Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

OBJECTIVE. Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. METHODS. Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. RESULTS. 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.     

Lymphocyte function—associated antigen 1 overexpression and t cell autoreactivity

Objective. To determine if DNA methylation inhibitors make T cells autoreactive by inducing lymphocyte function—associated antigen type 1 (LFA–1) (CD11a/CD18) overexpression. Methods. T cell clones were treated with 3 distinct DNA methylation inhibitors or were stably transfected with a CD18 cDNA in a mammalian expression vector, and the effects on LFA–1 expression and activation requirements were examined. Results. LFA–1 overexpression, caused by DNA methylation inhibitors or by transfection, correlates with the development of autoreactivity. Conclusion. LFA–1 overexpression may contribute to T cell autoreactivity.     

Demethylation of ITGAL (CD11a) regulatory sequences in systemic lupus erythematosus

OBJECTIVE: Inhibition of T cell DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo, suggesting that T cell DNA hypomethylation may contribute to autoimmunity. The hypomethylation effects are due, in part, to overexpression of lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18). Importantly, T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, suggesting that the same mechanism could contribute to human lupus. The present study investigated the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus. METHODS: Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter and flanking regions in T cells from lupus patients and healthy subjects, and in T cells treated with DNA methylation inhibitors. "Patch" methylation of promoter sequences in reporter constructs was used to determine the functional significance of the methylation changes. RESULTS: Hypomethylation of specific sequences flanking the ITGAL promoter was seen in T cells from patients with active lupus and in T cells treated with 5-azacytidine and procainamide. Patch methylation of this region suppressed ITGAL promoter function. CONCLUSION: DNA methylation changes occur in specific sequences that regulate LFA-1 expression in lupus T cells and in the hypomethylation model, indicating that altered methylation of specific genes may play a role in the pathogenesis of lupus.     

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.

     

N-acetylprocainamide is a less potent inducer of T cell autoreactivity than procainamide

We have reported that an inhibitor of DNA methylation, 5-azacytidine, makes cloned, antigen-specific CD4+ T cells autoreactive, and that procainamide and hydralazine mimic this effect. Those results suggested that procainamide and hydralazine may induce autoimmunity by inhibiting DNA methylation and causing T cell autoreactivity. We report now that N-acetylprocainamide, a procainamide derivative that does not induce lupus, is also a DNA methylation inhibitor, but it is 100 times less potent than procainamide in inducing T cell autoreactivity.     

Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo

Objective. T cells treated with DNA methylation inhibitors overexpress lymphocyte function-associated antigen 1 (LFA-1), which results in autoreactivity, and the autoreactive cells cause a lupus-like disease in vivo, suggesting a mechanism by which some agents may cause drug-induced lupus. This study compared the effects of procainamide (Pca) and hydralazine (Hyd) with those of structural analogs, to determine if the degree of LFA-1 overexpression and T cell autoreactivity correlated with the ability of the agents to induce autoimmunity. Methods. Cloned murine T helper 2 cells were treated with Pca, N-acetylprocainamide, Hyd, Phthalazine, or hydroxyurea (HU). The treated cells were then compared for LFA-1 overexpression, autoreactivity, and the ability to induce autoimmunity in vivo. Results. Pca and Hyd were more potent than their analogs or HU in all 3 assays. Conclusion. The results support a relationship between LFA-1 overexpression, T cell autoreactivity, and autoimmunity, and suggest a mechanism by which Pca and Hyd, but not the analogs, may cause drug-induced lupus.     

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.

     

Abnormal DNA methylation in CD4+ T cells from people with latent autoimmune diabetes in adults

Aberrant DNA methylation in T cells has been linked to pathogenesis of autoimmune diseases. To investigate genomic and gene-specific DNA methylation levels in CD4⁺ T cells from patients with latent autoimmune diabetes in adults (LADA), and to investigate changes in the expression of genes that regulate methylation as well as the autoimmune-related gene FOXP3 in these patients. Global CD4⁺ T cell DNA methylation was measured in 15 LADA patients and 11 healthy controls using a methylation quantification kit. mRNA levels of DNA methytransferases (DNMTs), methyl-DNA binding domain proteins (MBDs) and FOXP3 were measured by real time PCR. Methylation of a FOXP3 regulatory element region was determined by bisulphite genomic sequencing. Genomic DNA methylation in CD4⁺ T cells from LADA patients was significantly increased compared to controls. DNMT3b mRNA levels were higher in CD4⁺ T cells from LADA patients than in controls. DNMT3b expression positively correlated with global DNA methylation in LADA CD4⁺ T cells. FOXP3 expression was decreased, and the FOXP3 promoter region was hypermethylated in CD4⁺ T cells from LADA patients compared with controls. DNA methylation levels are altered in CD4⁺ T cells from LADA patients, which may contribute to disease onset and progression by affecting the expression of autoimmune-related genes.     

Histone modifications and methyl-CpG-binding domain protein levels at the TNFSF7 (CD70) promoter in SLE CD4+ T cells

In systemic lupus erythematosus (SLE), T lymphocytes overexpress CD70 (TNFSF7 gene), leading to the synthesis of autoreactive IgGs. CD70 upregulation in SLE CD4(+) T cells is associated with hypomethylation of TNFSF7 promoter. In this study, we explored histone modifications in the TNFSF7 promoter region in SLE CD4(+) T cells, and characterized the effects of a DNA methyltransferase inhibitor (5-azaC) and a histone deacetylase inhibitor (TSA) on CD70 expression. We found that CD70 mRNA was significantly increased in active lupus CD4(+) T cells, and in control cells treated with 5-azaC, TSA, or both. Histone H3 acetylation and dimethylated H3 lysine 4 (H3K4me2) levels were significantly elevated in patients with lupus, and both factors correlated positively with disease activity. MeCP2 protein levels within the TNFSF7 promoter decreased in patients with active lupus. Treatment of CD4+ T cells with 5-azaC alone significantly raised H3K4 dimethyl levels at the TNFSF7 locus. TSA treatment significantly increased H3 and H4 acetylation levels, as well as levels of H3K4 dimethylation at the TNFSF7 locus. Treatment with 5-azaC plus TSA enhanced H3 acetylation levels. These findings indicate that aberrant histone modifications within the TNFSF7 promoter may contribute to the development of lupus by increasing CD70 expression in CD4(+) T cells.     

Decreased microRNA‐142‐3p/5p expression causes CD4+ T cell activation and B cell hyperstimulation in systemic lupus erythematosus

Objective

To examine the role of microRNA‐142‐3p/5p (miR‐142‐3p/5p) in the development of autoimmunity in patients with systemic lupus erythematosus (SLE).

Methods

MicroRNA‐142‐3p/5p expression levels were determined by real‐time quantitative polymerase chain reaction, and potential target genes were verified using luciferase reporter gene assays. The effects of miR‐142‐3p/5p on T cell function were assessed by transfection with miR‐142‐3p/5p inhibitors or mimics. Histone modifications and methylation levels within a putative regulatory region of the miR‐142 locus were detected by chromatin immunoprecipitation assay and bisulfite sequencing, respectively.

Results

We confirmed that miR‐142‐3p and miR‐142‐5p were significantly down‐regulated in SLE CD4+ T cells compared with healthy controls and observed that miR‐142‐3p/5p levels were inversely correlated with the putative SLE‐related targets signaling lymphocytic activation molecule–associated protein (SAP), CD84, and interleukin‐10 (IL‐10). We demonstrated that miR‐142‐3p and miR‐142‐5p directly inhibit SAP, CD84, and IL‐10 translation, and that reduced miR‐142‐3p/5p expression in CD4+ T cells can significantly increase protein levels of these target genes. Furthermore, inhibiting miR‐142‐3p/5p in healthy donor CD4+ T cells caused T cell overactivation and B cell hyperstimulation, whereas overexpression of miR‐142‐3p/5p in SLE CD4+ T cells had the opposite effect. We also observed that the decrease in miR‐142 expression in SLE CD4+ T cells correlated with changes to histone modifications and DNA methylation levels upstream of the miR‐142 precursor sequence.

Conclusion

The results of this study indicate that reduced expression of miR‐142‐3p/5p in the CD4+ T cells of patients with SLE causes T cell activity and B cell hyperstimulation.

     

Quantification of regulatory T cells in peripheral blood of patients with systemic lupus erythematosus

Regulatory T cells (Tregs) are supposed to stop immune responses in the course of immune activation. However, chronic activation of immune system in systemic lupus erythematosus (SLE) and many other autoreactive disorders are evidence of malfunction of this system. Therefore, it is plausible to quantify presence of these cells in different diseases. Forty-one patients with diagnosis of SLE were enrolled in this study. Patients were divided into two groups of patients with active and inactive disease based on the disease activity score. Flow cytometry analysis was used to determine the frequency of regulatory T cells in peripheral blood according to high expression of CD25 and intracellular Forkhead/winged-helix (Foxp3). Further 30 healthy individuals considered as control group. Significantly less CD4+CD25hi regulatory T cells were detected in active patients (P < 0.001) compared to healthy individuals. The percentage of CD4+CD25hi cells were inversely correlated with the SLEDAI disease score in patients with active disease (r = −0.837, P < 0.0001). Patients with active disease had lower frequencies of CD4+Foxp3+ cells. However, increased frequencies of CD4+Foxp3+ T cells were observed in peripheral blood of patients with inactive disease compared with active patients or healthy individuals (P < 0.010). Moreover, a significant difference between the proportion of CD4+CD25-Foxp3+ population in healthy controls and patients with active disease was shown (P < 0.0005). Presence of lower frequencies of Tregs in patients with SLE could be evaluated as an immune turbulence and could be employed as a target for immunotherapeutic manipulation. However, controversies need to be resolved.     

A possible role of apoptosis for regulating autoreactive responses in systemic lupus erythematosus

It has been reported that apoptotic cells are increased in the peripheral blood from patients with systemic lupus erythematosus (SLE), where dysfunctions of T helper 1 (Th1) cells are known. In order to study whether apoptosis of Th1 cells is associated with the pathogenesis of SLE, early apoptotic cells in various T-cell subsets were detected using fluorescence-labeled annexin V (AnV). AnV binding was most frequently observed in CD4+CCR5+ T cells, and AnV binding rate (%) in this subset was higher in SLE than in normal controls (14.7 +/- 2.6), although that in active SLE (43.6 +/- 7.3) tended to be lower than that in inactive SLE (48.0 +/- 6.8). CD95/Fas expression was also increased in both active and inactive SLE. In some SLE patients, AnV binding rate changed in inverse proportion to titer of the serum anti-DNA antibody and in proportion to serum complement activity. These data suggest that apoptosis in Th1 cells is important in the pathogenesis of SLE and might play a role in regulating over-activation or autoreactive responses by T cells.     

Autoreactive murine Th1 and Th2 cells kill syngeneic macrophages and induce autoantibodies.

D10 cells, a cloned Th2 line, become autoreactive following treatment with DNA methylation inhibitors like 5-azacytidine (5-azaC), and induce anti-DNA antibodies if injected into unirradiated syngenic mice. The mechanism by which the autoreactive cells break tolerance is unknown. To further define effector functions required, we asked if 5-azaC-treated Th1 cells could also induce autoimmunity. AE7 cells, a cloned Th1 line, were treated with 5-azaC and shown to become autoreactive and induce anti-DNA antibodies in vivo. Comparison of effector mechanisms demonstrated that the two cell lines secreted a distinct repertoire of cytokines, and that only killing of syngeneic M? was common to both AE7 and D10 cells. This suggests that M? killing may be an early step in the induction of anti-DNA antibodies, providing antigenic nucleosomes and decreasing clearance of apoptotic material. Secretion of cytokines promoting B cell differentiation may play a role, but no one cytokine is required.     

Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus

OBJECTIVE: In systemic lupus erythematosus (SLE) autoantibody production is T cell dependent. For a proper T and B cell interaction, signalling of costimulatory molecules on these cells is necessary. The expression of costimulatory molecules on peripheral blood lymphocytes in patients with SLE in conjunction with disease activity was measured to evaluate whether expression of costimulatory molecules in SLE is increased. METHODS: Thirteen patients with SLE with active disease, 10 patients with inactive disease, and 14 controls entered the study. In addition, samples from 10 of the 13 patients with active disease could be studied at a moment of inactive disease as well. Isolated peripheral blood lymphocytes were stained for the lymphocyte subset markers CD4, CD8, CD19, their respective activation markers CD25, HLA-DR, CD38, and the costimulatory molecules CD40L, CD28, CD40, CD80, and CD86. Expression was measured by flow cytometry. RESULTS: Peripheral blood lymphocytes of patients with SLE showed signs of increased activation at the moment of active disease. Almost all CD4+ T cells expressed CD28, both in patients and in controls. CD80 expression on CD19+ B cells was low in both groups and did not correlate with disease activity. In contrast, the percentage of CD19+ B cells expressing CD86 was increased in patients with SLE even in patients with inactive disease (p=0.04) and correlated with the SLEDAI score (p=0.0005) and levels of anti-dsDNA (p=0.006). No changes in CD40 or CD40L expression were found in the patients with SLE. CONCLUSION: In patients with SLE the expression of CD86 on CD19+ B cells is increased and is associated with disease activity, B cell activation, and levels of anti-dsDNA. The increased CD86 expression will render (autoreactive) B cells more susceptible for T cells. This can facilitate autoantibody production and might be a target for immunosuppressive treatments.     

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.

     

Subpopulations of primed t helper cells in rheumatoid arthritis

Objective. To analyze subsets of primed T helper cells, defined by expression of the CD45RB isoform of the leukocyte common antigen, in the blood and synovial fluid (SF) of patients with rheumatoid arthritis (RA). Methods. Three-color immunofluorescence was used to study CD45 isoform expression by peripheral blood and SF CD4+ T cells. Results. CD45 isoform expression in the peripheral blood of patients with either RA or reactive arthritis did not differ from that in healthy controls. SF T cells from both RA patients and reactive arthritis patients were almost exclusively primed (CD45RO+) cells. RA SF T cells expressed very low levels of CD45RB; this is the most highly differentiated subset of primed cells. Patients with acute reactive arthritis showed higher levels of CD45RB^bright cells in their synovial fluid. Conclusion. The highly selected cell population in SF, representing one subset of primed cells, may relate to the apparent functional abnormalities of cells from this site in patients with RA.     

Activation signal transduction by β1 integrin in T cells from patients with systemic lupus erythematosus

Objective

Beta 1 integrin is a representative adhesion molecule for cell–cell and cell–extracellular matrix interactions, and it provides costimulatory signals to T cells. However, the relevance of β1 integrin to T cell activation in systemic lupus erythematosus (SLE) remains unclear. We undertook this study to perform a quantitative and functional analysis of β1 integrin–mediated signaling to T cells in patients with SLE.

Methods

Expression of cell surface molecules was assessed by flow cytometric analysis. Engagement of β1 integrins was performed by crosslinking using a specific monoclonal antibody. To assess tyrosine kinases in β1 integrin–mediated signaling, the cells were transfected with a wild‐type (WT) focal adhesion kinase (FAK), a dominant‐negative truncation of the FAK, or a WT PTEN expression plasmid via nucleofection.

Results

Beta 1 integrin expression was significantly up‐regulated on peripheral blood T cells from patients with active SLE, particularly those with the complication of World Health Organization class IV nephritis, whereas CD28 was significantly decreased in patients with active SLE compared with normal individuals. Beta 1 integrin expression closely correlated with serum hypocomplementemia. Engagement of β1 integrin on T cells from patients with active SLE, but not on those from normal individuals, induced cell proliferation as well as CD40L expression on T cells. Up‐regulation of CD40L expression and T cell proliferation, induced by β1 integrin stimulation, were completely inhibited by transfection of the dominant‐negative truncations of FAK or WT PTEN.

Conclusion

These results suggest that engagement of β1 integrins on SLE T cells could induce FAK‐mediated signaling and subsequent CD40L expression and proliferation. Thus, the β1 integrin signaling cascade might serve to enhance autoreactive T cell activation.