Stat3-dependent induction of p19INK4D by IL-10 contributes to inhibition of macrophage proliferation

We have previously reported that IL-10 inhibits proliferation of normal bone marrow-derived macrophages and of the monocyte/ macrophage cell line J774. Activation of Stat3 was shown to be necessary and sufficient to mediate inhibition of proliferation. To investigate further the mechanism of growth arrest, we examined the effect of IL-10 on expression of cell cycle inhibitors. We found that IL-10 treatment increases expression of the cyclin-dependent kinase inhibitors p19(INK4D) and p21(CIP1) in macrophages. IL-10 cannot induce p19(INK4D) expression or block proliferation when Stat3 signaling is blocked by a dominant negative Stat3 or a mutant IL-10R alpha which does not recruit Stat3 in J774 cells, whereas p21(CIP1) induction is not affected. An inducibly active Stat3 (coumermycin-dimerizable Stat3-Gyrase B), which suppresses J774 cell proliferation, also induced p19(INK4D) expression. Sequencing of the murine p19(INK4D) promoter revealed two candidate Stat3 binding sites, and IL-10 treatment activated a reporter gene controlled by this promoter. These data suggest that Stat3-dependent induction of p19(INK4D) mediates inhibition of proliferation. Enforced expression of murine pl9(INK4D) cDNA J774 cells significantly reduced their proliferation. Use of antisense p19(INK4D) and analysis of p19(INK4D)-deficient macrophages confirmed that p19(INK4D) is required for optimal inhibition of proliferation by IL-10, and indicated that additional IL-10 signaling events contribute to this response. These data indicate that Stat3-dependent induction of p19(INK4D) and Stat3 independent induction of p21(CIP1) are important components of the mechanism by which IL-10 blocks proliferation in macrophages.