Interferon‐α accelerates murine systemic lupus erythematosus in a T cell–dependent manner

Objective

To investigate the mechanism by which interferon‐α (IFNα) accelerates systemic lupus erythematosus (SLE) in (NZB × NZW)F₁ (NZB/NZW) mice.

Methods

NZB/NZW mice were treated with an adenovirus expressing IFNα. In some mice, T cells were depleted with an anti‐CD4 antibody. The production of anti–double‐stranded DNA (anti‐dsDNA) antibodies was measured by enzyme‐linked immunosorbent assay and enzyme‐linked immunospot assay. Germinal centers and antibody‐secreting cells (ASCs) in spleens and IgG deposition and leukocyte infiltrates in kidneys were visualized by immunofluorescence staining. The phenotype of splenic cells was determined by flow cytometry. Finally, somatic hypermutation and gene usage in V_H regions of IgG2a and IgG3 were studied by single‐cell polymerase chain reaction.

Results

IFNα‐accelerated lupus in NZB/NZW mice was associated with elevated serum levels of IgG2 and IgG3 anti‐dsDNA antibodies and accumulation of many IgG ASCs in the spleen, which did not develop into long‐lived plasma cells. Furthermore, IgG2a and IgG3 antibodies in the mice were highly somatically mutated and used distinct repertoires of V_H genes. The induction of SLE in the mice was associated with an increase in B cell Toll‐like receptor 7 expression, increased serum levels of BAFF, interleukin‐6 (IL‐6), and tumor necrosis factor α, and induction of T cells expressing IL‐21. Although IFNα drove a T cell–independent increase in serum levels of IgG, autoantibody induction and the development of nephritis were both completely dependent on CD4+ T cell help.

Conclusion

These findings demonstrate that, although IFNα activates both innate and adaptive immune responses in NZB/NZW mice, CD4+ T cells are necessary for IFNα‐driven induction of anti‐dsDNA antibodies and clinical SLE.

     

Treatment of murine lupus using nucleosomal T cell epitopes identified by bone marrow–derived dendritic cells

Objective

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against intact nuclear structures, such as nucleosomes. The most prominent of these autoantibodies are those directed against double‐stranded DNA (dsDNA) and histones. The majority are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. Much evidence suggests that the nucleosome is a major candidate autoantigen in SLE. In this study, a novel strategy was used to identify the critical CD4+ T cell autoepitopes in nucleosomes. In addition, peptide‐based therapy was then performed in a lupus animal model.

Methods

Bone marrow (BM)–derived dendritic cells (DCs) were used to examine the self–T cell responses against nucleosomes and to characterize the T cell epitope(s) of nucleosomes in (NZB × NZW)F₁ (BWF₁) mice.

Results

Several potential auto–T cell epitopes of core histone proteins (H2A, H2B, H3, and H4) were identified. Nucleosome‐pulsed BM‐derived DCs elicited release of interleukin‐4 and interferon‐γ, representing a Th0 (i.e., mixed Th1 and Th2) pattern of cytokine production. In addition, intradermal immunization of BWF₁ mice with the H3^111–130 peptide not only suppressed the level of anti‐dsDNA and anti–single‐stranded DNA IgG, but also significantly delayed the progress of glomerulonephritis in lupus‐prone BWF₁ mice.

Conclusion

These results will help in understanding how pathogenic autoimmune responses develop in spontaneous SLE. This may potentially open the way to T cell–based immunotherapy for lupus.

     

Interferon‐α–inducible proteins are novel autoantigens in murine lupus

Objective

To investigate the spectrum of B cell autoimmunity in the recently described anti‐CD1–autoreactive T cell receptor (TCR)–transgenic murine lupus‐like (CD1 lupus‐like) model.

Methods

Lethally irradiated BALB/c/nu/nu mice were injected intravenously with donor BALB/c bone marrow and spleen cells expressing TCRα and TCRβ transgenes that recognize CD1d. Sera from adoptive host animals that developed lupus (i.e., CD1 lupus mice) were collected at serial time points and analyzed by Western blotting and immunoprecipitation, using protein extracts prepared from NIH3T3 mouse fibroblasts and EL‐4 lymphocytes, respectively. Sera obtained from older animals in several models of spontaneous lupus (NZB/NZW, MRL++, and MRL/lpr mice), unmanipulated BALB/c/nu/nu mice, and normal BALB/c mice were used as controls.

Results

Analyses demonstrated that the prominent targets of autoantibodies in the CD1 lupus‐like model are interferon‐α (IFNα)–inducible antigens. Biochemical and serologic characterizations identified one antigen as belonging to the interferon‐inducible 202 (Ifi202) subfamily of proteins within the Ifi200 family, and a second antigen as a member of the 70‐kd heat‐shock protein family. Autoantibodies directed against these antigens were rapidly produced at an early stage of disease. Anti‐p50 autoantibodies were present in sera from 7 (78%) of 9 CD1 lupus mice that developed severe kidney disease.

Conclusion

IFNα‐inducible proteins represent a novel class of autoantigens in murine lupus, and the findings suggest additional roles for IFNα in this disease. Since Ifi202 autoantigens are encoded by the murine non–major histocompatibility complex lupus‐susceptibility gene locus Ifi202, these data provide a link between recent advances in lupus genetics and the formation of autoantibodies.

     

Impairment of endothelial cell differentiation from bone marrow–derived mesenchymal stem cells: New insight into the pathogenesis of systemic sclerosis

Objective

Systemic sclerosis (SSc) is a disorder characterized by vascular damage and fibrosis of the skin and internal organs. Despite marked tissue hypoxia, there is no evidence of compensatory angiogenesis. The ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells was recently demonstrated. The aim of this study was to determine whether impaired differentiation of MSCs into endothelial cells in SSc might contribute to disease pathogenesis by decreasing endothelial repair.

Methods

MSCs obtained from 7 SSc patients and 15 healthy controls were characterized. The number of colony‐forming unit–fibroblastoid colonies was determined. After culture in endothelial‐specific medium, the endothelial‐like MSC (EL‐MSC) phenotype was assessed according to the surface expression of vascular endothelial growth factor receptors (VEGFRs). Senescence, chemoinvasion, and capillary morphogenesis studies were also performed.

Results

MSCs from SSc patients displayed the same phenotype and clonogenic activity as those from controls. In SSc MSCs, a decreased percentage of VEGFR‐2+, CXCR4+, VEGFR‐2+/CXCR4+ cells and early senescence was detected. After culturing, SSc EL‐MSCs showed increased expression of VEGFR‐1, VEGFR‐2, and CXCR4, did not express CD31 or annexin V, and showed significantly decreased migration after specific stimuli. Moreover, the addition of VEGF and stromal cell–derived factor 1 to cultured SSc EL‐MSCs increased their angiogenic potential less than that in controls.

Conclusion

Our data strongly suggest that endothelial repair may be affected in SSc. The possibility that endothelial progenitor cells could be used to increase vessel growth in chronic ischemic tissues may open up new avenues in the treatment of vascular damage caused by SSc.

     

系统性红斑狼疮患者骨髓间质干细胞对B淋巴细胞免疫抑制作用的初步研究

目的：探讨系统性红斑狼疮（SLE）患者骨髓间质干细胞（MSCs）体外对B淋巴细胞的免疫调节作用是否存在异常。方法：采用直接贴壁法分离培养正常人及SLE患者MSCs。免疫磁珠法分选正常人外周血B淋巴细胞,与MSCs共培养,流式细胞术检测B细胞凋亡及表面标志表达,3H掺入法检测B细胞增殖,酶联免疫吸附法（ELISA）检测培养上清中免疫球蛋白IgG、IgM和IgA水平。结果：①B细胞＋正常MSCs共培养组B细胞增殖（4307±308）cpm较单纯B细胞培养组（7059±346）cpm降低（P〈0.05）;②B细胞＋正常MSCs共培养组B细胞凋亡率（14±7）%低于单纯B细胞培养组（25±8）%（P〈0.05）;③B细胞＋正常MSCs共培养组B细胞表面CD86表达率（53±5）%较单纯B细胞培养组（66±10）%低（P〈0.05）;④与MSCs共培养后,B细胞分泌免疫球蛋白IgG、IgM和IgA较单纯B细胞培养显著减少（P〈0.05）;⑤B细胞＋狼疮MSCs共培养组与B细胞＋正常MSCs共培养组比较,B细胞增殖、凋亡、CD86表达和Ig分泌两组间差异无统计学意义。结论：MSCs在体外对B细胞有抑制作用,SLE患者骨髓MSCs与正常MSCs相比,对B细胞的表面刺激分子表达、增殖、Ig分泌有相似的抑制作用。     

Abnormality of bone marrow-derived mesenchymal stem cells in patients with systemic lupus erythematosus

Bone marrow-derived mesenchymal stem cells (MSCs) are key components of the hematopoietic microenvironment and provide support to hematopoiesis and modulate immune system. Several studies suggest that SLE may be seen as stem cell disorders. However, it is unclear that whether MSCs from SLE patients are defective. So in this research, we studied the biological character of bone marrow derived MSCs in patients with SLE, focused on their phenotype (morphology and immunophenotype), karyotype, cytokines expression and hematopoietic support of MSCs. Our results showed that MSCs from SLE patients and normal controls can be successfully culture-expanded, but the MSCs from SLE grew more slowly than those of normal controls (P < 0.05). Cells from both groups were positive for CD29, CD44 and CD105, and negative for CD14, CD34, CD45 and HLA-DR. MSCs from SLE have a normal karyotype. Both groups express IL-6, IL7, IL-11, macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) at mRNA level. While IL-6 and IL-7 were down-regulated in MSCs from SLE patient (P < 0.05) at mRNA level. The MSCs from SLE patients and normal controls were infused into ICR (Tac: Icr: Ha strain) mice after high-dose chemotherapy, with no adverse events in either group. Recovery of white blood cells, hemoglobin and platelet was more rapid (P < 0.05) compared with the group without MSCs infusion. We conclude that MSCs in patient with SLE have abnormalities compared with those in normal control. MSCs in patient with SLE may play an important role in the SLE pathogenesis.     

异基因骨髓间充质干细胞移植治疗系统性红斑狼疮11例临床分析

目的 探讨异基因骨髓问充质干细胞(MSC)移植治疗难治性系统性红斑狼疮(SLE)的疗效及安全性.方法 血缘相关供者骨髓中分离培养MSC移植治疗11例SLE患者,移植前予环磷酰胺(CTX)0.8～1.8 g,分2～3次静脉输注,输注后隔日予MSC移植,移植细胞数(1×106/kg体质量).评价患者移植前后的临床表现和实验室检查指标的改变.结果 异基因MSC移植后,11例SLE患者随访1～13个月,所有患者均无移植相关并发症.移植后1个月SLE疾病活动评分(SLEDAI)由12±5降低为6±3(11例,P＜0.01).尿蛋白定量(24 h)移植后1个月由(1989±842)mg(518～3690 mg)降低为(1118±700)mg(10例,P＜0.05);移植后2个月[(2478±797)mg vs (924±659)mg,5例,P＜0.05];移植后6个月[(2478±797)mg vs(522±151)mg,5例,P＜0.01].2例肾功能不全患者血肌酐轻度下降.5例低蛋白血症患者移植后1个月血清白蛋白上升[(28±6)g/L vs(32±7)g/L,5例,P＜0.05].移植后1个月血清补体C3升高[(0.50±0.12)g/L vs(0.75±0.10)g/L,9例,P＜0.01],抗核抗体(ANA)滴度降低.结论 异基因MSC移植治疗难治性SLE有效安全.MSC取材方便,扩增迅速,输注安全经济.异基因MSC移植治疗SLE的长期疗效评估需进一步随访观察.     

Coordinate overexpression of interferon‐α–induced genes in systemic lupus erythematosus

Objective

To study the contribution of interferon‐α (IFNα) and IFNγ to the IFN gene expression signature that has been observed in microarray screens of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE).

Methods

Quantitative real‐time polymerase chain reaction analysis of healthy control PBMCs was used to determine the relative induction of a panel of IFN‐inducible genes (IFIGs) by IFNα and IFNγ. PBMCs from 77 SLE patients were compared with those from 22 disease controls and 28 healthy donors for expression of IFIGs.

Results

Expression of IFNα‐inducible genes was significantly higher in SLE PBMCs than in those from disease controls or healthy donors. The level of expression of all IFIGs in PBMCs from SLE patients with IFNα pathway activation correlated highly with the inherent responsiveness of those genes to IFNα, suggesting coordinate activation of that cytokine pathway. Expression of genes preferentially induced by IFNγ was not significantly increased in SLE PBMCs compared with control PBMCs. IFNα‐regulated gene‐inducing activity was detected in some SLE plasma samples.

Conclusion

The coordinate activation of IFNα‐induced genes is a characteristic of PBMCs from many SLE patients, supporting the hypothesis that IFNα is the predominant stimulus for IFIG expression in lupus.

     

Tγ cells in systemic lupus erythematosus

Circulating T cells bearing receptors for the Fcportion of IgG (Tγ) were identified by sensitive immunofluorescent techniques with rabbit IgG b4 allotype/anti-b4 complexes. A twofold decrease in both proportion and absolute number of Tγ cells was found in patients with active systemic lupus erythematosus (SLE) relative to values obtained during disease remission. The reduction in Tγ cells was most evident in patients with severe hypocomplementemia. A deficit of Tγ cells in active patients was not demonstrated. The percentage of total T cells rosetting with sheep erythrocytes was reduced in peripheral blood of most patients regardless of disease activity status, but particularly during SLE exacerbation. Cells lacking intrinsic surface immunoglobulin, IgG Fc receptors, and receptors for sheep erythrocytes were increased. These cells, operationally termed null. exhibited an inverse linear relationship with T cells that was not apparent in regression analyses performed against other lymphocyte subpopulations. Such differences were not found for B cells and IgG receptor-bearing non-B/non-T cells which were present in normal proportions in virtually all patients. The origin and functional significance of these unusual lymphocyte subpopulation abnormalities are discussed.     

脐带间充质干细胞移植治疗系统性红斑狼疮的临床分析

目的 探讨脐带间充质干细胞(UC-MSCs)移植治疗重症难治性系统性红斑狼疮(SLE)的疗效及安全性.方法 UC-MSCs移植治疗12例SLE患者,评价患者移植前后临床表现和实验室检查指标的变化.结果 UC-MSCs移植后,12例SLE患者随访1～26个月.移植后1个月SLE疾病活动指数(SLEDAI)由(18±4)分降低为(10±4)分(12例,P＜0.01),3个月时持续降为(7±4)分(9例).UC-MSCs移植后1个月尿蛋白定量(24 h)由(3359±1248)mg降低为(2103±749)mg(9例,P＜0.01),移植后3个月[(1427±616)mg与(3342±1333)mg,8例,P＜0.01],2例患者随访至26个月尿蛋白持续下降.5例肾功能不全者血清肌酐由移植前(416±250)μmol/L降至移植后1个月(264±119)μmol/L(P＜0.05);移植后3个月降至(226±103)μmol/L,与移植前比差异有统计学意义(P＜0.05).10例低蛋白血症患者移植后1个月血清白蛋白显著上升[(24±4)g/L与(29±3)g/L,P=0.01],3个月上升至(30±4)g/L.4例患者移植后血小板显著上升,3例患者移植后血清补体C3升高.所有患者均无移植相关并发症.结论 UC-MSCs移植治疗重症SLE安全有效,长期疗效需进一步随访观察.     

Effects of anti‐CD154 treatment on B cells in murine systemic lupus erythematosus

Objective

To determine the immunologic effects of anti‐CD154 (CD40L) therapy in the (NZB × NZW)F₁ mouse model of systemic lupus erythematosus.

Methods

Twenty‐week‐old and 26‐week‐old (NZB × NZW)F₁ mice were treated with continuous anti‐CD154 therapy. Mice were followed up clinically, and their spleens were studied at intervals for B and T cell numbers and subsets and frequency of anti–double‐stranded DNA (anti‐dsDNA)–producing B cells. T cell–dependent immunity was assessed by studying the humoral response to the hapten oxazolone.

Results

IgG anti‐dsDNA antibodies decreased during therapy and disease onset was delayed, but immune tolerance did not occur. During treatment, there was marked depletion of CD19+ cells in the spleen; however, autoreactive IgM‐producing B cells could still be detected by enzyme‐linked immunospot assay. In contrast, few IgG‐ and IgG anti‐dsDNA–secreting B cells were detected. Eight weeks after treatment cessation, the frequency of B cells producing IgG anti‐dsDNA antibodies was still decreased in 50% of the mice, and activation and transition of T cells from the naive to the memory compartment were blocked. Anti‐CD154 treatment blocked both class switching and somatic mutation and induced a variable period of relative unresponsiveness of IgG anti‐dsDNA–producing B cells, as shown by decreased expression of the CD69 marker and failure to generate spontaneous IgG anti‐dsDNA–producing hybridomas. Treated mice mounted an attenuated IgM response to the hapten oxazolone and produced no IgG antioxazolone antibodies.

Conclusion

Anti‐CD154 is a B cell–depleting therapy that affects multiple B cell subsets. Activation of both B and T cells is prevented during therapy. After treatment cessation, autoreactive B cells progress through a series of activation steps before they become fully competent antibody‐producing cells. The general immunosuppression induced during treatment will need to be taken into account when using B cell–depleting regimens in humans.

     

Reversal of serologic,immunologic, and histologic dysfunction in mice with systemic lupus erythematosus by long‐term serial adipose tissue–derived mesenchymal stem cell transplantation

Objective

To investigate the efficacy of human adipose tissue–derived mesenchymal stem cell (AD‐MSC) transplantation in systemic lupus erythematosus (SLE) and to determine the optimal transplantation window for stem cells either before or after disease onset.

Methods

(NZB × NZW)F₁ mice with SLE were administered human AD‐MSCs (5 × 10⁵) intravenously every 2 weeks from age 6 weeks until age 60 weeks, while the control group received saline vehicle on the same schedule. Another experiment was carried out with a different initiation time point for serial transplantation (age 6 weeks or age 32 weeks).

Results

Long‐term serial administration (total of 28 times) of human AD‐MSCs ameliorated SLE without any adverse effects. Compared with the control group, the human AD‐MSC–treated group had a significantly higher survival rate with improvement of histologic and serologic abnormalities and immunologic function, and also had a decreased incidence of proteinuria. Anti–double‐stranded DNA antibodies and blood urea nitrogen levels decreased significantly with transplantation of human AD‐MSCs, and serum levels of granulocyte–macrophage colony‐stimulating factor, interleukin‐4 (IL‐4), and IL‐10 increased significantly. A significant increase in the proportion of CD4+FoxP3+ cells and a marked restoration of capacity for cytokine production were observed in spleens from the human AD‐MSC–treated group. In the second experiment, an early stage treatment group showed better results (higher survival rates and lower incidence of proteinuria) than an advanced stage treatment group.

Conclusion

Serial human AD‐MSC transplantation had beneficial effects in the treatment of SLE, without adverse effects. Transplantation of human AD‐MSCs before disease onset was preferable for amelioration of SLE and restoration of immune homeostasis.

     

Anti–β2‐glycoprotein I antibodies in pediatric systemic lupus erythematosus and antiphospholipid syndrome

Objective

To determine whether serum β₂‐glycoprotein I antibody (anti‐β₂GPI) detection improves identification of pediatric subjects at risk for antiphospholipid syndrome (APS).

Methods

Serum antiphospholipid antibodies (aPL) were identified by anticardiolipin enzyme‐linked immunosorbent assay (ELISA), lupus anticoagulant assays, and syphilis screening in children with primary APS, systemic lupus erythematosus (SLE), or SLE plus APS. Anti‐β₂GPI level and isotype were determined by β₂GPI ELISA and correlated with clinical manifestations and other aPL assays.

Results

One hundred‐ten subjects under 22 years of age and of mixed ethnicity were evaluated. Fifty‐seven had SLE (including 14 with APS), 25 had primary APS, 16 had SLE‐like APS, 6 were healthy children with aPL detected incidentally, 4 had other rheumatic diseases and 2 had other conditions. Anti‐β₂GPI were detected in 48% of SLE subjects and did not improve aPL detection over standard tests. Anti‐β₂GPI were associated with stroke (P = 0.014), but not with other APS manifestations, and were rarely detected in primary APS. Among subjects with APS manifesting as chronic thrombocytopenia, anti‐β₂GPI distinguished subjects with SLE from those with primary APS.

Conclusions

With the exception of stroke, anti‐β₂GPI detection does not improve identification of pediatric APS over that of traditional aPL assays. Anti‐β₂GPI are rare in pediatric primary APS, but may predict evolution of chronic thrombocytopenia to SLE.

     

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

Objective

To investigate interferon‐γ (IFNγ) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNγ receptor (IFNγR) expression, STAT‐1 expression and phosphorylation, and the regulation of IFNγ‐inducible genes.

Methods

Fluorocytometry was used to investigate expression of STAT‐1, pSTAT‐1, CD95, HLA–DR, class I major histocompatibility complex (MHC), IFNγ‐inducible 10‐kd protein (IP‐10), monokine induced by IFNγ (Mig), and IFNγR in PBMCs from SLE patients and healthy individuals. STAT‐1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNα or IFNγ. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNγ‐inducible genes IP‐10 and Mig shortly after preparation or after stimulation with IFNγ in monocytes.

Results

STAT‐1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN‐inducible expression of CD95 and HLA–DR. STAT‐1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNγ‐inducible genes, such as IP‐10 or Mig, was increased in SLE monocytes. While STAT‐1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNα stimulation, incubation with IFNγ induced STAT‐1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT‐1 expression upon IFNγ stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNγ was also confirmed on the mRNA level, where expression of the IFN‐inducible, STAT‐1–dependent genes IP‐10 and Mig was more efficiently increased in SLE cells. However, IFNγR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.

Conclusion

In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNγ in this disease.