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Rheumatology 2008;47:789–794 Advance Access publication 3 April 2008 doi:10.1093/rheumatology/ken108 Altered frequency and migration capacity of CD4+CD25+ regulatory T cells in systemic lupus erythematosus H.-Y. Lee1,, Y.-K. Hong1,, H.-J. Yun1, Y.-M. Kim1, J.-R. Kim2 and W.-H. Yoo1 Objectives. To determine the frequency and chemokine receptor-related migratory capacity of CD4+CD25+ regulatory T cells (Tregs) and their association with clinical parameters in patients with SLE. Methods. The expression of CD4, CD25, FoxP3 and CCR4 was examined with flow cytometry after staining with fluorescence-conjugated antibodies in 20 patients with SLE, 20 patients with RA and 21 age- and sex-matched healthy controls. For analysis of migration capacity in 24-well chemotaxis chambers, sorted cells were stimulated with ligands of CCR4, CCL17 and CCL22 and analysed with FACScan. Correlations between the number of Tregs and CCR4+ Treg cells and clinical parameters were analysed. Results. The numbers of Tregsbright and CCR4+ Tregsbright were significantly decreased in the patients with SLE compared with healthy controls. The number of Tregsbright was negatively correlated with the levels of anti-dsDNA antibody and the number of CCR4+ Tregsbright had a positive correlation with the levels of C3. Percentage of migrated Tregsbright by CCL17 or CCL22 was significantly decreased in the patients with SLE compared with healthy controls. Conclusions. These results suggest that altered frequency of Tregs and CCR4+ Tregsbright and decreased migratory capacity of Tregs might be involved in the pathogenesis of SLE and indicate that targeting the Tregs can be a new therapeutic strategy in SLE. KEY WORDS: Regulatory T lymphocytes, Systemic lupus erythematosus, CCR4, Cell migration. the modulation of local immune responses [14, 15]. Specific expression of CCR4 and CCR8 on Tregs may allow their migration towards inflammatory sites leading to inhibition of antigen presenting cell (APC) function or suppression of responding T cells [16]. However, little is known of the mechanisms about the migration of Tregs to sites where they maintain suppressive effectiveness in SLE. The aim of this work was to perform a quantitative and functional analysis of Tregs in patients with SLE, including the expression of chemokine receptor and chemokine receptor ligand-mediated chemotaxis capacity. Introduction SLE is a systemic autoimmune disease characterized by a wide spectrum of clinical manifestations, the loss of tolerance to selfantigens and the production of autoantibodies [1]. The pathogenesis in SLE is dependent upon CD4+ T cells, which was well established from human studies [2] and animal models [3]. Therefore, a thorough examination of T-cell tolerance is crucial in understanding this disease. Evidence indicates that central tolerance remains intact in murine models of SLE [4, 5], therefore it is generally assumed that the periphery is the site of the critical breakdown of tolerance. Many recent studies have suggested that CD4+CD25+ regulatory T cells (Tregs) exhibit regulatory/immunosuppressive activity by actively preventing the activation and effector function of autoreactive T cells that have escaped tolerance, and thus play a critical role in the maintenance of self-tolerance [6, 7]. One possible explanation of the emergence of autoimmunity in SLE could relate to the deficient function of Tregs. Even though there are controversial data regarding the number and function of Tregs in SLE, recent reports have indicated that patients with SLE have a low number of Tregs in their peripheral blood [8] and many works suggested a defective function of Tregs in this condition [9–11]. However, the exact defects of Tregs are unknown and the pathogenic role of Tregs in SLE remains to be fully delineated. There is agreement among many investigators of increased recruitment of Tregs at the sites of inflammation compared with peripheral blood [12, 13]. Functional assays revealed an increased suppressive potency, indicating Tregs to be important players in Materials and methods Patients and controls This study enrolled 20 patients (3 males and 17 females, Table 1) with the diagnosis of SLE according to 1982 revised ACR criteria for SLE [17], 20 patients (3 males and 17 females) with the diagnosis of RA according to 1987 ACR criteria [18], and 21 ageand sex-matched healthy controls without history of autoimmune diseases. We excluded patients with a history of infection within 3 weeks and comorbidities, such as diabetes mellitus. Ethical approval to carry out the study was obtained from the local Institutional Review Board. Informed consent was also obtained according to the Declaration of Helsinki. Clinical characteristics of the patients with SLE are described in Table 1. Disease activity of SLE was assessed using the levels of C3 (mg/dl), and the titre of anti-dsDNA antibody (IU/ml). None of them showed any abnormalities on physical examination, chest radiography or in lung function tests. No allergic disease was seen in patients with SLE and RA or healthy controls. Peripheral blood mononuclear cell isolation and CD4+ T-cell sorting 1 Department of Internal Medicine and 2Department of Orthopedic Surgery, Chonbuk National University Medical School and Research Institute of Clinical Medicine, Jeonju, Jeonbuk, Korea. A peripheral blood sample of 10–20 ml was obtained in healthy donors, patients with SLE and RA after provision of informed consent. Peripheral bloods were diluted 1: 2 with phosphate buffer solution (PBS, pH 7.4) and layered on Ficoll–Paque Plus (Amersham Biosciences, AB Uppsala, Sweden) with centrifugation at 48C for 30 min. Peripheral blood mononuclear cells (PBMCs) Submitted 28 November 2007; accepted 13 February 2008. Correspondence to: W.-H. Yoo, Division of Rheumatology, Department of Internal Medicine, Chonbuk National University Medical School and Research Institute of Clinical Medicine, #634-18, Geumam-Dong, Deokjin-Gu, Jeonju, Jeonbuk 561–712, Korea. E-mail: [email protected] H.-Y. Lee and Y.-K. Hong equally contributed to this work. 789 ß The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected] H.-Y. Lee et al. 790 TABLE 1. Clinical characteristic of the patients with SLE included in this study No. Sex/age Disease duration (yrs) C3 (mg/dl) Anti–dsDNA Ab (IU/ml) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M/16 M/18 M/22 F/17 F/19 F/19 F/21 F/28 F/29 F/31 F/34 F/36 F/37 F/38 F/41 F/43 F/44 F/45 F/47 F/51 0.5 7.0 4.0 1.5 6.5 6.0 4.5 13.0 2.0 11.0 7.0 10.0 6.0 9.0 4.0 9.0 8.0 14.0 0.5 15.0 21 80 73 120 81 39 81 95 71 103 45 95 86 80 88 83 112 93 48 107 100.0 46.0 9.4 6.0 42.0 36.0 89.0 16.0 7.0 8.0 100.0 84.0 7.4 3.0 10.0 52.0 3.0 7.0 105.0 5.0 Organ involvement H, S A, H, V A, H A, S A, C, H, A, H, S A, H, S A, H, R, A, H, S A, H, R, A, C, H, A, S A, C, H, A, H, S A, H APS, H A, H, S H A, P, S A, H, R S, V APS S V WBC (count/l) 4530 4210 5900 7790 5860 2320 3210 8470 4240 7110 3670 9410 7540 7540 5770 5900 3350 7130 3910 6040 A: articular; C: CNS; H: haematological; R: renal; S: skin; V: vasculitis. were collected from the interphase of Ficoll–Paque and serum layers and washed twice with PBS. CD4+ T cells were isolated from PBMCs by a step of negative sorting, using a cocktail of streptavidin-conjugated CD8, CD11b, CD16, CD19, CD36, CD41a, CD56, CD123, CD235a and TCR antibodies. PBMCs were incubated with cocktail antibodies at room temperature for 15 min, and magnetic beads coupled with a monoclonal antibody at room temperature for 30 min (Human T Lymphocyte Separation Kit; BD Biosciences, San Jose, CA). CD4+ T cells were purified using magnetic board with >90–95% purity as assessed by flow cytometric counting of CD4+ T lymphocytes (data not shown). Flow cytometric analysis CD4+CD25+ T cells (Tregs) and CCR4+CD4+CD25+ T cells (CCR4+ Tregs) from PBMCs (1 106 cells) were stained with combinations of the following monoclonal antibodies at 48C for 30 min: FITC-labelled anti-CD4, phycoerythrin (PE)-labelled antiCD25, PE-cy7-labelled anti-CCR4 from BD Pharmingen, San Diego, CA and then cells were washed twice with cold flow cytometry staining buffer (0.5% BSA, 0.1% sodium azide, 2 mM EDTA). To examine the expression of FoxP3, PBMCs stained with anti-CD4 and anti-CD25 were fixed with fixation/permeabilization kit (eBiosciences, San Diego, CA, cat. 00-5123-43) and then washed twice with permeabilization buffer (eBiosciences, cat. 00-8333-56). These cells were stained with APC-conjugated anti-FoxP3 antibody (eBiosciences, cat. 17-4777-71). Stained cells were analysed with a FACSCalibur, Cellquest software (Becton Dickinson, CA, USA). Chemotaxis assays To examine the migratory capacity of Tregs, chemotaxis assays were conducted in 24-well chemotaxis chambers (Costar, Cambridge, MA, USA) with polyvinylpyrrolidine-free polycarbonate membranes (5 m pore size) [19]. The bottom chamber of each well was filled with 600 l of agonist at the appropriate concentration (diluted in RPMI 1640 and 0.1% BSA) and carefully overlaid with the polycarbonate membrane. Human chemokines TARC (CCL17, R&D Systems, Minneapolis, MN) and MDC (CCL22, R&D Systems) were used, 100 and 50 ng/ml, respectively. Sorted CD4+ T cells were washed twice and resuspended in RPMI 1640 medium and 0.1% BSA at 5 105 cells/ml, and 100 l of the cell suspension was added to the top chambers. The chambers were incubated for 2 h in a 5% CO2humidified incubator at 378C, and the cells migrated across the membrane into the lower chamber were counted with a FACScan (Becton Dickinson, CA, USA) for 60 s at a flow rate of 60 l/min, as previously described [20]. Statistical analysis All results are expressed as mean SE. Data were analysed using the SigmaPlot program, version 10.0 (Systat Software, GmbH Erkrath, Germany). Statistical analysis was performed using the unpaired t-test or paired t-test for comparisons between groups. Correlations between two clinical parameters were evaluated using the SPSS program, version 12.0 (SPSS, Chicago, IL, USA) by Pearson r-test. Differences were considered significant if P-values were 0.05 or less. Results Decreased frequency of CD4+CD25high/bright Tregs in peripheral blood from patients with SLE Frequency of Tregs in PBMCs from 20 patients with SLE, 20 patients with RA and 21 age- and sex-matched healthy controls was evaluated by flow cytometry. As shown in Fig. 1A, patients with SLE had statistically significantly decreased absolute number of Tregs (125.4 14.8 cells/l, P < 0.0001) compared with healthy controls (378.3 31.0 cells/l). Because it has been demonstrated that the brightest 2% of the CD25+ population contains most of the Tregs [19], the CD25+ brightest subset was studied further. Notably, the absolute numbers of Tregsbright were significantly decreased in patients with SLE (18.1 2.4 cells/l, P < 0.0001) and patients with RA (43.9 5.4 cells/l, P ¼ 0.001) compared with healthy controls (68.6 5.1 cells/l) (Fig. 1B). Because lymphocytopenia is a characteristic haematological feature of SLE [1], percentage of Tregs is more important than the absolute number of Tregs. Percentage of Tregs were significantly decreased in patients with SLE (15.5 0.2%, P ¼ 0.002) compared with healthy controls (22.1 0.9%). However, there was no significant difference between patients with RA and healthy controls (Fig. 1C and D). Also, the percentage of Tregsbright was significantly decreased in patients with SLE (2.2 0.2%, P < 0.0001) and patients with RA (2.1 0.2%, P < 0.0001) compared with healthy controls (3.8 0.3%) (Fig. 1D). Constitutive expression of the transcriptional repressor, FoxP3, is characteristic of Tregs and it is one of the most specific markers of Tregs. We examined the expression of FoxP3 in CD4+ T lymphocytes from healthy controls and patients with SLE by intracellular staining. The number and percentage of FoxP3+CD4+ T cells also decreased in patients with SLE and patients with RA compared with healthy controls (data not shown). As shown in Fig. 2, CD4+CD25high Tregs (Tregshigh) from normal healthy donors expressed high levels of FoxP3 by flow cytometry (41.6 5.1%, P < 0.03), whereas Tregshigh from patients with SLE expressed significantly less FoxP3 protein (30.2 6.5%, P < 0.03). Furthermore, CD4+CD25bright Tregs (Tregsbright) from normal healthy donors expressed uniformly much higher levels of FoxP3 by flow cytometry (82.6 5.2%) than Tregsbright from subjects with SLE with statistical significance (58.1 6.3%, P < 0.0001). CD4+CD25 T cells from normal healthy donors or patients with SLE did not express this transcription factor that governs Treg function. Decreased frequency of CCR4+ Tregs in peripheral blood from patients with SLE To determine whether patients with SLE have altered function in Tregs, we analysed the expression of chemokine receptor, CCR4+ on Tregs or Tregshigh/bright lymphocytes. Absolute number of CCR4+ Tregs and CCR4+ Tregshigh/bright were decreased in patients with SLE compared with healthy controls, but not significantly different in RA patients (data not shown). As shown in Fig. 3A, the percentage of CCR4+ Tregs was significantly Altered frequency and functions of lupus regulatory T cells A B P < 0.0001 140 NS 1000 800 600 400 200 0 Normal (n = 21) C RA (n = 20) P = 0.001 120 100 80 60 40 20 0 SLE (n = 20) Normal (n = 21) D P = 0.002 45 40 35 30 25 20 15 10 RA (n = 20) SLE (n = 20) P < 0.0001 8 NS Tregsbright (%/CD4+T cells) Tregs (%/CD4+T cells) P < 0.0001 Tregsbright(cell AN*/µl) Tregs (cell AN*/µl) 1200 791 P < 0.0001 7 6 5 4 3 2 1 0 5 Normal (n = 21) RA (n = 20) SLE (n = 20) Normal (n = 21) RA (n = 20) SLE (n = 20) *AN, absolute number FIG. 1. Absolute numbers and percentage of Tregshigh/bright in peripheral blood from patients with SLE, RA and healthy controls. Absolute numbers of Tregs (A), and Tregsbright (B) were compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). Percentage of Tregs (C) and Tregsbright (D) were also compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). PBMCs were stained with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and then analysed by flow cytometry. Results are expressed as dot plot and mean S.E. P < 0.0001 FoxP3+ cells (%) 100 Normal (n = 21) SLE patients (n = 20) 80 P < 0.03 60 40 20 NS 0 CD4+CD25– T cells Tregshigh Tregsbright FIG. 2. Percentage of FoxP3 expression in the peripheral blood from patients with SLE and healthy controls. Percentage of FoxP3+ cells was compared between patients with SLE (n ¼ 21) and healthy controls (n ¼ 21) in CD4+CD25 T lymphocytes, Tregs, Tregshigh and Tregsbright. The expression of FoxP3 was examined by intracellular staining with APC-conjugated anti-FoxP3 antibody after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody. The results were analysed by flow cytometry. Results are expressed as mean SE. Tregshigh, Tregsbright and Tregshigh/bright indicated CD4+CD25high, CD4+CD25bright and CD4+CD25high/bright, respectively. decreased in patients with SLE (27.9 2.8%, P < 0.018) compared with healthy controls (35.5 1.4%). The percentage of CCR4+ Tregsbright also significantly decreased in patients with SLE (34.6 3.1%, P ¼ 0.0004) compared with healthy controls (49.2 2.1%) (Fig. 3B). However, these differences were not seen between patients with RA and healthy controls. Relationship between Tregsbright, CCR4+ Tregsbright and clinical characteristics in patients with SLE The percentage and frequency of Tregsbright was compared in patients with SLE according to involved organs and treatment modalities. The percentage and frequency of Tregsbright and CCR4+ Tregsbright between SLE patients with (n ¼ 7) or without (n ¼ 13) renal involvements was not statistically significantly different (data not shown). There were no significant differences in the percentage and frequency of Tregs and CCR4+ Tregs in patients with SLE according to the other clinical parameters (data not shown). We examined the correlation between disease activity of SLE (C3, anti-dsDNA antibody) and the percentage, absolute number of Tregsbright, CCR4+ Tregsbright in peripheral blood of patients with SLE. The number of Tregsbright was negatively correlated with levels of anti-dsDNA antibody with statistical significance (P ¼ 0.01), but not with the levels of C3 (data not shown). As shown in Fig. 4A, the absolute number of CCR4+ Tregsbright was positively correlated with the levels of C3 (P ¼ 0.01). The number of CCR4+ Tregsbright was relatively low in the patients with high level of anti-dsDNA antibody without statistical significance (Fig. 4B). Increased Tregsbright and CCR4+ Tregsbright by high-dose glucocorticoid in patients with active SLE The frequency and function of Tregs might be related with disease activity of SLE. To define these results more, we compared the percentages of Tregs and CCR4+ Tregs before and 2 months after H.-Y. Lee et al. 792 A 70 NS CCR4+Tregsbright (%/CD4+ T cells) CCR4+ Tregs (%/CD4+ T cells) B P = 0.018 60 50 40 30 20 10 0 Normal (n = 21) RA (n = 20) P = 0.0004 80 NS 70 60 50 40 30 20 10 0 SLE (n = 20) Normal (n = 21) RA (n = 20) SLE (n = 20) FIG. 3. Percentage of (A) CCR4+ Tregs and (B) Tregsbright in CD4+ T lymphocytes in peripheral blood from patients with SLE, RA and healthy controls. Percentage of CCR4+ Tregs and Tregsbright among CD4+ T lymphocytes was compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). PBMCs were stained with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and PE-cy7-conjugated anti-CCR4, and then analysed by flow cytometry. Results are expressed as mean SE. Tregsbright indicated CD4+CD25bright. B 40 CCR4+ Tregs (absolute no./µl) CCR4+ Tregs (absolute no./µl) A r = 0.597 P = 0.01 n = 20 35 30 25 20 15 10 5 0 0 20 40 60 80 100 120 140 40 r = – 0.21 NS n = 20 35 30 25 20 15 10 5 0 0 C3 (mg/dl) 20 40 60 80 100 anti-dsDNA Ab. (IU/ml) 120 FIG. 4. Correlation of CCR4+ Tregsbright in peripheral blood with C3 (A) and anti-dsDNA antibody levels (B) in patients with SLE. The relationships between the percentage of CCR4+ Tregs in peripheral blood of the patients with SLE and disease activity markers of SLE, level of C3 and anti-dsDNA antibody, were examined. PBMCs were obtained from patients with SLE and analysed by three-colour flow cytometry after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and PE-cy7-conjugated anti-CCR4 antibody. The level of C3 and anti-dsDNA antibody was determined at the time of blood sampling. Tregsbright indicated CD4+CD25bright. therapy with high-dose glucocorticoid (1.0 g methylprednisolone/ day for 3 days and then maintenance dose of prednisolone, >0.5 mg/kg/day) in four patients with SLE with high disease activity. As shown in Fig. 5A, the percentage of Tregsbright increased statistically significantly after high-dose glucocorticoid therapy (1.1 0.4%) compared with the percentage of Tregsbright before therapy (0.9 0.4%, P ¼ 0.02). There was also statistically significant increase in the percentage of CCR4+ Tregsbright (Fig. 5B) after high-dose glucocorticoid therapy. Decreased CCR4 ligand-mediated migration of lupus Tregsbright To evaluate the functional abnormality of chemotactic activity in Tregs, we examined the migration capacity of Tregs using CCL22 and CCL17 with sorted CD4+ lymphocytes in the patients with SLE (n ¼ 6) and healthy controls (n ¼ 6). The number of migrated Tregs among sorted CD4+ T cells was evaluated by flow cytometry and the results were expressed as percentage of migrated cells compared with the number of migrated cells without stimulation with CCR4 ligands, CCL22 and CCL17 (The result was 100% and expressed as control). As represented in Fig. 6, the number of migrated Tregs by CCL22 among sorted CD4+ T lymphocytes was significantly decreased in patients with SLE (209.3 9%, P ¼ 0.02) compared with healthy controls (259.7 14.2%), but not statistically significantly decreased with CCL17. Notably, percentage of migrated Tregsbright by CCL22 in sorted CD4+ T lymphocytes was significantly decreased in patients with SLE (220.8 22.5%) compared with healthy controls (415.5 24.8%, P ¼ 0.0002). The percentage of migrated Tregsbright by CCL17 was also significantly decreased in patients with SLE (196 17.1%) compared with healthy controls (268.3 21.9%, P ¼ 0.03) (Fig. 6). Discussion In the present study, we demonstrated that (i) the percentage of Tregs (especially Tregshigh/bright, FoxP3+ Tregsbright and CCR4+) in peripheral blood was significantly decreased in patients with SLE compared with healthy controls; (ii) the percentages of Tregs and CCR4+ Tregs were significantly increased with high-dose glucocorticoid therapy in patients with SLE; and (iii) Tregs have decreased migration capacity to CCR4 ligands in patients with SLE compared with healthy controls. The present study showed that patients with SLE had significantly decreased percentage of Tregs in peripheral blood in association with disease activity as described previously [20–22]. These results suggest that decrease in the frequency of Tregs might play a specific pathogenic role in SLE. At this point, it is very important to answer the question ‘What are the causes and roles Altered frequency and functions of lupus regulatory T cells A CCR4+ Tregsbright (%/CD4+ T cells) B 3.0 Tregsbright (%/CD4+ T cells) 793 P < 0.02 2.5 2.0 1.5 1.0 0.5 0.0 Before* After* 100 P < 0.05 90 80 70 60 50 40 30 20 Before* After* *High-dose glucocorticoid treatment + FIG. 5. Effects of high-dose glucocorticoid on (A) the percentage of Tregs of CD4 T lymphocytes and (B) CCR4 expression on Tregsbright in patients with active SLE (n ¼ 4). PBMCs were obtained from patients with active SLE before and 2 months after the therapies with high-dose glucocoticoid and maintenance dose of prednisolone (>0.5 mg/kg/day) and analysed by three-colour flow cytometry after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and PE-cy7conjugated anti-CCR4 antibody. Tregsbright indicated CD4+CD25bright. Asterisk indicates high-dose glucocorticoid treatment. 500 Normal (n = 6) SLE patients (n = 6) P = 0.0002 Migrated cells (%) 400 P = 0.02 P = 0.03 NS 300 200 100 Control 0 CCL22 CCL17 Tregs CCL22 CCL17 Tregsbright FIG. 6. Percentage of migrated CD4+ T cells and Tregs by CCR4 ligands in sorted CD4+ T lymphocytes from healthy controls and patients with SLE. Sorted CD4+ T lymphocytes (3 105 cells/well) were added to the upper wells of a 24-well chemotaxis chamber, the lower wells of which contained CCR4 ligands: CCL22 and CCL17 (used at 50 and 100 ng/ml, respectively). The chambers were incubated for 2 h at 378C, and cells migrating across a 5 m pore size membrane into the lower chamber were counted with a FACScan for 60 s at a flow rate of 60 l/min after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody, as described in ‘Materials and methods’ section. Results are shown as migration index, which represents the ratio between cells migrated to the lower chamber in the presence of the agonistic chemokines and cells migrated in response to the medium alone. To define the percentage of Tregs in migrated cells, migrated CD4+ T cells were stained with the aforementioned antibodies and analysed with FACScan. Percentage of migrated Tregs and Tregsbright by CCR4 ligands was compared between healthy controls (n ¼ 6) and patients with SLE (n ¼ 6). The horizontal line at a chemotactic percentage of 100 represents unstimulated basal migration and is shown for ease of comparison with stimulated levels. Results are expressed as mean S.E. Tregsbright are indicated as CD4+CD25bright. of decreased Tregs in SLE?’ with further studies. One possible interpretation is the cell reallocation during the inflammatory process that is required to study about the kinetics of Tregs during inflammatory conditions. Notably, the generation of Tregs in the immune system is genetically controlled and it has been suggested that genetic defects primarily affect the development of Tregs and can be a primary cause of autoimmune disorders in humans [23, 24]. It has also been suggested that the impaired proliferation of peripheral Tregs is one of the cause of reduced levels of Tregs, as observed in vitro [25, 26]. The other question is what are the functional defects of Tregs in SLE? It was reported that Tregs isolated from patients with active SLE expressed reduced levels of FoxP3 and poorly suppressed CD4+ effector T cells in vitro [27]. Lupus Tregs have also increased expression of glucocorticoid-induced tumour factor receptor (GITR), TNF receptor II (TNFRII) and decreased expression of CCR4 [27]. Among them, altered expression of chemokine receptors suggests that defective chemotactic activity of Tregs at the inflammatory sites might be involved in the pathogenesis of autoimmunity. However, there were no studies about chemokine receptor expression and chemotactic abnormalities of Tregs in SLE. The efficient operation of the immune system is critically dependent on a complex series of cellular interactions and movements to specific locations that are mediated by leucocyte expression of surface adhesion and chemoattractant receptors. For example, CCR4+CD4+ T cells preferentially migrated into the renal tissue of the patients with lupus nephritis and thus the maldistribution of CCR4+ T cells might be involved in the pathogenesis of lupus nephritis [28]. A decreased proportion of CD4+FoxP3+ Tregs in the inflammatory infiltrate in the skin of patients with cutaneous lupus erythematosus has been reported [29]. Recent study demonstrated that chemokines and their receptors, especially CCR4, CCR8, were involved in the guidance of Tregs to the sites of inflamed areas to attenuate T-cell activation [30]. Previous studies also revealed that CD4+CD25high T cells are critical players in the modulation of local immune responses [13, 14, 31]. To our knowledge, this is the first study to demonstrate that patients with SLE have decreased CCR4+ Tregs in their peripheral blood. The frequency of CCR4+ Tregs was correlated with the disease activity marker, C3, suggesting their important role in the pathogenesis of SLE. Further studies are needed to confirm the effects of decreased CCR4+ Tregs on the balance between pro-inflammatory cells and Tregs in the local inflammation site, including kidney. Chemokine receptor expression is exquisitely regulated depending on the stage of activation and differentiation of T cells and coordinates tissue localization and encounters with APCs [32, 33]. Glucocorticoid is commonly used for the treatment of autoimmune diseases including SLE. Although there were previous reports that this agent down-regulates the expression of chemokine receptor expression in CD4+ T lymphocytes [34, 35], the effects on Tregs were not reported until now. To our knowledge, this is the first in vivo study to demonstrate that high-dose glucocorticoid therapy increased the frequency of CCR4+ Tregs in patients with SLE in peripheral blood. Further studies about the effects of glucocorticoid on the expression of chemokine receptors, response to chemokines and chemotactic capacity are needed to define the in vivo system. H.-Y. Lee et al. 794 In this study, we first demonstrated that Tregs have decreased expression of CCR4 and migration capacity in patients with SLE. However, the exact roles of defects of migration due to decreased CCR4+ Tregs are uncertain. To define these, next study will be the determination of infiltration pattern of Th2 lymphocytes and Tregs in inflamed tissues in SLE patients with organ involvement, such as lupus nephritis. Despite the first description about the alteration of chemokine receptors and migration capacity of Tregs in the patients with SLE, this study has some limitations. First, the total number of patients with SLE and healthy controls is too small to represent the exact abnormalities of Tregs in patients with SLE. Second, the enrolled patients with SLE have lower disease activity than in other studies to represent as a pathogenetic abnormality in SLE. Third, the discrepancies from previous studies may be due to differences in reagents, assays and lack of highly purified Tregs. And, lastly, we studied in vitro system without other inflammatory cells, such as APCs. Tregs regulate immune responses directly through interaction with T effector cells or indirectly through modification of APCs. Thus, we next sought to determine the migration capacity of Tregs of the patients with SLE with the above system. In summary, we provided evidence for decreased frequency of Tregs, chemokine receptor (CCR4) expression and that the chemotactic response profile of Tregs play a specific pathogenic role in SLE. Based on these results, it becomes conceivable to design strategies to manipulate the recruitment of Tregs to achieve tolerance in autoimmunity and suppress the abnormal immune inflammatory reactions in SLE. Rheumatology key messages This article provided evidence for decreased frequency of Tregs, chemokine receptor (CCR4) expression and that the chemotactic response profile of Tregs play a specific pathogenic role in SLE. The strategies to manipulate the recruitment of Tregs to achieve tolerance in autoimmunity and suppress the abnormal immune inflammatory reactions can be a new modality for SLE therapy. Acknowledgements Funding: This work was supported by the ERC programme of MOST/KOSEF (grant R11-2000-075-02003-0) and the Fund of Chonbuk National University Hospital Research Institute of Clinical Medicine. Disclosure statement: The authors have declared no conflicts of interest. References 1 Vila LM, Alarcon GS, McGwin G Jr, Bastian HM, Fessler BJ, Reveille JD. Systemic lupus erythematosus in a multiethnic US cohort, XXXVII: association of lymphopenia with clinical manifestations, serologic abnormalities, disease activity, and damage accrual. Arthritis Rheum 2006;55:799–806. 2 Voll RE, Roth EA, Girkontaite I et al. Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production. 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