Macrophage activation--enhanced capacity to kill, in a cell that otherwise mostly scavenges--is essential for host survival from infection and contributes to containment of tumours. Both microbes and tumour cells, therefore, may be under pressure to inhibit or reverse the activation of macrophages. This reasoning led to the demonstration of macrophage deactivating factors from both microbes and tumour cells. In some circumstances the host itself probably requires the ability to deactivate macrophages. Macrophages are essential to the healing of wounds and repair of tissues damaged by inflammation. Yet the cytotoxic products of the activated macrophages can damage endothelium, fibroblasts, smooth muscle and parenchymal cells (reviewed in ref. 6). Thus, after an inflammatory site has been sterilized, the impact of macrophage activation on the host might shift from benefit to detriment. These concepts led us to search for macrophage deactivating effects among polypeptide growth factors that regulate angiogenesis, fibrogenesis and other aspects of tissue repair. Among 11 such factors, two proteins that are 71% similar proved to be potent macrophage deactivators: these are transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2.

BackgroundWe hypothesized that gp91phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O2-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91phox knockout mice (gp91phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91phox generation.MethodsUnilateral TBI was induced in gp91phox-/- and wild-type (Wt) mice (C57/B6J) (25-30 g). The expression and roles of gp91phox after TBI were investigated using immunoblotting and staining techniques. Levels of O2- and peroxynitrite were determined in situ in the mouse brain. The activated phenotype in microglia that expressed gp91phox was determined in a microglial cell line, BV-2, in the presence of IFNγ or IL-4.ResultsGp91phox expression increased mainly in amoeboid-shaped microglial cells of the ipsilateral hemisphere of Wt mice after TBI. The contusion area, number of TUNEL-positive cells, and amount of O2- and peroxynitrite metabolites produced were less in gp91phox-/- mice than in Wt. In the presence of IFNγ, BV-2 cells had increased inducible nitric oxide synthase and nitric oxide levels, consistent with a classical activated phenotype, and drastically increased expression of gp91phox.ConclusionsClassical activated microglia promote ROS formation through gp91phox and have an important role in brain damage following TBI. Modulating gp91phox and gp91phox -derived ROS may provide a new therapeutic strategy in combating post-traumatic brain injury.

The NADPH oxidase 1 (Nox1) is a gp91phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O2−) of 160 nmol/mg protein/h and expressed the Nox1, p22phox, p67phox, and Rac1 mRNAs, but not the gp91phox, Nox4, p47phox, p40phox, and Rac2 mRNAs. They also expressed novel homologues of p47phox and p67phox (p41nox and p51nox, respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1, p22phox, p51nox, and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O2− (<2 nmol/mg protein/h). Cotransfection of p41nox and p51nox cDNAs in T84 cells enhanced PMA-stimulated O2− release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O2− release in association with the induction of Nox1 protein. The enhanced O2− production by cotransfection of p41nox and p51nox vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-β-activated kinase 1 and TGF-β-activated kinase 1-binding protein 1. A potent inhibitor for NF-κB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O2− production and induction of Nox1 protein. These results suggest that p41nox and p51nox are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.

Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O 2 ؊ generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47 phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67 phox , p47 phox , and p40 phox , which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47 phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47 phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) ␤II rather than an effect of GT on PKC ␤II activity, suggesting a failure of PKC ␤II to associate with the substrate, p47 phox , on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.The best-known NADPH oxidase occurs in phagocytes and provides large quantities of reactive oxygen species (ROS) to oxidatively modify microbes as a microbicidal mechanism. The phagocyte NADPH oxidase is a highly regulated multisubunit enzyme composed of p67 phox , p47 phox , p40 phox , and Rac2 in the cytosol, as well as flavocytochrome b 558 , which consists of gp91 phox and p22 phox , in the membrane (4,9,15,29,36,39,57). The enzyme is dormant in resting cells, with these phox (for phagocyte oxidase) components being distributed to their cellular compartments. However, once phagocytes encounter invading microbes or soluble stimulants, such as phorbol myristate acetate (PMA), cytosolic phox components and Rac2 migrate to the membrane to assemble with flavocytochrome b 558 , forming the active enzyme. The activated flavocytochrome b 558 works as the catalytic redox center, where electrons are transferred from NADPH to molecular oxygen to generate O 2 Ϫ , a precursor of ROS. Recently, increasing attention has been directed toward the Nox/Duox family in nonphagocytes (33), which is composed of homologues of gp91 phox . Although the rates of ROS production are quite low in nonphagocytes, their ROS are believed to play important roles in cell signaling, hypoxic response, the immune system, etc.Over the past decade, the biochemistry of phagocyte NADPH oxidase has been intensively studied, particularly concerning the roles of Src homology 3 (SH3) domains and proline-rich regions (PRR), as well as those of other domains recently identified in...

pylori lipopolysaccharide activates Rac1 and transcription of NADPH oxidase Nox1 and its organizer NOXO1 in guinea pig gastric mucosal cells.