The qRT-PCR experiments identified PKC as the dominant PKC isoform in FLT3L- and GM-CSF/IL-4-induced BMiDCs and splenic iDCs. M5/114.15.2), APC-CD86 (clone GL1) or respective IgG control monoclonal antibodies. CD86 (A) and Balaglitazone MHC II (B) expression in CD11c+ dendritic cells were determined using flow cytometry. production was monitored using L-012 chemiluminescence. The diagram represents the mean fold change of L-012 chemiluminescence area under the curve obtained in three independent experiments. generation in PMA-treated immature DCs precedes or occurs simultaneously with PKC activation. (A) generation was measured in vehicle- Balaglitazone and PMA-treated Nox2y/+ (wild type) and Nox2y/? BMiDCs using L-012 chemiluminescence (stimulation of Nox2 activity and downstream redox signaling promotes DC macropinocytosis of antigens. PKC/Nox2-mediated antigen macropinocytosis stimulates maturation of DCs and secretion of T-cell stimulatory cytokines. These findings may contribute to a better understanding of the regulatory mechanisms in DC macropinocytosis and downstream regulation of T-cell-mediated responses. receptor-mediated endocytosis and phagocytosis (4). Importantly, the endocytic process by which antigens are internalized not only determines the intracellular trafficking of the antigen but also influences the type of T-cell epitope being presented on MHC molecules (6). Previous studies showed that macropinocytosis is distinct in many ways from receptor-mediated endocytosis and phagocytosis (7). Indeed, phagocytosis and receptor-mediated endocytosis are strictly ligand-receptor-driven processes (4, 8), while macropinocytosis is characterized by receptor-independent internalization of extracellular fluid and pericellular solutes (4, 9). Phagocytosis is initiated by recognition and binding of the particle to Balaglitazone the plasma membrane, followed by localized actin remodeling, formation of a phagocytic cup around the particle and its subsequent internalization into the phagosome (7). Unlike phagocytosis, receptor-mediated endocytosis is largely an Balaglitazone actin-independent process in mammalian cells (10). In receptor-mediated endocytosis, specific cell surface receptors, such as C-type lectin receptors, Fc and Fc receptors mediate antigen internalization by DCs (11). On the contrary, macropinocytosis involves particle-independent, global activation of the actin cytoskeleton resulting in extensive plasma membrane ruffling over the entire surface of the cell. Some of the membrane ruffles curve into O-shaped macropinocytotic cups and close or fuse with the non-extended plasma membrane, leading to macropinosome formation and non-specific internalization of extracellular fluid and associated solutes (4, 7, 9). Previous studies demonstrated that membrane ruffling and macropinocytosis can be stimulated by various growth factors, including epidermal growth factor (12) and hepatocyte growth factor (HGF) (13), cytokines (14, 15), and phorbol esters (15, 16). Although the precise signaling mechanisms responsible for stimulation of macropinocytosis in DCs and other cell types are incompletely defined, phosphatidylinositol phosphates have been shown to play an important role (17). Plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of a number of actin-binding proteins and, thus, plays an important role in controlling submembranous actin polymerization and reorganization during Balaglitazone macropinocytosis (10). PI(4,5)P2 is phosphorylated to PI(3,4,5)P3 by phosphatidylinositol-3-kinase (PI3K) followed by recruitment and BIRC3 activation of small GTPase Rac1 and Rab5 to mediate cup closure and initiate macropinosome formation (10, 18). Furthermore, PI(4,5)P2 is a substrate for phospholipase C, which produces two important signaling molecules: diacylglycerol (DAG) and inositol trisphosphate (IP3) (17). Recent studies by our lab and others have demonstrated that DAG-mediated protein kinase C (PKC) activation in macrophages plays an important role in macropinocytosis (15, 17). The PKC family has been categorized into three groups, namely the DAG/Ca2+-dependent classical (, , and ), DAG-dependent novel (, , , and ), and DAG/Ca2+-independent atypical (, , , and ) PKC isoforms. Importantly, the PKC isoforms differ in their mechanism of activation, substrates, and signaling in the cell (19C21). The specific PKC isoform(s) mediating DC macropinocytosis of antigens and the signaling mechanisms downstream of PKC leading to macropinocytosis are currently unknown. The NADPH oxidases (Noxs) are transmembrane proteins that transfer electrons across biological membranes to reduce oxygen to superoxide anion or its dismuted form, hydrogen peroxide (H2O2) (22). The Nox family consists of seven members, namely Nox1CNox5, dual oxidase (DUOX) 1, and DUOX2. Nox2, the prototype isoform of the Nox family, consists of flavocytochrome b558, an integral membrane heterodimer comprising gp91and p22generation (26). Although signal transduction mediators upstream of Nox2 activation, such as PKC and Rac1, play an important role in macropinocytosis (10) and Nox2 activators also stimulate macropinocytosis (13, 15, 26, 27), the role of Nox2 in DC macropinocytosis has not been previously investigated. The goal of the present study was to identify the specific PKC isoform(s) involved in DC macropinocytosis. We also tested the hypothesis that Nox2 activation downstream of PKC contributes to DC macropinocytosis of antigens, leading to their maturation and secretion of T- cell stimulatory cytokines. The data shown herein demonstrate the involvement of a previously unidentified mechanism by which DAG-inducible PKC activation of Nox2 activity promotes DC macropinocytosis of antigens. PKCCNox2-mediated antigen macropinocytosis induced MHC II translocation to.
The qRT-PCR experiments identified PKC as the dominant PKC isoform in FLT3L- and GM-CSF/IL-4-induced BMiDCs and splenic iDCs