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.     

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 1α) 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. Conclusion. The model of 5-azaC-induced autoreactivity may have relevance to SLE.     

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.     

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.     

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.     

Hydralazine may induce autoimmunity by inhibiting extracellular signal-regulated kinase pathway signaling

OBJECTIVE: To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal-regulated kinase (ERK) pathway signaling. METHODS: The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c-Jun N-terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti-DNA antibodies were measured by enzyme-linked immunosorbent assay. RESULTS: In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen-activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti-double-stranded DNA antibodies in a murine model of drug-induced lupus, similar to the effect of hydralazine. CONCLUSION: Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine-induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic lupus.     

Antibodies to Hmg Proteins in Patients with Drug-Induced Autoimmunity

Objective. To determine the prevalence of auto-antibodies to high-mobility group (HMG) proteins in sera from patients with drug-induced lupus (DIL). Methods. Forty-two patients who developed auto-antibodies and/or lupus after treatment with procainamide or other drugs were tested for HMG autoantibodies by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Results. Twenty-eight of the 42 sera (67%) bound HMG-14 and/or HMG-17. In comparison, 9 of 42 (21%) bound HMG-1 and/or HMG-2. There was a good correlation between ELISA results and binding on immunoblots. Conclusion. The high prevalence of antibodies to the nucleosomal core HMGs (HMG-14 and HMG-17) in DIL patients adds evidence implicating nucleosomes as immunogens in drug-induced autoimmunity.     

Protein kinase Cδ oxidation contributes to ERK inactivation in lupus T cells

Objective

CD4+ T cells from patients with active lupus have impaired ERK pathway signaling that decreases DNA methyltransferase expression, resulting in DNA demethylation, overexpression of immune genes, and autoimmunity. The ERK pathway defect is due to impaired phosphorylation of T⁵⁰⁵ in the protein kinase Cδ (PKCδ) activation loop. However, the mechanisms that prevent PKCδ T⁵⁰⁵ phosphorylation in lupus T cells are unknown. Others have reported that oxidative modifications, and nitration in particular, of T cells as well as serum proteins correlate with lupus disease activity. We undertook this study to test our hypothesis that nitration inactivates PKCδ, contributing to impaired ERK pathway signaling in lupus T cells.

Methods

CD4+ T cells were purified from lupus patients and controls and then stimulated with phorbol myristate acetate (PMA). Signaling protein levels, nitration, and phosphorylation were quantitated by immunoprecipitation and immunoblotting of T cell lysates. Transfections were performed by electroporation.

Results

Treating CD4+ T cells with peroxynitrite nitrated PKCδ, preventing PKCδ T⁵⁰⁵ phosphorylation and inhibiting ERK pathway signaling similar to that observed in lupus T cells. Patients with active lupus had higher nitrated T cell PKCδ levels than did controls, which correlated directly with disease activity, and antinitrotyrosine immunoprecipitations demonstrated that nitrated PKCδ, but not unmodified PKCδ, was refractory to PMA‐stimulated T⁵⁰⁵ phosphorylation, similar to PKCδ in peroxynitrite‐treated cells.

Conclusion

Oxidative stress causes PKCδ nitration, which prevents its phosphorylation and contributes to the decreased ERK signaling in lupus T cells. These results identify PKCδ as a link between oxidative stress and the T cell epigenetic modifications in lupus.

     

CD19 hyperexpression augments Sle1‐induced humoral autoimmunity but not clinical nephritis

Objective

B cell hyperactivity is a common denominator in murine and human systemic lupus erythematosus. Some susceptibility genes in lupus are associated with B cell hyperactivity, but others are clearly not. While the Sle1 lupus susceptibility locus of NZM2410/NZW origin leads to chromatin‐focused autoimmunity, genetically engineered overexpression of CD19 leads to “generalized” B cell hyperactivity. We undertook this study to determine the degree to which generalized B cell hyperactivity can amplify lupus pathogenesis.

Methods

To elucidate the impact of generalized B cell hyperactivity on Sle1‐triggered autoimmunity, B6 mice bearing the human CD19 transgene were rendered congenic for the Sle1^z genetic locus and phenotyped for serologic, cellular, and pathologic evidence of lupus.

Results

As expected, B6.Sle1.hCD19^Tg/Tg mice, homozygous at Sle1 and bearing the hCD19 transgene, exhibited high levels of IgM and IgG anti‐DNA/antiglomerular autoantibodies, skewed B cell subsets, and profoundly activated B and T cells. Despite exhibiting glomerular IgM, IgG, and complement deposits, these mice did not exhibit accelerated mortality or any clinical evidence of renal dysfunction.

Conclusion

Generalized B cell hyperactivity may augment humoral autoimmunity, but this may not suffice to engender end‐organ disease in lupus. These findings allude to the presence of an additional distal checkpoint that dissociates pathogenic autoantibody formation and renal immunoglobulin deposition from the progression to clinical nephritis in lupus.

     

Hydralazine may induce autoimmunity by inhibiting extracellular signal–regulated kinase pathway signaling

Objective

To determine whether hydralazine might decrease DNA methyltransferase (DNMT) expression and induce autoimmunity by inhibiting extracellular signal–regulated kinase (ERK) pathway signaling.

Methods

The effect of hydralazine on DNMT was tested in vitro using enzyme inhibition studies, and in vivo by measuring messenger RNA (mRNA) levels and enzyme activity. Effects on ERK, c‐Jun N‐terminal kinase, and p38 pathway signaling were tested using immunoblotting. Murine T cells treated with hydralazine or an ERK pathway inhibitor were injected into mice and anti‐DNA antibodies were measured by enzyme‐linked immunosorbent assay.

Results

In vitro, hydralazine did not inhibit DNMT activity. Instead, hydralazine inhibited ERK pathway signaling, thereby decreasing DNMT1 and DNMT3a mRNA expression and DNMT enzyme activity similar to mitogen‐activated protein kinase kinase (MEK) inhibitors. Inhibiting T cell ERK pathway signaling with an MEK inhibitor was sufficient to induce anti–double‐stranded DNA antibodies in a murine model of drug‐induced lupus, similar to the effect of hydralazine.

Conclusion

Hydralazine reproduces the lupus ERK pathway signaling abnormality and its effects on DNMT expression, and inhibiting this pathway induces autoimmunity. Hydralazine‐induced lupus could be caused in part by inducing the same ERK pathway signaling defect that occurs in idiopathic 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.

     

The NZM2410‐derived lupus susceptibility locus Sle2c1 increases Th17 polarization and induces nephritis in fas‐deficient mice

Objective

Sle2 is a lupus susceptibility locus that has been linked to glomerulonephritis in the NZM2410 mouse. By itself, Sle2 does not induce any autoimmune pathology but results in the accumulation of B‐1a cells. This study was designed to assess the contribution of Sle2 to the pathogenesis of autoimmunity.

Methods

Sle2 or its subcongenic intervals (Sle2a, Sle2b, and Sle2c1) were bred to Fas‐deficient B6.lpr mice. Lymphoid phenotypes, which were focused on T cells, were assessed by flow cytometry, and histopathologic changes were compared between cohorts of B6.Sle2.lpr congenic mice and B6.lpr mice of ages up to 6 months.

Results

Sle2 synergized with lpr, resulting in a greatly accelerated lymphadenopathy that largely targeted T cells and mapped to the Sle2c1 locus. This locus has been identified as the main contributor to B‐1a cell expansion. Further analyses showed that Sle2c1 expression skewed the differentiation and polarization of Fas‐deficient T cells, with a reduction of the CD4+CD25+FoxP3+ regulatory T cell subset and an expansion of the Th17 cells. This was associated with a high number of T cell infiltrates that promoted severe nephritis and dermatitis in the B6.Sle2c1.lpr mice.

Conclusion

These results show that Sle2c1 contributes to lupus pathogenesis by affecting T cell differentiation in combination with other susceptibility loci, such as lpr. The significance of the cosegregation of this phenotype and B‐1a cell expansion in Sle2c1‐expressing mice in relation to the pathogenesis of lupus is discussed.

     

Constitutive overexpression of BAFF in autoimmune‐resistant mice drives only some aspects of systemic lupus erythematosus–like autoimmunity

Objective

To determine whether overexpression of BAFF can promote systemic lupus erythematosus (SLE)–like autoimmunity in mice that are otherwise autoimmune‐resistant.

Methods

We used class II major histocompatibility complex (MHC)–deficient C57BL/6 (B6) mice as a model of resistance to SLE and Sles1‐bearing B6 mice as a model of resistance to the autoantibody‐promoting capacity of the Sle1 region. We generated BAFF‐transgenic (Tg) counterparts to these respective mice and evaluated lymphocyte phenotype, serologic autoimmunity, renal immunopathology, and clinical disease in the BAFF‐Tg and non‐Tg mouse sets.

Results

Although constitutive BAFF overexpression did not lead to B cell expansion in class II MHC–deficient B6 mice, it did lead to increased serum IgG autoantibody levels. Nevertheless, renal immunopathology was limited, and clinical disease did not develop. In B6 and B6.Sle1 mice, constitutive BAFF overexpression led to increased numbers of B cells and CD4+ memory cells, as well as increased serum IgG and IgA autoantibody levels. Renal immunopathology was modestly greater in BAFF‐Tg mice than in their non‐Tg counterparts, but again, clinical disease did not develop. Introduction of the Sles1 region into B6.Sle1.Baff mice abrogated the BAFF‐driven increase in CD4+ memory cells and the Sle1‐driven, but not the BAFF‐driven, increase in serum IgG antichromatin levels. Renal immunopathology was substantially ameliorated.

Conclusion

Although constitutive BAFF overexpression in otherwise autoimmune‐resistant mice led to humoral autoimmunity, meaningful renal immunopathology and clinical disease did not develop. This raises the possibility that BAFF overexpression, even when present, may not necessarily drive disease in some SLE patients. This may help explain the heterogeneity of the clinical response to BAFF antagonists in human SLE.

     

Brief Report: Increased expression of a short splice variant of CTLA‐4 exacerbates lupus in MRL/lpr mice

Objective

CTLA‐4 is a negative regulator of the immune response expressed by regulatory T (Treg) cells and activated T cells. Polymorphisms in the CTLA4 gene have been associated with autoimmune diseases, including systemic lupus erythematosus. Disease‐associated polymorphisms have been shown to affect the production of the different CTLA‐4 variants through an effect on alternative splicing. This study was undertaken to evaluate the role of the 1/4 CTLA‐4 isoform in lupus‐prone mice.

Methods

We generated an MRL/lpr mouse strain that transgenically overexpresses a short isoform of CTLA‐4 (1/4 CTLA‐4) by backcrossing C57BL/6.1/4CTLA‐4–transgenic mice to the MRL/lpr strain for 9 generations. A new antibody was generated to detect the expression of the 1/4 CTLA‐4 isoform. Routine methods were used to evaluate kidney damage, humoral immunity, and cellular immunity.

Results

Expression of the 1/4 CTLA‐4 isoform accelerated autoimmune disease. Transgenic mice died earlier, had more severe renal disease, and had higher titers of anti–double‐stranded DNA antibodies than wild‐type MRL/lpr mice. The acceleration of autoimmunity and disease pathology associated with the presence of the short (1/4) isoform of CTLA‐4 was linked to increased numbers of activated T cells and B cells and heightened interferon‐γ production, but not to altered expression of the full‐length CTLA‐4 molecule or Treg cell numbers.

Conclusion

Our results indicate that the presence of the alternatively spliced 1/4 CTLA‐4 isoform can further promote autoimmunity and autoimmune pathology in lupus‐prone mice and suggest that altered splicing of CTLA4 contributes to the expression of autoimmune disease.

     

SLP-76-ADAP adaptor module regulates LFA-1 mediated costimulation and T cell motility

Although adaptor ADAP (FYB) and its binding to SLP-76 has been implicated in TcR-induced “inside-out” signaling for LFA-1 activation in T cells, little is known regarding its role in LFA-1-mediated “outside-in” signaling. In this study, we demonstrate that ADAP and SLP-76-ADAP binding are coupled to LFA-1 costimulation of IL-2 production, F-actin clustering, cell polarization, and T cell motility. LFA-1 enhancement of anti-CD3-induced IL-2 production was completely dependent on SLP-76-ADAP binding. Further, anti-CD3 was found to require CD11a ligation by antibody or ICAM1 to cause T cell polarization. ADAP augmented this polarization induced by anti-CD3/CD11a, but not by anti-CD3 alone. ADAP expression with LFA-1 ligation alone was sufficient to polarize T cells directly and to increase T cell motility whereas the loss of ADAP in ADAP−/− primary T cells reduced motility. A mutant lacking SLP-76-binding sites (M12) blocked LFA-1 costimulation of IL-2 production, polarization, and motility. LFA-1-ADAP polarization was also dependent on src kinases, Rho GTPases, phospholipase C, and phosphoinositol 3-kinase. Our findings provide evidence of an obligatory role for the SLP-76-ADAP module in LFA-1-mediated costimulation in T cells.     

Histone-specific Th0 and Th1 clones derived from systemic lupus erythematosus patients induce double-stranded DNA antibody production

Objective. To investigate whether histone-specific T helper (Th) cells that are able to induce anti-doublestranded DNA (anti-dsDNA) antibodies can be isolated from patients with systemic lupus erythematosus (SLE) and to characterize the cytokine secretion pattern of such Th clones. Methods. Peripheral blood mononuclear cells from SLE patients and healthy donors were stimulated with autologous apoptotic cell material or purified histones, expanded with interleukin-2 (IL-2), and cloned by limiting dilution. Histone reactivity of clones was examined by histone-specific proliferation and cytokine release. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) and CTLL-2 bioassay. Induction of anti-dsDNA antibodies was measured in cocultures of autologous B cells and Th clones by ELISA. Results. Numerous histone-specific T cell receptor (TCR) α/β+ Th clones were established from 2 of 3 patients with active SLE and from 1 of 2 healthy individuals. Most Th clones secreted IL-2, interferon-γ (IFNγ), and IL-4, whereas some produced predominantly IL-2 and lFNγ. Th clones that could stimulate the production of anti-dsDNA antibodies were derived from SLE patients and from a healthy individual. Conclusion. Th cells specific for histones may play an important role in the pathogenesis of SLE by inducing autoantibodies to dsDNA. Both Th1 and Th2 cytokines may be involved in the pathogenesis of SLE. The presence of histone-specific Th cells in a healthy individual indicates the importance of peripheral tolerance for preventing autoimmunity to nuclear antigens.     

Induction of differentiation in HL60 cells by the reduction of extrachromosomally amplified c-myc.

Oncogene amplification in tumor cells results in the overexpression of proteins that confer a growth advantage in vitro and in vivo. Amplified oncogenes can reside intrachromosomally, within homogeneously staining regions (HSRs), or extrachromosomally, within double minute chromosomes (DMs). Since previous studies have shown that low concentrations of hydroxyurea (HU) can eliminate DMs, we studied the use of HU as a gene-targeting agent in tumor cells containing extrachromosomally amplified oncogenes. In a neuroendocrine cell line (COLO 320), we have shown that HU can eliminate amplified copies of c-myc located on DMs, leading to a reduction in tumorigenicity in vitro and in vivo. To determine whether the observed reduction in tumorigenicity was due to differentiation, we next investigated whether HU could induce differentiation in HL60 cells containing extrachromosomally amplified c-myc. We compared the effects of HU, as well as two other known differentiating agents (dimethyl sulfoxide and retinoic acid), on c-myc gene copy number, c-myc expression, and differentiation in HL60 cells containing amplified c-myc genes either on DMs or HSRs. We discovered that HU and dimethyl sulfoxide reduced both c-myc gene copy number and expression and induced differentiation in cells containing c-myc amplified on DMs. These agents failed to have similar effects on HL60 cells with amplified c-myc in HSRs. By contrast, retinoic acid induced differentiation independent of the localization of amplified c-myc. These data illustrate the utility of targeting extrachromosomal DNA to modulate tumor phenotype and reveal that both HU and dimethyl sulfoxide induce differentiation in HL60 cells through DM elimination.     

Analyses of serum levels of type 1, type 2 and type 3 cytokines reveal multiple abnormalities in lupus‐prone (NZB × NZW) F1 mice

Aim: Inherent in vivo cytokine milieu may help protect normal subjects from developing clinical autoimmunity, whereas changes in cytokine milieu may contribute to the development of lupus‐like autoimmunity. Method: Here, we measured circulating levels of the three prototypic cytokines, type 1 (IFN‐γ and IL‐2), type 2 (IL‐4) and type 3 (TGFβ) in the NZB/NZW F1 model of lupus and MHC‐matched nonautoimmune mice. Results: Our results demonstrate that circulating IFN‐γ and IL‐4 levels were higher and active TGFβ levels were lower in lupus‐prone animals than in nonautoimmune mice. Conclusion: Such an in vivo cytokine milieu may contribute to the development of lupus in NZB/NZW F1 mice and to the nonautoimmune phenotype in normal animals.