Daniel W. Siemsen

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.

Fractionation and Characterization of Biologically-active Polysaccharides from Artemisia tripartita

The leaves of Artemisia species have been traditionally used for prevention and treatment of a number of diseases. In this study, five polysaccharide fractions (designated A-I-A-V) were isolated from the leaves of Artemisia tripartita Rydb. by the sequential use of hot-water extraction, ethanol precipitation, ultra-filtration, and chromatography. The homogeneity and average molecular weight of each fraction were determined by high performance size-exclusion chromatography. Sugar composition analysis revealed that Artemisia polysaccharides consisted primarily of xylose, glucose, arabinose, galactose, and galactosamine. Moreover, all fractions contained at least 3.4% sulfate, and fractions A-II-A-V contained an arabinogalactan type II structure. All fractions exhibited macrophage-activating activity, enhancing production of intracellular reactive oxygen species and release of nitric oxide, interleukin 6, interleukin 10, tumor necrosis factor alpha, and monocyte chemotactic protein 1. In addition, all fractions exhibited scavenging activity for reactive oxygen species generated enzymatically or produced extracellularly by human neutrophils. Finally, fractions A-I and A-V exhibited complement-fixing activity. Taken together, our results provide a molecular basis to explain at least part of the beneficial therapeutic effects of Artemisia extracts, and suggest the possibility of using Artemisia polysaccharides as an immunotherapeutic adjuvant.

Neutrophil isolation from nonhuman species.

The development of new advances in the understanding of neutrophil biochemistry requires effective procedures for isolating purified neutrophil populations. Although methods for human neutrophil isolation are now standard, similar procedures for isolating neutrophils from many of the nonhuman species used to model human diseases are not as well developed. Since neutrophils are reactive cells, the method of isolation is extremely important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animals (bovine, equine, ovine), small animals (murine and rabbit), and nonhuman primates (cynomolgus macaques), and describe optimized details for obtaining the highest cell purity, yield, and viability. We also describe methods to verify phagocytic capacity in the purified cell populations using a flow cytometry-based phagocytosis assay.

Antibody Imprint of a Membrane Protein Surface PHAGOCYTE FLAVOCYTOCHROME b

Structural features of the integral membrane protein flavocytochrome b (Cyt b) were discovered using an antibody “imprint” of the Cyt bsurface. Amino acid sequences were selected from a random nonapeptide phage-display library by their affinity for the monoclonal antibody 44.1 binding site, which recognizes the native conformation of the p22 subunit of Cyt b. Transferred nuclear Overhauser effect spectroscopy and rotating frame Overhauser effect spectroscopy NMR were used to study the antibody-bound conformation of a synthetic peptide derived from phage-displayed sequences. The NMR data supported the phage-display analysis suggesting the existence of a complex epitope and allowed the modeling of the close spatial proximity of the epitope components 29TAGRF33 and183PQVNPI188 from discontinuous regions of p22. Although these regions are separated by two putative membrane-spanning domains and are 150 residues apart in the sequence, they appear to combine to form a complex epitope on the cytosolic surface of the transmembrane protein. NMR constraints, measured from the antibody-bound conformation of a composite peptide mimetic of the Cyt b epitope, and one constraint inferred from the phage-display results, were used to demonstrate the close proximity of these two regions. This information provides a low resolution view of the tertiary structure of the native discontinuous epitope on the Cytb surface. Given additional antibodies, such imprint analysis has the potential for producing structural constraints to help support molecular modeling of this and other low abundance or noncrystallizable proteins.

Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses

Peroxynitrite, a potent oxidant generated in inflammatory tissues, can nitrate tyrosine residues on a variety of proteins. Based on previous studies suggesting that actin might be a potential target for peroxynitrite‐mediated nitration in neutrophils, we investigated the effects of peroxynitrite on actin function. We show here that peroxynitrite and the peroxynitrite generator (SIN‐1) modified actin in a concentration‐dependent manner, resulting in an inhibition of globular‐actin polymerization and filamentous‐actin depolymerization in vitro. The effects of peroxynitrite were inhibited by the pyrrolopyrimidine antioxidant PNU‐101033E, which has been shown previously to specifically block peroxynitrite‐mediated tyrosine nitration. Furthermore, spectrophotometric and immunoblot analysis of peroxynitrite‐treated actin demonstrated a concentration‐dependent increase in nitrotyrosine, which was also blocked by PNU‐101033E. Activation of neutrophils in the presence of a nitric oxide donor (S‐nitroso‐N‐acetylpenicillamine) resulted in nitration of exogenously added actin. Nitrated actin was also found in peroxynitrite‐treated neutrophils, suggesting that actin may be an important intracellular target during inflammation. To investigate this issue, we analyzed the effect of peroxynitrite treatment on a number of actin‐dependent neutrophil processes. Indeed, neutrophil actin polymerization, migration, phagocytosis, and respiratory burst activity were all inhibited by SIN‐1 treatment in a concentration‐dependent manner. Therefore, the ability of peroxynitrite to inhibit actin dynamics has a significant effect on actin‐dependent, cellular processes in phagocytic cells and may modulate their host defense function.

Inhibition of the human neutrophil NADPH oxidase by Coxiella burnetii

Coxiella burnetii is an obligate intracellular Gram-negative pathogen. A notable feature of C. burnetii is its ability to replicate within acidic phagolysosomes; however, the mechanisms utilized in evading host defenses are not well defined. Here, we investigated human neutrophil phagocytosis of C. burnetii (Nine Mile, phase II; NMII) and the effect of phagocytosed organisms on neutrophil reactive oxygen species (ROS) production. We found that opsonization with immune serum substantially enhanced phagocytosis of NMII. Human neutrophils phagocytosing opsonized NMII generated very little ROS compared to cells phagocytosing opsonized Staphylococcus aureus, Escherichia coli, or zymosan. However, phagocytosis of NMII did not affect the subsequent ROS response to a soluble agonist, indicating inhibition was localized to the phagolysosome and was not a global effect. Indeed, analysis of NADPH oxidase assembly in neutrophils after phagocytosis showed that translocation of cytosolic NADPH oxidase proteins, p47(phox) and p67(phox), to the membrane was absent in cells phagocytosing NMII, as compared to cells phagocytosing S. aureus or activated by phorbol myristate acetate. Thus, phagocytosed NMII is able to disrupt assembly of the human neutrophil NADPH oxidase, which represents a novel virulence mechanism for this organism and appears to be a common mechanism of virulence for many intracellular pathogens.

Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b

Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4‐hydroxynonenal (4‐HNE). 4‐HNE is a lipid peroxidation product generated by free radical attack on ω‐6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4‐HNE‐protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X‐linked, flavocytochrome b‐deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4‐HNE‐protein adducts and gp91‐phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation‐dried neutrophils. No 4‐HNE‐protein adducts were detected in flavocytochrome b‐deficient cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91‐phox‐positive cells from both the normal and heterozygous CGD carrier, significant 4‐HNE‐protein adduct labeling was observed, primarily in the phagosomes. When data from single‐ and doublelabeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4‐HNE‐protein adduct‐positive and 56% gp91‐phox‐positive, while cells from the normal father were >97% positive for both 4‐HNE‐protein adducts and gp91‐phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 ± 2%, and 0% positive for the fathers, mothers, and sons cells, respectively, and demonstrated that 4‐HNE‐protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4‐HNE and resulting 4‐HNE‐protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins. J. Leukoc. Biol. 57: 415–421; 1995.

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.

Inhibition of the Neutrophil NADPH Oxidase by Adenosine Is Associated with Increased Movement of Flavocytochrome b Between Subcellular Fractions

Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by the NADPH oxidase in fMLF-stimulated neutrophils. Although much is known about the pharamacology and signal transduction of this effect, it is not known how adenosine affects assembly and localization of the NADPH oxidase components within the neutrophil. We report here that adenosine pretreatment of fMLF-stimulated neutrophils results in decreased plasma membrane/secretory granule content of the flavocytochrome b components (p22phox and gp91phox) of the NADPH oxidase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upregulation of secretory and specific granule surface markers, confirming that degranulation was not impaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in fMLF-stimulated neutrophils. These data suggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocytosis and/or redistribution of flavocytochrome b between various subcellular compartments.