AN INVESTIGATION OF THE TEMPORAL INDUCTION OF CYTOKINE MESSENGER-RNASIN LPS-CHALLENGED THIOGLYCOLATE-ELICITED MURINE PERITONEAL-MACROPHAGES USING THE REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

Objective and Design: A comprehensive study to standardise interleukin (IL)-1 alpha, -1 beta, -6, -10, -12 and tumour necrosis factor alpha (TNF alpha) mRNA detection in murine peritoneal macrophages, using the reverse transcription polymerase chain reaction (RT-PCR) was carried out. Subjects: Thioglycollate-elicited peritoneal exudate cells were h arvested from female BALB/c mice and the adherent macrophage fraction isolated for use. Treatment: Peritoneal macrophages (1 x 10(6)) were i ncubated in the presence or absence of lipopolysaccharide (LPS; at a f inal concentration of 1 mu g/ml) for 0, 1, 2, 3, 4, 5 and 24 h. Method s: Culture supernatants and cells were harvested at each time point, t he secreted cytokine protein levels quantified by sandwich immunoassay s and the cytokine mRNA levels assessed by RT-PCR. Results: The IL-6 m RNA was not expressed in detectable amounts in the macrophages, unless challenged with LPS. TNF alpha, IL-1 alpha, IL-1 beta, IL-10 and IL-1 2 mRNAs were expressed in both stimulated and unstimulated macrophages . The levels of the PCR products and thus mRNAs of all the cytokines i ncreased with LPS stimulation, maximal levels being achieved 3 to 5 h post stimulation. Conclusions: RT-PCR produced consistent results, ind icating that this technique could be used to investigate the effect of biological mediators and novel pharmacological agents on cytokine mRN A levels in macrophages.