Detection, cDNA cloning and sequencing of canine interleukin 12

In canine peripheral blood mononuclear cells (PBMC) the mRNAs coding for both subunits of canine interleukin 12 (IL-12) were identified using reverse transcription polymerase chain reaction (RT-PCR). Stimulation of canine PBMC with Staphylococcus aureus strain Cowan plus Concanavalin A for 5 h resulted in significant mRNA synthesis. Likewise, inactivated vaccinia virus induced IL-12 mRNA synthesis, however with different kinetics. The complete nucleotide sequence for both IL-12 subunits was determined using rapid amplification of cDNA ends (RACE)-PCR and cloning of amplified specific cDNAs. Computer-aided amino acid (aa) sequence comparison of both canine IL-12 subunits revealed more than 80% identity with the amino acid sequences of six other mammalian species. Closest relationship was found to human, porcine, bovine and cervine IL-12. However, no reactivity was found with antibodies directed against human IL-12, when supernatants of stimulated canine PBMC were tested. Supernatants of canine PBMC stimulated for IL-12 release also induced interferon gamma (IFN-gamma) mRNA as detectable by RT-PCR; however, it was not clear whether IFN-gamma mRNA synthesis was due to an IL-12 specific effect or other stimuli. As to the stimulating effect of IL-12 on canine IFN-gamma mRNA synthesis, recombinant human IL-12 was found to be a good inducer. Since IL-12 is regarded a major regulatory molecule of T-cell-mediated immune response and cell growth our work on the cloning and sequencing of this cytokine from dogs lays the basis for future investigations on the biological and possible therapeutic role of canine IL-12.