Roles of CCR2 and CXCR3 in the T cell-mediated response occurring during lupus flares

OBJECTIVE: Infiltrating lymphocytes have been demonstrated to play an important role in the tissue injury that occurs in systemic lupus erythematosus (SLE). Inflammatory chemokines control lymphocyte traffic through their interaction with T cell chemokine receptors. In this study we assessed the expression of chemokine receptors on T cell subsets of patients with active or inactive SLE. METHODS: Forty-four SLE patients (40 women and 4 men) were included in the study. The patients were divided according to their SLE Disease Activity Index (SLEDAI), which resulted in a group of patients with inactive SLE (n = 27) and a group with active SLE (n = 17). The control group was composed of 22 healthy blood donors. A disease control group consisted of 18 patients infected with human immunodeficiency virus. Expression of chemokine receptors CCR1, CCR2, CCR5, CXCR3, CXCR4, and CX3CR1 was assessed on whole blood samples by immunofluorescence analysis. RESULTS: On T lymphocytes, significant differences between the SLE patients and controls were observed only in the expression of CCR2 and CXCR3. On monocytes, no significant differences in CCR2 expression were observed between the healthy controls and the SLE patients. The proportion of CD8+,CCR2+ T cells was significantly lower in the SLE patients compared with the controls (mean +/- SD 2.3 +/- 1.3% and 3.5 +/- 3.2% in the active and inactive SLE groups, respectively, versus 21 +/- 24% in controls; P < 0.0001 for both). The CD4+,CCR2+ subset was represented similarly among the controls and patients with inactive SLE (16.7 +/- 5.8% and 12.8 +/- 8.1%, respectively) but was depleted in patients with active SLE (7.1 +/- 4.4%; P < 0.0001 versus controls). The active SLE group expressed significantly lower circulating levels of CD4+,CCR2+ T cells than did the inactive disease group (P = 0.007). A negative correlation was found between the proportion of CD4+,CCR2+ T cells and the SLEDAI (r = -0.43, P = 0.005, by Spearman's correlation). Proportions of CD8+,CXCR3+ T cells were similar between the SLE groups and the control group (58 +/- 22.6% in active SLE, 47.1 +/- 20% in inactive SLE, and 59.4 +/- 17.3% in controls). The proportion of CXCR3-expressing CD4+ T cells was decreased in the active disease group (23.5 +/- 3.2% versus 39.9 +/- 12.5% in controls; P = 0.008) but not in the inactive disease group (34.8 +/- 9.5%). A trend toward a significant negative correlation was observed between the decreased proportion of CD4+,CXCR3+ T cells and the SLEDAI (P = 0.08). Following in vitro activation of purified CD4 T cells, only CCR2 was internalized, whereas expression of CXCR3 was retained in activated CD4 cells. CONCLUSION: The numbers of circulating CD4+,CXCR3+ and CD4+,CCR2+ T cells are selectively decreased during SLE flares. A decrease in the number of circulating CD4+ T cells expressing CCR2 and/or CXCR3 could serve as a biomarker of the SLE flare.     

CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients

Objective

The high frequency of CD4+ T cells in interstitial infiltrates of patients with lupus nephritis suggests a contribution of these cells to local pathogenesis. The aim of this study was to examine the role of CXCR3 and the chemokine CXCL10 in recruiting these cells into the kidney and to determine whether the infiltrating T cells could be monitored in the urine to provide a reliable biomarker for acute lupus nephritis.

Methods

The frequencies of CD3+ T cells, CXCR3+ cells, and CXCL10+ cells were determined by immunohistochemical and immunofluorescence analyses of kidney sections from 18 patients with lupus nephritis. The frequency of CXCR3+CD4+ T cells was determined by flow cytometry of peripheral blood and urine from 38 patients with systemic lupus erythematosus (SLE), and the values were compared with disease activity as determined by the Systemic Lupus Erythematosus Disease Activity Index.

Results

In renal biopsy tissues from patients with lupus nephritis, a mean of 63% of the infiltrating cells expressed CXCR3, ∼60% of them were T cells, and the CXCR3+ cells colocalized with CXCL10‐producing cells. In biopsy tissues from SLE patients with acute nephritis, ∼50% of the urinary CD4+ T cells were CXCR3+, as compared with 22% in the peripheral blood, and the frequency of urinary CXCR3+CD4+ T cells correlated with disease activity. Moreover, the number of urinary CD4+ T cells reflected nephritis activity, and elevation above 800 CD4+ T cells per 100 ml of urine sharply delineated active from inactive nephritis.

Conclusion

CXCR3+ T cells are recruited into the inflamed kidneys, are enriched in the urine, and are a valuable marker of nephritis activity in SLE. They also present a potential target for future therapies.

     

Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus

Objective

CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up‐regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE.

Methods

Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti‐CXCL12 antibody.

Results

CD19+ B cells and CD4+ T cells from SLE patients displayed a >2‐fold increase (P = 0.0001) and >3‐fold increase (P < 0.0001), respectively, in median CXCR4 expression compared with that in controls (n = 7–8). Moreover, CXCR4 expression on B cells was 1.61‐fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n = 8) than in patients with SLEDAI scores ≤10 (n = 16) (P = 0.0008), 1.71‐fold higher in patients with class IV LN (n = 5) than in patients with other classes of LN (n = 7) (P = 0.02), and 1.40‐fold higher in patients with active neuropsychiatric SLE (NPSLE) (n = 6) than in patients with inactive NPSLE (n = 18) (P = 0.01). CXCL12 was significantly up‐regulated in the tubules and glomeruli of kidneys in patients with LN (n = 14), with the percentage of positive cells correlating positively with the severity of LN.

Conclusion

CXCR4 appears to be up‐regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.

     

Coexpression of chemokine receptors CCR5, CXCR3, and CCR4 and ligands for P‐ and E‐selectin on T lymphocytes of patients with juvenile idiopathic arthritis

Objective

To investigate P‐ and E‐selectin ligand coexpression with chemokine receptors (CKRs) on T cells in the synovial fluid (SF) and blood of children with juvenile idiopathic arthritis (JIA).

Methods

Sixteen patients with polyarticular or persistent oligoarticular JIA (ages 5.3–15.1 years) were studied. SF and venous blood were collected, and immunostaining for the expression of CCR4, CCR5, CXCR3, and P‐ or E‐selectin ligands was performed.

Results

Compared to blood, SF was greatly enriched for CD4+ T cells bearing CCR5, CCR4, CXCR3, and both P‐ and E‐selectin ligand. Twenty‐five percent of the CD4+ T cells in SF expressed both CCR5 and CCR4, some also coexpressing CXCR3. Such cells were rare in blood. Half of the few CCR5+ T cells in blood coexpressed P‐ or E‐selectin ligand, a phenotype that was enriched up to 50‐fold in SF. A minority of CCR4+ and CXCR3+ cells in blood (∼25%) coexpressed selectin ligand; these were enriched 4–8‐fold in SF. Most CCR4‐expressing CD4+ T cells expressed both E‐selectin ligand and cutaneous lymphocyte antigen.

Conclusion

CCR4‐, CCR5‐, CXCR3‐, and selectin ligand–expressing CD4+ T cells preferentially accumulate in the joints of children with JIA. The marked enrichment of CCR5+ T cells coexpressing P‐selectin and/or E‐selectin ligand in CD4+ SF T cells suggests that the few such cells in blood selectively migrate to inflamed joints via endothelial P‐ and E‐selectin– and CCR5‐activating chemokines. The predominance of CCR4‐expressing CD4+ T cells coexpressing E‐selectin ligand suggests that such cells migrate not only to areas of cutaneous inflammation, as previously reported, but also to the joints in JIA. Combined targeting of CCR5‐ and E‐selectin–dependent mechanisms may be a relevant treatment strategy.

     

Engraftment of peripheral blood mononuclear cells from systemic lupus erythematosus and antiphospholipid syndrome patient donors into BALB‐RAG‐2−/−IL‐2Rγ−/− mice: A promising model for studying human disease

Objective

To construct a humanized mouse model of systemic lupus erythematosus (SLE) that resembles the human disease in order to define the pathophysiology and targets for treatments.

Methods

We infused peripheral blood mononuclear cells (PBMCs) from SLE patients into BALB‐ RAG‐2^−/−IL‐2Rγ^−/− double‐knockout (DKO) mice, which lack T cells, B cells, and natural killer cells. PBMCs from 5 SLE patients and 4 normal donors were infused intravenously/intraperitoneally at a density of 3–5 × 10⁶ cells per animal into nonirradiated 4–5‐week‐old mice. We evaluated the engraftment of human CD45+ cells and monitored the plasma levels of human IgG, anti–double‐stranded DNA (anti‐dsDNA) antibody, and anticardiolipin antibody (aCL), as well as proteinuria and kidney histology.

Results

There was 100% successful engraftment in 40 DKO mice infused with human PBMCs. In the PBMC fraction from SLE PBMC–infused DKO (SLE‐DKO) mice and normal donor PBMC–infused DKO (ND‐DKO) mice, an average of 41% and 53% human CD45+ cells, respectively, were observed at 4 weeks postengraftment, with 70–90% CD3+ cells. There were fewer CD3+CD4+ cells (mean ± SEM 5.5 ± 2.1%) and more CD3+CD8+ cells (79.4 ± 3.6%) in the SLE‐DKO mice as in the SLE patients from which the PBMCs were derived. CD19+ B cells and CD11c+ monocytic cells were found in the spleen, lung, liver, and bone marrow. There was no significant difference in plasma levels of human IgG and anti‐dsDNA antibodies between SLE‐DKO and ND‐DKO mice. Levels of aCL were significantly higher in all SLE‐DKO mice infused with PBMCs from an SLE patient who had high titers of aCL. SLE‐DKO mice had proteinuria, human IgG deposits in the kidneys, and a shorter life span. In SLE‐DKO mice engrafted with PBMCs from the aCL‐positive patient, we found microthrombi and infiltration of CD3+, CD8+, and CD19+ cells in the glomeruli, recapitulating the human antiphospholipid syndrome in these mice.

Conclusion

We established a novel humanized SLE‐DKO mouse exhibiting many of the immunologic and clinical features of human SLE.

     

Expression of CD44 variant isoforms CD44v3 and CD44v6 is increased on T cells from patients with systemic lupus erythematosus and is correlated with disease activity

Objective

To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations.

Methods

Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearson's correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxon's rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls.

Results

Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Expression of CD44v3 and CD44v6 was also higher on total T cells and CD4+ and CD8+ T cells from SLE patients compared with controls (P ≤ 0.03). Cell surface levels of CD44v3 on total T cells, CD4+ T cells, and CD8+ T cells as well as cell surface expression of CD44v6 on total T cells and CD4+ T cells were correlated with the SLEDAI score (P < 0.05). The presence of lupus nephritis was associated with the expression of CD44v6 on total T cells, CD4+ T cells, and CD4−CD8− T cells (P < 0.05). Positivity for anti–double‐stranded DNA antibodies was associated with the expression levels of CD44v6 on T cells (P < 0.05).

Conclusion

These results indicate that expression levels of CD44v3 and CD44v6 on T cells may represent useful biomarkers of SLE activity.

     

MRL/Mp CD4+,CD25− T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: A novel defect of T cell regulation in systemic lupus erythematosus

Objective

To investigate the hypothesis that loss of suppression mediated by peripheral CD4+,CD25+ regulatory T cells is a hallmark of systemic lupus erythematosus (SLE).

Methods

Mice of the MRL/Mp strain were studied as a polygenic model of SLE. Following immunomagnetic selection, peripheral lymphoid CD25+ and CD25− CD4+ T cells were cultured independently or together in the presence of anti‐CD3/CD28 monoclonal antibody–coated beads. Proliferation was assessed by measuring the incorporation of tritiated thymidine.

Results

While MRL/Mp CD4+,CD25+ regulatory T cells showed only subtle abnormalities of regulatory function in vitro, syngeneic CD4+,CD25− T cells showed significantly reduced sensitivity to suppression, as determined by crossover experiments in which MRL/Mp CD4+,CD25− T cells were cultured with H‐2–matched CBA/Ca CD4+,CD25+ regulatory T cells in the presence of a polyclonal stimulus.

Conclusion

Our findings highlight a novel defect of peripheral tolerance in SLE. Identification of this defect could open new opportunities for therapeutic intervention.

     

Reactivity of γ/δ T cells to human 60‐kd heat‐shock protein and their cytotoxicity to aortic endothelial cells in Takayasu arteritis

Objective

Increased numbers of circulating γ/δ T cells with a restricted T cell receptor repertoire, as well as colocalization of the expression of heat‐shock protein Hsp60/65 and γ/δ T cells in the arterial lesions of patients with Takayasu arteritis (TA), indicate that γ/δ T cells may react to Hsp60 and cause damage to the arterial endothelium. In this study we investigated the proliferative responses of γ/δ T cells to human Hsp60 and their cytotoxicity to human aortic endothelial cells (ECs) in patients with TA.

Methods

Blood samples were obtained from 12 patients with TA, 8 patients with systemic lupus erythematosus (SLE) (as disease controls), and 10 healthy control subjects. Proliferative responses of circulating γ/δ T cells to human Hsp60 were detected by flow cytometry–based bromodeoxyuridine incorporation assay. Cytotoxicity of the γ/δ T cells to human aortic ECs was analyzed by colorimetric lactate dehydrogenase release assay.

Results

The γ/δ T cells of 11 of 12 patients with TA exhibited reactivity to Hsp60, whereas none of the γ/δ T cells from patients with SLE or healthy controls showed reactivity (both P < 0.001). The mean ± SD proliferative response of γ/δ T cells in patients with TA was 21.4 ± 11.3%, compared with 4.2 ± 1.2% in patients with SLE and 4.01 ± 1.82% in healthy controls (both P < 0.001). In addition, compared with the control groups, the γ/δ T cells of patients with TA had increased spontaneous cytotoxicity to aortic ECs (22.1 ± 15.0% versus 9.6 ± 2.13% in SLE patients and 8.1 ± 4.7% in healthy controls; both P < 0.005), which was further enhanced following stimulation of γ/δ T cells with Hsp60. The cytotoxicity of the γ/δ T cells was significantly inhibited by treatment of these cells with concanamycin A and anti–Fas ligand–blocking antibodies.

Conclusion

The results show that γ/δ T cells in patients with TA are reactive to Hsp60 and exhibit cytotoxicity to aortic ECs, suggesting a key role of Hsp60 and γ/δ T cells in the pathogenesis of TA.

     

Expansion of toll‐like receptor 9–expressing B cells in active systemic lupus erythematosus: Implications for the induction and maintenance of the autoimmune process

Objective

Toll‐like receptors (TLRs) are pattern‐associated receptors in innate immunity that may be involved in the recognition of self antigens and the production of pathogenic autoantibodies. This study was undertaken to examine the expression and function of various TLRs in subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE).

Methods

The expression of TLRs in PBMCs from 50 SLE patients with active disease (SLE Disease Activity Index [SLEDAI] score ≥8; n = 26) or inactive disease (SLEDAI score <8; n = 24) and 20 healthy controls was studied by flow cytometry. TLR expression was assessed on various subpopulations of PBMCs (TLR‐2 and TLR‐4 by membrane staining; TLR‐3 and TLR‐9 by intracellular staining). TLR function was accessed by stimulating PBMCs with specific ligands.

Results

The proportion of B cells and monocytes expressing TLR‐9 was higher among patients with active SLE (mean ± SD 49.5 ± 24.4% and 30.7 ± 24.1%, respectively) than among patients with inactive disease (22.8 ± 19.6% and 14.3 ± 8.4%, respectively; P = 0.02 and P = 0.03). Among B cells, the proportion of plasma cells and memory B cells expressing TLR‐9 was increased in patients with active SLE. Increased percentages of TLR‐9–expressing B cells correlated with the presence of anti–double‐stranded DNA antibodies (P = 0.007). Treatment with serum from patients with active disease increased the percentage of TLR‐9–expressing plasma cells in serum from healthy controls. Enhanced induction of HLA–DR after TLR‐9 stimulation was documented in B cells from patients with active disease.

Conclusion

In patients with active SLE, the proportion of peripheral blood memory B cells and plasma cells expressing TLR‐9 is increased. Endogenous nucleic acids released during apoptotic cell death may stimulate B cells via TLR‐9 and contribute to SLE pathogenesis.

     

Selective lymphocyte chemokine receptor expression in the rheumatoid joint

Objective

In patients with rheumatoid arthritis (RA), chemokines and their receptors are important for lymphocyte trafficking into the inflamed joint. This study was undertaken to characterize the expression of chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CXCR3, and CX₃CR1 in normal (NL) peripheral blood (PB), RA PB, and RA synovial fluid (SF).

Methods

Using flow cytometry, immunohistochemistry, and 2‐color immunofluorescence, we defined the expression of chemokine receptors on CD3+ T lymphocytes in RA synovial tissue (ST), RA SF, RA PB, and NL PB.

Results

The percentage of CD3+ lymphocytes expressing CCR2, CCR4, CCR5, and CX₃CR1 was significantly elevated in RA PB compared with that in NL PB, while the percentage of CD3+ lymphocytes expressing CCR5 was significantly enhanced in RA SF compared with that in NL and RA PB. In contrast, similar percentages of CD3+ lymphocytes in NL PB, RA PB, and RA SF expressed CCR6 and CXCR3. Immunohistochemistry of RA ST showed lymphocyte expression of CCR4, and 2‐color immunofluorescence staining revealed RA ST CD3+ lymphocytes intensely immunoreactive for CXCR3, suggesting that these 2 receptors may be particularly important for CD3+ lymphocyte trafficking to the inflamed joint. In comparisons of chemokine receptor expression on naive (CD45RA+) and memory (CD45RO+) CD3+ lymphocytes, there were greater percentages of memory CD3+/CD4+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD4+ lymphocytes in RA PB and RA SF, and greater percentages of memory CD3+/CD8+ lymphocytes expressing CCR4, CCR5, and CXCR3 than naive CD3+/CD8+ lymphocytes in RA SF, suggesting receptor up‐regulation upon lymphocyte activation. In contrast, percentages of CD3+/CD8+ memory lymphocytes expressing CX₃CR1 were significantly less than percentages of naive CD3+/CD8+ lymphocytes in RA PB, suggesting that this receptor may be down‐regulated upon lymphocyte activation. A major difference between the RA PB and NL PB groups was significantly more CCR4+ memory leukocytes and memory CCR5+/CD3+/CD8+ lymphocytes in RA PB than NL PB, further suggesting that these receptors may be particularly important for lymphocyte homing to the RA joint.

Conclusion

These results identify CCR4, CCR5, CXCR3, and CX₃CR1 as critical chemokine receptors in RA.

     

MicroRNA‐126 regulates DNA methylation in CD4+ T cells and contributes to systemic lupus erythematosus by targeting DNA methyltransferase 1

Objective

To identify microRNA genes with abnormal expression in the CD4+ T cells of patients with systemic lupus erythematosus (SLE) and to determine the role of microRNA‐126 (miR‐126) in the etiology of SLE.

Methods

MicroRNA expression patterns in CD4+ T cells from patients with SLE and healthy control subjects were analyzed by microRNA microarray and stem loop quantitative polymerase chain reaction (qPCR). Luciferase reporter gene assays were performed to identify miR‐126 targets. Dnmt1, CD11a, and CD70 messenger RNA and protein levels were determined by real‐time qPCR, Western blotting, and flow cytometry. CD11a, CD70, and EGFL7 promoter methylation levels were detected by bisulfite sequencing. IgG levels in T cell–B cell cocultures were determined by enzyme‐linked immunosorbent assay.

Results

The expression of 11 microRNA was significantly increased or decreased in CD4+ T cells from patients with SLE relative to that in CD4+ T cells from control subjects. Among these, miR‐126 was up‐regulated, and its degree of overexpression was inversely correlated with Dnmt1 protein levels. We demonstrated that miR‐126 directly inhibits Dnmt1 translation via interaction with its 3′–untranslated region, and that overexpression of miR‐126 in CD4+ T cells can significantly reduce Dnmt1 protein levels. The overexpression of miR‐126 in CD4+ T cells from healthy donors caused the demethylation and up‐regulation of genes encoding CD11a and CD70, thereby causing T cell and B cell hyperactivity. The inhibition of miR‐126 in CD4+ T cells from patients with SLE had the opposite effects. Expression of the miR‐126 host gene EGFL7 was also up‐regulated in CD4+ T cells from patients with SLE, possibly in a hypomethylation‐dependent manner.

Conclusion

Our data suggest that miR‐126 regulates DNA methylation in CD4+ T cells and contributes to T cell autoreactivity in SLE by directly targeting Dnmt1.

     

Disease trends over time and CD4+CCR5+ T-cells expansion predict carotid atherosclerosis development in patients with systemic lupus erythematosus

Background and Aim

Patients with Systemic Lupus Erythematosus (SLE) present increased cardiovascular mortality compared to the general population. Few studies have assessed the long-term development and progression of carotid atherosclerotic plaque in SLE patients. Our aim was to investigate the association of clinical and laboratory markers of disease activity and classical cardiovascular risk factors (CVRF) with carotid atherosclerosis development in SLE patients in a prospective 5-year study.

CD4+CD25+ regulatory T cells in rheumatoid arthritis: Differences in the presence,phenotype, and function between peripheral blood and synovial fluid

Objective

In mice, CD4+CD25+ regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4+CD25+ regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA).

Methods

The presence and phenotype of CD4+CD25+ regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4+CD25− and CD4+CD25+ T cells with anti‐CD3 monoclonal antibodies and antigen‐presenting cells, followed by proliferation and cytokine detection.

Results

The percentage of CD4+CD25+ T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4+CD25+ regulatory T cells from controls. In SF, however, ∼40–50% of CD4+CD25+ T cells expressed an activated phenotype, i.e., CD69+, class II MHC+, OX‐40+, with high levels of CTLA‐4 and glucocorticoid‐induced tumor necrosis factor receptor. These synovial CD4+CD25+ T cells displayed an increased suppressive capacity compared with blood CD4+CD25+ T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4+CD25+ T cell–mediated suppression than were responder cells from PB.

Conclusion

We demonstrate that CD4+CD25+ regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.

     

The role of T helper cell subsets in pathogenesis of Systemic Lupus Erythematosus and their relation to disease activity

The chemokine receptors expression dictates the spectrum of action of chemokines. One possible role of chemokines in autoimmune diseases, such as Systemic Lupus Erythematosus (SLE), is to facilitate the migration of lymphocytes to specific target organs thus accounting for the accumulation of T-cells in different organs, and subsequent disease manifestations. In the present study we investigated the surface expression of the chemokine receptors CXCR3 and CCR5 on CD4-positive T-helper lymphocytes in patients with active SLE, patients in remission, and in healthy subjects. Furthermore, a possible correlation between these cytokine receptors and SLE disease activity index (SLEDAI) was investigated. The study included 48 patients; all met at least four of the 11 American College of Rheumatology (ACR) diagnostic criteria for SLE. They were subdivided according to their SLEDAI into 2 groups: active SLE group of 28 patients with SLEDAI (> or = 6), and 20 SLE patients in remission with SLEDAI (< 6). Twenty age and sex matching healthy subjects were included as controls. We found that patients in the active SLE group had significantly elevated the mean expressions of CXCR3 and CCR5 on the surface of CD4+ T-lymphocytes than in the SLE patients in remission and healthy controls. The SLE patients in remission were found to have substantial reduced CD4+ CXCR3+ expression in comparison to healthy controls. Interestingly, a significant positive correlation was found between SLEDAI and each mean level of CD4+ CXCR3 and CD4+ CCR5+ in all SLE patients. In conclusion, this study revealed that the surface expression of the chemokine receptors CXCR3 and CCR5 on CD4+ T cells were increased markedly in patients with active SLE more than SLE patients in remission and the healthy subjects, this increase correlated positively with the SLEDAI. A larger study should be conducted to examine the role of CXCR3 and CCR5 expression in predicting disease activity in SLE patients.     

The Vλ–Jλ repertoire of patients with systemic lupus erythematosus manifests characteristics of the natural antibody repertoire

Objective

To understand in detail the mechanisms of autoantibody production in patients with systemic lupus erythematosus (SLE), we performed a comprehensive analysis of the normal human immunoglobulin light chain V_λ repertoire and compared it with the V_λ repertoire in SLE patients.

Methods

The SLE V_λ repertoire of B cells obtained from 3 SLE patients was analyzed and compared in detail with the V_λ repertoire of IgM+ B cells obtained from 3 human fetal spleens and IgM+,CD5+ B cells obtained from 2 normal adults. Conventional IgM+,CD5– B cells obtained from normal adults were used as controls. V_λ–J_λ rearrangements were amplified from the genomic DNA of individual B cells by polymerase chain reaction.

Results

The expressed V_λ repertoire of SLE patients contained several similarities with the expressed repertoire of the fetus and the adult CD5+ B cells. The V_λ genes 3L and 1G were overexpressed in the fetus, the adult CD5+ B cells, and the patients with SLE. The selection for rearrangements with restricted junctional diversity by utilization of homology‐mediated joining, together with diminished N nucleotide addition, was a prominent feature of fetal, adult CD5+, and SLE B cell repertoires. Furthermore, profound expansion of V_λ clones with identical third complementarity‐determining regions was observed in the adult CD5+, fetal, and SLE B cell repertoires. Notably, significant numbers of expanded adult CD5+ B cells, fetal, and SLE V_λ clones utilized homology‐mediated joining at the V_λ–J_λ junctions.

Conclusion

These data demonstrate that the SLE V_λ–J_λ repertoire manifests characteristics of normal adult IgM+,CD5+ and fetal B cell populations that are known to be enriched for the production of natural autoantibodies.

     

Lower expression levels of the programmed death 1 receptor on CD4+CD25+ T cells and correlation with the PD‐1.3A genotype in patients with systemic lupus erythematosus

Objective

A genetic polymorphism in the programmed death 1 (PD‐1) gene encoding the coinhibitory PD‐1 immunoreceptor, PD‐1.3A, is associated with systemic lupus erythematosus (SLE). The aim of this study was to assess PD‐1 receptor expression in patients with SLE, in comparison with relatives and unrelated healthy controls, and to identify correlations of lower expression levels of PD‐1 receptor with the PD‐1.3A genotype.

Methods

Patients with SLE, patients' relatives, and unrelated healthy control subjects from Iceland and Sweden were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated with anti‐CD3/anti‐CD28, and PD‐1 expression was analyzed by flow cytometry. PD‐1.3A/G genotyping was performed using polymerase chain reaction–restriction fragment length polymorphism analysis.

Results

PD‐1 expression on PBMCs was induced after antibody stimulation, showing increases of 2.1‐fold in SLE patients, 3.1‐fold in relatives, and 5.1‐fold in healthy controls. The frequency of PD‐1+ cells was significantly lower in SLE patients compared with relatives and healthy controls. PD‐1 expression on PD‐1+ cells and on CD4+CD25+ T cells was significantly lower in SLE patients and relatives compared with healthy controls. PD‐1 expression was significantly elevated on CD25^high cells. Levels of PD‐1 expression on CD25^high and CD25^intermediate cells were significantly lower in SLE patients compared with healthy controls. PD‐1 was expressed on both FoxP3− and FoxP3+ cells. Lower expression of PD‐1 was significantly correlated with the PD‐1.3A/G genotype.

Conclusion

The results demonstrate significantly lower PD‐1 receptor expression in SLE patients and their relatives and reveal a significant correlation of lower PD‐1 expression with the PD‐1.3A allele. Thus, PD‐1.3A may contribute to abnormalities in PD‐1 receptor expression on CD4+CD25+ T cells in patients with SLE, providing support for an important role of the PD‐1 pathway in SLE and, possibly, in other autoimmune diseases.

     

N‐acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: A randomized,double‐blind,placebo‐controlled trial

Objective

Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double‐blind, placebo‐controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N‐acetylcysteine (NAC).

Methods

A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3‐month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty‐two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls.

Results

NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4−CD8− T cells (mean ± SEM 1.35 ± 0.12‐fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti‐DNA production (P = 0.049).

Conclusion

This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.