Calcium signaling in systemic lupus erythematosus T cells: A treatment target. (Arthritis Rheum. 2011;63:2058-2066.)
Abstract
Objective: 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.
Methods: 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.
Results: 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.
Conclusion: 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.
Suppression of skin and kidney disease by inhibition of spleen tyrosine kinase in lupus-prone mice. (Arthritis Rheum. 2010; 62:2086-2092.)
Abstract
Objective: Spleen tyrosine kinase (Syk) is involved in membrane-mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB x NZW)F(1) lupus-prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus-prone mice and whether they result in improvement in skin disease and modification of established disease.
Methods: Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow.
Results: We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks.
Conclusion: Syk inhibition suppresses the development of lupus skin and kidney disease in lupus-prone mice, suppresses established disease in lupus-prone mice, and may represent a valuable treatment for patients with SLE.
Fc epsilon receptor type I gamma chain replaces the deficient T cell receptor zeta chain in T cells of patients with systemic lupus erythematosus. (Arthritis Rheum. 2001; 44:1114-1121.)
Abstract
Objective: T cells from the majority of patients with systemic lupus erythematosus (SLE) express significantly lower levels of T-cell receptor zeta chain, a critical signaling molecule. However, TCR/CD3 triggering of SLE T cells shows increased phosphorylation of downstream signaling intermediates and increased [Ca2+]i response, suggesting the presence of alternative signaling mechanisms. We investigated whether Fcepsilon receptor type I gamma chain (FcepsilonRIgamma) could substitute for TCR zeta chain and contribute to T cell signaling in SLE.
Methods: T cells were purified from the peripheral blood of 21 patients with SLE and 5 healthy volunteers. The expression of FcepsilonRIgamma was investigated using immunoblotting, reverse transcriptase-polymerase chain reaction, and flow cytometry methods. Involvement of the FcepsilonRIgamma in T-cell signaling was studied by immunoprecipitation and/or immunoblotting after TCR/CD3 stimulation.
Results: Western blotting and densitometric analysis showed that the expression of FcepsilonRIgamma in SLE T cells was 4.3-fold higher than in normal T cells (P<0.001). Flow cytometric analyses of T lymphocyte subsets revealed that the proportions of FcepsilonRIgamma+,CD3+, FcepsilonRIgamma+,CD4+, and FcepsilonRIgamma+, CD8+ cells were significantly greater in SLE patients than in healthy controls (P<0.001). Immunoprecipitation of SLE T cell lysates with an anti-FcepsilonRIgamma antibody showed that FcepsilonRIgamma associates with the tyrosine kinase Syk and the CD3epsilon chain, suggesting that FcepsilonRIgamma is functionally involved in TCR signaling.
Conclusion: These results demonstrate that the FcepsilonRIgamma chain is expressed at high levels in a large proportion of SLE T cells. The increased expression of FcepsilonRIgamma chain in SLE T cells may account in part for the aberrant antigen receptor-initiated signaling and contribute to the diverse cellular abnormalities found in this disease.
Suppression of autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-dependent protein kinase IV. (Arthritis Rheum. 2011; 63:523-529.)
Abstract
Objective: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with aberrant immune cell function. Treatment involves the use of indiscriminate immunosuppression, which results in significant side effects. SLE T cells express high levels of calcium/calmodulin-dependent protein kinase type IV (CaMKIV), which translocates to the nucleus upon engagement of the T cell receptor-CD3 complex and accounts for abnormal T-cell function. The purpose of this study was to determine whether inhibition of CaMKIV would improve disease pathology.
Methods: We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at Week 8 or Week 12 of age and continuing through Week 16 and evaluated skin lesions, proteinuria, kidney histopathology, proinflammatory cytokine production, and costimulatory molecule expression. We also determined the effect of silencing of CAMK4 on interferon-γ (IFNγ) expression by human SLE T cells.
Results: CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of nephritis and skin disease, decreased expression of the costimulatory molecules CD86 and CD80 on B cells, and suppression of IFNγ and tumor necrosis factor–α production. In human SLE T cells, silencing of CAMK4 resulted in suppression of IFNγ production.
Conclusion: We conclude that suppression of CaMKIV mitigates disease development in lupus-prone mice by suppressing cytokine production and costimulatory molecule expression. Specific silencing of CAMK4 in human T cells results in similar suppression of IFNγ production. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.
Impaired nonrestricted cytolytic activity in systemic lupus erythematosus: Involvement of a pathway independent of Fas, tumor necrosis factor, and extracellular ATP that is associated with little detectable perforin. (Arthritis Rheum. 1997; 40:1130-1137.)
Abstract
Objective: To determine the cytolytic effector pathway involved in impaired generation of nonrestricted cytolytic activity in systemic lupus erythematosus (SLE).
Methods: Peripheral blood mononuclear cells (PBMCs) from normal subjects and SLE patients were stimulated in vitro with anti-CD3 monoclonal antibody (MAb) and interleukin-2 to promote the generation of nonrestricted cytolytic activity. On Day 13 of culture, the PBMCs were assayed for cytolytic activity against Fas-Daudi cells and Fas+ Jurkat cells. The effects on cytotoxicity of calcium chelation, antagonist anti-Fas MAb, tumor necrosis factor (TNF)–alpha and –beta, and ATP were measured. Intracellular perforin expression was determined by intracellular staining, and perforin messenger RNA levels were determined by quantitative competitive reverse transcription-polymerase chain reaction.
Results: We demonstrated the existence of a cytolytic pathway that is independent of Fas, TNF alpha, TNF beta, and ATP, but is dependent upon extracellular calcium. Despite its calcium dependence, this pathway is associated with low to undetectable levels of perforin.
Conclusion: Impaired generation of nonrestricted cytolytic activity in SLE is likely due to decreased activity of this Fas-, TNF alpha–, TNF beta–, ATP-independent pathway associated with very low levels of perforin.
Dysregulated expression of CXCR4/CXCL12 in subsets of patients with systemic lupus erythematosus. (Arthritis Rheum. 2010;62:3436-3446.)
Abstract
Objective: CXCR4 is a chemokine with multiple effects on the immune system. In murine lupus models, we demonstrated that monocytes, neutrophils, and B cells overexpressed CXCR4 and that its ligand, CXCL12, was up-regulated in diseased kidneys. We undertook this study to determine whether CXCR4 expression was increased in peripheral blood leukocytes from patients with systemic lupus erythematosus (SLE) and whether CXCL12 expression was increased in kidneys from patients with SLE.
Methods: Peripheral blood leukocytes from 31 SLE patients, 8 normal controls, and 9 patients with rheumatoid arthritis were prospectively analyzed by flow cytometry for CXCR4 expression. Biopsy samples (n = 14) from patients with lupus nephritis (LN) were immunostained with anti-CXCL12 antibody.
Results: CD19+ B cells and CD4+ T cells from SLE patients displayed a >2-fold increase (P=0.0001) and >3-fold increase (P<0.0001), respectively, in median CXCR4 expression compared with that in controls (n=7–8). Moreover, CXCR4 expression on B cells was 1.61-fold higher in patients with SLE Disease Activity Index (SLEDAI) scores >10 (n=8) than in patients with SLEDAI scores ≤10 (n=16) (P=0.0008), 1.71-fold higher in patients with class IV LN (n=5) than in patients with other classes of LN (n=7) (P=0.02), and 1.40-fold higher in patients with active neuropsychiatric SLE (NPSLE) (n=6) than in patients with inactive NPSLE (n=18) (P=0.01). CXCL12 was significantly up-regulated in the tubules and glomeruli of kidneys in patients with LN (n=14), with the percentage of positive cells correlating positively with the severity of LN.
Conclusion: CXCR4 appears to be up-regulated in multiple leukocyte subsets in SLE patients. The heightened expression of CXCR4 on B cells in active NPSLE and of CXCL12 in nephritic kidneys suggests that the CXCR4/CXCL12 axis might be a potential therapeutic target for SLE patients with kidney and/or central nervous system involvement.
Expression of CD44 variant isoforms CD44v3 and CD44v6 is increased on T cells from patients with systemic lupus erythematosus and is correlated with disease activity. (Arthritis Rheum. 2010; 62:1431-1437.)
Abstract
Objective: To quantify the expression of CD44 and variant isoforms CD44v3 and CD44v6 on T cells from patients with systemic lupus erythematosus (SLE), and to assess correlations of the level of expression of these molecules with disease manifestations.
Methods: Information on clinical and demographic characteristics was collected, and blood samples were obtained from 72 patients with SLE and 32 healthy control subjects matched to the patients by sex, race, and age. Expression of CD44 and variants CD44v3 and v6 on T cell subsets was determined by flow cytometry, and Pearson’s correlations of their expression levels with clinical variables, SLE Disease Activity Index (SLEDAI) scores, and presence of lupus nephritis were determined. Wilcoxon’s rank sum tests and conditional multivariable regression analyses were applied to identify differences in the expression of CD44 between patients with SLE and healthy controls.
Results: Expression of CD44 was higher on CD4+ and CD8+ T cells from SLE patients compared with controls (P<0.03). Expression of CD44v3 and CD44v6 was also higher on total T cells and CD4+ and CD8+ T cells from SLE patients compared with controls (P<0.03). Cell surface levels of CD44v3 on total T cells, CD4+ T cells, and CD8+ T cells as well as cell surface expression of CD44v6 on total T cells and CD4+ T cells were correlated with the SLEDAI score (P<0.05). The presence of lupus nephritis was associated with the expression of CD44v6 on total T cells, CD4+ T cells, and CD4-CD8- T cells (P<0.05). Positivity for anti-double-stranded DNA antibodies was associated with the expression levels of CD44v6 on T cells (P<0.05).
Conclusion: These results indicate that expression levels of CD44v3 and CD44v6 on T cells may represent useful biomarkers of SLE activity.