The human prolactin-inducible protein (PIP) or gross cystic disease fluid protein -15 (GCDFP-15) is a 15 kD protein secreted by human breast cancer cells and is abundant in fluids from gross cystic breast disease. Previous results from our laboratory showed that PIP KO mice had significantly lower numbers of CD4+ T cells in their secondary lymphoid organs, and these cells are impaired in their ability to differentiate into Th1 cells in vitro and in vivo leading to failure to control Leishmania major infection. In the present study, we further assessed the role of PIP in adaptive immunity by comparing cytokine production and intracellular signaling events in macrophages from WT and PIP KO mice following IFN-γ and lipopolysaccharide (LPS) stimulation. We show that although the expressions of IFN-γR and TLR4 on macrophages from KO and WT mice were comparable, PIP KO macrophages were significantly impaired in producing proinflammatory cytokines following IFN-γ and LPS stimulation. This was associated with impaired phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducers of activation of transcription (STATs) proteins in IFN-γ and LPS-stimulated macrophages from PIP KO mice. Interestingly, the expression of suppressors of cytokine signaling (SOCS) 1 and 3 proteins, known to suppress IFN-γ and LPS signaling, was higher in PIP KO macrophages compared to those from WT mice. Collectively, our studies clearly show that deficiency of PIP significantly affects intracellular signaling events leading to proinflammatory cytokine production in macrophages, and further confirm a role for PIP as important immunoregulatory protein involved in host defense. / October 2015