DETECTION OF INVIVO-INDUCED IL-1 MESSENGER-RNA IN MURINE CELLS BY FLOW-CYTOMETRY (FC) AND FLUORESCENT INSITU HYBRIDIZATION (FISH)

Flow cytometry (FC) and fluorescent in situ hybridization (FISH) were used to detect in vivo induced IL-1 alpha (a) messenger RNA. Spleen cells and thioglycollate-induced peritoneal exudate cells (PEC) were harvested from Balb/C mice three hours after intraperitoneal injection of 20 ug LPS. Cells from each population were phenotyped for MAC or IA(d), fixed in 4% paraformaldehyde and then permeabilized with 70% ETOH. RNA-RNA FISH was performed by incubating suspended cells at 37-degrees-C for 17 hours with biotinylated sense IL-1a, antisense IL-1a or antisense IL-2 probes in 50% formamide. Hybridized cells were washed in 2X SSC, treated with RNAse, stained with avidin conjugated to fluorescein (FITC) or allophycocyanin (APC) and analyzed immediately by FC. Initially, avidin-FITC was used to detect hybridized probe. Dual fluorescent FC analysis of IL-1a mRNA expression in LPS stimulated IA(d+) cells showed an increase in mean fluorescent intensity (MFI) of 51 log channels (0.25 logs) when compared to unstimulated cells. Additionally, induction of specific IL-1a mRNA expression in cells from LPS treated animals was illustrated by increases in percent positive cells (24%) and in equivalent soluble fluorescein molecules (ESFM) bound (47%) when compared to cells from vehicle treated mice. Unhybridized cells and cells hybridized with control antisense IL-2 probe did not exhibit increases in MFI or ESFM. In subsequent experiments on MAC+ PEC, the use of avidin-APC to detect bound probe resulted in a greater separation between the FISH signals of antisense IL-1a and control sense probe (121 log channels, 0.6 logs) than that seen with FITC. These results were confirmed in studies on stimulated spleen cells in which strong fluorescent signals (> 0.5 logs above the MFI of the sense probe) were obtained with antisense IL-1a riboprobe in IA(d+) cells as well as the total cell population. These findings demonstrate that hybridization signals for a specific cytokine mRNA, induced in vivo, can be detected in defined, selected cell populations using dual fluorescent cytofluorometry.