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 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.