Detection by in situ hybridization and phenotypic characterization of cells expressing IL-6 mRNA in human stimulated blood

Abstract

IL-6 has manifold biologic functions in immune and inflammatory responses and is produced by a variety type of cells. In this work, we used the whole blood culture to identify the cells expressing IL-6 gene/protein after various stimulation. When the whole blood was incubated with LPS or Con A, much IL-6 activity, measured by the growth promoting assay using a murine IL-6-dependent hybridoma clone, was detected in the plasma as early as 4 h of culture and continued to increase with time, reaching a plateau around 12 h. Immunocytochemical analysis with anti-rIL-6 antiserum revealed that a proportion of mononuclear cells (MNC) contained intracytoplasmic IL-6 in LPS- or Con A-stimulated blood. Northern blot analysis for MNC from the blood stimulated with these stimuli showed that their transcripts for IL-6 peaked at 4 h, then rapidly declined and was undetectable after 24 h of stimulation. In situ hybridization technique with radiolabeled antisense RNA probe for IL-6 demonstrated that a fraction of MNC from LPS- as well as Con A-stimulated blood expressed IL-6 mRNA. With the combined use of in situ hybridization and immunofluorescence by corresponding mAb, it was confirmed that IL-6 mRNA expressing cells in stimulated blood were exclusively monocytes. In the whole blood culture, it was shown that expression of IL-6 mRNA by monocytes was inhibited by dexamethasone, but not by cyclosporin A. These results suggest that monocytes are the major cells expressing IL-6 gene/protein in the circulation after exposure to external stimuli.