CYTOKINE MESSENGER-RNA LEVELS IN UNMANIPULATED (EX-VIVO) AND IN-VITROSTIMULATED MONKEY PBMCS USING A SEMIQUANTITATIVE RT-PCR AND HIGH-SENSITIVITY FLUORESCENCE-BASED DETECTION STRATEGY

To investigate the spectrum of cytokines expressed by peripheral blood mononuclear cells (PBMC) from cynomolgus macaques (Macaca fasciculari s), we used a semi-quantitative RT-PCR to determine levels of mRNA cod ing for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TNF-alpha. The PCR products were labelled and quantified using a nov fluorescent tag TOTO-1 (thiazole orange dimer) and an automated fluorescence-based electrophoretic instrument. Using this assay, the base line levels of cytokine mRNA expression in unmanipulated PBMCs (ex vivo) from 10 hea lthy monkeys were compared with the mRNA levels for the same cytokines in PBMC samples from two pre-immunized monkeys following culture with previously defined optimal concentrations of purified protein derivat ive (PPD), tetanus toroid (TT) and the mitogen concanavalin A (con-A). While transcripts for IL-2, IL-4 and IFN-gamma were either low or not detected in unmanipulated PBMCs, varying levels of IL-1 beta, IL-5, I L-10, and TNF-alpha were readily detected in the same samples. With th e exception of IL-10, the mitogen con-A induced the highest levels of cytokine expression, followed by levels induced by culture with TT. Th e levels of cytokine expression induced by PPD however, were not signi ficantly elevated, despite the fact that the cells showed marked proli ferative responses. This assay is a simple and convenient method for e valuating the levels of cytokine expression in small PBMC samples and will allow for the concurrent evaluation of immune profiles with funct ional immune analyses. (C) 1996 Academic Press Limited