Dysregulation of p56lck kinase in patients with systemic lupus erythematosus.

In this study we investigated one of the possible mechanisms of p56lck down-regulation in peripheral blood lymphocytes (PBLs) from Systemic Lupus Erythematosus (SLE) patients and we correlated p56lck dysregulation with accelerated apoptosis in SLE PBLs. PBLs from SLE patients and healthy donors were isolated. p56lck protein expression and lck mRNA level were estimated by immunoblotting and RT-PCR, respectively. FACS analysis was used to evaluate the apoptosis and p56lck levels in apoptotic and non-apoptotic PBLs. A non-radioactive Tyrosine Kinase Assay Kit was used to measure p56lck activity. Our results demonstrated that PBLs from SLE patients displayed lower levels of lck mRNA and p56lck protein as compared to healthy donors. The apoptosis of fresh or cultured PBLs was enhanced in SLE patients, especially in anti-DNA negative group. The expression of p56lck was inverse correlated with apoptosis of fresh and cultured SLE PBLs, especially in anti-DNA negative patients. Double staining FACS analysis showed that p56lck expression was lower in apoptotic than in non-apoptotic PBLs. p56lck specific activity was directly correlated to apoptosis in SLE PBLs. While the low expression of p56lck may be the result of lower degree of synthesis, the increased specific activity could directly correlated to the extent of apoptosis in SLE PBLs. Based on our observations, we assume that the p56lck dysregulation could play a role in SLE pathogenesis.     

Interleukin-2 receptor expression in peripheral blood lymphocytes from systemic lupus erythematosus patients: relationship to clinical activity

Deficient interleukin-2 (IL-2) production and other T-cell dysfunctions have been demonstrated in active systemic lupus erythematosus (SLE). The generation of IL-2 receptors is known to be important to the growth and differentiation of T and B lymphocytes. This study investigated IL-2 receptor expression in peripheral blood lymphocytes (PBL) from patients with active and inactive SLE. PBL from 27 SLE patients, diagnosed by revised ARA criteria, were assayed for IL-2 receptor expression, IL-2 and immunoglobulin (Ig) production. PBL from SLE patients with active disease spontaneously expressed increased numbers of IL-2 receptors compared to those with inactive disease (P less than 0.01) and normal donors (P less than 0.01). There was no significant increase in IL-2 receptors expression in PBL from active SLE patients in response to mitogenic stimulation with PHA compared to inactive SLE patients and normal donors. There was negligible IL-2 production in response to mitogenic stimulation and increased spontaneous IgG production by PBL from active SLE patients compared to normal donors (P less than 0.001). Purified B cells isolated from active SLE patients showed significant spontaneous IL-2 receptor expression when compared to spontaneous IL-2 receptor expression by normal B cells (P = 0.005). Therefore, in addition to derangements in Ig and IL-2 production, the level of spontaneous expression of IL-2 receptors may represent a cellular indicator of disease activity, and hence, may be a useful parameter in monitoring disease activity in SLE patients. The significance of the increased IL-2 receptor expression on B cells of active SLE patients is unknown, but may represent a marker of polyclonal activation of these cells.     

系统性红斑狼疮患者CD4+T细胞上CD137的表达

目的:探讨系统性红斑狼疮(Systemic lupus erythematosus,SLE)患者CD4 T细胞上共刺激分子CD137的表达及其作用机制。方法:应用流式细胞术检测30例系统性红斑狼疮患者和20例正常对照者外周血T细胞活化前后CD137的表达。结果:活动期SLE患者CD4 T细胞表达的CD137明显高于稳定期及正常对照组(表达百分率分别为21.56±4.08、3.01±0.09和1.24±0.12,P<0.01),稳定期SLE患者表达的CD137与正常对照组比较无统计学差异(P>0.05)。但是活动期和稳定期SLE患者的CD4 T细胞用抗CD3单抗体外刺激活化后表达的CD137均显著高于正常对照组(表达百分率分别为56.25±9.11、27.26±3.41和13.17±1.54,P<0.01)。另外,活动期SLE患者CD4 T细胞活化后表达的CD137与补体呈负相关关系(r=-0.447,P<0.05),与IgG和24小时尿蛋白定量呈正相关关系(r=0.451,P<0.05,r=0.245,P<0.05)。结论:活动期系统性红斑狼疮患者T细胞活化前后CD137的表达均显著增高,而且CD4 T细胞活化后CD137表达水平可能提示病情和肾脏受累程度。     

Elevated gene expression of argininosuccinate synthetase in peripheral lymphocytes from systemic lupus erythematosus (SLE) patients.

Argininosuccinate synthetase (ASS) is a rate-limiting enzyme of urea cycle and functions primarily in the liver, whereas ASS activity is hardly detected in normal lymphocytes. In this study, we examined the level of ASS gene expression in peripheral blood lymphocytes (PBL) from human SLE patients by amplification of reverse-transcribed mRNA using the polymerase chain reaction. We have demonstrated that (a) approximately 40% of SLE patients exhibited 2.5 to 5 times higher expression of ASS gene in PBL than those of healthy PBL and (b) the elevation of ASS gene expression of PBL significantly correlates with the active pathogenesis of SLE patients according to the criteria of Japanese Ministry of Health and Welfare (p < 0.001 by student's two-tailed t-test). Thus, it is suggested that ASS gene expression is a promising marker of hyperactivated lymphocytes uniquely generated in patients with systemic autoimmune disease.     

Protooncogene expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus as an indicator of the disease activity

In the present study, we examined the various protooncogene expressions in PBMC (peripheral blood mononuclear cell) of systemic lupus erythematosus (SLE) patients to determine if they could be an indicator for the disease activity. We divided SLE patients into "very active," "active," and "remitting" states according to the clinical symptoms in addition to the laboratory data peculiar to SLE. In addition, we determined the amount of circulating immune complex (IC) as one of the representative laboratory indicators for the disease activity. We found a positive correlation with either c-myc or c-myb expression and the amounts of IC and clinical disease activity. The degree of c-myc and c-myb expression was significantly reduced along with or prior to the amelioration of clinical symptoms and improvement as determined by laboratory data under treatment with prednisolone and/or azathioprine administration. The degree of c-myc and c-myb gene expression had no direct relation to the presence of particular clinical sign(s) or autoantibody. The expression of the c-raf gene was found in SLE and other systemic autoallergic patients although it showed no correlation with the disease activity. No significant expression of c-src, c-ras, c-fos, c-fgr, c-fps, c-fes, c-fms, c-yes, c-rel, c-abl, c-mos, c-sis, and c-erb B genes was found in the patients. c-myc and c-myb expression as having pathogenic and clinical significance is discussed.     

IL-18对系统性红斑狼疮患者淋巴细胞凋亡和P53蛋白表达的影响

目的　研究系统性红斑狼疮 (SLE)患者外周血淋巴细胞在体外IL 1 8刺激培养下细胞凋亡及P53蛋白表达情况。方法　AnnexinV联合PI染色定量法及免疫荧光染色法 ,分析了 44例SLE患者和 30例正常人外周淋巴细胞在体外IL 1 8刺激培养后凋亡发生率 ,凋亡相关基因P53蛋白的表达以及淋巴细胞凋亡发生与疾病活动性的相关性。结果　在IL 1 8刺激培养作用下 ,活动期SLE淋巴细胞凋亡发生率较正常人显著增高 (P <0 .0 1 ) ,而静止期则无明显变化 (P >0 .0 5)。P53蛋白表达在活动期SLE较正常人显著性下降 (P <0 .0 1 ) ,静止期无明显变化 (P >0 .0 5)。P53的表达与疾病活动指数SLEDAI之间有明显的相关性 (P <0 .0 1 )。结论　IL 1 8可引起SLE患者PBL凋亡率的增高 ,表明IL 1 8在体内凋亡或凋亡相关性免疫机制中起着一定的作用     

Activity of gamma-glutamyl transpeptidase in peripheral blood lymphocytes in systemic lupus erythematosus

In the population of peripheral blood lymphocytes from systemic lupus erythematosus (SLE) patients activity of gamma-glutamyl transpeptidase (GGTP) was lower than in healthy individuals. However, when erythrocyte-rosette forming cells (E-RFC) and non erythrocyte-rosette forming cells (non E-RFC) were separated by E-RFC method it was found that in SLE patients the activity of GGTP was increased in E-RFC and decreased in the population of non E-RFC. The altered activity of GGTP may be due to impaired surface membranes and changes in activation of these cells in SLE patients.     

Upregulated BclGL expression enhances apoptosis of peripheral blood CD4 T lymphocytes in patients with systemic lupus erythematosus

Increased lymphocyte apoptosis has been suggested to contribute to the development of systemic lupus erythematosus (SLE), but the critical factors involved in the apoptotic pathways are still unknown. By long serial analysis of gene expression (LongSAGE) profiles and microarray analyses, a novel apoptosis-related gene BclG_L expression was found significantly increased in peripheral blood CD4⁺ T cells of SLE patients, which was correlated with the enhanced CD4⁺ T cells apoptosis, anti-nuclear antibody (ANA) titer and proteinuria. In vitro, BclG_L expression could be specially upregulated by SLE serum stimulation and positively correlated with induced CD4⁺ T cell apoptosis. Enforcing BclG_L overexpression by lentivirus could directly enhance CD4⁺ T cell apoptosis, but these apoptosis-inducing effects could be partially inhibited by knockdown of BclG_L expression. Collectively, these results indicate that increased BclG_L expression may contribute to the aberrant CD4⁺ T cell apoptosis which causes an inappropriate immune response and impaired homeostasis in SLE.     

Electron microscopic study of distinctive structures in peripheral blood lymphocytes obtained from twins with systemic lupus erythematosus.

Peripheral blood lymphoid cells obtained from nine twin pairs (six monozygotic and three dizygotic) in which one or both twins had systemic lupus erythematosus (SLE) were examined by electron microscopy for the occurrence of two distinctive intracytoplasmic structures-tubuloreticular structures (TRS) and tubular crystalloids (TC). TRS were found in 0.8 to 14.8% of lymphoid-cell cross sections in 9 of 11 twins with SLE and 2 clinically well but serologically abnormal twins. Lymphoid cells of twins both clinically and serologically normal did not exhibit TRS, although their monozygotic or dizygotic SLE-positive counterparts possessed these structures. Thus, the expression of TRS was more consistent with an acquired than inborn trait and appeared to correlate with disease and serologic manifestations of SLE. TC were found in 1.7 to 7.9% of lymphoid-cell cross sections in every twin examined. No correlation was recognized between clinical or laboratory data and the frequency of TC-bearing cells. The significance and the ultrastructural development of TC in the peripheral blood lymphoid cells are briefly discussed.     

Nuclear phosphotyrosyl-protein with DNA-binding ability in peripheral blood mononuclear cells from systemic lupus erythematosus patients.

This study examined the phosphorylation of cytoplasmic and nuclear proteins in peripheral blood mononuclear cells (PBMC) of systemic lupus erythematosus (SLE) patients. The cytoplasmic and nuclear protein kinase activity in PBMC from SLE patients was at least five-fold higher than that of normal healthy subjects. PBMC of SLE patients produced different nuclear endogenous substrates on phosphorylation and also displayed distinct protein kinase activity. Nuclear phosphoproteins, with human PBMC DNA-binding ability, of 38 kD and 70 kD were detected from both SLE patients and normal healthy subjects, while the 40 kD phosphoprotein, with tyrosine as the main phosphorylation residue, was found only in SLE patients. Other nuclear phosphoproteins, and most of the detected cytoplasmic phosphoproteins, were present in higher levels in both normal PBMC with mitogen stimulation, such as PHA, and SLE PBMC. The expression level of the 40 kD nuclear phosphotyrosyl-protein showed a positive correlation with the clinical disease activity of SLE. These results suggest that PBMC from SLE patients had distinct tyrosine protein kinase (TPK) activity and/or a different endogenous substrate of nuclear DNA-binding proteins in tyrosine phosphorylation. The possible significance of tyrosine phosphorylation in PBMC of SLE patients in the pathogenesis, and its clinical meaning, are discussed.     

Antibody-dependent direct cytotoxicity of human lymphocytes. I. Studies on peripheral blood lymphocytes and sera of patients with systemic lupus erythematosus.

Antibody-dependent direct cytotoxicity (ADDC) is generally believed to be unrelated to T-cell function in experimental animals. The role of ADDC in humans and its clinical usefulesss was evaluated in patients with systemic lupus erythematosus (SLE) and normal controls. Peripheral blood lymphocytes from patients with active SLE were unable to lyse antibody-coated target cells in vitro to the same degree as lymphocytes from patients with inactive SLE and controls. Sera from patients with active SLE suppressed ADDC by lymphocytes derived from normal controls and this abnormality was not corrected by overnight incubation or by extensive washing of lymphocyte preparations. Although there was poor correlation between ADDC and the proportions of B cells and null cells in effector lymphocyte populations from SLE patients and controls, it is concluded that this assay provides another means of determining immune competence in man.     

系统性红斑狼疮病人血T,B淋巴细胞Bcl—2的表达

目的：探讨Ｂｃｌ－２在系统性红斑狼疮（ＳＬＥ）发病机制中作用。方法：采用流式细胞仪双标记法检测３１例ＳＬＥ病人外周血Ｔ、Ｂ细胞Ｂｃｌ－２蛋白表达。结果：发现活动期ＳＬＥ病人活动期ＳＬＤ病人ＣＤ３＾＋、ＣＤ４＾＋和ＣＤ８＾＋Ｔ细胞Ｂｃｌ－２蛋白表达明显高于非活动期ＳＬＥ病人、其他疾病组和正常对照组。ＣＤ１９＾＋Ｂ细胞Ｂｃｌ－２蛋白表达在各组之间并无统计学差异。ＣＤ３＾＋Ｔ细胞Ｂｃｌ－２蛋白表达的平均     

系统性红斑狼疮患者外周血淋巴细胞表达BLyS和CD38的变化

目的:研究系统性红斑狼疮(SLE)患者外周血淋巴细胞表达BLyS和CD38的变化。方法:收集22名SLE患者和14名健康人外周血淋巴细胞, 用流式细胞仪检测外周血淋巴细胞表达BLyS和CD38的变化。结果:SLE患者外周血BLyS⁺淋巴细胞、CD19⁺淋巴细胞和CD19⁺CD38⁺淋巴细胞显著增加, BLyS⁺淋巴细胞增加与CD19⁺CD38⁺淋巴细胞增加呈正相关(r=0.434, P<0.05).结论:SLE患者外周血淋巴细胞表达BLyS和CD19⁺B淋巴细胞表达CD38均显著增加, 且二者增加呈正相关。     

Activation profile of Toll‐like receptors of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease associated with aberrant activation of T and B lymphocytes for the production of inflammatory cytokines and autoreactive antibodies. Animal studies of SLE have indicated that Toll‐like receptors (TLR) are important in the pathogenesis of murine lupus. In the present clinical study, differential protein expressions of TLR‐1–9 of monocytes and different lymphocyte subsets from patients with SLE and normal control subjects were determined by flow cytometry. Results showed that the expression of intracellular TLRs (TLR‐3, ‐8, ‐9) and extracellular TLRs (TLR‐1, ‐2, ‐4, ‐5, ‐6) were elevated in monocytes, CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and B lymphocytes of SLE patients compared to control subjects (all P < 0·001). Moreover, cell surface expression of TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes, and TLR‐6 on B lymphocytes, were correlated positively with SLE disease activity index (SLEDAI) (TLR‐4 on CD4⁺ T lymphocytes and CD8⁺ T lymphocytes: r = 0·536, P = 0·04; r = 0·713, P = 0·003; TLR‐6 in B lymphocytes: r = 0·572, P = 0·026). In concordance with the above results, there is an observable increased relative induction (%) of inflammatory cytokine interleukin (IL)‐1β, IL‐6, IL‐10 and IL‐12, chemokines CCL2, CXCL8, CCL5 and CXCL10 from peripheral blood mononuclear cells (PBMC) upon differential stimulation by PolyIC (TLR‐3 ligand), lipopolysaccharide (TLR‐4 ligand), peptidoglycan (TLR‐2 ligand), flagellin (TLR‐5 ligand), R837 (TLR‐7 ligand) and CpG DNA (TLR‐9 ligand) in SLE patients compared to controls. These results suggest that the innate immune response for extracellular pathogens and self‐originated DNA plays immunopathological roles via TLR activation in SLE.     

Abnormal expression of BAFF and its receptors in peripheral blood and skin lesions from systemic lupus erythematosus patients

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by abnormal T and B cells. B-cell activating factor (BAFF) has been suggested to play a crucial role in lupus by promoting the proliferation, differentiation, and survival of B cells. Increased serum levels of BAFF have been found in patients with lupus. However, the expression of BAFF and its receptors on immune cells and in skin has not been systematically reported before. Here, we report that SLE patients showed increased levels of BAFF on circulating CD3⁺ T cells and B-cell maturation antigen (BCMA) on CD14⁺ monocytes and dramatically increased expression of BAFF in lupus skin lesions compared with those of healthy controls. TACI was undetectable on circulating immune cells. An increased serum level of BAFF was also confirmed in lupus patients in this study. Our findings may provide a better understanding of the pathogenesis and predictors of BAFF antibody treatment response, as well as potential targets for skin therapies.