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

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.

Conflict of interest statement

None of the authors has potential financial conflicts that could compromise the claims made herein.

Copyright © 2011 by the American College of Rheumatology.

Figures

Figure 1. The calcium-NFAT pathway is hyperactive in MRL/lpr T cells

Lymph node cells were isolated from MRL/lpr and MRL/MpJ mice. (A) The cells were stimulated in vitro with anti-CD3 antibody and anti-mouse cross-linking antibody, and the calcium tracing was recorded. Blue tracing: MRL/lpr CD3+CD4-CD8- cells; green: MRL/lpr CD4+ cells; red: MRL/MpJ CD4+ cells. (B) The time to reach the highest intracellular calcium concentration after the addition of the cross-linking antibody (n=3) is depicted here. (*): statistically significant difference. (C) Western blot of nuclear and cytoplasmic lysates from non-stimulated cells using anti-NFATc1 and actin antibodies are depicted here. (One out of three independent experiments). (D) MRL/lpr mice (n=3) ages 15-17 weeks and MRL/MpJ mice (n=2) of the same age were sacrificed. Spleen and lymph node CD3+CD4+ and CD3+CD4-CD8-, and CD19+ cells were isolated. The T cells were pretreated with tacrolimus 100 μg/mL or PBS for 30 min prior to activation. Then T cells were cultured for 13 days with B cells in the presence of anti-CD3/anti-CD28 antibodies. The IgG was measured in the supernatants. The IgG production of control T:B cell cultures was arbitrarily set at 1 and all IgG values are plotted as fold increase over control.

Figure 2. Dipyridamole blocks the expression of CD154 by T cells and does not affect B cell activation

(A) Normal T cells were activated in vitro for 18 hours with anti-CD3 and anti-CD28 plate bound antibodies in the presence of absence of dipyridamole or tacrolimus in the concentrations indicated. Tartaric acid 0.4% was used as control. CD154 levels in the supernatant were measured by ELISA (summary of three independent experiments). (*) denotes statistically significant differences. (B) Normal B cells were isolated from leukapheresed blood and activated in vitro with an anti-IgM antibody for 18 hours. The cells were stained with an anti-CD86-APC conjugated antibody and the expression of CD86 was measured using flow cytometry (one out of three independent experiments).

Figure 3. Dipyridamole blocks the expression of CD154 by SLE T cells and the production of immunoglobulin by SLE PBMC

(A) T cells from SLE patients (n=9) were activated in vitro for 18 hours with anti-CD3/anti-CD28 plate bound antibodies in the presence of dipyridamole 50μM, tacrolimus 0.01 mg/mL or tartaric acid 0.4% (control). CD154 levels in the supernatant were measured with ELISA. (B) T cells from SLE patients (n=9) were activated in vitro for 18 hours with anti-CD3/anti-CD28 plate bound antibodies in the presence of dipyridamole 50μM or tartaric acid 0.4% (control). The surface CD154 was measured using flow cytometry and the percentage of CD154 expressing CD4+ T cells are plotted here. (C) PBMC from SLE patients (n=11) were activated with anti-CD3/CD28 antibodies in the presence of tartaric acid 0.4% or dipyridamole 50μM as described in the Patients and Methods. The IgG was measured in the culture supernatant using an ELISA. (*): statistically significant difference.

Figure 4. Dipyridamole blocks the expression of pro-inflammatory cytokines by SLE T cells

T cells from SLE patients (n=9) were activated in vitro for 18 hours with anti-CD3 and anti-CD28 plate bound antibodies in the presence of dipyridamole 50 μM or tartaric acid 0.4 % (control). The supernatants were tested using a flow cytometry based method (see Materials and Methods) for various cytokine concentrations. (A) IFN-γ, TNF-α, IL-4 and IL-2 levels are shown here. (B) IL-17 levels are shown here. (C) IL-6 levels are shown here. (*): statistically significant difference between dipyridamole and tartaric acid treated activated T cells.

Figure 5. Dipyridamole delays the progression of kidney disease and prevents the emergence of skin ulcers in MRL/lpr mice

5 mice were treated with three times weekly dipyridamole 50 mg/kg i.p. and 5 mice with Tartaric acid 0.4%. The progression of proteinuria (A) and pyuria (B) as measured by a semiquantitave method (described in Patients and Methods) is depicted here. (C) Dipyridamole-treated animals (top panel) did not develop the skin ulcers that control treated mice developed (lower panel). (D) H&E stain (4×) of the ulcerative lesion from an MRL/lpr mouse and a higher power (20×) exhibiting vacuolar changes at the epithelial basement in healthy appearing skin close to the ulcer.

Figure 6. Dipyridamole treatment of MRL/lpr mice leads to a relative decrease in the number of CD3+CD4-CD8- cells in the spleens of treated animals and a decrease in IL-6 in their serum

(A) Splenocytes from MRL/lpr mice treated with control (upper panel) or dipyridamole (lower panel) for 3 weeks were isolated and stained for CD3, CD4 and CD8. The cells depicted here are gated on CD3. (B) The IL-6 was measured in the serum of the MRL/lpr mice treated with dipyridamole or control at the end of the 3-week treatment using flow cytometry. (*): statistically significant difference.