Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity.

Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O2-). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the Km for NADPH, but increased the Vmax for O2- production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity.

Phox activity of differentiated PLB-985 cells is enhanced, in an agonist specific manner, by the PLA2 activity of Prdx6-PLA2.

Peroxiredoxin 6-phospholipase A(2) (Prdx6-PLA(2) ) is a bi-functional enzyme with peroxi-redoxin (Prdx) and phospholipase A(2) (PLA(2) ) activities. To investigate its impact on phagocyte NADPH oxidase (phox) activity in a neutrophil model, the protein was knocked down in PLB-985 cells using stable expression of a small hairpin RNA (shRNA) and phox activity was monitored after cell differentiation. The knockdown cells had reduced oxidase activity in response to stimulation with the formylated peptide fMLF, but the response to the phorbol ester PMA was unchanged. Reintroduction of shRNA-resistant Prdx6-PLA(2) into the knockdown cells by stable transfection with a Prdx6-PLA(2) expression plasmid restored the fMLF response, as did reintroduction of Prdx6-PLA(2) mutated in the Prdx active site; reintroduction of PLA(2) active site mutants, however, failed to restore the response. Thus, the PLA(2) activity of Prdx6-PLA(2) in intact cells mediates its ability to enhance phox activity in response to fMLF. In combination with previous publications by other groups, our work indicates that various PLA(2) isoforms can enhance oxidase activity but they are differentially important in different cell types and in the response to different agonists.

IFN-γ alters the expression of diverse immunity related genes in a cell culture model designed to represent maturing neutrophils.

The cytokine interferon-γ (IFN-γ) is approved as a drug to treat chronic granulomatous disease (CGD) and osteopetrosis and is also used in hyperimmunoglobulin E syndromes. Patients with CGD have defects in proteins of the NOX2 NADPH oxidase system. This leads to reduced production of microbicidal ROS by PMNs and recurrent life threatening infections. The goal of this study was to better understand how IFN-γ might support phagocyte function in these diseases, and to obtain information that might expand potential uses for IFN-γ. Neutrophils mature in the bone marrow and then enter the blood where they quickly undergo apoptotic cell death with a half-life of only 5-10 hours. Therefore we reasoned that IFN-γ might exert its effects on neutrophils via prolonged exposure to cells undergoing maturation in the marrow rather than by its brief exposure to short-lived circulating cells. To explore this possibility we made use of PLB-985 cells, a myeloblast-like myeloid cell line that can be differentiated into a mature, neutrophil-like state by treatment with various agents including DMSO. In initial studies we investigated transcription and protein expression in PLB-985 cells undergoing maturation in the presence or absence of IFN-γ. We observed IFN-γ induced differences in expression of genes known to be involved in classical aspects of neutrophil function (transmigration, chemotaxis, phagocytosis, killing and pattern recognition) as well as genes involved in apoptosis and other mechanisms that regulating neutrophil number. We also observed differences for genes involved in the major histocompatibility complex I (MHCI) and MHCII systems whose involvement in neutrophil function is controversial and not well defined. Finally, we observed significant changes in expression of genes encoding guanylate binding proteins (Gbps) that are known to have roles in immunity but which have not as yet been linked to neutrophil function. We propose that changes in the expression within these classes of genes could help explain the immune supportive effects of IFN-γ. Next we explored if the effect of IFN-γ on expression of these genes is dependent on whether the cells are undergoing maturation; to do this we compared the effects of IFN-γ on cells cultured with and without DMSO. For a subset of genes the expression level changes caused by IFN-γ were much greater in maturing cells than non-maturing cells. These findings indicate that developmental changes associated with cell maturation can modulate the effects of IFN-γ but that this is gene specific. Since the effects of IFN-γ depend on whether cells are maturing, the gene expression changes observed in this study must be due to more than just prolonged application of IFN-γ and are instead the result of interplay between cell maturation and changes caused by the chemokine. This supports our hypothesis that the effects of IFN-γ on developing neutrophils in the bone marrow may be very different from its effects on mature cells in the blood. Collectively the findings in this study enhance our understanding of the effects of IFN-γ on maturing myeloid cells and indicate possible mechanisms by which this cytokine could support immune function.

INF-γ Enhances Nox2 Activity by Upregulating phox Proteins When Applied to Differentiating PLB-985 Cells but Does Not Induce Nox2 Activity by Itself.

BACKGROUND: The cytokine and drug interferon-γ enhances superoxide anion production by the antimicrobicidal Nox2 enzyme of neutrophils. Because mature neutrophils have a short lifespan, we hypothesized that the effects of interferon-γ on these cells might be mediated by its prolonged exposure to differentiating neutrophil precursors in the bone marrow rather than its brief exposure to mature circulating neutrophils. Effects of INF-Γ on NOX2 activity: To address this possibility we exposed the myeloid PLB-985 cell line to interferon-γ for 3 days in the presence of dimethyl sulfoxide which induces terminal differentiation of these cells. Interferon-γ was found to enhance superoxide production by Nox2 in a concentration dependent manner. In contrast, application of interferon-γ alone for 3 days failed to induce detectible Nox2 activity. Additionally, application of interferon-γ for 3 hours to pre-differentiated PLB-985 cells, which models studies using isolated neutrophils, was much less effective at enhancing superoxide anion production. Effects of INF-Γ on phox protein levels: Addition of interferon-γ during differentiation was found to upregulate the Nox2 proteins gp91phox and p47phox in concert with elevated transcription of their genes. The p22phox protein was upregulated in the absence of increased transcription presumably reflecting stabilization resulting from binding to the elevated gp91phox. Thus, increased levels of gp91phox, p47phox and p22phox likely account for the interferon-γ mediated enhancement of dimethyl sulfoxide-induced Nox2 activity. In contrast, although interferon-γ alone also increased various phox proteins and their mRNAs, the pattern was very different to that seen with interferon-γ plus dimethyl sulfoxide. In particular, p47phox was not induced thus explaining the inability of interferon -γ alone to enhance Nox2 activity. Short application of interferon-γ to already differentiated cells failed to increase any phox proteins. CONCLUSIONS: Our findings indicate that interferon-γ has complex effects on phox protein expression and that these are different in cells undergoing terminal differentiation. Understanding these changes may indicate additional therapeutic uses for this cytokine in human disorders.

Therapeutic options for transfusion related acute lung injury; the potential of the G2A receptor.

Priming of polymorphonuclear leukocytes (PMNs) enhances their adhesion to endothelium, the release of their granule content and their production of reactive oxygen species. These effects are etiological in transfusion related acute lung injury (TRALI) and many clinically important mediators of TRALI prime PMNs. A priming activity that develops over time in stored blood products has been shown to be due to the accumulation of lysophospatidylcholines (lyso-PCs) and has been found to be related clinically to TRALI. Lyso- PCs prime PMNs activating the G2A receptor and several inhibitors of this receptor, which could potentially be therapeutic in TRALI, have been identified. Recent work has described early steps in the signaling from the G2A receptor which has revealed potential targets for novel antagonists of lyso-PC mediated priming via G2A. Additionally, characterization of the process by which lyso-PCs are generated in stored blood products could allow development of inhibitors and additive solutions to block their formation in the first place.