ABSTRACT
Bacterial lipopolysaccharide (LPS) elicits responses by macrophages that help the body repel infections. Recent evidence indicates that phosphatidylinositol 3-kinase (PI 3-kinase) may mediate some of these responses. Here, we show that exposing macrophages to LPS rapidly increased membrane-associated PI 3-kinase activity and also elevated p70 S6 kinase activity. Inhibitors of PI 3-kinase or the mammalian target of rapamycin (mTOR) fully blocked p70 S6 kinase activation, implying that this kinase is controlled by PI 3-kinase and mTOR. These inhibitors also substantially reduced LPS-induced nitric oxide (NO) production. This inhibition was, in part, attributable to impaired LPS-stimulated secretion of interferon-beta, an autocrine co-factor for NO production. However, the addition of exogenous interferon-beta did not fully restore NO production, indicating that the NO response was being inhibited by another mechanism as well. Together, these data suggest that PI 3-kinase, mTOR, and possibly p70 S6 kinase mediate LPS-induced NO production by regulating the secretion of interferon-beta and by a second undefined mechanism.
Subject(s)
Interferon-beta/metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Nitric Oxide/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Kinases , Androstadienes/pharmacology , Animals , Cell Line , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Membrane/metabolism , Chromones/antagonists & inhibitors , Chromones/pharmacology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Interferon-beta/antagonists & inhibitors , Interferon-beta/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Macrophages/cytology , Macrophages/enzymology , Macrophages/metabolism , Mice , Mice, Knockout , Morpholines/antagonists & inhibitors , Morpholines/pharmacology , Nitrites/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Protein-Tyrosine Kinases/deficiency , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Ribosomal Protein S6 Kinases/metabolism , Sirolimus/antagonists & inhibitors , Sirolimus/pharmacology , TOR Serine-Threonine Kinases , Tumor Necrosis Factor-alpha/metabolism , WortmanninABSTRACT
Macrophage Fcgamma receptors (FcgammaRs) mediate the uptake and destruction of antibody-coated viruses, bacteria, and parasites. We examined FcgammaR signaling and phagocytic function in bone marrow-derived macrophages from mutant mice lacking the major Src family kinases expressed in these cells, Hck, Fgr, and Lyn. Many FcgammaR-induced functional responses and signaling events were diminished or delayed in these macrophages, including immunoglobulin (Ig)G-coated erythrocyte phagocytosis, respiratory burst, actin cup formation, and activation of Syk, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases 1 and 2. Significant reduction of IgG-dependent phagocytosis was not seen in hck(-)(/)-fgr(-)(/)- or lyn(-)(/)- cells, although the single mutant lyn(-)(/)- macrophages did manifest signaling defects. Thus, Src family kinases clearly have roles in two events leading to FcgammaR-mediated phagocytosis, one involving initiation of actin polymerization and the second involving activation of Syk and subsequent internalization. Since FcgammaR-mediated phagocytosis did occur at modest levels in a delayed fashion in triple mutant macrophages, these Src family kinases are not absolutely required for uptake of IgG-opsonized particles.
