An increased frequency of autoantibody-inducing CD4+ T cells in pre-diseased lupus-prone mice

Pathogenic autoantibody production in murine models of lupus is dependent on autoreactive CD4+ helper T cells. However, the mechanisms which permit the selection and maintenance of this autoantibody-inducing CD4+ T-cell repertoire are currently unknown. We hypothesized that the peripheral CD4+ T-cell repertoire of lupus-prone mice was enriched with autoantibody-inducing specificities. To test this, we utilized the splenic focus assay to determine if pre-diseased lupus-prone (NZB x NZW)F(1) mice have an elevated frequency of autoreactive CD4+ T lymphocytes capable of supporting autoantibody production. The splenic focus limiting dilution assay permits anti-nuclear antibodies to be generated from contact-dependent T-B interactions in vitro. We show that young, pre-diseased lupus-prone mice have an elevated frequency of autoantibody-inducing CD4+ T cells. Interestingly, these autoantibody-inducing CD4+ T-cell responses are also present in the thymus. Therefore, an elevated frequency of autoantibody-inducing CD4+ T cells predisposes lupus-prone mice to the development of autoantibodies.     

sIL－2R与SLE的临床表现和自身抗体的相关性

用双抗体夹心ＥＬＩＳＡ检测６６例ＳＩＥ病人血清叽ｓＩＬ－２Ｒ水平，ＳＬＥ病人显著高于正常人，疾病活动期高于非活动期。血清ｓＩＬ－２Ｒ水平与ＡＮＡ、抗ｄｓ－ＤＮＡ抗体、抗Ｓｍ、ＳＳ－Ａ、ＳＳ－Ｂ抗体无关，而与ＳＬＥ患者的发热、贫血、白细胞减少、关节受累及肾脏损害相关，且与ＳＬＥ疾病活动性和ＥＳＲ成正相关，与补体Ｃ＿３、Ｃ＿４和ＣＨ＿（５０）成负相关。提示血清ｓＩＬ－２Ｒ水平是监测ＳＬＥ疾病活动性的一个良好指标。     

Anti-idiotype and immunosuppressant treatment of murine lupus.

The effect of the administration of a xenogeneic anti-idiotype antibody (anti-Id33) to a cross-reactive idiotype (Id33) present on anti-dsDNA antibody was examined in 6-week-old (NZB/NZW) F1 (BWF1) female mice. The administration of anti-Id33 led to a transient reduction in immunoglobulins expressing Id33, followed by a rise at 30 and 34 weeks that was significantly higher than in untreated mice (P less than 0.05). Likewise, anti-dsDNA antibody levels were significantly higher at 10 and 18 weeks than in untreated mice (P less than 0.01). No differences were seen in survival to 40 weeks, proteinuria or the severity of glomerulonephritis. Concurrent administration of cyclosporin A (CyA) with anti-Id33 markedly ameliorated glomerular injury and proteinuria and improved survival. By contrast, glomerular injury, proteinuria and survival were worse in mice treated with cyclophosphamide plus anti-Id33, compared with untreated mice. Neither CyA nor cyclophosphamide treatment, when given with anti-Id33 altered serum levels of anti-dsDNA, anti-ssDNA or Id33+ immunoglobin, compared with untreated mice. The different effects of CyA and cyclophosphamide on T lymphocytes and their discrepant effects on glomerular injury when given with anti-Id33 in this model lead us to postulate a role for T lymphocytes in the glomerular injury of BWF1 lupus.     

Differential effects of sex hormones on autoantibody production and proteinuria in chronic graft-versus-host disease-induced experimental lupus nephritis

In patients with systemic lupus erythematosus, the female-to-male ratio is as high as 10:1. Sex hormones are thought to play a role in this difference in susceptibility. In a previous study, we demonstrated a high susceptibility of female mice to the development of glomerulonephritis after induction of chronic graft-versus-host disease (GVHD), compared with male mice. In order to unravel further this gender-related difference (C57Bl/10*DBA/2)F¹ hybrid mice were either castrated or ovariectomized and treated with 17β-ethinyloestradiol or testosterone-decanoate preceding the induction of chronic GVHD. Testosterone-decanoate reduced significantly the development of albuminuria in females. In contrast, proteinuria of 17β-ethinyloestradiol-treated female mice was in the same range as that of sham-operated mice. Autoantibody levels against glomerular basement membrane, renal tubular epithelium, dsDNA and ssDNA, as determined by ELISA, were higher in 17β-ethinyloestradiol-treated female mice than in all other groups. Immunofluorescence studies showed the presence of immunoglobulin and complement deposits in glomeruli of all animals, without significant differences between the experimental groups. Our findings confirm earlier observations, in that testosterone-decanoate is shown to be an inhibitory compound, whereas 17β-ethinyloestradiol has stimulating properties in autoimmunity. Moreover, our results show for the first time differential hormonal effects on autoantibody levels and proteinuria in experimental lupus nephritis.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

Antigen DNA isolated from immune complexes in plasma of patients with systemic lupus erythematosus hybridizes with the Escherichia coli lac Z gene.

Antigen DNA was isolated from immune complexes in plasma of three patients with active systemic lupus erythematosus (SLE) using affinity column. The antigen DNA thus obtained was subjected to hybridization experiments in order to investigate its origin. Unexpectedly, plasmid pUC18 used as a probe was found to hybridize with the antigen DNA, pUC18 was then cleaved into three fragments with the restriction enzyme HaeII. A 445-bp fragment containing lac Z DNA hybridized with the antigen DNA. Finally, the lacZ DNA itself was found to hybridize with the antigen DNA. These data strongly suggest that the antigen DNA obtained from three patients is of bacterial origin.     

IL-10RA基因多态性及其与系统性红斑狼疮关系的研究

目的:确定SLE模型小鼠IL-10RA基因变异及其与SLE表现型是否存在关联。方法:用微卫星遗传标记及数量性状位点(QTL)分析方法确定SLE模型小鼠B/W F1的SLE易感基因精确染色体定位并选取候选易感基因,对候选易感基因进行测序分析,选取有基因序列异常的候选易感基因进行PCR-SSCP分析,确定候选易感基因碱基序列变异位点与抗染色质抗体、抗DNA抗体,抗组蛋白抗体及蛋白尿等SLE表现型的相关关系。结果:QTL分析结果表明B/W F1×NZB小鼠抗染色质抗体易感基因与NZW型IL-10RA基因紧密连锁;测序分析发现IL-10RA基因编码区有18处碱基变异,其中7处碱基变异将导致编码氨基酸的变异;抗染色质抗体、抗DNA抗体,抗组蛋白抗体及蛋白尿等SLE表现型与NZW型IL-10RA基因密切相关。另一种SLE模型小鼠MRL的IL-10RA基因存在相同变异。结论:NZW小鼠IL-10RA基因编码区碱基序列存在变异,B/W F1×NZB小鼠SLE表现型与NZW小鼠第9染色体IL-10RA编码区碱基变异相关,提示IL-10RA可能是SLE模型小鼠的一个SLE易感基因。     

New therapies for systemic lupus erythematosus

In the past 40 years, prognosis for patients with systemic lupus erythematosus (SLE) has improved, with 10-year survival now approximately 90%. This is due probably to a combination of earlier disease diagnosis and diagnosis of milder disease, due in part to availability of multiple serological tests for SLE, use of steroids and other immunosuppressive agents, and availability of renal dialysis and transplantation. Despite this, however, the potential for significant morbidity and mortality remains in the group of patients with partially responsive or treatment resistant disease. More recently, advancements in the understanding of molecular mechanisms involved in the pathogenesis of SLE have translated to the development of novel therapies, offering possible alternatives to this patient cohort. Discussion of these pharmacological options and ongoing research forms the basis of this review.     

Suppressive effect of hyperbaric oxygenation on immune responses of normal and autoimmune mice.

We studied the effect of hyperbaric oxygenation (HBO) on immune responses in normal and autoimmune mice. Mice were exposed to HBO in an animal chamber at a pressure of 252.5 kPa for 1 h and once a day for 5 days. The immunization of C3H/He mice with sheep erythrocytes induced marked anti-sheep erythrocyte antibody response on day 7. However, this response was markedly suppressed in HBO-treated mice. The suppression is dependent on the duration of HBO and it works on the early and the late stage of antibody responses. HBO suppresses the development of both sheep erythrocyte-specific B cells and helper T cells after the immunization. Then, we tried to expose autoimmune mice to HBO. Spontaneous immunoglobulin production of NZB and MRL/lpr spleen cells was also significantly suppressed by HBO. Furthermore, long term HBO exposure results in the suppression of the development of autoimmune symptoms such as proteinuria, facial erythema and lymphadenopathy in MRL/lpr mice. All these results suggest that HBO is applicable for the treatment of autoimmune diseases.