Combination of nifedipine and subtherapeutic dose of cyclosporin additively suppresses mononuclear cells activation of patients with rheumatoid arthritis and normal individuals via Ca2+–calcineurin–nuclear factor of activated T cells pathway
M-C. Lu, Division of Allergy, Immunology, and Rheumatology, Buddhist Dalin Tzu Chi General Hospital no. 2, Min-Sheng Road, Dalin Town, Chia-Yi, Taiwan 62247. E-mail: firstname.lastname@example.org
Abnormal Ca2+-mediated signalling contributes to the pathogenesis of rheumatoid arthritis (RA). However, the potential implication of calcium channel blocker in RA remained unknown. We hypothesized that nifedipine, an L-type calcium channel blocker, combined with a calcineurin inhibitor, could suppress T cell activation via targeting different level of the Ca2+ signalling pathway. The percentage of activated T cells and the apoptotic rate of mononuclear cells (MNCs) was measured by flow cytometry. The MNC viability, cytokine production, cytosolic Ca2+ level and activity of the nuclear factor of activated T cells (NFAT) were measured by enzyme-linked immunosorbent assay (ELISA). The NFAT-regulated gene expression, including interleukin (IL)-2, interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF), was measured by real-time polymerase chain reaction (PCR). We found that the percentage of activated T cells in anti-CD3 + anti-CD28-activated MNC was higher in RA patients. High doses of nifedipine (50 µM) increased MNCs apoptosis, inhibited T cell activation and decreased T helper type 2 (Th1) (IFN-γ)/Th2 (IL-10) cytokine production in both groups. The Ca2+ influx was lower in anti-CD3 + anti-CD28-activated MNC from RA patients than healthy volunteers and suppressed by nifedipine. When combined with a subtherapeutic dose (50 ng/ml) of cyclosporin, 1 µM nifedipine suppressed the percentage of activated T cells in both groups. Moreover, this combination suppressed more IFN-γ secretion and NFAT-regulated gene (GM-CSF and IFN-γ) expression in RA-MNCs than normal MNCs via decreasing the activity of NFATc1. In conclusion, we found that L-type Ca2+ channel blockers and subtherapeutic doses of cyclosporin act additively to suppress the Ca2+-calcineurin-NFAT signalling pathway, leading to inhibition of T cell activity. We propose that this combination may become a potential treatment of RA.
Ca2+ influx is crucial for T cell activation upon antigen stimulation . A biphasic Ca2+ influx occurs in T cells after T cell receptor binding to an antigen presented by major histocompatibility complex molecules. The initial elevation in the intracellular Ca2+ concentration is due to the release of Ca2+ from the endoplasmic reticulum store. Subsequently, there is a lesser but more prolonged influx from extracellular sources. This second influx is necessary for the activation of transcription factors involved in T cell activation . The pathway from Ca2+ influx to T cell activation contains the following two steps: (1) increased intracellular Ca2+ activates calmodulin kinase and calcineurin; and (2) calcineurin stimulates nuclear factor of activated T cells (NFAT) translocation into the nucleus where NFAT induces the expression of several cytokine genes . Previous studies have shown that L-type Ca2+ channels play important roles in T cell activation and proliferation [4–6]. Base on this, L-type calcium channel blockers have been demonstrated therapeutic potential in the treatment of systemic lupus erythematosus (SLE) , asthma , atherosclerosis  and glomerulonephritis .
Rheumatoid arthritis (RA) is a common and disabling chronic inflammatory disease. Animal studies suggest that abnormal Ca2+ signalling induced by up-regulation of the calcineurin pathway contributed to the pathogenesis of chronic arthritis . In clinical practice, calcineurin inhibitors such as cyclosporin and FK-506 have been used as potent disease-modifying anti-rheumatic drugs (DMARDs) in the treatment of RA . Based on these observations, we hypothesized that an L-type calcium channel blocker combined with a calcineurin inhibitor could suppress the Ca2+ signalling pathway at different levels, and the combination therapy could become a potent therapy for the treatment of RA.
Materials and methods
Twelve patients fulfilling the 1987 American College of Rheumatology (ACR) revised criteria for the classification of RA  were recruited into this study. Twelve healthy volunteers served as a control group. All the participants signed declarations of informed consents approved by the Local Internal Review Board and Ethics Committee of Buddhist Dalin Tzu Chi General Hospital, Chia-Yi, Taiwan (no. B09702019). Blood samples were collected at least 12 h after the last dosage of corticosteroids or immunosuppressants in order to minimize the effects from the drugs on the in-vitro studies. Patients were excluded from this study if they had recently (within 4 weeks) been on calcium channel blockers, cyclosporin or FK-506.
Preparation of mononuclear cells (MNCs) from the peripheral blood of RA patients and controls
Heparinized venous blood obtained from RA patients and healthy volunteers was mixed with a 2% dextran solution (mol. wt. 464 000 daltons; Sigma-Aldrich Chemical Company, St Louis, MO, USA) at a ratio of four parts blood to one part dextran, and the mixture was incubated at room temperature for 30 min. A leucocyte-enriched supernatant was collected and layered over a Ficoll-Hypaque density gradient solution (specific gravity 1·077; Pharmacia Biotech, Uppsala, Sweden). After centrifugation at 250 g for 25 min, MNCs were aspirated from the interface.
In order to mimic T cell activation, freshly prepared MNCs (1 × 106 /ml) were cultured in 96-well flat-bottomed microtitre plates precoated with 1 µg anti-human CD3 and 1 µg anti-human CD28 (BioLegend, San Diego, CA, USA) with different concentrations of nifedipine (0, 1, 10 or 50 µM) in the presence or absence of a subtherapeutic dose (50 ng/ml) of cyclosporin at 37°C in 5% CO2 for 72 h. After culture, cells were pelleted by centrifugation at 300 g and the use for subsequent analysis included the percentage of activated T cells, MNCs apoptosis, cell viability and the DNA binding activity of NFATc1. The supernatant was concomitantly collected and stored at −80°C for the measurement of cytokines.
Detection of HLA-DR+CD3+ activated T cells by flow cytometry
To determine the percentage of activated T cells, cells were incubated with either 20 µl of fluorescein isothiocyanate (FITC)-labelled anti-CD3/phycoerythrin (PE)-labelled anti-human leucocyte antigen D-related (HLA-DR) (BD Biosciences, Franklin Lakes, NJ, USA) or a 20 µl Simultest control (BD Biosciences), as per the manufacturer's protocol. Then, the percentage of HLA-DR+CD3+ cells in CD3+ cells was determined by fluorescence activated cell sorter (FACScan) flow cytometry (Becton Dickinson, San Jose, CA, USA) and analysed using Lysis II software (Becton Dickinson).
Detection of apoptosis by flow cytometry
The percentage of apoptosis in MNC was determined by double-staining with FITC-annexin V and propidium iodide (PI) kit (BD Biosciences) and analysed by flow cytometry.
Cell viability and proliferation using the mitochondrial dehydrogenase cleavage assay
After initial treatment, 10 µl water-soluble tetrazolium salts (WST-1) (Roche Applied Science, Basel, Switzerland) was added to each well and the plate was then incubated for 30 min. The intensity of colour formation was detected using an enzyme-linked immunosorbent assay (ELISA) microplate reader.
Measurement of the cytosolic free Ca2+ levels
The cytosolic free Ca2+ levels were measured using the Fluo-4 direct calcium assay kit (Molecular Probes, Eugene, OR, USA), according to the manufacturer's recommendations. In brief, peripheral blood mononuclear cells (PBMCs) at 1·25 × 105 cells/well in 96-well plates were loaded with Fluo-4 calcium reagent at 37°C for 60 min. Then PBMCs were loaded with different concentrations of nifedipine (0, 1, 10, 50 µM) for 10 min. PBMCs were then stimulated with 0·2 µg anti-human CD3 and 0·2 µg anti-human CD28 and fluorescence was measured immediately by microplate reader (Anthos Zenyth 3100, Cambridge, UK). The fold change of fluorescence intensity was calculated by (fluorescence intensity)/(baseline fluorescence intensity) of each well.
Measurement of nuclear NF-ATc1 DNA binding activity
Nuclear extract-protein extraction reagent (NE-PER) and cytoplasmic extraction reagents (Pierce Biotechnology, Rockford, IL, USA) were used to prepare nuclear extracts from stimulated MNCs, according to the manufacturer's instructions. The DNA binding activity of NF-ATc1 in the nuclear extract was detected with a sensitive multi-well colorimetric assay kit (Active Motif, Carlsbad, CA, USA).
Quantitative analysis of interleukin (IL)-2, interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF) genes expression by real-time polymerase chain reaction (PCR)
The mRNA was isolated by QIAamp RNA Blood Mini kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The RNA expression level of IL-2, IFN-γ and GM-CSF mRNA was quantified by real-time PCR using a one-step reverse transcription (RT)–PCR kit (TaKaRa, Shiga, Japan). The primers for IL-2 were forward: 5′-CCCAAGAAG-GCCACAGAACT-3′ and reverse: 5′-TGCTGATTAAGTCCCTGGGTCTTA-3′; for IFN-γ, forward: 5′-CTTTAAAGATGACCAGACCATCCA-3′ and reverse: 5′-ATCTCGTTTCTTTTTGTTGCTATTGA-3′; for GM-CSF, forward: 5′-ATGTTTGACCTCCAGGAGCC-3′ and reverse: 5′-GGTGATAATCTGGGTTGCACA-3′; for 18S ribosomal RNA, forward: 5′-GCCCGAAGCGTTTACTTTGA-3′ and reverse: 5′-TCCATTATTCCTAGCTGCGGTATC-3′. The condition for quantitative PCR was 42°C for 5 min and 95°C for 10 s for reverse transcription, followed by 40 cycles of 95°C for 5 s and 34°C for 34 s. All reactions were performed in duplicate on an ABI Prism 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The expression of 18S ribosomal RNA was used as endogenous control for data normalization. The threshold cycle (Ct) is defined as the cycle number at which the change of fluorescence intensity crosses the threshold of 0·2. The value of each Ct was first normalized by 18S ribosomal RNA. The residual gene expression of each cytokine gene was defined as 2–▵Ct × 100%. The ▵Ct was defined to the Ct value of MNCs culture in different concentrations of nifedipine with 50 ng/ml cyclosporin or 50 ng/ml cyclosporin alone minus the Ct value of gene expression from MNCs culture in medium only. The residual gene expression of IL-2, IFN-γ and GM-CSF was averaged and presented as mean residual expression of NFAT-regulated genes .
The concentrations of IFN-γ (a representative Th1 cytokine) and IL-10 (a representative Th2 cytokine) in culture supernatants were determined by their respective ELISA kits (BD Biosciences), according to the manufacturer's protocol.
All data were represented as mean ± standard deviation (s.d.). Statistical significance was assessed by either paired or unpaired Mann–Whitney U-tests. A P-value of less than 0·05 was considered statistically significant.
Demographic and clinical data of RA patients and controls
The demographics of the RA patients and the healthy volunteers are shown in Table 1.
Table 1. Demographics and clinical data of rheumatoid arthritis (RA) patients and healthy volunteers.
Healthy volunteers (n = 12)
RA patients (n = 12)
F, female; M, male; RF, rheumatoid factor; ACPA, anti-citrullinated protein antibody; CRP, C-reactive protein; s.d., standard deviation.
Age (mean years ± s.d.)
50·3 ± 14·3
57·4 ± 10·7
1·17 ± 1·09
Effect of nifedipine on the percentage of activated T cells, apoptosis and cell viability of MNCs in RA patients and healthy volunteers
After stimulation with anti-CD3 + anti-CD28, the percentage (%) of activated T cells in the MNCs from RA patients was significantly higher than the control MNCs (Fig. 1a; 85·8 ± 6·0% versus 72·8 ± 12·3%, P = 0·0094). The activated T cells from RA patients seemed more susceptible to nifedipine than control MNCs, as 10 µM nifedipine was able to suppress T cell activation effectively in RA MNCs, while 50 µM nifedipine was required to suppress T cell activation in control MNCs (Fig. 1a). Nifedipine at 50 µM could increase the apoptotic rate in anti-CD3 + anti-CD28-activated MNC from both RA patients and healthy controls (Fig. 1b), whereas no affect was seen on cell viability and proliferation in RA patients and healthy controls analysed by WST-1 assay (Fig. 1c).
Effects of nifedipine on IFN-γ and IL-10 secretion by anti-CD3 + anti-CD28-activated MNCs
To investigate whether nifidipine acts on the secretion of Th1/Th2 cytokines by stimulated MNCs, IFN-γ and IL-10 were measured as representative cytokines. As shown in Fig. 2a, a tendency for stimulated RA MNCs to produce less IFN-γ but more IL-10 (Fig. 2b) than control MNCs. Nifedipine at a concentration of 50 µM could suppress IFN-γ secretion effectively from both RA and control MNCs. In contrast, 1 µM nifedipine was able to inhibit RA MNCs IL-10 secretion significantly, whereas 10 µM nifedipine was required to suppress IL-10 secretion from control MNCs (Fig 2b).
Effects of the combination treatment on T cell activation, apoptosis and proliferation of MNCs in RA patients and healthy volunteers
Because cyclosporin can potently inhibit calcineurin (a downstream molecule tightly controlled by Ca2+ influx) activation, we hypothesized that the combination of nifedipine with a subtherapeutic dose of cyclosporin (50 ng/ml) may lead to greater immunosuppressive effects on T cell activation, as the combination therapy targets different steps in the Ca2+-medicated signalling pathway. As expected, we found that T cell activation was suppressed by 50 ng/ml cyclosporin in both RA and control MNCs (Fig. 3a). Compared to MNCs treated with cyclosporin alone, the addition of 1 µM nifedipine further inhibited T cell activation in both RA (57·58 ± 10·59% versus 67·89 ± 14·99%, P = 0·0077) and control (55·17 ± 8·54% versus 61·83 ± 10·73%, P = 0·0109) MNCs (Fig. 3a). The subtherapeutic dose of cyclosporin (50 ng/ml) did not elicit apoptosis in anti-CD3 + anti-CD28-activated MNCs from RA patients and healthy control, but did in the presence of 50 µM nifedipine (Fig. 3b). In addition, no difference of cell viability and proliferation in anti-CD3 + anti-CD28-activated MNCs from RA patients and healthy controls in different concentrations (1–50 µM) of nifedipine + 50 ng/ml cyclosporin assessed by WST-1 assay.
Effects of the combination treatment on IFN-γ and IL-10 secretion by anti-CD3 + anti-CD28-activated MNC
Next, we assessed the effects of the combination of nifedipine and cyclosporin on IFN-γ and IL-10 cytokine secretion. Cyclosporin (50 ng/ml) suppressed the secretion of both IFN-γ and IL-10 considerably in anti-CD3 + anti-CD28-activated MNCs from RA patients and controls (Fig. 4a). In MNCs co-treated with cyclosporin, the addition of 1 µM nifedipine suppressed IFN-γ secretion further in RA MNCs, but 10 µM nifedipine was required to suppress IFN-γ secretion in control MNCs (Fig. 4a). Conversely, 10 µM nifedipine + 50 ng/ml of cyclosporin suppressed IL-10 secretion significantly by RA MNCs, whereas only 1 µM nifedipine + 50 ng/ml cyclosporin was required to attain the same effect in control MNCs (Fig. 4b).
Effect of the nifedipine on the cytosolic Ca2+ level in anti-CD3 + anti-CD28-activated MNCs
Two representative cases demonstrated that the cytosolic Ca2+ level raised in MNCs after activated by anti-CD3 + anti-CD28 and nifedipine (1–50 µM) could suppress Ca2+ influx effectively in MNCs from the RA patients and healthy controls (Fig. 5a,b). In addition, the maximum cytosolic Ca2+ level in the MNCs from RA patients was significantly lower than the control MNCs (Fig. 5c).
Effect of combination treatment on NFATc1 DNA binding activity and NFAT-regulated gene expression, including IL-2, IFN-γ and GM-CSF in anti-CD3 + anti-CD28-activated MNC
Because NFATc1 is a transcription factor in the downstream of Ca2+ influx and becomes a part of the calcineurin signalling pathway, we attempted to determine whether changes in NT-ATc1 activation could be a potential mechanism induced by the combination of low-dose cyclosporin (50 ng/ml) and nifedipine. As shown in Fig. 6a, cyclosporin (50 ng/ml) alone suppressed nuclear NFATc1 DNA binding activity in anti-CD3 + anti-CD28-activated MNCs of RA patients and controls. We also found that the combination of a subtherapeutic dose of cyclosporin (50 ng/ml) and increasing concentrations of nifedipine further decreased nuclear NFATc1 DNA binding activity in activated MNCs from both groups compared with 50 ng/ml cyclosporin alone (Fig. 6a). Recent studies demonstrated that the clinical effectiveness of calcineurin inhibitors correlated well with the inhibition of NFAT-regulated gene (such as IL-2, IFN-γ and GM-CSF) transcription [17,18]. After normalization, we found that there was no statistical difference in IL-2 expression between RA and control MNC in different concentrations of nifedipine (0, 1, 10 or 50 µM) in the presence of 50 ng/ml cyclosporin or culture median alone (Fig. 6b,c). However, in control MNCs, cyclosporin (50 ng/ml) alone suppressed IFN-γ expression and cyclosporin (50 ng/ml) + 50 µM nifedipine was required to suppress GM-CSF expression (Fig. 6b). In RA-MNCs, cyclosporin (50 ng/ml) alone could suppress GM-CSF expression and cyclosporin (50 ng/ml) + 1 µM nifedipine could suppress IFN-γ expression effectively (Fig. 6c). Finally, we found that 50 ng/ml cyclosporin did not inhibit significantly the mean NFAT-regulated gene expression. However, the addition of only 1 µM nifedipine with cyclosporin could suppress the NFAT-regulated gene expression significantly (Fig. 6d). These results suggest that nifedipine plus cyclosporin inhibited Th1/Th2 cytokine production and T cell activation via the Ca2+–calcineurin–NFATc1 signalling pathway.
Our study demonstrated that a low dose of nifedipine plus a subtherapeutic dose (50 ng/ml) of cyclosporin exhibited an additive immunosuppressive effect on T cell activation through the Ca2+–calcineurin–NFAT pathway. Nifedipine, an L-type calcium channel blocker, is a popular anti-hypertensive medication in clinical practice due to its minimal side effects. The calcineurin inhibitors, cyclosporin and FK-506, are well-known DMARDs that are often used in the treatment of RA . However, the use of cyclosporin is limited due to its common adverse effects, which include hypertension, hyperuricaemia, renal toxicity and neurotoxicity. Previous clinical studies have demonstrated that the addition of an L-type calcium channel blocker did not bring about any additional adverse effects in renal transplant patients treated with cyclosporin. In fact, the combination even revealed a small, but significant, renal protective effect that was independent of the anti-hypertensive effect [19,20]. Moreover, an in-vitro study revealed that L-type calcium channel blockers, particular nifedipine, could inhibit several proinflammatory cytokines including IL-1, IL-6 and tumour necrosis factor (TNF)-α secretion in normal peripheral MNCs after stimulation . Therefore, the combination of nifedipine and cyclosporin appears to be a potential therapy for RA.
Our previous report suggested that a high dose of nifedipine (50 µM) could suppress Th1/Th2 cytokine production and increased apoptosis of anti-CD3 + anti-CD28-activated MNCs from SLE patients . In this study, we found further that a high concentration of nifedipine (50 µM, equal to 17·3 mg/kg) could suppress T cell activation and promote apoptosis and Th1/Th2 cytokine secretion in both RA and control MNCs. These results are consistent with the previous studies that 50 µM nifedipine is able to suppress T cell activation significantly [4,22]. However, Zhang et al.  have reported that less than 1 µM nifedipine is needed to suppress L-type calcium channel-induced vascular tone in cardiovascular systems. As the cardiovascular system is more sensitive than T cells to nifedipine, the use of nifedipine at levels sufficient to suppress T cell activation (≧50 µM) would render patients at risk for developing bradycardia and hypotension . This discrepancy is derived from expression of different ion channel subtypes as the L-type Ca2+ channels expressed on lymphocytes are from a different subfamily in excitatory cardiac tissue [25,26]. It is expected that development of a lymphocyte-specific L-type calcium blocker would be of great value in specifically suppressing immune activation. Alternatively, using a combination therapy approach would be beneficial in clinical practice. Cyclosporin, a calcineurin inhibitor, inhibits the Ca2+-calcineurin-mediated signalling pathway. In the present study, we found that 1 µM nifedipine did not inhibit T cell activation and IFN-γ secretion in RA MNCs. However, after combined with a subtherapeutic concentration of cyclosporin (50 ng/ml, therapeutic range: 100–400 ng/ml), 1 µM nifedipine (equal to 0·35 mg/kg) could additively inhibit T cell activation and IFN-γ secretion in RA MNCs. We propose that this additive effect is derived from inhibition at different levels of the Ca2+ signalling pathway, namely the suppression of the Ca2+ influx by nifedipine and the inhibition of calcineurin by cyclosporin.
Our study also suggested that the percentage of activated T cells was higher in the MNCs from RA patients than controls after stimulation with anti-CD3 + anti-CD28, which was consistent with a previous clinical study . Abnormal T cell functions were well characterized in patients with RA . RA-MNCs were more susceptible to Ca2+ channel blocker-induced suppression of T cell activation than control MNCs (nifedipine had a significant effect at 10 µM in RA MNCs versus 50 µM in control MNCs; Fig. 1a). We speculated that the decreased expression of calmodulin  and attenuated TCR-induced Ca2+ response [30–32] caused by prolonged exposure to TNF-α may play a critical role. As shown in Fig. 5, this attenuated Ca2+ response would render RA-T cells more sensitive to L-type calcium channel blockade.
Recently, monitoring NFAT-regulated genes such as IL-2, GM-CSF and IFN-γ expression has become a sensitive functional marker for cyclosporin-medicated immunosuppression. We note that the expression levels of IL-2 did not decrease after the addition of 50 ng/ml cyclosporin as the DNA binding activity of NFATc1 dose. In fact, the expression of IL-2 was controlled by several transcription factors other than NFAT . The p300/CREB binding protein (CBP)  and nuclear factor (NF) 90  could be activated specifically after CD28 co-stimulation. Although 50 ng/ml cyclosporin + 50 µM nifedpine could suppress the DNA binding activity of NFATc1 effectively, the magnitude of decreased NFATc1 activity was not enough to cause a significant reduction of IL-2 gene expression. In contrast, the expression of IFN-γ and GM-CSF decreased in parallel with the decrease of DNA binding activity of NFATc1. It is interesting that, in normal MNC, the addition of 50 ng/ml cyclosporin could suppress the expression of IFN-γ and 50 ng/ml cyclosporin + 50 µM nifedpine was required to suppress GM-CSF expression. However, in RA-MNC, the addition of 50 ng/ml cyclosporin could suppress the expression of GM-CSF, and 50 ng/ml cyclosporin + 1 µM nifedpine suppressed IFN-γ expression effectively. This suggested that there might be abnormal signal transduction in RA-T cells. Finally, the combination of 50 ng/ml cyclosporin + 1 µM nifedpine could effectively lower the mean residual NFAT-regulated gene expression. The mean residual NFAT-regulated gene expression which comes from the average of residual gene expression of IL-2, IFN-γ and GM-CSF becomes a biological marker to evaluate the functional effectiveness of calcineurin inhibitors . Clinically, the mean residual NFAT-regulated gene expression correlated with the development of infections and malignancy in transplant patients [37,38]. Indeed, the mean residual NFAT-regulated gene expression decreased in MNCs cultured in 50 ng/ml cyclosporin + 1 µM nifedipine. Although our results suggest that there might be an addictive immunosuppressive effect by the combination of a subtherapeutic dose cyclosporin and nifedipine at a cellular level, further animal studies and clinical trials are needed for confirmation.
In conclusion, our study revealed that a high dose of nifedipine (17·3 mg/kg) could suppress T cell activation and Th1/Th2 cytokine secretion effectively in MNCs from RA patients and controls. In combination with a subtherapeutic dose of cyclosporin (50 ng/ml), a therapeutic dose of nifedipine (0·35 mg/kg) could suppress T cell activation in RA and control MNCs, and Th1 cytokine secretion in RA MNCs effectively. This drug combination may be a potential therapeutic regimen for RA treatment in the future.
This work was supported by a grant from the National Science Council (NCS 99–2314-B-303-003) and the Buddhist Dalin Tzu-Chi General Hospital (DTCRD98-02), Taiwan.