Nifedipine protects against overproduction of superoxide anion by monocytes from patients with systemic sclerosis

Twelve non-smoking patients with SSc were included (three men and nine women); their mean age was 56 (±10 SD) years and the mean disease duration was 8 ± 5 years. Each of the patients had been hospitalised for systematic follow-up of the disease. SSc was classified as limited cutaneous or diffuse cutaneous according to the criteria of LeRoy and colleagues [12]. The clinical features of their disease were assessed as recommended [13]; results are detailed in Table 1.

The initial evaluation included biological tests performed on the morning of admission, after 1 hour of rest at room temperature; patients stopped taking calcium-channel blockers 3 days before admission [10]. This wash-out period was long enough for DTCCAs to have ceased to have an effect because the half-lives of nifedipine and nicardipine lie between 6 and 11 h and these drugs are converted into inactive metabolites. Patients were also evaluated 2 weeks later, when they were receiving stable treatment (including 20 mg of nifedipine three times a day). As before, this was done in the morning, after 1 h of rest at room temperature. All patients gave written informed consent. The control subjects were 10 healthy non-smokers from the laboratory staff (8 women and 2 men, mean age 48 ± 9 years).

Blood samples (20 ml) were collected on EDTA. Whole blood was diluted 1:2 in RPMI 1640 solution (Eurobio, Les Ulis, France), layered on 15 ml of Ficoll-Hypaque (relative density 1.077; Eurobio) and centrifuged at 150 g for 25 min at room temperature to separate mononuclear cells from red blood cells and polymorphonuclear neutrophils. Mononuclear cells collected at the interface were washed twice in RPMI 1640 solution and separated into lymphocytes and monocytes by gradient centrifugation with 47% Percoll (Pharmacia, Uppsala, Sweden) at 150 g for 25 min. The purity of the monocyte population (more than 90%) was assessed by May–Grunwald–Giemsa staining. Cells were diluted in RPMI and used immediately.

O₂^•- was quantified by the cytochrome c reduction assay at 550 nm [14] with a Uvikon^® spectrophotometer equipped with a thermostat-controlled cuvette holder and a magnetic stirrer. In brief, 840 µl of monocytes (2 × 10⁶ cells) were incubated at 37°C with 80 µM cytochome c in 1 ml of PBS with stirring before stimulation with 10 µl of PMA (100 nM). Absorbance was read against a reference blank curve containing PBS and cytochrome c at 5, 10 and 15 min. Results are expressed in nmoles of O₂^•- per 10⁶ cells using 21.2 mM/cm as the extinction coefficient. For in vitro evaluation of the effects of DTCCAs, control monocytes were preincubated for 30 min with the desired concentration of drugs or vehicle (dimethyl sulphoxide, 100 µM) before stimulation with PMA.

The respiratory burst of control and nifedipine-treated monocytes induced by formyl-Met-Leu-Phe (fMLP) was measured by a sensitive fluorimetric assay for hydrogen peroxide under standard conditions [15] using the Amplex Red fluorescent probe (Molecular Probes, Eugene, OR, USA). The increase in the fluorescent signal (excitation and emission wavelengths of 530 and 590 nm respectively) was monitored continuously after the stimulation of monocytes with 1 µM fMLP. Results are expressed as the percentage of control values, representing the total hydrogen peroxide production.

Phosphorylated proteins were detected by using the fluorescent Pro-Q-Diamond dye (Molecular Probes), which can directly detect phosphate groups attached to tyrosine, serine or threonine residues in gels. In brief, monocytes from healthy donors were treated with or without nifedipine and stimulated for 10 min with 100 nM PMA (which induces the activation of PKC). Reactions were stopped with ice-cold buffer and cell lysates were prepared by incubating monocytes for 30 min in 0.5 ml of ice-cold extraction buffer (350 nM NaCl, 20 mM Hepes-KOH, pH 7.9, 1 mM EDTA, 0.1 mM EGTA, 20% glycerol, 1% Nonidet P40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulphonyl fluoride, 20 mM ß-glycerophosphate, 0.1 mM Na₃VO₄ and 1 µg/ml each of aprotinin, pepstatin and leupeptin). Cells were then disrupted by sonication (three times 5 s) and proteins were delipidated and desalted before being subjected to SDS gel electrophoresis. Protein phosphorylation was also studied after treatment of a cytosolic fraction of resting monocytes with PMA for 10 min. Protein samples (150 µg of a 1 mg/ml protein sample) were treated in vitro with nifedipine for 30 min before stimulation of PKC with a mixture of diacylglycerol and calcium. This was done with the PKC Biotrak^® enzyme assay (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) in accordance with the manufacturer's instructions. Proteins (50 µg) were separated by SDS gel electrophoresis. Proteins were fluorescently stained by fixing the gels overnight in 50% methanol and 10% trichloracetic acid. The gels were washed with deionised water for 10–20 min, stained with Pro-Q-Diamond for 180 min and destained by three washes in 4% acetonitrile in 50 mM sodium acetate, pH 4, for 2 hours. Gels were scanned with a fluorimager, a Typhoon 9400 laser scanner (Amersham Biosciences), with excitation at 532 nm and a 580 nm band pass emission filter for Pro-Q-diamond dye detection. Phosphorylated proteins were quantified densitometrically with the Image Quant software. Results represent the net phosphorylation induced by PMA, expressed as percentages of the control PMA lane (taken as 100%).

The study was approved by local ethics committee, and all patients gave written informed consent.

Monocytes from patients with SSc (n = 12) not treated by nifedipine produced a significantly greater amount of O₂^•- than controls (n = 10) after stimulation ex vivo with the PKC activator PMA for 15 min (9.1 ± 1.7 versus 5.4 ± 0.7 nmol per 10⁶ cells; P < 0.01; Fig. 1a). This difference was also observed when monocytes were stimulated with PMA for 10 min (7.6 ± 2.3 versus 4.3 ± 0.8 nmol per 10⁶ cells; P < 0.01) but not when they were stimulated for 5 min (2.7 ± 0.8 versus 2.1 ± 0.4 nmol per 10⁶ cells; not significant). These observations suggest that the biochemical modifications affecting monocytes from patients with SSc might not affect the kinetics of NADPH oxidase but rather its late regulatory mechanisms.

Monocytes from patients treated with nifedipine (60 mg/day) for 14 days produced a significantly smaller amount of O₂^•- than monocytes from untreated patients with SSc after stimulation with PMA for 5 min (1.0 ± 0.7 versus 2.7 ± 0.8 nmol per 10⁶ cells; P < 0.05), 10 min (1.6 ± 1.4 versus 7.6 ± 2.3 nmol per 10⁶ cells; P < 0.001) and 15 min (1.8 ± 1.0 versus 9.1 ± 1.7 nmol per 10⁶ cells; P < 0.001; Fig. 1a). Disease subsets, in particular the cutaneous subtype, did not influence baseline O₂^•- production by monocytes or the decrease in O₂^•- production with nifedipine treatment.

To determine whether monocytes were directly altered by nifedipine, monocytes from healthy donors were treated in vitro with nifedipine for 30 min before stimulation with PMA. Nifedipine induced a concentration-dependent inhibition of O₂^•- production at concentrations of 10 and 50 µM (n = 5) (Fig. 1b). PMA induction was inhibited by 50% (IC₅₀) by approximately 3–5 µM nifedipine. This inhibitory effect of nifedipine was not due to any cytotoxic effect, as determined by the trypan blue exclusion test (cell death less than 5%). The effects of nifedipine were next studied on the monocyte respiratory burst induced by an inflammatory agonist such as fMLP. However, fMLP induced very weak monocyte O₂^•- production in standard conditions, so respiratory burst was measured with a fluorimetric assay. Treatment of monocytes for 30 min with 5 and 10 µM nifedipine significantly inhibited hydrogen peroxide production by 55 ± 7% and 90 ± 9%, respectively, relative to controls (n = 3).

Next we studied whether other calcium-channel blockers alter the O₂^•- production of healthy monocytes (n = 5) (Fig. 1c). A strong and similar inhibitory effect was observed with nifedipine or nicardipine, but a less marked inhibitory effect was observed with diltiazem. To investigate whether the inhibitory effect of nifedipine was due to the scavenging of free radicals, O₂^•- was generated by the xanthine/xanthine oxidase system in the presence of various concentrations of nifedipine. In these conditions, nifedipine did not affect O₂^•- generation, ruling out a scavenger effect (data not shown).

The production of O₂^•- by phagocytes is dependent on the activation of PKC, a family of isoenzymes that phosphorylate various proteins including some components of the NADPH oxidase. Because PMA directly activates various PKC isoforms, we studied the effect of nifedipine on PKC-dependent protein phosphorylation in healthy monocytes. Phosphorylated proteins were directly detected in polyacrylamide gels by using Pro-Q-Diamond, a fluorescent dye that binds to phosphate groups on proteins. In the absence of PMA, nifedipine inhibited the basal phosphorylation of various proteins in monocytes (Fig. 2a). In monocytes not treated with nifedipine, stimulation with PMA markedly increased the phosphorylation state of various proteins and nifedipine strongly decreased the PMA-induced phosphorylation of proteins. Densitometric analysis of 10 major phosphorylated protein bands with molecular masses of between 60 and 15 kDa showed that nifedipine inhibited the net protein phosphorylation induced by PMA in a concentration-dependent manner. The inhibition of phosphorylation was also investigated in this cell system under the same conditions with the classical PKC inhibitor calphostin; the inhibition of protein phosphorylation had a quite similar profile (Fig. 2b) with closed results on densitometric analysis.

The inhibitory effects of nifedipine were also demonstrated in a cell-free system in which the activity of PKCs present in the cytosolic fraction of resting monocytes was directly stimulated in the presence of a mixture of calcium and diacylglycerol. In these conditions, treatment of the cytosolic fraction with nifedipine abolished PKC-dependent protein phosphorylation (Fig. 2c).

To determine whether PKC is a direct target of nifedipine, we investigated kinase activity in vitro with a biologically active PKC recombinant that was pretreated with or without various concentrations of nifedipine or with other classical PKC inhibitors such as calphostin or GF109203X [16]. Nifedipine (1, 10 and 50 µM) dose-dependently inhibited PKC activity with an inhibition reaching about 80%. Similar inhibition was observed with calphostin and GF109203X (n = 3; Fig. 3). These results strongly suggest that nifedipine might directly inhibit PKC.