Minute Numbers of Contaminant CD8+ T Cells or CD11b+CD11c+ NK Cells Are the Source of IFN-gamma in IL-12/IL-18-stimulated Mouse Macrophage Populations

Macrophages were reported to be strong producers of interferon gamma (IFN-gamma) after stimulation by interleukin 12 (IL-12) plus IL-18, which gave rise to a novel concept of auto-crine macrophage activation. Here, we show that peritoneal exudate and bone marrow-derived mouse macrophages generated by conventional techniques contain small quantities of CD11b(+)CD11c(+)CD31(+)DX5(+)NK1.1(+) natural killer (NK) cells or CD3(+)CD8(+)TCRbeta(+) T cells, respectively. Intracellular cytokine staining, purification of macrophages by sorting, and the analysis of macrophages from alymphoid RAG2(-/-)gamma-chain(-/-) mice revealed that the high amount of IFN-gamma protein in the supernatants of unseparated IL-12/IL-18-stimulated macrophage populations originates exclusively from the contaminating lymphoid cells. Notably, IL-12/IL-18 still induced IFN-gamma mRNA in highly purified macrophages from wild-type mice and in macrophages from RAG2(-/-)gamma-chain(-/-) mice, whereas nuclear translocation of signal transducer and activator of transcription 4 (STAT4) and production of IFN-gamma protein were no longer detectable. These results question the concept of autocrine macrophage activation by secreted IFN-gamma, suggest differences in the expression of IFN-gamma mRNA and protein between macrophages and lymphoid cells, and illustrate that the limited purity of most myeloid cell populations (</= 98%) might lead to false conclusions.