Neutrophils play an essential role in the body's innate defense against pathogens and are one of the primary mediators of the inflammatory response. To defend the host, neutrophils use a wide range of microbicidal products, such as oxidants, microbicidal peptides, and lytic enzymes. The generation of microbicidal oxidants by neutrophils results from the activation of a multiprotein enzyme complex known as the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is responsible for transferring electrons from NADPH to O2, resulting in the formation of superoxide anion. During oxidase activation, cytosolic oxidase proteins translocate to the phagosome or plasma membrane, where they assemble around a central membrane-bound component known as flavocytochrome b. This process is highly regulated, involving phosphorylation, translocation, and multiple conformational changes. Originally, it was thought that the NADPH oxidase was restricted to phagocytes and used solely in host defense. However, recent studies indicate that similar NADPH oxidase systems are present in a wide variety of nonphagocytic cells. Although the nature of these nonphagocyte NADPH oxidases is still being defined, it is clear that they are functionally distinct from the phagocyte oxidases. It should be noted, however, that structural features of many nonphagocyte oxidase proteins do seem to be similar to those of their phagocyte counterparts. In this review, key structural and functional features of the neutrophil NADPH oxidase and its protein components are described, including a consideration of transcriptional and post-translational regulatory features. Furthermore, relevant details about structural and functional features of various nonphagocyte oxidase proteins will be included for comparison.

Macrophages play an important role in the pathogenesis of chronic inflammatory disease. Activation of these phagocytes induces the production of proinflammatory cytokines, such as IL-1 and TNF-alpha and the generation of reactive oxygen species (ROS), such as superoxide anion (O2*-). Recently, we found that TNF-alpha treatment of human monocytic cells (MonoMac1) and isolated human monocytes resulted in up-regulation of the NADPH oxidase gene, neutrophil cytosolic factor 2 (NCF2). These results suggested that TNF-alpha, produced by activated macrophages, could serve as an autocrine/paracrine regulator of the oxidase, resulting in increased and/or prolonged production of O2*-. To gain a better understanding of the mechanisms involved in NADPH oxidase regulation by TNF-alpha, we evaluated transcriptional regulation of oxidase genes in MonoMac1 cells and human monocytes. We show that TNF-alpha-treated cells have increased levels of mRNA and up-regulated expression of NADPH oxidase subunits p47(phox), p67(phox), and gp91(phox), as well as increased oxidase activity. Pharmacological inhibitors of NF-kappaB activation blocked TNF-alpha-induced up-regulation of NCF1, NCF2, and CYBB message, which correlated with a reduction in expression of the corresponding oxidase proteins and decreased O2*- production. These data demonstrate that the increase in and/or maintenance of O2*- production in TNF-alpha-treated MonoMac1 cells and monocytes are a result, in part, of transcriptional up-regulation of three essential NADPH oxidase genes via the NF-kappaB pathway. This novel finding supports a model, whereby TNF-alpha-dependent activation of NF-kappaB up-regulates phagocyte NADPH oxidase activity, leading to enhanced ROS production and further NF-kappaB activation, potentially contributing to sustained oxidant production in chronic inflammation.

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox , is up-regulated by TNF-␣, and we recently mapped a region in the NCF2 promoter that was required for this TNF-␣-dependent response. Because this TNF-␣-responsive region (TRR) lacked recognizable transcription factor binding elements, we performed studies to identify factors involved in regulating NCF2 via the TRR. Using the TRR sequence as bait in a yeast one-hybrid screen, we identified the zinc finger transcription factor Pleomorphic Adenoma Gene-Like 2 (PLAGL2) as a candidate regulator of NCF2 expression. PLAGL2-specific antibodies were generated that detected the native and SUMO1-modified forms of endogenous PLAGL2. EMSA and DNA-binding protein affinity purification analyses demonstrated specific binding of in vitro-translated as well as endogenously expressed PLAGL2 to the TRR, and chromatin immunoprecipitation assays demonstrated enhanced binding of endogenous PLAGL2 to the TRR in vivo with TNF-␣ treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67 phox protein expression, and subsequent superoxide production in response to TNF-␣. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-␣ treatment. These data clearly identify PLAGL2 as a novel regulator of NCF2 gene expression as well as NADPH oxidase activity and contribute to a greater understanding of the transcriptional regulation of NCF2.The NADPH oxidase is a multicomponent, enzyme complex consisting of both membrane-bound and cytosolic components and is found in all phagocytic leukocytes, such as monocytes and neutrophils, where it plays an essential role in host defense against pathogens (reviewed in Ref. phox are sufficient for a functional oxidase; however, in vivo the system is clearly more complicated (reviewed in Ref. 6). Although the specific role of p67 phox in enzyme function is still being determined, it has been proposed that p67phox is an NADPH-binding protein (7), and Dang et al. (8) subsequently showed that p67 phox was able to directly bind NADPH. In addition, several groups have shown that p67 phox binding to flavocytochrome b 558 induces conformational changes in flavocytochrome b 558 , resulting in initiation of electron flow (9 -11). The physiological importance of p67 phox in NADPH oxidase function is demonstrated by certain forms of chronic granulomatous disease (CGD), which are caused by autosomal mutations in NCF2 (12). Patients with CGD have an inactive NADPH oxidase, making them susceptible to recurrent bacterial and fungal infections (reviewed in Ref. 13).Regulation of p67 phox expression appears distinct from other phox proteins, as p67 phox is the last phox protein to be expressed during cellular differentiation, and its expression correlates the closest with the acquisition of oxidase activity (14). Thus, it has been proposed that p67 phox may be the rate-limiting cofactor in NADPH oxidase activation (10,15,16). A number of stud...

The phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is a multiprotein enzyme that catalyzes the production of microbicidal oxidants. Although oxidase assembly involves association of several membrane and cytosolic oxidase proteins, one of the cytosolic cofactors, p67phox, appears to play a more prominent role in final activation of the enzyme complex. Based on the importance of p67phox, we investigated transcriptional regulation of the p67phox gene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein-1 (AP-1) was essential for basal transcriptional activity. As p67phox can be up-regulated by tumor necrosis factor alpha (TNF-alpha), which activates AP-1, we hypothesized that TNF-alpha might regulate NCF2transcription via AP-1. In support of this hypothesis, we show here that NCF2 promoter-reporter constructs are up-regulated by TNF-alpha but only when AP-1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2 mRNA and p67phox protein are up-regulated by TNF-alpha in various myeloid cell lines as well as in human monocytes. It was surprising that mutagenesis of the AP-1 site in NCF2 promoter constructs did not eliminate TNF-alpha induction, suggesting additional elements were involved in this response and that AP-1 might play a more indirect role. Indeed, we used NCF2 promoter-deletion constructs to map a novel TNF-alpha-responsive region (TRR) located between -56 and -16 bp upstream of the translational start site and demonstrated its importance in vivo using transcription factor decoy analysis. Furthermore, DNase footprinting verified specific binding of factor(s) to the TRR with AP-1 binding indirectly to this region. Thus, we have identified a novel NCF2 promoter/enhancer domain, which is essential for TNF-alpha-induced up-regulation of p67phox.

Although acquired bone marrow failure (BMF) is considered a T cell-mediated autoimmune disease, possible innate immune defects as a cause for systemic immune deviations in response to otherwise innocuous infections, have not been extensively explored. In this regard we recently demonstrated an important role of type-I-IFNs in protecting hematopoiesis during systemic stress responses to the opportunistic fungal pathogen Pneumocystis in lymphocyte-deficient mice. Mice deficient in both lymphocytes and type-I-IFN-receptor (IFrag−/− mice) develop rapidly progressing BMF due to accelerated bone marrow cell apoptosis associated with innate immune deviations in the bone marrow in response to Pneumocystis lung infection. However, the communication pathway between lung and bone marrow eliciting the induction of BMF in response to this strictly pulmonary infection has been unclear. Here we report that absence of an intact type-I-IFN-system during Pneumocystis lung infection not only causes BMF in lymphocyte-deficient mice but also transient bone marrow stress in lymphocyte-competent mice. This is associated with an exuberant systemic IFN-γ response. IFNγ neutralization prevented Pneumocystis lung infection-induced bone marrow depression in type-I-IFN-receptor-deficient (IFNAR−/−) mice, and prolonged neutrophil survival time in bone marrow from IFrag−/− mice. IL-1β and upstream regulators of IFNγ, IL-12 and IL-18, were also upregulated in lung and serum of IFrag−/− mice. In conjunction there was exuberant inflammasome-mediated caspase-1-activation in pulmonary innate immune cells required for processing of IL-18 and IL-1β. Thus, absence of type-I-IFN-signaling during Pneumocystis lung infection may result in deregulation of inflammasome-mediated pulmonary immune activation causing systemic immune deviations triggering BMF in this model.