CD5 expression in B cells from patients with systemic lupus erythematosus

The recently recognized importance of B cells in systemic lupus erythematosus (SLE) raises the question as to whether those expressing CD5 predominate over the remaining B lymphocytes in the pathophysiology of this disease. Owing to their B B-cell receptor (BCR) polyspecificity, autoantibody production has been originally ascribed to CD5-positive B1 lymphocytes. Instead, it has since been established that high-affinity autoantibodies derive from CD5-negative B2 cells. Even worse, in the light of current findings, CD5-positive B cells have been considered to play a paradoxical role in preventing, rather than inducing, autoimmunity. In this context, there is evidence that the membrane expression of CD5 is regulated, and, to this end, a genetic mechanism has been described, based on the selection between exon 1A (E1A) and exon 1B (E1B). The full-length protein variant, encoded by E1A-cd5, translocates the phosphatase SHP-1 to the vicinity of the BCR, raises its threshold, and thereby limits the response of autoreactive B cells. In contrast, the truncated variant, encoded by E1B-cd5, remains in the cytoplasm, along with SHP1. Normally, EIB E1B is silenced by methylation and its product degraded in the proteosomes. Hence, a defect in the DNA methyl transfer favors the development of SLE, by preventing the effects of SHP-1.     

系统性红斑狼疮外周血单个核细胞CD40L的表达增高

目的:了解系统性红斑狼疮(SLE)患者外周血单个核细胞(PBMCs)的白细胞分化抗原40配体(CD40L)表达,探讨其在发病中的作用。方法:分离SLE患者和正常人PBMCs,采用流式细胞术,检测其在正常状况和应用植物凝集素(PHA)及地塞米松(Dex)后,CD40L的表达水平,并进行比较;分析SLE患者CD40L的表达水平和狼疮活动指数(SLEDAI)的相关性。结果:活动期SLE患者PBMCs的CD40L阳性细胞百分率(%)明显高于对照组,且高于静止期SLE患者;应用PHA处理24h后,3组PBMC表达CD40L均明显增加,但活动期SLE患者增加更明显;应用地塞米松后,SLE患者(活动期和静止期)PBMCs的CD40L表达明显减少,对照组无明显改变;SLE患者(活动期和静止期)CD40L的表达水平和SLEDAI均呈明显正相关。结论:CD40L在SLE患者PBMCs的表达增加,和疾病活动度有关;其受PHA和Dex调控,在SLE发病和病程中起重要作用。     

CD5+ B cells from individuals with systemic lupus erythematosus express granzyme B

Recently, we reported that IL‐21 induces granzyme B (GzmB) and GzmB‐dependent apoptosis in malignant CD5⁺ B cells from patients with chronic lymphocytic leukemia. Several autoimmune diseases (AD) are associated with enhanced frequencies of CD5⁺ B cells. Since AD are also associated with elevated IL‐21 and GzmB levels, we postulated a link between CD5⁺ B cells, IL‐21 and GzmB. Here, we demonstrate that IL‐21 and GzmB serum levels are highly correlated in subjects with systemic lupus erythematosus (SLE) and that freshly isolated CD5⁺ SLE B cells constitutively express GzmB. IL‐21 directly induced GzmB expression and secretion by CD5⁺ B cells from several AD and from cord blood in vitro, and the simultaneous presence of BCR stimulation strongly enhanced this process. Furthermore, IL‐21 suppressed both viability and expansion of CD5⁺ B cells from SLE individuals. In summary, our study may explain the elevated levels of IL‐21 and GzmB in SLE and other AD. Moreover, our data suggest that IL‐21 may have disease‐modifying characteristics by inducing GzmB in CD5⁺ B cells and by suppressing their expansion. Our results provide the rationale for further evaluation of the therapeutic potential of IL‐21 in certain AD such as SLE.     

Der p 2 can induce bystander activation of B cells derived from patients with systemic lupus erythematosus

Although many patients with SLE also have allergies, the immunological events triggering the onset and progression of the clinical manifestations of SLE by allergens have yet to be clarified. A total of three autoantigens, phosphoglycerate kinase 1 (PGK-1), triosephosphate isomerase (TIM) and enolase were identified by autologous serum in B cell lysate derived from HDM allergic SLE patients after Der p 2 stimulation. Autoantigen, TRIM-21 expression were also significantly increased in B cells derived from HDM allergic SLE patients. In PBMCs derived from SLE patients, the concentration of anti-PGK-1 was significantly upregulated after Der p 2 stimulation compared to HDM allergic without SLE patients and healthy subjects. Inflammatory related cytokines and chemokines include IL-1β, IL-6, IL-8, CXCL5 could be upregulated after Der p 2 stimulation in PBMCs derived from HDM allergic SLE patients. In conclusion, our data demonstrated that long-term allergen exposure could be a contributing factor in the development of SLE.     

Identification of DNA-reactive B cells in patients with systemic lupus erythematosus

Autoreactive B cells play a central role in systemic lupus erythematosus (SLE). Characterization of DNA-reactive B cells in the blood of lupus patients has been limited by the low frequency of the population. Using a tetrameric configuration of a peptide mimetope of DNA, we identified peptide-reactive B cells in peripheral blood. Antibodies derived from these B cells bound to peptide and were largely cross-reactive to dsDNA. This methodology enables us to track the development of autoreactive B cells, which recognize peptide and dsDNA, in individual patients with SLE and permits the isolation of autoreactive B cells for further characterization.     

Cytokine-independent progression of immunoglobulin production in vitro by B lymphocytes from patients with systemic lupus erythematosus.

B lymphocytes from patients with systemic lupus erythematosus (SLE) secreted high levels of immunoglobulin spontaneously when cultured in vitro. Addition of the cytokines interleukin-2, interleukin-4 and interleukin-6 either alone or in combination failed to augment spontaneous immunoglobulin synthesis. Percoll-separated low-density SLE B lymphocytes matured into immunoglobulin-secreting cells also independent of exogenous interleukins. During maturation these cells became enlarged and less dense, and began to express CD23. This was in contrast to normal B cells, which did not secrete immunoglobulin spontaneously but synthesized IgM after interleukin stimulation. These results indicate that in vitro immunoglobulin synthesis by SLE B cells is already initiated in these cells and progresses independently of further stimulatory manoeuvres.     

Dysfunctional B cells in systemic lupus erythematosus

The classical view of B cells in the biology of autoimmune responses to infectious and self-antigens (Ag) that they promote immunity primarily by producing antibodies (Ab) is far from being complete. Indeed, studies over the last decade suggest that B cells have extraordinarily diverse functions within the immune system other than Ab production, which could contribute to autoimmunity. They normally play a role in the development of lymphoid architecture, regulating dentritic cells (DC) and T cell subsets function through cytokine production, and in activation of T cells. Receptor editing is also important in B cells which aids in immunity to infection and, possibly, prevention of autoimmunity. Both abnormalities in the distribution of B cells subsets and clinical benefit response to B cell depletion in autoimmune diseases, including systemic lupus erythematosus (SLE), highlight their pivotal function. Transgenic (Tg) animal models have shown that sensitivity of B cells to B cell Ag receptor (BCR) cross-linking is correlated to autoimmunity. Indeed, negative signaling by CD5 and other molecules, such as CD22, in maintaining tolerance through recruitment of src-homology two domain-containing protein tyrosine phosphatase-1 (SHP-1) has also been documented. In fact, we have now reached a newer area whereby B cells returned as an important contributor to autoimmune disorders.     

Spontaneous production of bone-resorbing lymphokines by B cells in patients with systemic lupus erythematosus

The culture supernatant of B cells from patients with active systemic lupus erythematosus (SLE) who had never been treated with corticosteroids had bone-resorbing activity (BRA) which stimulated the⁴⁵Ca release from prelabeled murine fetal bones. Then we studied the characteristics and the relationship of this BRA with several lymphokines previously reported. The BRA was eluted as three peaks at approximately 17,000, 35,000, and 80,000 daltons by Sephacryl S-200 column chromatography. Recombinant (r)IL 1, rIL 1, and rTNF possessed BRA, but rIL 4 and rIL 6 did not. Furthermore, the BRA from SLE B cells was absorbed with anti-IL 1 antibody but not with anti-IL 1 and anti-TNF antibody. Therefore, the fact that SLE B cells produce BRA which corresponds to IL 1 and IL 1 produced by B cells might be one of the causes of bone destruction in SLE patients.     

The paradox of CD5-expressing B cells in systemic lupus erythematosus

The pathophysiological relevance of B cells for systemic lupus erythematosus (SLE), particularly those expressing the T-cell marker CD5, raises the question as to how they operate upon autoimmune processes. Based on their production of low-affinity multispecific antibodies (Abs), CD5(+) B lymphocytes, also referred to as B1 cells, have originally been endowed with the autoAb making. It has since been established that high-affinity Abs to double-stranded DNA are not generated by these cells, but rather by B2 cells. It does not appear that they have the exclusive rights to the production of pathogenic autoAbs. In the light of recent findings, CD5 plays a paradoxical role in preventing autoimmunity. Hence, misguided signaling through CD5 could lead to autoimmunity. This provocative view differs from the na?ve interpretation that the increased levels of B1 cells in SLE represent a direct source of autoAbs responsible for damaging organs.     

Cross-reactivity of IgG anti-DNA-secreting B cells in patients with systemic lupus erythematosus

This study is the first to analyze the cross-reactivity of in vivo activated B cells from patients with systemic lupus erythematosus. A chamber ELIspot assay was used to determine whether lymphocytes secreting antibodies that bound to DNA or 2,4,6-trinitrophenol (TNP)-keyhole limpet-hemocyanin (KLH) could simultaneously bind to the unrelated antigens actin or ovalbumin. IgM anti-DNA-, IgM anti-TNP-KLH- and IgG anti-TNP-KLH-secreting B cells from patients and controls showed similar levels of cross-reactivity (ranging from 6% to 23%, depending upon the antibody isotype and antigen pair examined). In general, IgG-producing cells were less cross-reactive than IgM producers from the same individual (on the average threefold, p < 0.001). In contrast, IgG anti-DNA-secreting B cells from lupus patients (i) showed no decrease in cross-reactivity when compared to IgM anti-DNA-secreting cells and (ii) were significantly more cross-reactive than control IgG anti-DNA-secreting cells and IgG anti-TNP-KLH secreting cells from patients (p < 0.001). The degree of IgG anti-DNA cross-reactivity correlated with disease activity (r = 0.52, p < 0.02). The implications of these findings with respect to repertoire expression and disease pathogenesis are discussed.     

IL-10 production in B cells is confined to CD154+ cells in patients with systemic lupus erythematosus

The immunological hallmark of SLE is B cell hyperactivity. CD154 (CD40-L) is normally expressed in activated T cells, and plays an important role in T-B interactions. Its expression is increased in SLE T cells. Additionally, its expression on B cells leads to the development of SLE-like disease in a transgenic model. IL-10 is a key cytokine in the disturbed SLE immune system. The aim of this work was to explore the relation between IL-10 and CD154 expression in SLE B cells. We studied 11 SLE patients and 10 healthy volunteers. Mononuclear cells were isolated from peripheral blood and cultured in the presence or absence of Cowan I Strain Staphylococcus (CSS). Surface CD154 and intracytoplasmic IL-10 expression were quantified with flow cytometry. In basal conditions, CD154 expression was not different in patients and controls. B cell stimulation did not cause a significant increase in CD154 expression in control B cells. However, its expression increased 2 times in B cells obtained from SLE patients. IL-10 expression was confined to CD154(+) cells. Our results show that IL-10 production is intimately linked to CD154 expression in B cells, and that the IL-10(+)CD154(+) B cell subset increases abnormally when SLE-derived cells are stimulated with CSS.     

Aberrant expression of the costimulatory molecule CD40 ligand on monocytes from patients with systemic lupus erythematosus

CD40 ligand (CD40L, CD154) is overexpressed on T and B cells in systemic lupus erythematosus (SLE). Monocytes have been shown to contribute to immune-mediated pathology in SLE and to express CD40L under certain conditions. Therefore, we studied CD40L expression on lupus monocytes ex vivo, as well as after activation in vitro. A highly significant sevenfold increase in the frequency of CD40L-expressing peripheral monocytes from 23 SLE patients, compared to 16 healthy individuals (mean percentage of CD40L(+)CD14(+) among CD14(+) cells, 11.7 versus 1.6), was found by flow cytometry. Increased CD40L expression on monocytes correlated significantly with disease activity, elevated gamma-globulin serum levels, as well as increased CD40L expression on T cells. CD40L expression by lupus monocytes was verified at both the mRNA and protein levels, while LPS stimulation was found to upregulate CD40L mRNA accumulation and surface protein expression. CD40L expression on activated lupus monocytes within anti-CD3-stimulated, mononuclear cell cultures was also enhanced compared to control-derived monocytes. These novel findings underscore the multiplicity of pathways through which monocytes may contribute to SLE pathology and suggest that T cell-independent CD40L-mediated cell to cell interactions may be also involved in humoral immune activation in SLE.     

CD38 polymorphisms in Spanish patients with systemic lupus erythematosus

The ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (CD38) gene is a positional and functional candidate gene for the susceptibility to systemic lupus erythematosus (SLE) because CD38 gene maps in the described SLE risk region 4p15 and CD38 molecule is a leukocyte activation antigen and ectoenzyme involved in numerous immune functions. The aim of this study was to investigate the possible association of the polymorphisms located at positions 182 of intron 1 (C/G) and 418 (C/T, located in exon 3) of the CD38 gene with the susceptibility and clinical features of SLE. Genotyping of 276 Spanish patients with SLE and 194 controls was performed by polymerase chain reaction amplification-refractory mutation system techniques. No association between the polymorphisms studied and the susceptibility to SLE was found. However, when patients were stratified according to their clinical manifestations, a significant increase of CC individuals and a significant decrease of CG individuals among patients with discoid rash (67.9% vs. 53.1% in controls p = 0.02, pc > 0.05, odds ratio [OR] = 1.87, 95% confidence interval [95% CI] 1.05-3.35; and 23.5% vs. 40.2% in controls, p = 0.008, pc = 0.024, OR = 0.46 95% CI 0.24-0.85) were found. Logistic regression analysis identified CC genotype as an independent risk factor for discoid rash among patients with SLE (p = 0.01, OR = 2.23, 95% CI 1.19-4.18). In conclusion, a slight contribution of the polymorphism located in intron 1 of the CD38 gene in the clinical features of SLE could be postulated.     

Mitogenic responses to lipopolysaccharide by B lymphocytes from patients with systemic lupus erythematosus

Peripheral blood lymphocytes from 43 patients with systemic lupus erythematosus (SLE) and from age- and sex-matched normal controls were cultured with lipopolysaccharide (LPS) to examine the response to the polyclonal B-cell activator. Lymphocytes from active SLE patients incorporated 4840 +/- 471 (mean +/- SE) cpm in response to LPS, whereas lymphocytes from inactive SLE patients incorporated 6906 +/- 897 cpm. In contrast, lymphocytes from normal individuals incorporated 7452 +/- 1126 cpm. Ig synthesis of lymphocytes from active SLE in response to LPS stimulation was also less than that of normal individuals. The helper T-cell function of active SLE, as examined by co-culturing irradiated SLE lymphocytes with unirradiated normal lymphocytes, was normal. These results thus suggested that a defect of B lymphocytes exists in active SLE patients. This B-cell defect and T suppressor cells apparently play an important role in the pathogenesis of SLE.     

系统性红斑狼疮患者的Th2偏移不受CD40/CD154影响

目的 探讨系统性红斑狼疮(SEE)患者外周血Th极化状况的改变以及其受CD40/CD154和地塞米松(Dex)的影响.方法 使用ELISA法检测SEE患者和对照组血浆IFN-γ和IL-10水平,分离、培养外周血单个核细胞(PBMC),应用CD40 mAb和Dex进行干预,使用ELISA法检测培养液上清IFN-γ和IL-10水平.结果 SLE患者血浆IFN-γ水平明显低于对照组(P<0.01,P<0.05),活动期组血浆IL-10水平明显高于缓解期组和对照组(均P<0.01);CD40 mAb对PBMC培养液上清IFN-γ和IL-10水平无影响(均P>0.05);Dex可使SLE患者PBMC培养液上清IFN-γ水平升高(P<0.05,P<0.01),Dex可以明显降低SEE患者和对照组PBMC培养液上清IL-10水平(均P<0.05).结论 SEE患者血浆IFN-γ水平降低、IL-10水平增高,存在Th2偏移;此种改变不受CD40/CD154影响但能被Dex抑制.     

B cells and atherosclerosis in systemic lupus erythematosus

Introduction: Cardiovascular (CV) events, as a result of accelerated atherosclerosis, are an important cause of mortality in patients with Systemic lupus erythematosus (SLE). The etiology of SLE is multifactorial and still unclear; among other potential culprits, excessive B cell activation seems to play a crucial role. Accumulating evidence supports a contributory role of B cells in the pathogenesis of atherosclerosis as well.

Areas covered: This article focuses on the contribution of both B cells and autoantibodies in the pathogenesis of atherosclerosis in both general and lupus populations. Review of the published literature on experimental models has also been performed.

Expert opinion: Distinct B cell subsets seem to exhibit separate effects on the progression of atherosclerosis, with B2 B cells displaying a mainly atherogenic phenotype, while B1 B cells are mostly viewed as atheroprotective. Selective B2 inhibition by anti-B cell therapies seems a promising therapeutic strategy against atherosclerosis development in the setting of lupus.     

Regulatory T cells in patients with systemic lupus erythematosus

Regulatory T cells have an important role in the control of self-reactivity, and in the pathogenesis of autoimmune inflammatory conditions. The aim of this work was to perform a quantitative and functional analysis of regulatory T cells in patients with systemic lupus erythematosus (SLE). We studied twenty-three patients with SLE (19 active, 4 inactive), and twenty-seven healthy subjects as well as fifteen patients with rheumatoid arthritis (RA). The following cell subsets were analyzed in peripheral blood mononuclear cells by flow cytometry: CD4+CD25+, CD4+CD25(bright), CD4+Foxp3+ (Treg cells), CD8+CD28- (Ts cells), CD4+IL-10+ (Tr1 cells), and CD4+TGF-beta+ (Th3 cells). In addition, the in vitro suppressive activity of CD4+CD25+ lymphocytes was tested. We found no significant differences in the levels of all regulatory cell subsets studied in SLE patients compared to controls and RA patients. However, a defective regulatory function of CD4+CD25+T cells was observed in a significant fraction (31%) of patients with SLE. Our data indicate that although approximately one third of patients with SLE show an abnormal immunosuppressive function of Treg lymphocytes, their levels of the different regulatory T cell subsets in peripheral blood are not significantly different from those found in controls.     

HLA-DP-positive T cells in patients with systemic lupus erythematosus

HLA-DP+ T cells in peripheral blood from 23 patients with systemic lupus erythematosus (SLE) were examined using two-colour flow cytometry analysis. A marked increase of HLA-DP+ T cells was observed in patients with SLE (20.5-98.7%; 59.8 +/- 20.8%) in comparison to normal subjects (1.3-20.6%; 11.1 +/- 7.2%), and the ratio of these cells greatly exceeded that of the HLA-DR+ T cells (6.5-49.1%; 21.5 +/- 12.7%). This high frequency of HLA-DP+ T cells in patients with active SLE decreased with prednisolone therapy. When the lymphocytes from normal subjects were stimulated with PHA in vitro, HLA-DP+ T cells increased from 1.8 to 59.2%. Therefore, it appears that the HLA-DP antigen expression on T cells is a practical marker for monitoring changes in the proportion of activated T cells in patients with SLE during the course of therapy.     

Nuclear deposits of immunoglobulins in skin of patients with systemic lupus erythematosus.

Skin nuclear speckled IgG deposition was noted in seven patients. The patients' clinical courses satisfied at least four of the preliminary criteria of the American Rheumatism Association for the diagnosis of systemic lupus erthematosus (SLE). Examination of approximately 700 additional biopsies from patiets with SLE, connective tissue and various skin diseases as well as normal individuals, failed to demonstrate similar nuclear immunoglobulin deposition. Sera from these seven patients had higher titre, complement fixing anti-nuclear antibodies (ANA) of IgG class which produced a speckled nuclear fluorescent pattern. In addition, the sera of all seven patients demonstrated by gel double diffusion precipitating antibodies against nuclear ribonucleoprotein (RNP) or Sm antigens.