In situ hybridization and cytofluorometric analysis of cytokine mRNA during in vitro activation of human T cells.
We present an original method for in situ hybridization (ISH) using non isotopic probes and flow cytometry analysis that permits rapid detection of lymphokine transcripts at single cell level in an in vitro activated human Jurkat T cell line and in peripheral blood T cell subsets. After PMA and either ionomycin or ConA stimulation, cells were fixed and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualised using FITC-conjugated anti-DIG antibody, and the resultant signal was analysed by flow cytometry. IL-2 mRNA was first detected in activated Jurkat T cells. Addition of cycloheximide 4 hours after the beginning of stimulation increased both the frequency of labelled cells and the amount of mRNA per cell, as determined by the mean fluorescence intensity. The specificity and sensitivity of IL-2 mRNA detection were tested by comparison with Northern blot analysis and in situ hybridization (ISH) with immuno-cytochemical staining. IL-2 and IFN-gamma mRNA were detectable in PBMC as early as 3 hours after in vitro stimulation with PMA and ionomycin. The frequency of positive cells and the amount of mRNA per cell peaked at 6-8 hours, when the percentages of IL-2 and IFN-gamma mRNA-containing cells reached 30-40% and 15-20%, respectively. The two lymphokines were expressed in both CD4+ and CD8+ T cells, but the frequency of IL-2 expressing cells and the amount of IL-2 mRNA per cell were higher in CD4+ (60%) than in CD8+ T cells (25%), whereas IFN-gamma were preferentially transcribed by CD8+ T cells (40%). The results obtained by this method were in accordance with the data obtained by Northern blot analysis, with cellular protein content estimated by immuno-fluorescence staining, and with IL-2 titration by bioassay. We compared the performance of this method with ISH using radioactive probes.
Morvan
P Y
PY
Groupe Universitaire de Recherche en Immunologie Fondamentale et Appliquée, Université de Rennes 1, France.
Picot
C
C
Dejour
R
R
Gillot
E
E
Genetet
B
B
Genetet
N
N
D000818:Animals; D001681:Biological Assay; D002460:Cell Line; D003124:Colorimetry; D003513:Cycloheximide / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000235:genetics*; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics*; D008213:Lymphocyte Activation; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*
D007376:Interleukin-2; D012333:RNA, Messenger; D007371:Interferon-gamma; D003513:Cycloheximide