The functional consequences of partial calcineurin inhibition in human peripheral blood mononuclear leucocytes

Abstract

Calcium-dependent signal transduction is essential to the induction of cytokine expression by stimuli acting through the T cell receptor. In vitro, the immunosuppressant cyclosporine (CyA) blocks this pathway by inhibition of calcineurin (CN) phosphatase activity. But in vivo, patients on CyA have only 50% inhibition of CN and can mount cytokine responses. To simulate this state of partial inhibition, we studied the responses of human peripheral blood mononuclear leucocytes (PBL) in vitro at low CyA concentrations. PBL were challenged in vitro with calcium ionophores or anti-CD3 monoclonal antibody. The induction of IFN-γ (interferon-gamma) and IL-2 (interleukin 2) steady-state mRNA was studied by Northern blotting and reverse transcriptase-polymerase chain reaction. IFN-γ protein was assessed in a radiolabelled antibody binding assay or by ELISA (enzyme-linked immunosorbent assay). CN was assessed by dephosphorylation of a 32P-serine labelled 19 amino acid substrate. CyA inhibited CN with an IC 50 (concentration giving 50% inhibition) of 10 ng/ml (95% confidence interval, CI = 8–13 ng/ml). Likewise, the induction of IFN-γ and IL-2 mRNA by calcium ionophore A23187 was inhibited with IC 50 of 14 ng/ml (95% CI = 8–27 ng/ml) and 32 ng/ml (95% CI = 5–178 ng/ml), respectively, while the IC 50 for inhibition of IFN-γ protein secretion was 8 ng/ml (95% CI = 9–18 ng/ml). Partial inhibition of CN also altered the threshold for IFN-γ induction. CyA 10 ng/ml inhibited IFN-γ induction by anti-CD3 monoclonal antibody OKT3 significantly more at low OKT3 concentrations (10 ng/ml, mean ± SEM = 72 ± 9% inhibition) compared to high OKT3 concentrations (1000 ng/ml, 47 ± 6%, p < 0.01). Similar results were seen using high and low concentrations of A23187. Finally, cells pretreated with CyA recovered the ability to respond to high concentrations of A23187 (5 μM) faster than low concentrations (0.5 μM). We conclude that the principal defect in lymphocytes with partial CN inhibition is a reduction in maximum cytokine output which is closely related to the degree of CN inhibition. In addition, there is significantly greater inhibition of weak stimuli compared to maximal stimuli. These defects may explain why patients on CyA can have a reduction in immune responsiveness but still retain protection from infection.