Cyclosporine inhibition of leukocyte calcineurin is much less in whole blood than in culture medium

Recent reports have shown that cyclosporine (CsA)-treated patients have abundant calcineurin phosphatase (CN) activity in vivo despite CsA blood concentrations that would be completely inhibitory in vitro. We sought to determine whether this disparity was a result of altered distribution of CsA in culture medium (CM) compared with whole blood (WB). CN activity was measured in peripheral blood leukocytes (PBL) that had been exposed in vitro to CsA in either WE or CRI. Cells from both groups were also stimulated with OKT3 to determine the effect of CsA on the induction of IFN-gamma synthesis. CsA accumulation in PBL was determined by liquid scintillation counting of PBL exposed to H-3-CsA. The IC50 for CsA inhibition of CN activity was significantly lower for PBL in CRI (2 mu g/L) compared with PBL in WE (102 mu g/L, P less than or equal to 0.005). Likewise, for CsA inhibition of IFN-gamma induction, the IC50 was 18 mu g/L for PBL in CM compared with 690 mu g/L for PBL in WE (P less than or equal to 0.005). The shift in IC50 was accompanied by a 10-fold increase in H-3-CsA in PBL in CM compared with PBL in WB. We conclude that PBL exposed to CsA in CM accumulate significantly more CsA than PBL in WE. The result is that CsA inhibition of CN activity and cytokine induction appears at least an order of magnitude greater than its true effect in biologic fluids. This disparity is due to partitioning of CsA to irrelevant CsA binding sites, which are abundant in WE and in vivo, but absent in culture medium.