Increased serum IL-10 in lupus patients promotes apoptosis of T cell subsets via the caspase 8 pathway initiated by Fas signaling

We sought to investigate the expression of Fas and FasL on T cell surface and caspase 8 involvement in T cell apoptosis promoted by serum IL-10 in systemic lupus erythematosus(SLE) patients.Cells and sera were obtained from 35 SLE patients.Apoptosis of T cells in patients with SLE was increased and associated with the SLE disease activity index(SLEDAI).Elevated expression of Fas and FasL on T cell surface contributed to increased apoptosis of T cells.Increased IL-10 in the sera of SLE patients was capable of inducing Fas and FasL expression on CD4~+T cell surface,promoting apoptosis of this cell subset.Decreased IL-10 serum levels and low expression of Fas were found in 5 patients of the first follow-up group after 2-month treatment.In another group with one-year treatment,the SLEDAI declined to inactive scores.Serum IL-10 was decreased significantly,and expression of Fas and FasL on T cells was also reduced.Declined apoptosis was predominant only in CD4~+T cell subset.When sera with high level of IL-10 were used to culture PBMCs from healthy controls,activated caspase 8 was elevated in CD3~+T,CD4~+T and CD8~+T cells.The study showed that serum IL-10 induced apoptosis of T cell subsets via the caspase8 pathway initiated by Fas signaling.Increased apoptosis of T cells contributes to autoantigen burden,which is pathogenic in the development of SLE.     

系统性红斑狼疮患者血清IgG对骨髓造血干/祖细胞抑制和凋亡的研究

目的：观察系统性红斑狼疮（SLE）患者血清IgG类抗体体外造血抑制活性及其与造血细胞过度凋亡的关系.方法：应用甲基纤维素集落培养法、原位末端标记及流式细胞术观察系统性红斑狼疮患者血清IgG在体外抑制正常骨髓造血干/祖细胞的增殖及促进正常CD34＋细胞的凋亡.结果：活动期SLE患者血清IgG体外可明显抑制正常骨髓粒-巨噬系祖细胞（CFU-GM）和红系祖细胞（CFU-E）的增殖.这种IgG可特异吸附于正常CD34＋细胞表面,加速CD34＋细胞凋亡,上调CD34＋细胞Fas抗原表达水平.结论：活动期SLE患者血清IgG在体外可抑制正常骨髓造血干/祖细胞的增殖.机制与其上调CD34＋ Fas抗原表达水平而促进CD34＋细胞凋亡有关.     

Diminished expression of CD59 on activated CD8+ T cells undergoing apoptosis in systemic lupus erythematosus and Sjögren's syndrome

The present study was undertaken to examine the phenotype of T cells undergoing in vitro apoptosis in patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS). Compared with normal controls, we found diminished expression of CD59 antigen (one of the cell‐surface complement‐regulatory proteins) on CD8⁺ T cells, but not on CD4⁺ T cells, from patients with SLE and SS. Three‐colour immunofluorescence analysis revealed that these CD59^dim CD8⁺ T cells were activated T cells, expressing both human leucocyte antigen (HLA)‐DR and CD45RO antigens. In addition, these CD59^dim CD8⁺ T cells were more susceptible to in vitro apoptosis than CD59^bright CD8⁺ T cells. In two patients with active lupus, the percentage of CD59^dim CD8⁺ T cells was significantly decreased after steroid therapy. These findings suggest that decreased expression of CD59 antigen on in vivo‐activated CD8⁺ T cells may be correlated with disease activity and may be involved in activation‐induced apoptosis in patients with SLE and SS.     

Soluble Fas molecule in the serum of patients with systemic lupus erythematosus

The serum level of soluble Fas (sFas) molecules in 35 patients with SLE was determined by enzyme-linked immunosorbent assay (ELISA) and its relation to other lymphocyte activation markers and clinical parameters was examined. The level of sFas increased significantly compared to that in normal subjects, consistent with previous reports. There was a significant correlation between the level of sFas and that of sCD4, suggesting some relation between sFas and activation of CD4⁺ T cells. Patients with lymphopenia tended to have low levels of sFas, making it possible to hypothesize that sFas protects against apoptosis. Although the change in the level of sFas with steroid therapy was variable, some relation to the differential activation of T cell subsets was suggested.     

Elevated soluble Fas/APO-1 (CD95) levels in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours

Soluble Fas (sFas) is produced as translation products of alternative mRNA splicing, and antagonizes the membranous Fas molecule in Fas/Fas ligand interactions. We investigated the serum sFas levels in 64 Japanese silicosis patients with no clinical symptoms of autoimmune diseases or malignant tumours, using ELISA for sFas. The serum sFas levels in the silicosis patients were significantly higher than those in healthy volunteers. Elevated serum sFas levels were also detected in patients with systemic lupus erythematosus but, unexpectedly, no difference was observed in sFas levels between progressive systemic sclerosis patients and healthy volunteers. On the other hand, there was no significant difference in the expression of Fas on peripheral blood lymphocytes between the patients with silicosis and age-matched healthy volunteers. These observations provided the first evidence that serum sFas levels are elevated in silicosis patients without clinical symptoms of autoimmune diseases or malignant tumours. It remains to be clarified whether patients with elevated sFas levels have a tendency to develop autoimmune diseases later, or whether some other distinct factor(s) is necessary to initiate the progression of autoimmune diseases.     

HLA DRB1*1503 allelic haplotype predominance and associated immunodysregulation in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, relapsing, and remitting disease affecting primarily African American females of child bearing age. Familial aggregation of this disease suggests that at least part of the susceptibility for this disease is genetic, although environmental and hormonal influences are also likely to play a role. Early studies of genetic susceptibility to SLE revealed several of the major histocompatibility complex molecules, namely HLA DR, to be linked to SLE. Meta-analysis of genome scans has yielded loci significant for lupus patients, one of which includes the MHC region.Regulatory T cells are immunoregulatory cells that modulate activated immune cells. These cells play a large role in homeostasis of the immune responses and maintenance of immunologic tolerance, i.e., prevention of autoimmunity. Decreased numbers of regulatory T cells have been described in many autoimmune diseases, including systemic lupus erythematosus.Autoantibody production in systemic lupus erythematosus and the resulting immune complex formation and complex deposition into tissues are arguably the central core of immune dysregulation leading to disease manifestations and symptoms. Inability of the immune system to recognize and inhibit autoreactive immune cells in this particular autoimmune disease may be the result of inappropriate numbers and function of regulatory T cells.This study aims to characterize the immune cell population in patients from our community suffering from systemic lupus erythematosus and to prove that these patients exhibit a unique cellular profile compared to healthy age, race and gender matched control subjects. Surprisingly, our findings demonstrate that patients from the local Mississippi area exhibit increased proportions of CD25⁺ FoxP3⁺ regulatory T cells and CD25⁺ FoxP3⁻ T cells (of CD45⁺ CD3⁺ CD4⁺ helper T cells) as compared to healthy controls.HLA tissue-typing of these lupus patients revealed a prominent subgroup (~ 30%) of patients possessing the HLA DRB1*1503 allele. The investigation of this subgroup demonstrated regulatory T cell composition similar to that of the total lupus group and to that of the non-HLA DRB1*1503 subgroup.Genetic analysis for molecular gene expression levels of various lupus-associated genes by real-time PCR demonstrated a unique profile as compared to healthy controls. Increased gene expression of FoxP3 together with decreased gene expression levels of GATA3, TNFAIP3, and TNFSF4 suggest that variations in gene products compared to healthy controls may be playing a role in the immune cell dysregulation and disproportionate CD25⁺ FoxP3⁺ regulatory T cells.     

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.     

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25^+/highCD127^Ø/lowFoxP3⁺, and effector T cells were defined as CD25⁺CD127⁺FoxP3^Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4⁺TREG and CD28⁺TREG cells and an increased frequency of CD40L⁺TREG cells. There was a decrease in the TREG/effector-T ratio for GITR⁺, HLA-DR⁺, OX40⁺, and CD45RO⁺ cells, and an increased ratio of TREG/effector-T CD40L⁺ cells in patients with SLE. In addition, CD40L⁺TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.     

Elevated Double Negative T Cells in Pediatric Autoimmunity

Purpose

Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated.

Methods

We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3⁺CD56^?TCRαβ⁺CD4^?CD8^?) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls.

Results

Sixteen cases (29.6 %) had elevated DNTs (≥2.5 % of CD3⁺CD56^?TCRαβ⁺ cells) compared to 1 (3.6 %) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls.

Conclusion

DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity.     

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.     

The IL-12-STAT4 axis in the pathogenesis of human systemic lupus erythematosus

Generation of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). As demonstrated in a number of lupus mouse models, recent evidence suggests that both GC and extrafollicular pathways contribute to the generation of autoantibodies also in human SLE, and that CD11c⁺ IgD⁻CD27⁻(double negative:DN) B cells play a central role in the latter pathway. In this mini-review, the author will first briefly summarize the features of CD11c⁺ DN B cells in human SLE, and discuss how the IL-12-STAT4 axis might contribute to the generation of autoantibodies in SLE. In addition, various types of CD4⁺ helper T cell subsets promoting the generation of autoantibodies in SLE will be described, and finally it will be discussed how these recent discoveries contribute to understanding of SLE pathogenesis and treatment of SLE patients.     

The associations of circulating CD4+CD25high regulatory T cells and TGF-β with disease activity and clinical course in patients with adult-onset Still's disease

Objective. To determine circulating levels of CD4⁺CD25^high regulatory T (Treg) cells and transforming growth factor-β (TGF-β) in patients with adult-onset Still's disease (AOSD) and to examine the associations with disease activity and clinical course of this disease. Methods. The frequencies of circulating CD4⁺CD25^high Treg cells in 52 active AOSD patients, 42 active systemic lupus erythematosus (SLE) patients, and 22 healthy controls (HCs) were determined using flow cytometry analysis. Levels of serum TGF-β and soluble interleukin-2 receptor (sIL-2R) were measured by enzyme-linked immunosorbent assay. Results. Significantly lower levels of circulating CD4⁺CD25^high Treg cells and serum TGF-β were found in AOSD patients and SLE patients than those found in HCs. Levels of circulating CD4⁺CD25^high Treg cells and TGF-β were inversely correlated with disease activity scores for AOSD patients and SLE patients. Circulating CD4⁺CD25^high Treg cell frequencies were positively correlated with serum TGF-β levels for patients with both diseases. Levels of circulating CD4⁺CD25^high Treg cells and TGF-β significantly increased, paralleling clinical remission and the decrease in levels of C-reactive protein and soluble interleukin-2 receptor after effective therapy in AOSD patients. AOSD patients with monocyclic course had significantly higher levels of circulating CD4⁺CD25^high Treg cells and TGF-β compared to those with polycyclic and chronic articular course. Conclusion. Diminished levels of circulating CD4⁺CD25^high Treg cells and TGF-β, and inverse correlation with disease activity in patients with AOSD and SLE might be involved in the pathogenesis of both diseases. Increased levels of circulating CD4⁺CD25^high Treg cells or TGF-β might be associated with a favorable clinical course in AOSD patients.     

Human umbilical cord derived mesenchymal stem cells promote interleukin‐17 production from human peripheral blood mononuclear cells of healthy donors and systemic lupus erythematosus patients

Inflammation instigated by interleukin (IL)‐17‐producing cells is central to the development and pathogenesis of several human autoimmune diseases and animal models of autoimmunity. The expansion of IL‐17‐producing cells from healthy donors is reportedly promoted by mesenchymal stem cells derived from fetal bone marrow. In the present study, human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) were examined for their effects on lymphocytes from healthy donors and from patients with systemic lupus erythematosus (SLE). Significantly higher levels of IL‐17 were produced when CD4⁺ T cells from healthy donors were co‐cultured with hUC‐MSCs than those that were cultured alone. Blocking experiments identified that this effect might be mediated partially through prostaglandin E₂ (PGE₂) and IL‐1β, without IL‐23 involvement. We then co‐cultured hUC‐MSCs with human CD4⁺ T cells from systemic lupus erythematosus patients. Ex‐vivo inductions of IL‐17 by hUC‐MSCs in stimulated lymphocytes were significantly higher in SLE patients than in healthy donors. This effect was not observed for IL‐23. Taken together, our results represent that hUC‐MSCs can promote the IL‐17 production from CD4⁺ T cells in both healthy donor and SLE patients. PGE₂ and IL‐1β might also be partially involved in the promotive effect of hUC‐MSCs.     

Analysis of FOXP3<Superscript>+</Superscript> regulatory T cell subpopulations in peripheral blood and tissue of patients with systemic lupus erythematosus

Regulatory T cells (Tregs) are critical mediators of immune tolerance, yet their involvement in the autoimmune disease systemic lupus erythematosus (SLE) is incompletely understood. We analyzed CD4⁺ T cell subpopulations with Treg-related phenotypes and their association with disease activity in peripheral blood (PB) and tissues of patients with SLE. In detail, we quantified subpopulations regarding CD25, FOXP3, CD62L, CCR6, CD27, CD45RA, and CD45RO expression in PB from 31 patients with SLE divided into two disease activity groups and 32 healthy controls using flow cytometry. CD4⁺ and FOXP3⁺ T cells in skin and kidney biopsies of patients with SLE were quantified by immunohistochemistry. CD4⁺CD25^+/++FOXP3⁺ and CD4⁺CD25⁺CD45RA^?/CD45RO⁺ T cell frequencies were significantly higher in PB from patients with active compared to inactive SLE. The fraction of CD4⁺CD25⁺⁺FOXP3⁺ Tregs and CD4⁺CD25⁺CD45RA⁺/CD45RO^? naïve Tregs was not significantly different between these groups. CD4⁺CD25⁺⁺ Tregs from active SLE patients comprised significantly less CD27⁺ cells and more CCR6⁺ cells compared to patients with inactive SLE. The percentage of CD4⁺FOXP3⁺ T cells among inflammatory infiltrates in skin and kidney biopsies of SLE patients was not different from other inflammatory skin/kidney diseases. In conclusion, although CD4⁺FOXP3⁺ T cell frequencies in the inflamed tissues of SLE patients were comparable to other inflammatory diseases, distinct T cell subpopulations appeared misbalanced in PB of patients with active SLE. Here, cells phenotypically resembling activated T cells, but not Tregs, were increased compared to patients with inactive SLE. Within Tregs of patients with active SLE, markers related to Treg function and homing were altered.     

Serum levels of soluble Fas ligand in patients with silicosis

Certain patients with silicosis have been reported to exhibit immunological abnormalities such as the appearance of antinuclear antibodies and the occurrence of autoimmune diseases. Fas ligand (FasL) is a type II membrane protein which induces apoptosis by binding to its membrane receptor, Fas. FasL is converted to a soluble form by a metalloproteinase-like enzyme. We have already found serum soluble Fas (sFas) levels in silicosis patients as well as in patients with systemic lupus erythematosus (SLE) to be significantly higher than those in healthy volunteers. To examine further the role of the Fas/FasL system in silica-induced immunological abnormalities, we investigated serum soluble FasL (sFasL) levels in silicosis patients with no clinical symptoms of autoimmune diseases, using ELISA for sFasL. Although the serum sFasL levels in patients with SLE were significantly higher than those in healthy volunteers and showed a slight positive correlation with serum sFas levels, those in silicosis patients exhibited no significant difference from those in healthy volunteers, and there was no correlation with serum sFas levels. However, sFasL levels were elevated in silicosis patients with slight dyspnoea or normal PCO2 among various clinical parameters of silicosis. It may be speculated that the immunological disturbances presented by the abnormalities of apoptosis-related molecules in silicosis patients do not occur with a similar degree of respiratory involvement. Further studies are required to clarify which kinds of factors are involved in silicosis patients who exhibit immunological abnormalities.     

Renal‐infiltrating CD11c+ cells are pathogenic in murine lupus nephritis through promoting CD4+ T cell responses

Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE), causing morbidity and mortality in 40–60% of SLE patients. The pathogenic mechanisms of LN are not completely understood. Recent studies have demonstrated the presence of various immune cell populations in lupus nephritic kidneys of both SLE patients and lupus‐prone mice. These cells may play important pathogenic or regulatory roles in situ to promote or sustain LN. Here, using lupus‐prone mouse models, we showed the pathogenic role of a kidney‐infiltrating CD11c⁺ myeloid cell population in LN. These CD11c⁺ cells accumulated in the kidneys of lupus‐prone mice as LN progressed. Surface markers of this population suggest their dendritic cell identity and differentiation from lymphocyte antigen 6 complex (Ly6C)^low mature monocytes. The cytokine/chemokine profile of these renal‐infiltrating CD11c⁺ cells suggests their roles in promoting LN, which was confirmed further in a loss‐of‐function in‐vivo study by using an antibody‐drug conjugate (ADC) strategy targeting CX₃CR1, a chemokine receptor expressed highly on these CD11c⁺ cells. However, CX₃CR1 was dispensable for the homing of CD11c⁺ cells into lupus nephritic kidneys. Finally, we found that these CD11c⁺ cells co‐localized with infiltrating T cells in the kidney. Using an ex‐ vivo co‐culture system, we showed that renal‐infiltrating CD11c⁺ cells promoted the survival, proliferation and interferon‐γ production of renal‐infiltrating CD4⁺ T cells, suggesting a T cell‐dependent mechanism by which these CD11c⁺ cells promote LN. Together, our results identify a pathogenic kidney‐infiltrating CD11c⁺ cell population promoting LN progression, which could be a new therapeutic target for the treatment of LN.     

Th22, but not Th17 Might be a Good Index to Predict the Tissue Involvement of Systemic Lupus Erythematosus

Purpose

T-helper (Th) cells abnormalities are considered to be associated with the pathogenesis of Systemic lupus erythematosus (SLE). Recently, The Th22 cells have been identified and implicated in the pathogenesis of autoimmune diseases such as Rheumatoid arthritis (RA), although therir role in Systemic lupus erythematosus (SLE) remains unclear. The present study intends to investigate their roles in SLE.

Methods

Clinical data were collected in 65 SLE patients and 30 healthy controls. The patients were divided into active and inactive groups. CD4⁺IFN-γ^?IL-17^?IL-22⁺Tcells (Th22 cells),CD4⁺ IFN-γ^?IL-22^?IL-17⁺T cells (Th17 cells),and CD4⁺ IFN-γ⁺ (Th1 cells) were assayed by flow cytometry. Serum interleukin-22 (IL-22) and IL-17 levels were measured by enzyme-linked immunosorbent assay.

Results

The main observation focused on increased Th22 cells in patients with sole lupus skin disease and decreased Th22 cells in patients with sole lupus nephritis. Likewise, concentrations of serum IL-22 were increased in patients with sole lupus skin disease, and decreased in patients with sole lupus nephritis. Additionally, there was a positive correlation between the percentage of Th22 cells and IL-22 production. The percentage of Th17 cells or concentration of serum IL-17 correlated positively with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI).

Conclusion

Th22 seems to be a more significant index to predict the tissue involvement of SLE than Th17, although Th17 may play a role in the activity of SLE.