Detection of cytokine gene expression in human monocytes and lymphocytes by fluorescent in situ hybridization in cell suspension and flow cytometry.
The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to detect TNFalpha-mRNA accumulation in human peripheral blood mononuclear cells (PBMC) and isolated monocytes is described. The fragment of the glyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a control probe. The hybridization signals were detected by staining with fluorescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-FITC, respectively. The cells were stimulated in vitro with lipopolysaccharide (LPS) for 0.5-6 h. The TNFalpha-mRNA was detected in monocytes 1 h after stimulation with LPS, and the highest accumulation was seen around 2 h. The TNFalpha-mRNA in stimulated PBMC was detected at the lower level peaking around 4 h. The TNFalpha-mRNA accumulation was lower in lymphocytes than in monocytes when PBMC were studied. There was no difference in the level of GAPDH-mRNA between unstimulated and stimulated cells. Finally, an enhanced accumulation of TNFalpha-mRNA was observed in PBMC from some patients with sepsis or cancer. Thus, this study shows that cytokine gene expression may be detected in cells ex vivo. This opens the possibility of studying the level of cytokine gene activation in PBMC of patients with diseases where the role of cytokines in their pathophysiology is implicated.
Wieckiewicz
J
J
Department of Clinical Immunology, Polish-American Institute of Paediatrics, Jagiellonian University, Medical College, 30-663 Cracow, Poland.
Krzeszowiak
A
A
Ruggiero
I
I
Pituch-Noworolska
A
A
Zembala
M
M
D001710:Biotin; D015342:DNA Probes; D004076:Digoxigenin; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012680:Sensitivity and Specificity; D018805:Sepsis / Q000235:genetics / Q000473:pathology; D013274:Stomach Neoplasms / Q000235:genetics / Q000473:pathology / Q000601:surgery; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D015342:DNA Probes; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D001710:Biotin; D012260:Ribonucleases; D004076:Digoxigenin