Fluvastatin effects on intracellular calcium increase and akt1/PKB activation.

<p>(A–F) Fluvastatin inhibits intracellular free calcium increase [Ca²⁺]_i induced upon engagement of NK cell activating receptors. (A). Effect of fluvastatin on calcium increase triggered by CD16 or NKG2D or DNAM1 or LFA1 with the specific mAbs followed by GAM; unmAb: unrelated mAb matched for isotype plus GAM. NK cells were cultured without drug (black), with fluvastatin (10 µM, red; 1 µM, blue; 0.1 µM, green; left panels) or in right subpanels with fluvastatin alone (red, 10 µM) or in combination with 1 mM of mevalonate (green) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fura-2 at 37°C and [Ca²⁺]_I analyzed on a spectrofluorimeter along time (800 sec). The arrows indicate the addition of GAM to achieve the optimal cross-linking for the indicated receptors. (B) Effect of fluvastatin on calcium increase triggered as in A. NK cells were cultured without drug (solvent), with fluvastatin alone (10 µM, fluva) or in combination with 1 mM of mevalonate (fluva+meva) or with mevalonate alone (meva) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fluo-4 for 45 min at 37°C; intracellular free calcium increase was assessed by flowcytometry upon engagement of the indicated surface receptors after 600 sec of incubation. Results are shown as forward scatter (FSC, x-axis) vs Log green fluorescence intensity (Fluo-4, Y-axis). Each subpanel is divided in four quadrants: lower left: very small cells/debries; lower right: cells with FSC of living NK cells; upper left: small cells/debries labelled with Fluo-4; upper right: NK cells strongly labelled with Fluo-4. In each subpanel in the upper right quadrant is shown the % of NK cells with high Fluo-4 fluorescence. Evaluation of Fluo-4 staining was performed after 10 min upon engagement of each molecule. (C–F). Percentage of NK cells (% responding cells), cultured with different doses of fluvastatin (C,E) or different combination of drugs (D,F), with an increase of intracellular calcium concentration upon engagement of the indicated receptors. Results are expressed as mean of responding cells ±SD of 6 independent experiments. *p<0.001. (G). Evaluation of activation of akt1/PKB in NK cells cultured as in A–F. Results are expressed as % of akt1/PKB activation as ratio between pakt1 and total akt1×100 in each condition at 5 min after stimulation with mAb to the indicated receptors and GAM. Basal: basal level of akt1/PKB activation in cultured NK cells. LY294002: akt1/PKB activation in the presence of the upstream PI3K inhibitor LY294002. *p<0.001 vs solvent.