Stimulatory factors for interleukin-12 production from murine splenic macrophages co-cultured with Babesia microti and Babesia rodhaini infected erythrocytes

Stimulatory factors for interleukin 12 (IL-12) production associated with Babesia microti and Babesia rodhaini infected erythrocytes were examined using an in vitro assay system established, since a remarkable increase of serum IL-12 concentration and differentiation of helper T cell (Th cell) into helper T cell type 1 (Th1 cell) was observed in early phase of infection with Babesia spp. in mice. To investigate direct stimulating activity of infected erythrocytes for IL-12 production, intact splenic macrophages were co-cultured with them and examined the expression of IL-12 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). Both B. microti and B. rodhaini infected erythrocytes elicited IL-12 mRNA expression in co-cultured macrophages. Since only the supernatant obtained from the cultured medium of infected erythrocytes showed an activity for IL-12 production from macrophages, the supernatant was collected, concentrated, and heated, followed by the collection of soluble fraction. As the heat stable soluble supernatant showed an IL-12 production activity, it was fractionated by gel filtration. The elution profile of the heat stable soluble supernatant from B. microti infected erythrocytes was quite different to that from B. rodhaini infected and non-infected erythrocytes. The differences of stimulatory activity were also observed in the fraction of eluate, especially Fraction 3, between B. microti infected erythrocytes, and B. rodhaini infected and non-infected erythrocytes. These results suggested that B. microti infected erythrocytes and/or B. microti itself released some factors to stimulate IL-12 production from splenic macrophages, resulted in the Th1 differentiation