Quantitative comparison of rapamycin and cyclosporine effects on cytokine gene expression studied by reverse transcriptase-competitive polymerase chain reaction.
We developed and applied a quantitative competitive polymerase chain reaction method to study the expression of various cytokine genes in Con A-stimulated murine splenocytes. This method relies on the use of competitive templates that differ from the target cytokine cDNA templates only by the introduction of a unique restriction endonuclease site in the center of each competitive template. After same-tube amplification using a single pair of oligonucleotide primers for both the target and competitive templates, restriction with the unique endonuclease yields 2 species that are readily resolved on agarose gel electrophoresis: full-length target and half-length competitor fragments. Using this method, we studied the time course and effects of CsA and rapamycin on IL-2, IFN-gamma, IL-4, and IL-10 gene expression following Con A stimulation. IL-2, IFN-gamma, and IL-10 share a common pattern of gene expression peaking at approximately 6 hr, while IL-4 gene expression peaks later, at approximately 20 hr. CsA very effectively inhibits expression of IL-2, IFN-gamma, and IL-4, but it inhibits IL-10 expression only 65%. Rapamycin inhibits IL-10 gene expression 100% and is less effective inhibiting the other cytokines. This pattern of inhibition is consistent with a calcium and protein kinase C independent pathway for IL-10 gene regulation and supports the notion that CsA and rapamycin may be used together to advantage.
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Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02215.
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D000818:Animals; D001483:Base Sequence; D016572:Cyclosporine / Q000494:pharmacology*; D016207:Cytokines / Q000032:analysis* / Q000235:genetics; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D015870:Gene Expression / Q000187:drug effects; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011090:Polyenes / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D020123:Sirolimus
D016207:Cytokines; D017931:DNA Primers; D007166:Immunosuppressive Agents; D011090:Polyenes; D012333:RNA, Messenger; D016572:Cyclosporine; D012194:RNA-Directed DNA Polymerase; D020123:Sirolimus