An investigation of the temporal induction of cytokine mRNAs in LPS-challenged thioglycollate-elicited murine peritoneal macrophages using the reverse transcription polymerase chain reaction

Abstract.

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 harvested from female BALB/c mice and the adherent macrophage fraction isolated for use. ¶Treatment: Peritoneal macrophages (1 × 10⁶) were incubated in the presence or absence of lipopolysaccharide (LPS; at a final concentration of 1 <mu>g/ml) for 0, 1, 2, 3, 4, 5 and 24 h. ¶Methods: Culture supernatants and cells were harvested at each time point, the secreted cytokine protein levels quantified by sandwich immunoassays and the cytokine mRNA levels assessed by RT-PCR. ¶Results: The IL-6 mRNA 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-12 mRNAs were expressed in both stimulated and unstimulated macrophages. The levels of the PCR products and thus mRNAs of all the cytokines increased with LPS stimulation, maximal levels being achieved 3 to 5 h post stimulation. ¶Conclusions: RT-PCR produced consistent results, indicating that this technique could be used to investigate the effect of biological mediators and novel pharmacological agents on cytokine mRNA levels in macrophages.