Thomas D. Batiuk

Calcineurin activity is only partially inhibited in leukocytes of cyclosporine-treated patients.

Measurement of the degree of immunosuppression induced clinically by drugs such as cyclosporine is an important but elusive goal. In lymphocytes in vitro, cyclosporine (CsA) blocks the phosphatase activity of the enzyme calcineurin, preventing cytokine induction. We sought to measure the degree of calcineurin blockade in patients on CsA. Calcineurin activity was measured in peripheral blood mononuclear cells (PBL) from stable CsA-treated renal transplant patients, compared with controls. Cytokine expression was assessed by challenging ex vivo PBL with calcium ionophore A23187 (5 microM) for 60 min and measuring interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) mRNA induction. In vitro, CsA inhibited both calcineurin activity and cytokine induction with an IC50 of 10-20 micrograms/L. In CsA-treated patients with therapeutic CsA levels (mean trough CsA blood level = 180 +/- 55 micrograms/L), calcineurin activity was detectable but reduced by 50% compared with controls (P < or = 0.001) and correlated with CsA trough levels (r = -0.390, P < or = 0.01). The induction of cytokine mRNA in such patients was not blocked, but was sensitive to CsA in vitro, suggesting that CsA is much less available in vivo in body fluids than it is for isolated cells in vitro. In lymphocytes of patients on CsA, calcineurin activity is reduced but 50% of the activity persists, permitting strong signals to trigger cytokine expression. Partial calcineurin inhibition may explain why the immune responsiveness of patients on CsA is reduced but still sufficient for host defense.

Cyclosporine inhibition of calcineurin activity in human leukocytes in vivo is rapidly reversible.

Despite increasing information about the mechanism of action of cyclosporine A (CsA), little is known about the way lymphocytes recover from CsA. Recovery is central to understanding the pharmacodynamics of CsA in vivo. We studied the recovery of calcineurin phosphatase (CN) activity in CsA-treated cells. Single dose kinetics in renal transplant patients showed that inhibition of CN activity in PBL increased and fell concomitant with CsA blood vessels. In vitro, control PBL treated with CsA 100 micrograms/l, washed, and resuspended in CsA-free medium showed little recovery (0-20%) after 24 h. Erythrocytes or anti-CsA Ab added to the recovery medium increased recovery to 50% within 4 h. Similar recovery was seen in the ability of cells to produce IFN-gamma after OKT3 stimulation. Recovery of CN activity was associated with the efflux of [3H]CsA, was not blocked by cycloheximide and was temperature sensitive. A cell line with high expression of surface P glycoprotein (PGP), showed rapid recovery. However, PGP blockade did not prevent recovery in PBL, indicating a different PGP-independent mechanism. In PBL, recovery from CsA is slow and limited in vitro, but rapid in vivo, where CsA equilibrates among a complex set of extralymphocytic binding sites.

EVIDENCE THAT CALCINEURIN IS RATE-LIMITING FOR PRIMARY HUMAN LYMPHOCYTE ACTIVATION

Cyclosporine (CsA) is both a clinical immunosuppressive drug and a probe to dissect intracellular signaling pathways. In vitro, CsA inhibits lymphocyte gene activation by inhibiting the phosphatase activity of calcineurin (CN). In clinical use, CsA treatment inhibits 50-75% of CN activity in circulating leukocytes. We modeled this degree of CN inhibition in primary human leukocytes in vitro in order to study the effect of partial CN inhibition on the downstream signaling events that lead to gene activation. In CsA-treated leukocytes stimulated by calcium ionophore, the degree of reduction in CN activity was accompanied by a similar degree of inhibition of each event tested: dephosphorylation of nuclear factor of activated T cell proteins, nuclear DNA binding, activation of a transfected reporter gene construct, IFN-gamma and IL-2 mRNA accumulation, and IFN-gamma production. Furthermore, the degree of CN inhibition was reflected by a similar degree of reduction in lymphocyte proliferation and IFN-gamma production in the allogeneic mixed lymphocyte cultures. These data support the conclusion that CN activity is rate-limiting for the activation of primary human T lymphocytes. Thus, the reduction of CN activity observed in CsA-treated patients is accompanied by a similar degree of reduction in lymphocyte gene activation, and accounts for the immunosuppression observed.

The Molecular Immunology of Acute Rejection: An Overview

Abstract Transplantation immunology began as an empirical science but can now take its place as a discipline with a strong theoretical and molecular basis. The Laws of Transplantation, formulated at the beginning of this century by Little and Tyzzer, can be paraphrased as ‘Isografts succeed; allografts fail’. As the century closes, the molecular basis of those laws is emerging. Achieving the full potential of the advances in immunology requires that transplant clinicians learn the molecular basis for their art and participate in the development and testing of new hypotheses; and that immunologists assess their ideas against the real world of the transplant recipient. This review will highlight areas of recent progress in transplant immunology and point out areas where clinical events remain poorly explained. Because many excellent reviews are available, we will primarily quote articles after 1990. Our approach to transplantation biology at the molecular level is summarized in Table 1.

Tissue Distribution of Calcineurin and its Sensitivity to Inhibition by Cyclosporine

The immunosuppressive activity of cyclosporine is mediated by inhibiting calcineurin phosphatase. However, calcineurin is widely distributed in other tissues. We examined the degree of calcineurin inhibition by cyclosporine in various tissues. In vitro, the cyclosporine concentration inhibiting 50% (IC50) of calcineurin was ∼ 10 ng/mL in human and mouse leukocytes suspensions. In vitro and in vivo IC50s of cyclosporine in homogenates of mouse kidney, heart, liver, testis, and spleen were also comparable (9–48 ng/mL). The maximum calcineurin inhibition by cyclosporine varied, from 83 to 95% of calcineurin activity in spleen, kidney, liver, and testis to 60% in heart and only 10% in brain. Maximum calcineurin inhibition was increased by the addition of cyclophilin A, indicating that cyclophilin concentrations were limiting in some tissues, at least in this assay. Western analysis of mouse tissues showed significantly less cyclophilin in heart than other tissues. cyclosporine concentrations per weight of tissue protein were highest in kidney and liver and lowest in brain and testis after oral dosing, with intermediate levels in spleen, heart, and whole blood. Thus each cyclosporine dose produces rapid and widespread inhibition of calcineurin in tissues, with differences in total susceptibility of each tissue.

Infectious disease prophylaxis in renal transplant patients: a survey of US transplant centers.

Definitive approaches to most infectious diseases following renal transplantation have not been established, leading to different approaches at different transplant centers. To study the extent of these differences, we conducted a survey of the practices surrounding specific infectious diseases at US renal transplant centers.

Quantitating immunosuppression : Estimating the 50% inhibitory concentration for in vivo cyclosporine in mice

Cyclosporine (CsA) blocks T cell responses in vitro by inhibiting the phosphatase activity of calcineurin (CN) and thus preventing the activation of cytokine transcription. In the study presented here, we measured the extent of inhibition of these functions in the tissues of CsA-fed mice. Mice fed increasing doses of CsA were assessed for CsA blood and tissue levels, spleen cell CN activity, ex vivo spleen cell cytokine induction by A23187, and in vivo interferon-gamma induction during an allogeneic response. The CN activity of spleen homogenates and cell suspensions and the ex vivo cytokine responses of spleen cells from CsA-treated mice were inhibited with a 50% inhibitory concentration (IC50) greater than 300 microg/L. The in vivo interferon-gamma response to an allogeneic ascites tumor was also inhibited by CsA treatment, with IC50s between 517 and 886 microg/L. The true IC50 for CsA in vivo may be even higher, as CsA levels in spleen and kidney were 4-fold higher than concomitant blood levels. We conclude that inhibition of CN activity by systemically administered CsA leads to a parallel reduction in cytokine gene induction in response to an allogeneic stimulus. In light of our previous clinical findings that therapeutic levels of CsA in renal transplant patients were associated with only partial inhibition of CN activity, these current results support the concept that partial CN inhibition can account for both the immunosuppression and the immunocompetence of CsA-treated patients.

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 WB or CM. 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 3H-CsA. The IC50 for CsA inhibition of CN activity was significantly lower for PBL in CM (2 micrograms/L) compared with PBL in WB (102 micrograms/L, P < or = 0.005). Likewise, for CsA inhibition of IFN-gamma induction, the IC50 was 18 micrograms/L for PBL in CM compared with 690 micrograms/L for PBL in WB (P < or = 0.005). The shift in IC50 was accompanied by a 10-fold increase in 3H-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 WB. 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 WB and in vivo, but absent in culture medium.

Calcineurin activity in children with renal transplants receiving cyclosporine.

BACKGROUND The major immunosuppressive effect of cyclosporine is through the inhibition of calcineurin, an enzyme important in the activation of T lymphocytes. In children, neither calcineurin activity nor its inhibition by cyclosporine (CsA) has been investigated. METHODS Calcineurin activity, was measured in stable pediatric renal transplant patients, with healthy children used as controls. Whole blood CsA concentrations were measured by monoclonal radioimmunoassay. Simultaneous calcineurin and CsA levels were measured before and 1, 2, 3.5, 5, and 12 hr after their routine morning CsA dose. RESULTS Calcineurin activity was approximately 50% inhibited at trough blood concentrations (148 microg/L); moreover, inhibition increased as CsA concentrations rose and declined as concentrations fell. Maximum calcineurin inhibition was about 70% at concentrations of about 431 microg/L. Linear regression analysis revealed a significant correlation between mean CsA blood concentration and the mean degree of inhibition of calcineurin activity (P=0.005, one-tailed). CONCLUSION We conclude that inhibition of calcineurin activity by CsA in pediatric renal transplant recipients correlates with CsA blood concentrations.

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

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-gamma (interferon-gamma) and IL-2 (interleukin 2) steady-state mRNA was studied by Northern blotting and reverse transcriptase-polymerase chain reaction. IFN-gamma 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 IC50 (concentration giving 50% inhibition) of 10 ng/ml (95% confidence interval, CI = 8-13 ng/ml). Likewise, the induction of IFN-gamma and IL-2 mRNA by calcium ionophore A23187 was inhibited with IC50 of 14 ng/ml (95% CI = 8-27 ng/ml) and 32 ng/ml (95% CI = 5-178 ng/ml), respectively, while the IC50 for inhibition of IFN-gamma protein secretion was 8 ng/ml (95% CI = 9-18 ng/ml). Partial inhibition of CN also altered the threshold for IFN-gamma induction. CyA 10 ng/ml inhibited IFN-gamma 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 microM) faster than low concentrations (0.5 microM). 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.