Drs. Ichinose and Juang contributed equally to this work.
Systemic Lupus Erythematosus
Suppression of autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-dependent protein kinase type IV
Article first published online: 28 JAN 2011
Copyright © 2011 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 63, Issue 2, pages 523–529, February 2011
How to Cite
Ichinose, K., Juang, Y.-T., Crispín, J. C., Kis-Toth, K. and Tsokos, G. C. (2011), Suppression of autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-dependent protein kinase type IV. Arthritis & Rheumatism, 63: 523–529. doi: 10.1002/art.30085
- Issue published online: 28 JAN 2011
- Article first published online: 28 JAN 2011
- Accepted manuscript online: 15 OCT 2010 11:56AM EST
- Manuscript Accepted: 5 OCT 2010
- Manuscript Received: 31 AUG 2010
- USPHS/NIH. Grant Number: R01-AI-49954
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.
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.
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.
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.
Autoantibodies, immune complexes, cytokines, and T lymphocytes contribute to tissue injury in systemic lupus erythematosus (SLE) (1, 2), and treatment involves the use of indiscriminate immunosuppressive drugs that have significant side effects. T cells from SLE patients have an altered pattern of gene expression that modifies their behavior and grants them increased inflammatory capacity (3). Circulating anti–T cell receptor (anti-TCR)–CD3 complex antibodies present in the sera of SLE patients contribute to the SLE T cell phenotype through a mechanism that involves the activation of calcium/calmodulin-dependent protein kinase type IV (CaMKIV) and its translocation to the nucleus where it affects the expression of genes (4). The proinflammatory cytokines interferon-γ (IFNγ) (5) and tumor necrosis factor α (TNFα) (6) have been shown to contribute to the immunopathogenesis of human and murine lupus.
Previous studies examining the role of B cells as autoantigen-presenting cells (APCs) in the activation of autoreactive T cells demonstrated that expression of CD86 and/or CD80 molecules by B cells are essential for breaking T cell tolerance to self antigens (7). The expression of CD86 and CD80 on the surface membrane of peripheral blood B cells from patients with SLE is increased (8) and may contribute to the increased ability of B cells to provide help to T cells. Moreover, the expression of CD86 and CD80 has been shown to be expressed in the glomeruli of various types of glomerulonephritis and is believed to contribute to tissue pathology (9, 10). Absence of CD86 and/or CD80 costimulation interfere with the spontaneous activation and the accumulation of memory CD4+ or CD8+ T lymphocytes in MRL/lpr mice and the development of nephritis, antibody production (11, 12), and skin disease (13).
We hypothesized that inhibition of CaMKIV should interfere with the development of autoimmunity and the expression of disease pathology. Accordingly, we treated MRL/lpr mice with KN-93, a known CaMKIV inhibitor (14–17). We found that CaMKIV inhibition with this small-molecule inhibitor resulted in significant suppression of proteinuria, nephritis, and IFNγ and antibody production, as well as the expression of CD86 and CD80 on the surface of B cells. In experiments using human SLE T cells, we found that silencing of CAMK4 resulted in suppression of IFNγ production.
MATERIALS AND METHODS
Female MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice were purchased from The Jackson Laboratory. MRL/lpr mice were treated with the CaMKIV inhibitor KN-93 (EMD Biosciences). The agent was administered by intraperitoneal injections at a dosage of 2.67 μg/gm of body weight per mouse 3 times a week. In a disease prevention experiment, KN-93 administration was started before the onset of proteinuria, when the mice were 8 weeks old. These mice received the agent every other week.
In a separate experiment, the effectiveness of KN-93 treatment of established disease was evaluated. KN-93 administration was started when mice were 12 weeks old and continued at a frequency of 3 times a week for 5 weeks. Mice in both experiments were euthanized at the end of their sixteenth week of age. All mice were maintained in our specific pathogen–free animal facility, and all experiments were approved by the Institutional Animal Care Committee of Beth Israel Deaconess Medical Center.
Mice in each group were placed overnight in a Nalgene metabolic cage to collect urine. Urine was examined using Multistix 10 SG reagent strips and a Clinitek Status Analyzer (Bayer Healthcare). Proteinuria was expressed on a scale of 0–4+, where 0 = none, 1+ = 30–100 mg/dl, 2+ = 100–300 mg/dl, 3+ = 300–2,000 mg/dl, and 4+ = >2,000 mg/dl (18, 19).
The kidneys and skin of the mice were removed, fixed in 10% buffered formalin, and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin (H&E) or periodic acid–Schiff for light microscopy. We evaluated the pathologic changes in the kidney and quantified the intensity of the disease in the glomerular, tubulointerstitial, and perivascular areas according to a previously described scoring system (20, 21).
Measurement of serum anti–double-stranded DNA (anti-dsDNA) antibody and cytokines.
The serum anti-dsDNA antibody concentration was with a mouse anti-dsDNA IgG enzyme-linked immunosorbent assay (ELISA) kit (Alpha Diagnostic). Serum IFNγ and TNFα concentrations were detected with ELISA kits (R&D Systems) according to the manufacturer's protocols.
Measurement of cytokines in cell supernatants.
Two million splenocytes were incubated in 1 ml of RPMI 1640 supplemented with 10% fetal calf serum and stimulated for 24 hours with phosphate buffered saline (PBS) or lipopolysaccharide (LPS; 1 μg/ml). At the end of the culture period, supernatants were collected. IFNγ and TNFα concentrations were detected with ELISA kits according to the manufacturer's protocols.
Single-cell suspensions prepared from the spleens of MRL/lpr mice were isolated and stimulated for 24 hours with LPS (1 μg/ml) or PBS. Spleen cells were stained with fluorochrome-conjugated CD80, CD86 (eBioscience), or CD19 (BioLegend) antibodies. Samples were acquired in a LSRII flow cytometer (BD Biosciences). Analysis was performed with FlowJo software version 7.5.3 (Tree Star). Thirty thousand T cells were acquired for analysis.
RNA extraction and polymerase chain reaction.
Human T cells from SLE patients and controls were homogenized and total RNA was extracted using an RNeasy Mini kit (Qiagen). Complementary DNA was produced using random primers from an equal amount of RNA. The following primers were designed using Primer3 software (22): for IFNγ, 5′-GCAGCCAACCTAAGCAAGAT-3′ (forward) and 5′-GGGTCACCTGACACATTCAA-3′ (reverse); for CaMKIV, 5′-TGTACACATGGATACCGCTC-3′ (forward) and 5′-TCTTCAGCTTCTCCTCTGCT-3′ (reverse); and for 18S ribosomal RNA, 5′-ACTCAACACGGGAAACCTCA-3′ (forward) and 5′-AACCAGACAAATCGCTCCAC-3′ (reverse).
Small interfering RNA (siRNA) transfection.
T cells were purified from the peripheral blood of patients with SLE and from age- and sex-matched control subjects with the use of RosetteSep (Stem Cell Technologies). T cells were electroporated in the presence of CAMK4 siRNA (Qiagen) or control siRNA using an Amaxa Nucleofector (Lonza). After 48 hours of incubation, cells were stimulated with PBS or plate-bound anti-CD3/anti-CD28 antibodies (2 μg/ml of CD3 and 2 μg/ml of CD28). After a 5-hour culture, supernatants were collected, and cells were lysed for RNA extraction. The IFNγ concentration was determined with the use of an ELISA kit (R&D Systems) according to the manufacturer's protocol.
Human SLE T cells.
Results are expressed as the mean ± SD. Kruskal-Wallis test with post hoc comparisons using Scheffe's test were used for intergroup comparisons of multiple variables. Statistical analyses were performed with StatView software (Abacus Concepts). P values less than 0.05 were considered statistically significant.
Blocking of disease development in MRL/lpr mice by inhibition of CaMKIV activity.
In order to determine if CaMKIV plays a role in the pathogenesis of lupus in the MRL/lpr mouse model, we treated 8-week-old mice with KN-93, a pharmacologic inhibitor of CaMKIV. KN-93 administration prevented the development of skin lesions (Figures 1A and B) and significantly reduced the size and weight of the spleens (Figures 1D and F) and lymph nodes (Figures 1E and G). The well-known expanded double-negative T cell population (CD3+CD4–CD8–) in the spleens of MRL/lpr mice decreased significantly after treatment with KN-93, whereas the CD8+ cell subset population increased (data available upon request from the author). Similarly, the percentage of CD62Llow (activated T cells) decreased significantly (data not shown). Furthermore, serum titers of anti-dsDNA antibodies decreased significantly in mice treated with KN-93 (P < 0.01) (Figure 2E). These results indicate that CaMKIV contributes to the increased lymphocyte activation that causes skin disease and spleen and lymph node enlargement in the MRL/lpr mouse.
Development of immune complex–mediated glomerulonephritis manifested by proteinuria and pyuria in MRL/lpr mice.
As shown in Figure 2, proteinuria and pyuria, indicating the presence of renal inflammation, were prevented by the administration of KN-93. Accordingly, inhibition of CaMKIV significantly decreased the degree of kidney damage, as assessed histologically (Figure 3). Mice treated with KN-93 developed significantly less damage in the glomerular, tubulointerstitial, and perivascular areas than did mice treated with PBS. Importantly, these results were observed when KN-93 was administered as a preventative in 8-week-old mice (before the onset of disease) as well as when disease onset preceded the initiation of treatment (Figures 2 and 3).
Reduced proinflammatory cytokine production and costimulatory molecule expression following inhibition of CaMKIV.
To define the mechanism through which KN-93 prevents disease in MRL/lpr mice, we analyzed the production of two essential inflammatory cytokines, IFNγ and TNFα. As shown in Figure 4, KN-93 treatment caused a significant reduction in serum levels of these mediators. Ex vivo stimulation of spleen cells isolated from MRL/lpr mice confirmed that KN-93 was able to block LPS-induced production of IFNγ and to significantly reduce the secretion of TNFα. Inhibition of CaMKIV in spleen cells also diminished the activation of B cells in response to LPS and decreased their capacity to stimulate T lymphocytes by abolishing the up-regulation of the costimulatory molecules CD80 and CD86 (Figure 5). Taken together, these data indicate that CaMKIV is a key element in the inflammatory response that drives autoimmunity in the MRL/lpr mouse.
Involvement of CaMKIV in the production of IFNγ in T cells from patients with SLE.
To determine the relevance of our findings in human disease, we analyzed the effect of CaMKIV inhibition on T cells obtained from patients with SLE and from healthy individuals. Production of IFNγ was higher in cells from patients with SLE than in cells from normal controls (Figure 6). Transfection with specific siRNA was able to knock down the expression of CAMK4 in T cells (Figures 6A and B). This inhibition caused a significant decrease in the production of IFNγ in cells from patients with SLE. These findings indicate that CaMKIV is involved in the expression of IFNγ in patients with SLE.
In the present study, we demonstrated that CaMKIV inhibition results in significant suppression of proteinuria, glomerulonephritis, IFNγ and antibody production, and decreased expression of the costimulatory molecules CD86 and CD80. The experiments described herein were designed after we had shown that SLE T cells express high levels of CaMKIV, which translocates to the nucleus upon engagement of the TCR–CD3 complex (4), in order to demonstrate the in vivo significance of the CaMKIV overexpression in the development of SLE. The successful treatment of MRL/lpr mice with the CaMKIV inhibitor KN-93, before and after initiation of the disease, suggests that CaMKIV contributes to the expression of autoimmunity and organ damage through mechanisms that are unrelated to thymic selection.
Several effector immune cell mechanisms are known to contribute to the pathogenesis of murine and human lupus (3). IFNγ has been demonstrated to be important in the expression of disease in the MRL/lpr mouse, and disruption of its production suppresses or eliminates the expression of the disease (23). Here, we show that inhibition of CaMKIV in the MRL/lpr mouse and silencing of CAMK4 in human lupus T cells significantly suppressed the expression of IFNγ (Figures 4 and 6).
It has been reported that lymphoid hyperplasia in MRL/lpr mice represents the expansion of double-negative T cells, which may derive, both in mice (24, 25) and in humans (26), from activated CD8 T cells. We evaluated whether KN-93 could influence CD4, CD8, double-negative T cell, and B cell populations in MRL/lpr mice. In our studies, we observed that while the CD8+ population increased, the double-negative subset decreased significantly in the spleens of MRL/lpr treated with KN-93 (data available upon request from the author).
In human SLE, CD86 and CD80 expression on resting and activated B cells is increased as compared to the expression on cells from normal individuals (8, 27). In the lupus-prone MRL/lpr mice, up-regulation of CD86 and CD80 expression has been reported (12, 13) and has been demonstrated to be important in the expression of the disease. Moreover, blockade of the engagement of CD86 and CD80 with CTLA-4Ig in (NZB × NZW)F1 mice prevented the development of a lupus-related disease. This treatment blocked autoantibody production and prolonged survival, even when it was delayed until the advanced stages of the disease (28). Our studies demonstrate that pharmacologic inhibition of CaMKIV using ex vivo B cells from MRL/lpr mice (Figure 5) resulted in decreased expression of CD86 and CD80.
KN-93 probably has global suppressive effects on the immune system that extend beyond the T cells in MRL/lpr mice. We have shown here that costimulatory molecule expression by B cells was decreased (Figure 5) along with the production of TNFα by spleen macrophages (Figure 4). In addition, we have found that interleukin-1β (IL-1β), IL-6, and TNFα production by human monocyte/macrophages is suppressed in the presence of KN-93 (data not shown).
Although KN-93 has been claimed to inhibit CaMKIV, it has been reported to inhibit CaMKI and CaMKII as well (14–17). It is possible that some of the observed effects may be due to the inhibition of several CaMK molecules. Because SLE T cells express increased amounts of CaMKIV and not other CaMK molecules (4), we used a CaMKIV-specific silencing approach to reproduce one of the claimed mechanisms of action, the IFNγ production. The fact that silencing CAMK4 suppressed IFNγ production by human SLE T cells supports our claim that CaMKIV inhibition is important in the mitigation of lupus pathology, and these claims are transferable to humans.
In conclusion, our experiments have provided evidence that increased IFNγ, TNFα, and autoantibody production, as well as increased expression of CD86 and CD80, represent pathways through which increased levels of CaMKIV may lead to autoimmunity and to relevant pathology. We have presented in vivo and in vitro evidence that KN-93 or another, more specific CaMKIV inhibitor may warrant further development for the treatment of patients with SLE. We have shown that KN-93 not only prevents, but also suppresses, established disease in MRL/lpr mice. The consistency between our human and murine data further strengthens this claim. Since CaMKIV is also expressed in neuronal cells and is important for the proper function of these cells, the timing and dosing will, as with any other drug, determine its therapeutic efficacy.
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Tsokos had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Ichinose, Juang, Tsokos.
Acquisition of data. Ichinose, Juang, Crispin, Kis-Toth.
Analysis and interpretation of data. Ichinose, Juang, Crispin, Kis-Toth, Tsokos.