MACROPHAGE-LIKE RAW-264 CELL-LINE AND TIME-RESOLVED FLUOROIMMUNOASSAY (TRFIA) AS TOOLS IN SCREENING DRUG EFFECTS ON CYTOKINE SECRETION

A screening system was set up to study the effects of drugs on cytokine secretion by macrophages in vitro. The system is based on the murine macrophage-like cell line RAW 264, which can be activated with lipopolysaccharide (LPS) to produce cytokines. The responsiveness of the RAW 264 cells was outlined by challenging them with different concentrations of LPS for 6 or 24 h. Substantial time- and dose-dependent increases were recorded for interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF alpha) secretions. A general procedure was established to construct time-resolved fluoroimmunoassays (TRFIA) from commercial immunochemicals produced originally for enzyme immunoassays. Practical measuring ranges of the non-competitive assays were 100 pg/ml-10 ng/ml for IL-1 beta and TNF alpha and 10 pg/ml-5 ng/ml for IL-6 and IL-5. The interleukin-5 (IL-5) assay was set up for unrelated human studies, but the others were used in the characterization of RAW 264 cytokine secretion. An immunosuppressive effect with dexamethasone phosphate could be achieved and recorded in the model system. Thus, the system offers a simple and easy-to-use model for screening immunomodulatory effects of drugs on the cytokine secretion of macrophages.