J. Wieckiewicz et al., DETECTION OF CYTOKINE GENE-EXPRESSION IN HUMAN MONOCYTES AND LYMPHOCYTES BY FLUORESCENT IN-SITU HYBRIDIZATION IN CELL-SUSPENSION AND FLOW-CYTOMETRY, INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE, 1(6), 1998, pp. 995-999
The use of digoxigenin (DIG)- and biotin-labelled dsDNA probes to dete
ct TNF alpha-mRNA accumulation in human peripheral blood mononuclear c
ells (PBMC) and isolated monocytes is described. The fragment of the g
lyceraldehyde-3-phosphate dehydrogenase GAPDH-cDNA was used as a contr
ol probe. The hybridization signals were detected by staining with flu
orescein isothiocyanate (FITC)-labelled anti-DIG antibody and avidin-F
ITC, respectively. The cells were stimulated in vitro with lipopolysac
charide (LPS) for 0.5-6 h. The TNF alpha-mRNA was detected in monocyte
s 1 h after stimulation with LPS, and the highest accumulation was see
n around 2 h. The TNFa-mRNA in stimulated PBMC was detected at the low
er level peaking around 4 h. The TNF alpha-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 stimul
ated cells. Finally, an enhanced accumulation of TNF alpha-mRNA was ob
served in PBMC from some patients with sepsis or cancer. Thus, this st
udy shows that cytokine gene expression may be detected in cells ex vi
vo. This opens the possibility of studying the level of cytokine gene
activation in PBMC of patients with diseases where the role of cytokin
es in their pathophysiology is implicated.