An investigation of the temporal induction of cytokine mRNAs in LPS-challenged thioglycollate-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 harvested from female BALB/c mice and the adherent macrophage fraction isolated for use.

TREATMENT

Peritoneal macrophages (1 x 10(6)) were incubated in the presence or absence of lipopolysaccharide (LPS; at a final concentration of 1 microgram/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.