations in culture supernatants and, on the other hand, lower frequencies of IL-10 expressing SmD183–119-specific memory T-cells were found in supernatants with higher anti-SmD183–119 and anti-dsDNA antibody concentrations (P = 0.014 and P = 0.035, respectively, Fig. 6A and B. Changes of the other cytokines did not show any correlation to the concentrations of antibody in culture supernatants.


Figure 6
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  		FIG. 6. Autoantibody levels in supernatants are influenced by the cytokine memory of SmD183–119-specific T-cells. Percentage differences in frequencies of Th cells expressing IL-10 correlated with anti-SmD183–119 (A) and anti-dsDNA (B) antibody concentrations in culture supernatants. Differences in the frequencies were calculated comparing the frequency of Th cells expressing IL-10 in the culture stimulated with SmD183–119 vs in the medium control. An increased frequency in SmD183–119-specific T-cells expressing IL-10 was associated with low anti-SmD183–119 and anti-dsDNA autoantibody levels in culture supernatants.

 

   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Acknowledgements
 References
 
The analysis of the T-cell memory for cytokine expression, both systemic and autoantigen specific, may help to understand the role of autoreactive T-cells in SLE. Among the autoantigens that are known to be specifically recognized by the T-cells in SLE, the SmD183–119 peptide has been described as an important autoantigen in murine and human lupus [26, 28–30]. SmD183–119-reactive T-cells have been identified and were associated with cardiopulmonary pathology in SLE patients [30]. Furthermore, SmD183–119-reactive T-cells have been shown to provide T-cell help for pathogenic anti-dsDNA and anti-Sm antibodies in various murine lupus models [29] (manuscript in preparation). Modulation of SmD183–119-reactive T-cells by immunization or by induction of high-dose tolerance influences anti-dsDNA antibody levels and survival in murine lupus [11, 28]. According to the pivotal role of SmD183–119 in lupus, T-cells are already primed in vivo for this ubiquitous autoantigen. The present cytokine memory before further ex vivo stimulation seems strongly reflected here by the similarity of the cytokine memory in both cultures either stimulated with SmD183–119 or IL-2 alone. Therefore, associations between disease activity and T-cells expressing TNF-{alpha} and IL-10 might represent cytokine memory of autoreactive T-cells such as SmD183–119-reactive T-cells. Furthermore, similarities between both cultures can also reflect ongoing stimulations with autoantigens and, therefore, permanent activation and enrichment of autoreactive T-cells as it is suggested by studies showing the presence of nucleosomes in B-cell cultures [31]. One limitation of the approach used in the present study is that the in vitro enrichment of autoantigen specific T-cells might yield different cytokine producers than in vivo encounter of autoantigen in the specific lupus microenvironment. Nevertheless, the association between cytokine memory of SmD183–119-reactive T-cells and disease activity, serum and supernatant autoantibody levels (Figs 4 and 5) support the role of SmD183–119 as an important autoantigen and suggest an in vivo priming by SmD183–119 that may be representative for other autoantigens in SLE.

Here, we observed an increased cytokine memory for TNF-{alpha} in cultures from SLE patients compared with those from healthy donors. Also, the frequencies of TNF-{alpha}-expressing memory T-cells correlated with disease activity. A role for TNF-{alpha}+ T-cells in disease pathogenesis is also suggested by the frequencies of TNF-{alpha}+ T-cells in the SmD183–119 driven ex vivo expansion in lupus patients. The higher the frequencies of memory T-cells expressing TNF-{alpha} responding to the SmD183–119 peptide the higher were the anti-dsDNA antibody levels in the corresponding patient. Despite divergent results concerning the role of TNF-{alpha} in lupus models [32, 33], TNF-{alpha} blockade has been successfully used in lupus patients suffering from nephritis [13]. Our results show that low frequencies of autoreactive TNF-{alpha}+ memory T-cells correlate to clinical benefit in SLE. The association between levels of anti-SmD183–119 antibodies in serum and the frequencies of IFN-{gamma}+ SmD183–119-specific memory T-cells further underlines the importance of proinflammatory cytokines in SLE.

The cytokine memory for IL-10 was impaired in SLE. Frequencies of IL-10 expressing Th memory cells were decreased in the SmD183–119-expanded Th cells of SLE patients compared with healthy donors. A reduced frequency of IL-10+ T-cells has been detected previously in SLE patients [34], but their functional role in lupus pathogenesis remains unclear. Here, we show that this reduction is not only symptomatic but also affects autoreactive Th cells. Higher frequencies of SmD183–119-specific memory Th cells expressing IL-10 were associated with low autoantibody levels in the supernatants. The data suggest an involvement of SmD183–119-reactive IL-10 memory T-cells in the regulation of autoantibodies in SLE.

IL-10 appears to be a key cytokine in the pathogenesis of SLE since IL-10 polymorphisms are among the strongest susceptibility genetic factors in SLE [35]. This cytokine inhibits activation and effector function of antigen-presenting cells [36]. Thus, IL-10 has the potential to limit and terminate inflammatory immune reaction 37 including those of lupus. A beneficial effect of IL-10 is supported by studies in an experimental murine lupus model, showing a protective role of IL-10 [10, 11].

Paradoxically, at first sight a pathogenetic role of IL-10 has also been postulated for lupus, based on the observation that systemic anti-IL-10 treatment is beneficial in murine and human lupus [38, 39]. This seemingly contradictory effect of IL-10 can be reconciled if one considers the source and time point of IL-10 production. IL-10 is expressed by various cells and IL-10 produced by non-T-cells appears to predominate [34]. While IL-10 producing T-cells may be protective by silencing Antigen Presenting Cell (APC) and Th1 cells as shown in in vitro studies [10, 11], IL-10 derived from other cells may drive autoantibody production by B-cells. Furthermore, as shown in animal studies, the role of IL-10 is influenced by the stage of disease [10]. Late in disease development, an increasing frequency of IL-10-expressing T-cells directed to the autoantigens is required to suppress the ongoing autoimmune process. This possible defense mechanism is reflected here by the association between SmD183–119-specific IL-10+ T-cells and disease activity as well as by our ongoing experiments in animal models showing a regulatoy function of IL-10 expressing autoreactive T-cells. During further progress of the disease, IL-10+ T-cells fail to suppress Th1 cells and APC efficiently, and consequently, IL-10 from Th cells and other cells augments differentiation of B-cells into plasma cells producing pathogenic autoreactive antibodies [10, 11]. This would explain both the previously described beneficial effect of anti-IL-10 on established and active disease and the protective effect of IL-10 on lupus aetiopathogenesis. It is to be shown, whether therapy with IL-10 memory Th cells may be superior to anti-IL-10 therapy as suggested by other groups in SLE [38, 39].

G.R: and F.H. have a patent for the commercial use of the SmD183–119 peptide (United States Patent 5,945,105) as diagnostic marker and for therapeutic use.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by grants from University research foundation of the Humboldt University, the Deutsche Forschungsgemeinschaft (Ri 1056/2-6, SFB 650, TP10) and by grants from ‘Wolfgang Schulze’ Foundation. We thank the statistician Joachim Listing from the German Rheumatology Research Centre for critical help and advices.

Grant supporters G.R. and F.H. have a patent DFG Ri 1056/2-6, SFB 650, TP 10, Grant from the Charité University, Foundation ‘Wolfgang Schulze’. The other authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Theocharis S, Sfikakis PP, Lipnick RN, Klipple GL, Steinberg AD, Tsokos GC. (1995) Characterization of in vivo mutated clones from patients with systemic lupus erythematosus. Clin Immunol Immunopathol 74:135–42.[CrossRef][Web of Science][Medline]
  2. Mosmann TR and Coffman RL. (1989) TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–73.[CrossRef][Web of Science][Medline]
  3. Murray LJ, Lee R, Martens C. (1990) In vivo cytokine gene expression in T-cell subsets of the autoimmune MRL/Mp-lpr/lpr mouse. Eur J Immunol 20:163–70.[Web of Science][Medline]
  4. Voll RE, Roth EA, Girkontaite I, et al. (1997) Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production. Arthritis Rheum 40:2162–71.[Web of Science][Medline]
  5. Muehlinghaus G, Cigliano L, Huehn S, et al. (2005) Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B-cells into plasma cells. Blood 105:3965–71.[Abstract/Free Full Text]
  6. Clinchy B and Moller G. (1994) Interleukin-4 induces in vitro migration in naive B-cells through direct mechanisms. Scand J Immunol 40:429–36.[CrossRef][Web of Science][Medline]
  7. Goodman DJ, Gaff C, Gerondakis S. (1993) The IL-4 induced increase in the frequency of resting murine splenic B-cells expressing germline Ig heavy chain gamma 1 transcripts correlates with subsequent switching to IgG1. Int Immunol 5:199–208.[Abstract/Free Full Text]
  8. Banchereau J, Briere F, Liu YJ, Rousset F. (1994) Molecular control of B lymphocyte growth and differentiation. Stem Cells 12:278–88.[Web of Science][Medline]
  9. Smith CI, Norberg R, Isenberg D, et al. (1988) Anti-DNA antibody synthesis after bone marrow transplantation: implications for IgG subclass restrictivity and pathogenicity of autoantibodies. Bone Marrow Transplant 3:583–8.[Web of Science][Medline]
  10. Yin Z, Bahtiyar G, Zhang G, et al. (2002) IL-10 regulates murine lupus. J Immunol 169:2148–55.[Abstract/Free Full Text]
  11. Beebe AM, Cua DJ, de Waal Malefyt R. (2002) The role of interleukin-10 in autoimmune disease: systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Cytokine Growth Factor Rev 13:403–12.[CrossRef][Web of Science][Medline]
  12. Riemekasten G, Langnickel D, Enghard P, et al. (2004) Immune tolerance to the SmD183–119 peptide postpones development of murine lupus and induces regulatory Tr1 cells. J Immunol 173:5835–42.[Abstract/Free Full Text]
  13. Aringer M, Graninger WB, Steiner G, Smolen JS. (2004) Safety and efficacy of tumor necrosis factor alpha blockade in systemic lupus erythematosus: An open-label study. Arthritis Rheum 50:3161–9.[CrossRef][Web of Science][Medline]
  14. Cassese G, Arce S, Hauser AE, et al. (2003) Plasma cell survival is mediated by synergistic effects of cytokines and adhesion-dependent signals. J Immunol 171:1684–90.[Abstract/Free Full Text]
  15. Hauser AE, Muehlinghaus G, Manz RA, et al. (2003) Long-lived plasma cells in immunity and inflammation. Ann NY Acad Sci 987:266–9.[Web of Science][Medline]
  16. Assenmacher M, Lohning M, Scheffold A, et al. (1998) Commitment of individual Th1-like lymphocytes to expression of IFN-gamma versus IL-4 and IL-10: selective induction of IL-10 by sequential stimulation of naive Th cells with IL-12 and IL-4. J Immunol 161:2825–32.[Abstract/Free Full Text]
  17. Richter A, Lohning M, Radbruch M. (1999) Instruction for cytokine expression in T helper lymphocytes in relation to proliferation and cell cycle progression. J Exp Med 190:1439–50.[Abstract/Free Full Text]
  18. Schneider S, Bruns A, Moewes B, et al. (2002) Simultaneous cytometric analysis of (auto)antigen-reactive T and B-cell proliferation. Immunobiology 206:484–95.[CrossRef][Web of Science][Medline]
  19. Helsloot J and Sturgess A. (1997) T-cell reactivity to Sjogren's syndrome related antigen La(SSB). J Rheumatol 24:2340–7.[Web of Science][Medline]
  20. Nagy G, Pallinger E, Antal-Szalmas P, et al. (2001) Measurement of intracellular interferon-gamma and interleukin-4 in whole blood T lymphocytes from patients with systemic lupus erythematosus. Immunol Lett 74:207–10.[Web of Science]
  21. Filaci G, Grasso I, Contini P, et al. (1996) DsDNA-, nucleohistone- and DNASE I-reactive T lymphocytes in patients affected by systemic lupus erythematosus: correlation with clinical disease activity. Clin Exp Rheumatol 14:543–50.[Web of Science][Medline]
  22. McCutcheon M, Wehner N, Wensky A, et al. (1997) A sensitive ELISPOT assay to detect low-frequency human T lymphocytes. J Immunol Meth 210:149–66.[CrossRef][Web of Science][Medline]
  23. Tan EM, Cohen AS, Fries AS, et al. (1982) The revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 25:1271–7.[Web of Science][Medline]
  24. Bombardier C, Gladman D, Urowitz MB, Caron D, Chang CH. the Committee on Prognosis Studies inSLE. (1992) Derivation of the SLEDAI: a disease activity index for lupus patients. Arthritis Rheum 35:630–40.[Web of Science][Medline]
  25. Atherton E. (1989) Solid phase peptide synthesis: a practical approach(IRL press, Oxford).
  26. Riemekasten G, Marell J, Trebeljahr G, et al. (1998) A novel epitope on the C-terminus of SmD1 is recognized by the majority of sera from patients with systemic lupus erythematosus. J Clin Invest 102:754–63.[Web of Science][Medline]
  27. Hiepe F, Kiessig S, Jahn HD, et al. (1986) A sensitive and class specific solid phase enzyme immunoassay for anti-DNA autoantibodies in supernatants of lymphocytes cultures and human hybridomas. Biomed Biochem Acta 45:K29–35.[Web of Science][Medline]
  28. Riemekasten G, Kawald A, Weiss C, et al. (2001) Strong acceleration of murine lupus by injection of the SmD1(83–119) peptide. Arthritis Rheum 44:2435–45.[CrossRef][Web of Science][Medline]
  29. Riemekasten G, Langnickel D, Ebling FM, et al. (2003) Identification and characterization of SmD183–119-reactive T-cells that give T-cell help for pathogenic anti-dsDNA antibodies. Arthritis Rheum 48:475–85.[CrossRef][Web of Science][Medline]
  30. Riemekasten G, Weiß C, Schneider S, et al. (2002) T-cell reactivity against the SmD183–119 C-terminal peptide in patients with systemic lupus erythematosus. Ann Rheum Dis 61:779–85.[Abstract/Free Full Text]
  31. Cabrespines A, Laderach D, Lebosse C, Bach JF, Koutouzov S. (1998) Isolation and characterization of apoptotic nucleosomes, free and complexed with lupus autoantibody generated during hybridoma B-cell apoptosis. J Auto Immun 11:19–27.
  32. Deguchi Y and Kishimoto S. (1991) Tumour necrosis factor/cachectin plays a key role in autoimmune pulmonary inflammation in lupus-prone mice. Clin Exp Immunol 85:392–5.[Web of Science][Medline]
  33. Gordon C, Ranges GE, Greenspan JS, Wofsy D. (1989) Chronic therapy with recombinant tumor necrosis factor-alpha in autoimmune NZB/NZW F1 mice. Clin Immunol Immunopathol 52:421–34.[CrossRef][Web of Science][Medline]
  34. Chang DM, Su WL, Chu SL. (2002) The expression and significance of intracellular T helper cytokines in systemic lupus erythematosus. Immunol Invest 31:1–12.[CrossRef][Web of Science][Medline]
  35. D'Alfonso S, Rampi M, Bocchio D, Colombo G, Scorza-Smeraldi R, Momigliano-Richardi P. (2000) Systemic lupus erythematosus candidate genes in the Italian population: evidence for a significant association with interleukin-10. Arthritis Rheum 43:120–8.[CrossRef][Web of Science][Medline]
  36. Waal Malefyt R, Abrams J, Bennett B, Figdor CG, de Vries JE. (1991) Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174:1209–20.[Abstract/Free Full Text]
  37. Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A. (2001) Interleukin-10 and the IL-10 receptor. Annu Rev Immunol 19:683–765.[CrossRef][Web of Science][Medline]
  38. Llorente L, Zou W, Levy Y, et al. (1995) Role of interleukin 10 in the B lymphocyte hyperactivity and autoantibody production of human systemic lupus erythematosus. J Exp Med 181:839–44.[Abstract/Free Full Text]
  39. Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M. (1994) Continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in NZB/W F1 mice. J Exp Med 179:305–10.[Abstract/Free Full Text]
Submitted 8 August 2005; revised version accepted 21 April 2006.
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