Isolation of Neutrophils from Nonhuman Species.

The development of new advances in understanding the role of neutrophils in inflammation requires effective procedures for isolating and purifying neutrophils. Methods for isolating human neutrophils are fairly standard, and some are covered in other chapters of this volume and previous editions. However, procedures for isolating neutrophils from nonhuman species used to model human diseases vary from those used in isolating human neutrophils and are not as well developed. Since neutrophils are highly reactive and sensitive to small perturbations, the methods of isolation are important to avoid isolation technique-induced alterations in cell function. We present methods here for reproducibly isolating highly purified neutrophils from large animal models (bovine, equine, ovine), small animal models (murine and rabbit), and nonhuman primates (cynomolgus macaques) and describe optimized details for obtaining the highest cell purity, yield, and viability.

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.

Modulation of PLAGL2 transactivation by positive cofactor 2 (PC2), a component of the ARC/Mediator complex.

The pleomorphic adenoma gene (PLAG) family of transcription factors regulates a wide range of physiological processes, including cell proliferation, tissue-specific gene regulation, and embryonic development, although little is known regarding the mechanisms that regulate PLAG protein activity. In this study, a yeast two-hybrid screen identified PC2, a component of the Mediator complex, as a PLAGL2-binding protein. We show that PC2 cooperates with PLAGL2 and PU.1 to enhance the activity of a known PLAGL2 target promoter (NCF2). The PLAGL2-binding element in the NCF2 promoter consisted of the core sequence of the bipartite PLAG1 consensus site, but lacked the G-cluster motif, and was recognized by PLAGL2 zinc fingers 5 and 6. Promoter and PLAGL2 mutants showed that PLAGL2 and PU.1 were required to bind to their respective sites in the promoter, and PC2 knockdown demonstrated that PC2 was essential for enhanced promoter activity. Co-immunoprecipitation and promoter-reporter studies reveal that the effect of PC2 on PLAGL2 target promoter activity was conferred via the C-terminus of PLAGL2, the region that is required for PC2 binding and contains the PLAGL2 activation domain. Importantly, chromatin immunoprecipitation analysis and PC2 knockdown studies confirmed that endogenous PC2 protein associated with the NCF2 promoter in MM1 cells in the region occupied by PLAGL2, and was required for PLAGL2 target promoter activity in TNF-alpha-treated MM1 cells, respectively. Lastly, the expression of another known PLAGL2 target gene, insulin-like growth factor II (IGF-II), was greatly diminished in the presence of PC2 siRNA. Together, the data identify PC2 as a novel PLAGL2-binding protein and important mediator of PLAGL2 transactivation.

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.

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.

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.

Role of NF-kappaB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-alpha.

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.

Binding of pleomorphic adenoma gene-like 2 to the tumor necrosis factor (TNF)-alpha-responsive region of the NCF2 promoter regulates p67(phox) expression and NADPH oxidase activity.

NCF2, the gene encoding the NADPH oxidase cytosolic component p67(phox), is up-regulated by TNF-alpha, and we recently mapped a region in the NCF2 promoter that was required for this TNF-alpha-dependent response. Because this TNF-alpha-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-alpha treatment. Knockdown of PLAGL2 protein inhibited up-regulation of NCF2 transcript, p67(phox) protein expression, and subsequent superoxide production in response to TNF-alpha. Furthermore, relative levels of native and SUMO1-modified endogenous PLAGL2 protein were modulated in a time-dependant manner in response to TNF-alpha 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.

Neutrophil isolation from nonhuman species.

Advances in the understanding of neutrophil biochemistry require the development of 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. Because 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 (cow, horse, sheep) and small (mouse, rabbit) animal models 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.

Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR.

The regulator VicR of the two-component regulatory system VicRK is essential in several Gram-positive bacteria. However, the authors were able to generate an unconditional vicR insertional mutant of group A Streptococcus. This mutant grew well in rich media but not in non-immune human blood and serum, had attenuated virulence, and was unstable in mice. Complementation of the mutant with vicR expressed in trans restored its phenotype to wild-type. A vicK deletion mutant had a phenotype similar to that of the vicR mutant. Phagocytosis and killing of the vicR mutant were normal, suggesting that VicRK does not regulate processes involved in evasion of host defence. Microarray analysis showed that vicR inactivation down-regulated the transcription of 13 genes, including putative cell wall hydrolase gene pcsB and spy1058-1060, which encode a putative phosphotransferase system enzyme II for carbohydrate transport, and upregulated the expression of five genes, including spy0183 and spy0184, which encode an osmoprotectant transporter OpuA. Consistent with microarray analysis, the vicR mutant took up more of the osmoprotectants betaine and proline and was sensitive to osmotic stress, indicating that vicR inactivation induced osmotic stress and increased susceptibility to osmotic pressure. Additionally, a spy1060 deletion mutant also displayed attenuated virulence. These results suggest that VicRK regulates processes involved in cell wall metabolism, nutrient uptake, and osmotic protection.

Variants of the 5'-untranslated region of human NCF2: expression and translational efficiency.

The NCF2 gene encodes p67(phox), an essential component of the multi-protein NADPH oxidase enzyme in phagocytic leukocytes, as well as in certain non-phagocytic cells. In humans, the NCF2 gene is expressed as multiple NCF2 variants that differ in the 5'-untranslated region (5'-UTR). Previously, we reported the presence of four NCF2 5'-UTR mRNA variants (designated as NCF2 exon 1, intron 1a, intron 1b and intron 1c). As each of the gene variants encodes an identical p67(phox) protein, the functional significance of these message variants was not apparent. In this study, we investigated the relative expression levels and tissue-specificity of NCF2 5'-UTR variant mRNAs and their translation efficiency and stability. NCF2 5'-UTR variant transcripts were differentially expressed in various cell lines and human tissues. In vitro translation assays indicated that the NCF2 5'-UTR variants also differed in their effects on the translation of a luciferase reporter mRNA and NCF2 mRNA. Notably, NCF2 intron 1 5'-UTR variants, which are the predominantly expressed variants found in vivo, strongly inhibited translation when compared to the NCF2 exon 1 5'-UTR variant. In contrast, RNA decay assays demonstrated that there was no significant difference between stability of NCF2 intron 1 transcripts and the exon 1 5'-UTR variant in HL-60, MonoMac 6, and U937 cells. Moreover, expression of the variant transcripts remained unchanged after neutrophil phagocytosis, and was similar in normal neutrophils and neutrophils from a patient with X-linked chronic granulomatous disease. These studies suggest that expression of p67(phox) is regulated through mechanisms that include modulation of transcription and translation.

Identification of a novel tumor necrosis factor alpha-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 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.

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.

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.

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.

Cloning and sequencing of rabbit leukocyte NADPH oxidase genes reveals a unique p67(phox) 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 p67(phox), the rabbit phox proteins were highly conserved. In contrast, rabbit p67(phox) had a very divergent C-terminus and was 17 amino acids longer than any other known p67(phox) 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 p67(phox) and a mutated rabbit p67(phox) 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 p67(phox) proteins were able to support oxidase activity, although the truncated form reproducibly supported a higher level of activity than full-length p67(phox). 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.