Immunophilins may limit calcineurin inhibition by cyclosporine and tacrolimus at high drug concentrations.
BACKGROUND
The immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK506 or FK) are qualitatively similar but differ in molar potency. Both drugs sterically inhibit the phosphatase activity of calcineurin (CN) but differ in molar potency. In our study we explored whether differential inhibition of CN explained the differences in molar potency of FK versus CsA.


METHODS
We compared their effects on NFATC2 dephosphorylation using Western analysis, interferon-gamma production using ELISA, and CN phosphatase activity using the CN assay in human peripheral blood leukocytes (PBL) and mouse spleen cell suspension.


RESULTS
The FK concentration inhibiting 50% (IC50) of all three activities was approximately 0.2 microg/ml in human PBL, versus 5-20 microg/ml for CsA. Although inhibition of interferon-gamma secretion and NFATC2 dephosphorylation was complete, inhibition of CN phosphatase activity was incomplete with both drugs at saturation, particularly with FK. Inhibition of CN phosphatase activity was incomplete whether FK treatment was in vivo in mouse or in vitro in various human and mouse tissues, especially brain. Exogenous FKBP12 or CyPA increased CN phosphatase inhibition, suggesting that incomplete inhibition of CN phosphatase activity reflected limiting amounts of active immunophilin.


CONCLUSIONS
These data contradict the prevailing assumption that immunophilins are abundant and not limiting for inhibition of CN by CsA or FK. Further, the observation that FK and CsA completely inhibit immune function without completely inhibiting CN suggests that the inhibition of immune function is not mediated by general CN inhibition but by inhibition of a subset of CN which is critical for lymphocyte activation.
Department of Microbiology and Immunology, University of Alberta, Edmonton, Canada.
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L
Kung
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PF
Halloran
D000818:Animals; D065095:Calcineurin Inhibitors; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020104:Immunophilins / Q000502:physiology*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007371:Interferon-gamma / Q000378:metabolism; D008213:Lymphocyte Activation; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology; D016559:Tacrolimus / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism
D065095:Calcineurin Inhibitors; D007166:Immunosuppressive Agents; D014157:Transcription Factors; D007371:Interferon-gamma; D016572:Cyclosporine; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases; D020104:Immunophilins; D016559:Tacrolimus