Blood Of Systemic Lupus Erythematosus Patients Research Articles

Abnormal Ultrastructure of the Platelet Plasma Membrane in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the body’s immune system mistakenly attacks healthy tissue. It can affect the skin, joints, kidneys, brain, and other organs. Blood cells, including platelets, are also involved in SLE and contribute to the pathogenesis of disease. We studied ultrastructure of platelets isolated from the blood of SLE patients and found that the plasma membrane was rough and shaggy compared to the normally smooth cell surface. These changes in the membrane morphology increased with the disease severity and were more pronounced when SLE was associated with the antiphospholipid syndrome, suggesting that platelets are strongly affected by the immune reactions underlying SLE.

Expression of orphan receptor GPR15/BOB up-regulated in systemic lupus erythematous/crosstalk with pro-inflammatory cytokines

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease that has protean manifestations and follows a relapsing and remitting course. More than 90% of cases of SLE occur in women, frequently starting at childbearing age. The pattern of tissue injury in SLE is primarily related to the formation of the immune complexes deposits that can induce the inflammatory response by activation of adhesion molecules on endothelium, resulting in the recruitment of pro inflammatory leukocytes. The goal of this study was to investigate the expression of orphan receptor GPR15/BOB in the lesion/ peripheral blood of SLE patients and non-SLE patients. Messenger RNA expression was examined by reverse-transcription polymerase chain reaction (RT-PCR) and GPR15/BOB protein by immunofluorescence. Cytokines were analyzed from blood serum using enzyme linked immunoassay (ELISA) for IL-6 and INF-α. Further; GPR15/BOB expression on peripheral blood leukocytes was analysed by flow cytometry. GPR15/BOB (P=0.002) expression levels were significantly increased in lupus patients compared to age-matched controls. African American women with lupus had a 2-fold increase in GPR15/BOB expression level when compared to their healthy controls or European American lupus patients. Serum levels revealed significant increases in expression of IL-6, IFN-γ and TNF-α in lupus patients with expression of GPR15/BOB compared to non-expression of GPR15/BOB -lupus patients. This study shows expression of GPR15/BOB is up-regulation in SLE patients and provides an opportunity for targeting with antagonist as a novel therapy for SLE.

ID: 209: Single cell interferon signatures in lupus patient monocytes reveal a differential impact of interferon signaling between monocyte subtypes

Type I interferon (IFN) is a primary pathogenic factor in human systemic lupus erythematosus (SLE). IFN signatures have been observed in immune cell populations. We examined gene expression in individual SLE patient monocytes in this study. CD14+ + CD16− classical monocytes (CLs) and CD14dimCD16+ non classical monocytes (NCLs) from SLE patients were purified by magnetic separation. The Fluidigm C1 System was used for single cell capture and target gene pre-amplification. Rt-PCR was used to quantify expression of 87 monocyte-related genes. An individual cell IFN score was generated based upon the expression of 17 IFN-induced genes. Monocytes from the same SLE patient blood sample demonstrated varying levels of IFN-induced gene expression. In CLs, high IFN score correlated with CD32a, IL1B, and IL8 expression. In NCLs, high IFN score was correlated with inflammatory mediators, including cytokines such as IL12, IL23, and IL15; the immune receptors CD36, CD32a, CD80, and TLR7; and inflammatory signaling genes such as RELA, STAT2, IRAK1, and MyD88. CD16 transcripts were detected in a small group of classical monocytes, despite a lack of surface CD16 expression. In these cells, CD16 expression was positively correlated with IFN score (p = 0.019). This study reveals diverse IFN responses of individual monocytes, supports the idea that IFN signaling has distinct effects upon classical and non-classical monocytes. IFN may contribute to the transition from classical to non-classical subtype in SLE. Single cell studies can reveal effects of IFN on single immune cells which may be masked in whole blood or mixed cell populations.

Single cell gene expression in lupus patient monocytes reveals a differential impact of interferon signaling between monocyte subtypes (TECH2P.920)

Abstract Background/Purpose Interferon (IFN) gene expression signatures have been observed in whole blood and mixed immune cell populations in systemic lupus erythematosus (SLE), but the significance of this IFN signaling in immune cell subsets is not well understood. We examined gene expression in individual SLE patient monocytes to explore the impact of increased IFN-induced transcription in single cells. Methods CD14++CD16- classical monocytes and CD14dimCD16+ non classical monocytes from SLE patients were purified by magnetic separation. Single cells were isolated, and gene expression in each individual cell was examined by rtPCR. Results Monocytes from the same SLE patient blood sample demonstrated varying levels of IFN-induced gene expression. In classical monocytes, high IFN score correlated with CD32a, IL1B, and IL8 expression. In non-classical monocytes, high IFN score was correlated with a larger number of inflammatory mediators, including cytokines such as IL12, IL23, and IL15; the immune receptors CD36, CD32a, CD80, and TLR7; and inflammatory signaling genes such as RELA, STAT2, IRAK1, IRAK4, and MyD88. Conclusion We observed striking diversity in the IFN responses of individual monocytes, demonstrating that IFN signaling has distinct effects upon classical and non-classical monocytes. Single cell studies can reveal effects of IFN on single immune cells and uncommon populations such as non-classical monocytes, which would be masked in whole blood or mixed cell populations.

Circulating follicular helper-like T cells in systemic lupus erythematosus: association with disease activity.

To assess circulating follicular helper T (Tfh)-like CD4+ T cells in patients with systemic lupus erythematosus (SLE) and determine their relationship to disease activity. Blood samples from patients with SLE, as well as blood samples from patients with Behçet's disease (BD) and healthy individuals as controls, were analyzed. In all samples, circulating Tfh-like cells were enumerated by flow cytometry, using, as markers, expression of CXCR5, inducible T cell costimulator (ICOS), and programmed death 1 (PD-1) protein, as well as secretion of interleukin-21 (IL-21). The frequency of circulating Tfh-like cells was compared to that of circulating plasmablasts (CD19+IgD-CD38+). In addition, the possible association of circulating Tfh-like cells with the SLE Disease Activity Index (SLEDAI) was evaluated. The subset of circulating Tfh-like T cells, identified as CXCR5(high) ICOS(high) PD-1(high) , was expanded in the blood of SLE patients compared to controls. Circulating Tfh-like cells were found to produce IL-21 and had lower expression of CCR7 as compared to that in circulating CXCR5(high) central memory T cells, thereby enabling their distinction. Expression of PD-1, but not ICOS or CXCR5, was significantly elevated in circulating Tfh-like cells from SLE patients compared to controls. PD-1 expression among CXCR5(high) circulating Tfh-like cells correlated with the SLEDAI, frequency of circulating plasmablasts, and anti-double-stranded DNA antibody positivity, but not with disease duration or past organ injury; rather, this cell profile appeared to be a reflection of current active disease. Circulating Tfh-like cells are associated with disease activity in SLE, suggesting that their presence indicates abnormal homeostasis of T cell-B cell collaboration, with a causal relationship that is central to disease pathogenesis. These findings also suggest that circulating Tfh-like cells provide a surrogate for aberrant germinal center activity in SLE, and that their PD-1 expression offers a tool for measuring disease activity and monitoring the response to therapies.

Generation of Tolerogenic Dendritic Cells from Systemic Lupus Erythematosus Patients

Generation of Tolerogenic Dendritic Cells from Systemic Lupus Erythematosus Patients

Higher level of IL-6 in Jaccoud's arthropathy secondary to systemic lupus erythematosus: a perspective for its treatment?

Jaccoud's arthropathy (JA) is a condition characterized clinically by 'reversible' joint deformities along with an absence of articular erosions on a plain radiograph. The main clinical entity associated with JA is systemic lupus erythematosus (SLE) with a prevalence of around 5 %. The aim of the present study was to compare the inflammatory markers including cytokine levels in blood of SLE patients with and without JA. Patients with diagnosis of SLE as defined by ACR criteria were screened and divided in two groups, one with JA and one control group without JA. Erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), complement C3 and C4 levels antinuclear antibodies (ANA), anti-dsDNA antibodies and serum levels of IL-2, IL-6, IL-10, IL-21, IL-22 and TNF-α were determined in all patients. Eighty female patients with SLE, 18 (22.5 %) with JA and 62 (77.5 %) without JA, were included in this study. JA patients had higher disease duration (p = 0.008), ESR (p < 0.001), CRP level (p = 0.002), ANA titer (p < 0.001) and dsDNA antibody level (p = 0.009). The serum levels of IL-2, IL-10, IL-21, IL-22 and TNF-α were not significantly different between the two groups (p > 0.05), but the level of IL-6 was higher in JA group (p < 0.001). The serum level of IL-6 might have a correlation with JA secondary to SLE.

OP0220 Pathogenic Role of IL-10 Producing Helper T Cells in Systemic Lupus Erythematosus

BackgroundIL-10 is a potent anti-inflammatory cytokine that contributes to the suppressive functions of regulatory T-cells, but it is also a well-known B cell growth and differentiation factor produced by CD4+...

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus.

IntroductionPolymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.MethodsThe following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10−D16low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.ResultsSLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10−CD16low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.ConclusionsOur results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.

SAT0178 IGA+ plasmablasts contribute to the expansion of peripheral plasmablasts in SLE

BackgroundActive systemic lupus erythematosus (SLE) is associated with increased levels of peripheral blood antibody-secreting plasmablasts which correlate with autoantibody production and disease activity. Plasmablasts which circulate in healthy individuals at...

Serum from patients with SLE instructs monocytes to promote auto-antigen specific IgG and IgA responses (P4028)

Abstract The persistent development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). Previous work using SLE serum demonstrated that monocyte differentiation into dendritic cells (DCs) is a type I IFN-dependent manner and such SLE-DCs activate T cells. However, whether SLE-DCs promote B cell responses has not been elucidated. Here, we demonstrate that SLE-DCs can efficiently stimulate naïve and memory B cells to differentiate into polyclonal IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. More importantly, when SLE-DCs were stimulated with SLE neutrophils, these SLE-DCs were able to induce ss-DNA specific IgG and IgA from naïve B cells. SLE-DC-mediated IgG-PB differentiation is dependent on BAFF and IL-10, whereas IgA-PB differentiation is mainly dependent on APRIL. Moreover, SLE-DCs as well as monocytes from SLE patients express CD138 and thus can trans-present CD138-bound APRIL to B cells, which is crucial for inducing IgA switching and PB differentiation. The expression of CD138-bound APRIL is not dependent of IFNα, explaining why DCs generated with IFNα (IFN-DCs) are less efficient at inducing IgA responses. Nonetheless, both SLE-DCs and IFN-DCs induce the expression of Bcl-XL in PBs and support their survival, thereby leading to increased Ig production. Taken together, our study suggests that a direct interplay between SLE-DCs and peripheral B cells contribute to SLE pathogenic autoantibody responses.

Increased interleukin-9 and CD4+IL-9+ T cells in patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin affecting all the organ systems. Apart from genetic and environmental factors, autoantibody and immune complex deposition as well as cytokine imbalances contribute to immune dysfunction. Interleukin‑9 (IL-9) is a Tcell-derived factor preferentially expressed by CD4+ Tcells and it has been characterized in human and murine systems. IL-9 targets cells of the lymphoid, myeloid and mast cell lineages, and is likely to contribute to the development of allergic and autoimmune diseases such as asthma, arthritis, multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Nevertheless, until recently there have been no studies on its role in SLE in humans. In the present study, the mRNA and serum IL-9 levels in the peripheral blood of SLE patients and healthy controls were assessed using real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Flow cytometry was used to analyze the percentages of CD4+IL-9+ Tcells in SLE patients. Moreover, differences between the groups and the effect of glucocorticoids were analyzed. The results showed that the plasma concentration and mRNA levels of IL-9 were significantly elevated in SLE patients compared with the healthy controls. The percentages of CD4+IL-9+ Tcells were also increased in SLE patients. In addition, serum IL-9 levels and the percentages of CD4+IL-9+ Tcells were correlated with the SLE disease activity index (SLEDAI). Additionally, the percentages of CD4+IL-9+ Tcells and serum IL-9 levels in 8untreated active SLE patients were decreased at 1, 2 and 3weeks after treatment with methylprednisolone. In conclusion, we provide evidence that IL-9 is increased in SLE patients. Moreover, it is described for the first time that high expression of IL-9 levels and the percentages of CD4+IL-9+ Tcells correlate with disease activity and severity. This suggests an important role of IL-9 in the pathogenesis of SLE.

Elevated apoptosis and impaired proliferation contribute to downregulated peripheral γ δ T cells in patients with systemic lupus erythematosus.

Objective. To investigate the frequency of peripheral γ δ T cells in patients with systemic lupus erythematosus (SLE) and its correlation with disease activity and to analyze the apoptotic status, proliferation ability, and intracellular cytokine profile of these cells. Methods. Flow cytometry was performed to detect the percentage and intracellular cytokine expression of peripheral γ δ T cells from SLE patients. Annexin-V/PI double staining was applied to determine the proportion of apoptotic γ δ and CD3+ T cells. γ δ T cell proliferation was analyzed by CFSE labeling technique. Results. The percentage and absolute number of γ δ T cells were remarkably decreased in active SLE patients compared to those in inactive patients and healthy controls, with γ δ T cell count negatively correlated with disease activity. Compared with healthy controls, peripheral γ δ T cells from active SLE patients exhibited higher apoptotic rate and lower proliferation ability, as well as elevated expression of intracellular IFN-γ, IL-4, IL-10, and TGF-β, but not IL-17 or Foxp3. Conclusion. Decreased γ δ T cells in the peripheral blood of SLE patients might be caused by upregulated apoptosis and downregulated cell proliferation. These γ δ T cells may secret both pro- and anti-inflammatory cytokines to perform their functions in SLE.

Safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: Results of a phase I, placebo‐controlled, double‐blind, dose‐escalation study

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies and inflammation in multiple organ systems. Elevation of messenger RNA levels of interferon (IFN)-regulated genes (IRGs) has been described in the peripheral blood of SLE patients and has been associated with disease activity. The safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of rontalizumab, a humanized IgG1 monoclonal antibody that neutralizes IFNα, were assessed in a phase I dose-escalation study of single and repeat doses of rontalizumab in adults with mildly active SLE. The present report describes the safety results and the impact of rontalizumab on expression of IRGs, IFN-inducible proteins, and autoantibodies. Patients were enrolled into dose groups ranging from 0.3 to 10 mg/kg, administered via intravenous (IV) or subcutaneous routes. Expression levels of 7 IRGs and IFN-inducible serum proteins were monitored as potential biomarkers for the PD activity of rontalizumab. An acceptable safety profile was demonstrated for rontalizumab in patients with SLE. Prespecified criteria for dose-limiting toxicity were not met. The incidence of serious adverse events was comparable across cohorts. The PK properties were as expected for an IgG1 monoclonal antibody and were proportional to dose. Following administration of rontalizumab, a rapid decline in the expression of IRGs was observed in the 3 mg/kg and 10 mg/kg IV cohorts, and this effect could be sustained with repeat dosing. There was no apparent decline in the levels of IFN-inducible proteins or levels of anti-double-stranded DNA and anti-extractable nuclear antigen autoantibodies following treatment with rontalizumab. The preliminary safety, PK profile, and observed PD effects of rontalizumab support further evaluation of its safety and efficacy in SLE.

Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation

The development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). SLE serum can induce monocyte differentiation into dendritic cells (DCs) in a type I IFN-dependent manner. Such SLE-DCs activate T cells, but whether they promote B cell responses is not known. In this study, we demonstrate that SLE-DCs can efficiently stimulate naive and memory B cells to differentiate into IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. SLE-DC-mediated IgG-PB differentiation is dependent on B cell-activating factor (BAFF) and IL-10, whereas IgA-PB differentiation is dependent on a proliferation-inducing ligand (APRIL). Importantly, SLE-DCs express CD138 and trans-present CD138-bound APRIL to B cells, leading to the induction of IgA switching and PB differentiation in an IFN-α-independent manner. We further found that this mechanism of providing B cell help is relevant in vivo, as CD138-bound APRIL is expressed on blood monocytes from active SLE patients. Collectively, our study suggests that a direct myeloid DC-B cell interplay might contribute to the pathogenesis of SLE.

Alterations in T-lymphocyte sub-set profiles and cytokine secretion by PBMC of systemic lupus erythematosus patients upon in vitro exposure to organochlorine pesticides

Chronic exposure to organochlorine pesticides (OCP) has been suspected of causing immunoregulatory abnormalities that eventually lead to development and progression of Systemic Lupus Erythematosus (SLE), but the role of these non-genetic stimuli has remained poorly understood. The objectives of the study were to quantify the levels of different OCP residues in the blood of SLE patients and to study the effects of in vitro treatment of peripheral blood mononuclear cells (PBMC) from these patients and healthy controls with OCP. Levels of different OCP residues in the blood were measured by gas-liquid chromatography. Isolated PBMC were treated in vitro with hexachlorocyclohexane (HCH), o,p’-dichlorodiphenyltrichloroethane (DDT), or phytohemagglutinin-M (PHA-M) for 72 h, then stained with different dye-labeled monoclonal antibodies to analyze alterations in T-lymphocytes using flow cytometry. Levels of different TH1 and TH2 cytokines were also estimated by ELISA. Significantly higher levels of p,p’-DDE and β-HCH were detected in the blood of SLE patients than in healthy controls. HCH exposure markedly increased the percentages of CD3+CD4+ T-lymphocytes and expression of CD45RO+ on CD4+ and CD8+ T-lymphocytes, but decreased CD4+CD25+ T-lymphocytes in SLE patients. DDT exposure increased the percentages of CD3+CD4+ T-lymphocytes and decreased those of CD4+CD25+ T-lymphocytes in SLE patients as compared to healthy controls. No significant responsiveness of patient PBMC to PHA-M stimulation was observed indicating suppression of T-lymphocytes by these OCP. Further, both HCH and DDT decreased the levels of IL-2 and IFNγ but had no effect on IL-4 levels in SLE patients. DDT also increased significantly the levels of IL-10 in patients. It is likely that higher levels and prolonged durations of exposure to HCH and DDT may significantly influence T-lymphocyte sub-sets and cytokine expression in vivo that could lead to the development or exacerbation of SLE.

Two systemic lupus erythematosus (SLE) global disease activity indexes—the SLE Disease Activity Index and the Systemic Lupus Activity Measure—demonstrate different correlations with activation of peripheral blood CD4+ T cells

Global disease activity measurement in systemic lupus erythematosus (SLE) patients is important for the clinical estimation and adjustment of therapy. By contrast, immune system activation plays a significant role in disease pathogenesis, with CD4+ lymphocytes acting as central cells in the immune response. We investigated which scale better correlates with immunologic changes in the blood of SLE patients, the SLE Disease Activity Index (SLEDAI) or the Systemic Lupus Activity Measure (SLAM) scale. Samples of peripheral blood were obtained from 45 SLE patients with different disease activity as assessed by the SLEDAI and the SLAM scales on the same day. We assessed the percentage of CD4+ T cells with activation-associated receptors: CD69, CD25int, CD95, HLA-DR, and CD4+ T cells with killing properties containing perforin and granzyme B. Our results indicated that the percentage of CD4+CD69+ and CD4+CD25(int) cells did not correlate with either the SLEDAI or the SLAM scale. Significant and positive correlations were observed between percentages of CD4+CD95+ and CD4+HLA-DR+ lymphocytes and SLE activity, but only when activity was measured using the SLAM scale, not with the SLEDAI scale. The percentage of CD4+perforin+ and CD4+granzyme B+ cells also strongly correlated with disease activity measured only with the SLAM scale. We conclude that the SLAM scale better reflects changes of immune system activity in SLE patients compared with the SLEDAI scale.

Calcium signaling in systemic lupus erythematosus T cells: A treatment target

Systemic lupus erythematosus (SLE) T cells display a hyperactive calcineurin/NF-AT pathway. The aim of this study was to determine whether this pathway is responsible for the aberrant SLE T cell function and to test the effectiveness of the recently recognized calcineurin inhibitor dipyridamole in limiting SLE-related pathology. T cells and mononuclear cells were isolated from the peripheral blood of SLE patients and healthy individuals. Murine cells were isolated from the spleens and lymph nodes of lupus-prone MRL/lpr mice and control MRL/MpJ mice. Cells were treated in vitro with tacrolimus, dipyridamole, or control. MRL/lpr mice were injected intraperitoneally with 50 mg/kg of dipyridamole 3 times a week for 3 weeks. MRL/lpr T cells, especially CD3+CD4-CD8- cells, displayed a robust calcium influx upon activation and increased levels of NF-ATc1. MRL/lpr T cells (both CD4+ and CD3+CD4-CD8- cells) provided help to B cells to produce immunoglobulin in a calcineurin-dependent manner. Dipyridamole treatment of SLE T cells significantly inhibited CD154 expression, interferon-γ, interleukin-17 (IL-17), and IL-6 production, and T cell-dependent B cell immunoglobulin secretion. Treatment of MRL/lpr mice with dipyridamole alleviated lupus nephritis and prevented the appearance of skin ulcers. NF-AT activation is a key step in the activation of SLE T cells and the production of immunoglobulin. Dipyridamole inhibits SLE T cell function and improves pathologic changes of the disease in lupus-prone mice. We propose that dipyridamole can be used in treatment regimens for patients with SLE.

Glucocorticoid induced TNFR-related protein (GITR) as marker of human regulatory T cells: expansion of the GITR+CD25- cell subset in patients with systemic lupus erythematosus

Regulatory T cells (T(REG)) represent a T cell subset able to modulate immune response by suppressing autoreactive T-lymphocytes. The evidence of a reduced number and an impaired function of this cell population in autoimmune/inflammatory chronic diseases led to the hypothesis of its involvement in the pathogenesis of these disorders. Glucocorticoid-induced TNFR-related protein (GITR) is a well known marker of murine T(REG) cells, but little is known in humans. The aim of this study was to investigate the characteristics of T(REG) cells in systemic lupus erythematosus (SLE) and the potential role of GITR as marker of human T(REG). Nineteen SLE patients and 15 sex- and age-matched normal controls (NC) were enrolled. CD4(+) T cells were magnetic sorted from peripheral blood by negative selection. Cell phenotype was analyzed through flow-cytometry using primary and secondary antibodies and real time polymerase-chain reaction (PCR) using TaqMan probes. The CD25(high)GITR(high) subset was significantly decreased in SLE patients with respect to NC (0.37±0.21% vs 0.72±0.19%; p<0.05). On the opposite, the CD25⁻GITR(high) cell population was expanded in the peripheral blood of SLE patients (3.5±2.25 vs 0.70±0.32%, p<0.01). Interestingly, FoxP3 at mRNA level was expressed in both CD25⁻GITR(high) and CD25(high)GITR(high) cells, suggesting that both cell subsets have regulatory activity. CD4(+)CD25⁻GITR(high) cells are increased in SLE as compared to NC. The expression of high level of GITR, but not CD25, on FoxP3+ cells appears to point to a regulatory phenotype of this peculiar T cell subset.

Changes in the number and biological ability of endothelial progenitor cells from peripheral blood of patients with systemic lupus erythematosus (SLE)

Objective To analyze the changes in number and biological ability of endothelial progenitor cells (EPCs) from peripheral blood of SLE patients. Methods Mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation from peripheral blood of 20 female SLE patients and 20 healthy female controls. EPCs were identified by double staining using antibodies to CD34 and CD133, or antibodies to CD133 and vascular endothelial growth factor receptor 2 (VEGFR2). Phycoerythrin (PE) conjugated antiCD34, fluorescein isothiocyanate (FITC) conjugated anti-CD133 and APC conjugated anti-VEGFR2 antibodies were used in a three color flow cytometric analysis to determine the percentage of EPCs in peripheral MNCs.The proliferation and migration ability of EPCs were measured by MTT assay and modified millicell chamber assay, respectively. The adhesion activity of EPCs was evaluated by counting the number of adherent cells.Results The percentage and proliferation rate of EPCs in peripheral MNCs from female SLE patients were significantly lower than those from the healthy controls(4.49% ± 1.66% vs 20.81% ± 4.14%, 23.11% ± 3.16%vs 35.65% ± 1.74%, both P ＜ 0.01 ). The migration and adhesion ability of EPCs from SLE patients was impaired compared with those from the healthy controls (12.00 ± 2.12 vs 23.60 ± 3.0 cells/field, 22.43 ± 4.43vs 36.43 ± 3.69 cells/filed, both P ＜ 0.01 ). Conclusion There is a decrease in the number and an impairment in biological ability of EPCs in SLE patients. Key words: Lupus erythematosus, systemic; Endothelium; Stem cells; Biology