Analysis of the Interrelationship between IL-12, TNF-α, and IFN-γ/Production during Murine Listeriosis

Abstract

IL-12, a recently described cytokine, is an important mediator in the early production of IFN-γ during infection. To evaluate the timing of IL-12 production, and its relationship to TNF-α and IFN-γ production during primary murine listeriosis, we measured cytokine mRNA and protein levels in C57B1/6 mice infected intravenously with Listeria monocytogenes (LM). IL-12 is a disulfide-linked heterodimer containing two chains (designated P35 and P40); however, bioactive cytokine production has been more closely linked with P40 expression. Consequently, we monitored mRNA and protein levels of P40 in the spleen as a marker for IL-12 production in vivo. Splenic P40 mRNA levels (assayed using RNase protection methods) were low in uninfected animals, but increased markedly beginning 15 to 18 hr after LM infection. In sublethally infected animals, P40 mRNA levels remained elevated for 5 days, returning to baseline with the resolution of infection. P40 protein (assayed using an antibody capture ELISA) could be detected in the spleens of LM-infected animals beginning around 18 hr postinfection confirming linkage between P40 mRNA accumulation and the generation of a protein product. In comparing P40 and IFN-γ mRNA levels in vivo, we found in each case that substantial increases in mRNA accumulation did not appear until 15-18 hr postinfection. In comparable studies using BALB/c animals, cytokine production began slightly earlier (between 12 and 15 hr) but once again P40 and IFN-γ mRNA levels increased in a coordinated manner. P40 mRNA (like IFN-γ and TNF-α mRNA) only accumulated in animals infused with live, virulent bacteria. Although we could detect no obvious lag between the time of onset of IL-12 and IFN-γ accumulation in vivo, infusions of anti-IL-12 antibodies markedly reduced IFN-γ expression implying that IL-12 production precedes and directs IFN-γ production. TNF-α production, on the other hand, was not diminished by anti-IL-12 treatment. Our studies demonstrate that IL-12 generation is an essential step in normal IFN-γ production during listeriosis, and suggest that IL-12, once produced, may begin enhancing IFN-γ production in vivo in less than 3 hr.