Katherine A. Gauss

Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases

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.

Role of NF‐κB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor‐α

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‐α and the generation of reactive oxygen species (ROS), such as superoxide anion (O2•–). Recently, we found that TNF‐α 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‐α, 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‐α, we evaluated transcriptional regulation of oxidase genes in MonoMac1 cells and human monocytes. We show that TNF‐α‐treated cells have increased levels of mRNA and up‐regulated expression of NADPH oxidase subunits p47phox, p67phox, and gp91phox, as well as increased oxidase activity. Pharmacological inhibitors of NF‐κB activation blocked TNF‐α‐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‐α‐treated MonoMac1 cells and monocytes are a result, in part, of transcriptional up‐regulation of three essential NADPH oxidase genes via the NF‐κB pathway. This novel finding supports a model, whereby TNF‐α‐dependent activation of NF‐κB up‐regulates phagocyte NADPH oxidase activity, leading to enhanced ROS production and further NF‐κB activation, potentially contributing to sustained oxidant production in chronic inflammation.

AP-1 is essential for p67phox promoter activity

The cytosolic NADPH oxidase cofactor p67phoxhas been shown to be one of the limiting factors in assembly andactivation of this multi‐protein enzyme complex and, therefore, must behighly regulated at the transcriptional level. In the present studies, we have further characterized the promoter for humanp67phox. Genomic sequence upstream of thetranslational start site (TLS; 2 kb) was cloned, and RACE was used toidentify and compare the transcriptional start site (TSS) in twomyeloid cell lines, HL‐60 and PLB‐985. Two major TSS were identifiedwithin the first intron for both cell lines, and one transcriptisolated from PLB‐985 cells started approximately 34 bp 5′ of exon 1and contained no intron 1 sequence. To identify regulatory regions ofthe promoter, a luciferase reporter was used to assay a series ofpromoter deletion constructs. The greatest transcriptional activity wasobserved for fragments containing at least 500 bp upstream of the TLS. Sequence analysis of the p67phox promoterrevealed consensus binding sites for previously described transcriptionfactors including AP‐1 and PU.1. Site‐directed mutagenesis of the AP‐1site demonstrated that this site was essential for basal transcription. EMSA, competition, and super‐shift assays showed that this site wasspecifically recognized by nuclear factors of the AP‐1 family. EMSAanalysis and promoter‐reporter assays with the PU.1 consensus sites atpositons ‐176, ‐283, and ‐328 demonstrate that PU.1 binds the site atposition ‐283 with high affinity. Mutagenesis of any one of the PU.1sites reduced the basal transcriptional activity by approximately 50%,demonstrating that, although none of these sites is singularlyresponsible for the basal transcriptional activity, all three sitesplay some role in the transcriptional activity of thep67phox promoter. In support of thisconclusion, mutagenesis of all three sites completely abrogatedtranscriptional activity.

Binding of Pleomorphic Adenoma Gene-like 2 to the Tumor Necrosis Factor (TNF)-α-responsive Region of the NCF2 Promoter Regulates p67phox Expression and NADPH Oxidase Activity

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox, is up-regulated by TNF-α, and we recently mapped a region in the NCF2 promoter that was required for this TNF-α-dependent response. Because this TNF-α-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-α treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67phox protein expression, and subsequent superoxide production in response to TNF-α. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-α 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.

Binding of pleomorphic adenoma gene-like 2 to the TNF-α-responsive region of the NCF2 promoter regulates p67phox expression and NADPH oxidase activity

NCF2, the gene encoding the NADPH oxidase cytosolic component p67phox, is up-regulated by TNF-α, and we recently mapped a region in the NCF2 promoter that was required for this TNF-α-dependent response. Because this TNF-α-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-α treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67phox protein expression, and subsequent superoxide production in response to TNF-α. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-α 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.

Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67phox homolog

The NADPH oxidase plays an important role in immune and nonimmune cell functions. Because rabbits represent an established model for studying a number of important disease processes that involve NADPH oxidase activity, we carried out studies to clone and sequence all five rabbit leukocyte NADPH oxidase genes. Comparison of the rabbit sequences with those of other species showed that, with the exception of p67phox, the rabbit phox proteins were highly conserved. In contrast, rabbit p67phox had a very divergent C‐terminus and was 17 amino acids longer than any other known p67phox homolog. This was surprising, given the high degree of conservation among all of the phox proteins sequenced previously. To evaluate the functional consequences of this difference, wild‐type rabbit p67phox and a mutated rabbit p67phox missing the C‐terminal 17 amino acids were expressed and analyzed in a cell‐free assay. Our results show that the full‐length and truncated rabbit p67phox proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full‐length p67phox. These studies contribute to our understanding of the nature of the leukocyte NADPH oxidase in different species and will be valuable in future research using the rabbit model.

Cloning and expression of bovine p47-phox and p67-phox: comparison with the human and murine homologs.

Neutrophils play an essential role in bovine cellular host defense, and compromised leukocyte function has been linked to the development of respiratory and mucosal infections. During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide anion‐generating complex known as the NADPH oxidase. Two of the essential cytosolic components of the NADPH oxidase are p47‐phox and p67‐phox. Currently, only the human and murine homologs of these proteins have been sequenced. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and express bovine p47‐phox and p67‐phox. Using polymerase chain reaction (PCR) cloning techniques and a bovine bone marrow cDNA library, we have cloned both of these bovine NADPH oxidase cytosolic components. Comparison of the bovine sequences with those of the human and murine homologs showed that they were highly conserved, but also revealed important information regarding key structural features of p47‐phox and p67‐phox, including location of putative phosphorylation sites. Functional expression of bovine p47‐phox and p67‐phox showed that these proteins could substitute for the human proteins in reconstituting NADPH oxidase activity in a cell‐free assay system, again demonstrating the high degree of conservation between human and bovine homologs. This study greatly contributes to our understanding of the potential structural/functional regions of p47‐phox and p67‐phox as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases. J. Leukoc. Biol. 67: 63–72; 2000.

Molecular analysis of the bovine anaphylatoxin C5a receptor

Recruitment of phagocytes to inflammatory sites involves the coordinated action of several chemoattractants, including the anaphylatoxin C5a. While the C5a receptor (C5aR) has been well characterized in humans and rodents, little is known about the bovine C5aR. Here, we report cloning of bovine C5R1, the gene encoding bovine C5aR. We also analyzed genomic sequence upstream of the C5R1 translation start site. Although the bovine C5aR amino acid sequence was well conserved among species, significant differences in conserved features were found, including major differences in the N terminus, intracellular loop 3, and transmembrane domain VII. Analysis of C5aR expression by flow cytometry and confocal microscopy demonstrated high levels of C5aR on all bovine neutrophils and a subset of bovine monocytes. C5aR was not expressed on resting or activated bovine lymphocytes, although C5aR message was present in these cells. C5aR was also expressed on a small subset of bovine mammary epithelial cells. Pharmacological analysis of bovine C5aR‐mediated responses showed that bovine C5a and C5adesArg both induced dose‐dependent calcium fluxes and chemotaxis in bovine neutrophils, with similar efficacy for both agonists. Treatment of bovine neutrophils with C5a or C5adesArg resulted in homologous desensitization of bovine C5aR and cross‐desensitization to interleukin 8 (IL‐8) and platelet‐activating factor (PAF); whereas, treatment with IL‐8 or PAF did not cross‐desensitize the cells to C5a or C5adesArg. Overall, these studies provide important information regarding distinct structural and functional features that may contribute to the unique pharmacological properties of bovine C5aR.

Identification of a novel tumor necrosis factor α-responsive region in the NCF2 promoter

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 α (TNF‐α), which activates AP‐1, we hypothesized that TNF‐α might regulate NCF2transcription via AP‐1. In support of this hypothesis, we show here that NCF2 promoter‐reporter constructs are up‐regulated by TNF‐α 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‐α 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‐α 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‐α‐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‐α‐induced up‐regulation of p67phox.

Molecular analysis of the bison phagocyte NADPH oxidase: cloning and sequencing of five NADPH oxidase cDNAs

During the host defense process, neutrophils migrate into infected tissues where they become activated, resulting in the assembly of a superoxide anion-generating complex known as the NADPH oxidase. Despite the importance of this system in animal host defense, almost nothing is known about the NADPH oxidase in neutrophils from wild ruminant species. In the present studies, we provide a molecular analysis of the bison leukocyte NADPH oxidase. Using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends, we cloned and sequenced the full-length cDNAs for five bison NADPH oxidase components: p22(phox), p40(phox), p47(phox) and p67(phox), and gp91(phox). When compared to other species, the deduced amino acid sequences of the bison homologs were most similar to those of bovine. Interestingly, a bison p40(phox) alternative splice product was isolated, which was similar to that observed for human p40(phox) in that the cDNAs contained sequence from intron 8. Consistent with the high degree of similarity between bison and bovine amino acid sequences, immunoblot analysis showed that the bison homologs migrated similarly to their bovine counterparts. Overall, these studies show that the bison and bovine NADPH oxidase genes are highly conserved between these two species, despite their divergence from a common ancestor over 1 million years ago.