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Activation of neutrophils by Ferula iliensisessential oils enriched in (E/Z)-propenyl sec-butyl disulfides is mediated by activation of transient receptor potential V1 channels. Essential oil extracts from Ferula iliensishave been used traditionally in Kazakhstan for treatment of inflammation and other illnesses. Because little is known about the biologic activity of these essential oils that contributes to their therapeutic properties, we analyzed their chemical composition and evaluated their phagocyte immunomodulatory activity. The main components of the extracted essential oils were (E)-propenyl sec-butyl disulfide (15.7–39.4%) and (Z)-propenyl sec-butyl disulfide (23.4–45.0%). Ferulaessential oils stimulated [Ca2+]imobilization in human neutrophils and activated ROS production in human neutrophils and murine bone marrow phagocytes. Activation of human neutrophil [Ca2+]iflux by Ferulaessential oils was dose-dependently inhibited by capsazepine, a TRPV1 channel antagonist, indicating that TRPV1 channels mediate this response. Furthermore, Ferulaessential oils stimulated Ca2+influx in TRPV1 channel–transfected HEK293 cells and desensitized the capsaicin-induced response in these cells. Additional molecular modeling with known TRPV1 channel agonists suggested that the active component is likely to be (Z)-propenyl sec-butyl disulfide. Our results provide a cellular and molecular basis to explain at least part of the beneficial therapeutic properties of FEOs.

PVL‐mediated priming of PMNs enhances the host innate immune response. CA‐MRSA infections are often caused by strains encoding PVL, which can cause lysis of PMNs and other myeloid cells in vitro, a function considered widely as the primary means by which PVL might contribute to disease. However, at sublytic concentrations, PVL can function as a PMN agonist. To better understand this phenomenon, we investigated the ability of PVL to alter human PMN function. PMNs exposed to PVL had enhanced capacity to produce O2−in response to fMLF, but unlike priming by LPS, this response did not require TLR signal transduction. On the other hand, there was subcellular redistribution of NADPH oxidase components in PMNs following exposure of these cells to PVL—a finding consistent with priming. Importantly, PMNs primed with PVL had an enhanced ability to bind/ingest and kill Staphylococcus aureus. Priming of PMNs with other agonists, such as IL‐8 or GM‐CSF, altered the ability of PVL to cause formation of pores in the plasma membranes of these cells. Microarray analysis revealed significant changes in the human PMN transcriptome following exposure to PVL, including up‐regulation of molecules that regulate the inflammatory response. Consistent with the microarray data, mediators of the inflammatory response were released from PMNs after stimulation with PVL. We conclude that exposure of human PMNs to sublytic concentrations of PVL elicits a proinflammatory response that is regulated in part at the level of gene expression. We propose that PVL‐mediated priming of PMNs enhances the host innate immune response.

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 p67phoxgene [neutrophil cytosolic factor 2 (NCF2)] and demonstrated previously that activator protein‐1 (AP‐1) was essential for basal transcriptional activity. As p67phoxcan 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 NCF2promoter‐reporter constructs are up‐regulated by TNF‐α but only when AP‐1 factors were coexpressed. Consistent with this observation, we also demonstrate that NCF2mRNA and p67phoxprotein 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 NCF2promoter 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 NCF2promoter‐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 NCF2promoter/enhancer domain, which is essential for TNF‐α‐induced up‐regulation of p67phox.

Neutrophils play an essential role in the cellular defense of the bovine mammary gland and compromised leukocyte function has been linked to the development of bovine mastitis. During mastitis, large numbers of leukocytes migrate into the mammary tissues where they become activated, resulting in the assembly of neutrophil membrane and cytosolic proteins to form a superoxide aniongenerating complex known as the NADPH oxidase. The key membrane‐associated component of the NADPH oxidase is flavocytochrome b, which is a heterodimer of p22‐phoxand gp91‐phox. Currently, only the human, porcine, murine, and rattus p22‐phoxand the human, porcine, and murine gp91‐phoxgene sequences are known. Because of the important role neutrophils play in bovine host defense, we carried out studies to clone, sequence, and analyze expression of bovine flavocytochrome b. Using polymerase chain reaction cloning techniques and a bovine spleen cDNA library we have cloned both of the bovine flavocytochrome bsubunits, p22‐phoxand gp91‐phox. Comparison of the bovine sequences with those of other species also revealed important information regarding key structural features of gp91‐phoxand p22‐phox, including location of putative glycosylation sites. This study greatly contributes to our understanding of the potential functional sites of the flavocytochrome bsubunits as well as providing information that can be used to study the role of neutrophils in bovine inflammatory diseases such as mastitis. J. Leukoc. Biol. 64: 114–123; 1998.

Cytosolic components of the phagocyte NADPH oxidase (p47phox, p67phox, and Rac2) translocate to the plasma membrane on cell activation where they interact with a membrane‐bound cytochrome bto generate superoxide anion. Phosphorylation reactions are known to be important for activity of NADPH oxidase. Translocation of Rac2, p47phox, and p67phoxwere all enhanced in formyl‐Met‐Leu‐Phe‐stimulated neutrophils treated with 50 nM of the protein phosphatase 1/2A inhibitor calyculin A. Rac translocation was blocked by the tyrosine kinase inhibitors genistein (50 μM) and herbimycin (17 μM), whereas movement of p47phoxand p67phoxwere not inhibited. Cell‐free analysis of Rac translocation also demonstrated that translocation of p47phoxand p67phoxwere not linked to the movement or availability of Rac2. Thus, Rac2 does not appear to regulate NADPH oxidase by controlling movements of the cytosolic components to the membrane‐associated enzyme but may exert its effect at the level of the assembled complex. Tyrosine kinase activity is required for translocation of Rac in the chemoattractant‐stimulated human neutrophil. J. Leukoc. Biol.58: 108–113; 1995.