Abstract
The addition of calcineurin inhibitors, including cyclosporine A (CsA) and FK-506 (tacrolimus), to transplant protocols has markedly reduced acute allograft rejection and prolonged patient survival. Although monitoring of serum drug levels has been shown to be a poor indicator of efficacy, there is little data on calcineurin enzymatic activity in humans. Therefore, we measured calcineurin in isolated CD3+/4+ T cells from 81 non-transplant controls and 39 renal allograft patients by using a 32PO4-labeled calcineurin-specific substrate. A gender difference was observed in the control cohort, with activity in males significantly higher than that in females (1073 ± 134 versus 758 ± 75 fmol/μg/min, respectively). Activity of both groups was comparably inhibited by 5 ng/ml tacrolimus (27 ± 4 versus 30 ± 4%). Calcineurin is a downstream target of the T-cell receptor (TCR). Therefore, activity was measured in isolated T cells after incubation with anti-CD3/CD28 antibodies to stimulate the TCR. Calcineurin activity increased significantly from 1214 ± 111 to 1652 ± 138 fmol/μg/min; addition of either tacrolimus or CsA (500 ng/ml) blocked CD3/CD28 stimulation. Despite therapeutic levels of tacrolimus and CsA (mean 11.4 and 172 ng/ml), basal calcineurin activity was significantly higher among renal transplant recipients than controls (1776 ± 175 versus 914 ± 78 fmol/μg/min). In contrast, anti-CD3/CD28 antibodies failed to stimulate calcineurin activity in transplant subjects. Finally, we found that basal and stimulated calcineurin activities are inversely related. Consistent with this finding, basal activity in resting T cells rose over time after transplant but stimulation fell (r2 = 0.785, p < 0.05). These data suggest that examination of TCR-stimulated calcineurin activity after renal transplantation may be useful for monitoring immunosuppression of individual patients.
Footnotes
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This work was supported in part by the National Institutes of Health [Grant R01-DK066422-01] (to J.L.G.); and by the Carols and Marguerite Mason Trust (to J.A.T.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.109.154096.
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ABBREVIATIONS: TCR, T-cell receptor; IL, interleukin; BMI, body mass index; CsA, cyclosporine A; FK-506, tacrolimus; ANOVA, analysis of variance.
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↵1 O.E.M. is on sabbatical leave from El Minia University, El Minia, Egypt.
- Received March 24, 2009.
- Accepted May 5, 2009.
- U.S. Government work not protected by U.S. copyright
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