Cytokine mRNA levels in unmanipulated (ex vivo) and in vitro stimulated monkey PBMCs using a semi-quantitative 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 fascicularis), we used a semi-quantitative RT-PCR to determine levels of mRNA coding 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 new 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 healthy 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 derivative (PPD), tetanus toxoid (TT) and the mitogen concanavalin A (con-A). While transcripts or IL-2, IL-4 and IFN-gamma were either low or not detected in unmanipulated PBMCs, varying levels of IL-1 beta, IL-5, IL-10, and TNF-alpha were readily detected in the same samples. With the exception of IL-10, the mitogen con-A induced the highest levels of cytokine expression, followed by levels induced by culture with TT. The levels of cytokine expression induced by PPD however, were not significantly elevated, despite the fact that the cells showed marked proliferative responses. This assay is a simple and convenient method for evaluating the levels of cytokine expression in small PBMC samples and will allow for the concurrent evaluation of immune profiles with functional immune analyses.
Benveniste
O
O
Service de Neuropathologie expérimentale et Neurovirologie, Institut Paris-Sud sur les Cytokines, Fontenay aux Roses, France.
Vaslin
B
B
Villinger
F
F
Le Grand
R
R
Ansari
A A
AA
Dormont
D
D
D000818:Animals; D001331:Automation; D001500:BCG Vaccine; D001483:Base Sequence; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis*; D017931:DNA Primers; D015870:Gene Expression; D007371:Interferon-gamma / Q000096:biosynthesis; D007375:Interleukin-1 / Q000096:biosynthesis; D016753:Interleukin-10 / Q000096:biosynthesis; D007376:Interleukin-2 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D007378:Interleukins / Q000096:biosynthesis*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology*; D008252:Macaca fascicularis; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D012016:Reference Values; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D001500:BCG Vaccine; D016207:Cytokines; D017931:DNA Primers; D007375:Interleukin-1; D007376:Interleukin-2; D015850:Interleukin-6; D007378:Interleukins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma