The recruitment of p47 and Rac2G12V at the phagosome is transient and phosphatidylserine dependent.

in Biology of the Cell (2013)

BACKGROUND INFORMATION: During phagocytosis, neutrophils internalise pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH ... [more ▼]

BACKGROUND INFORMATION: During phagocytosis, neutrophils internalise pathogens in a phagosome and produce reactive oxygen species (ROS) by the NADPH oxidase to kill the pathogen. The cytosolic NADPH oxidase subunits p40phox , p47phox , p67phox and Rac2 translocate to the phagosomal membrane to participate in enzyme activation. The kinetics of this recruitment and the underlying signalling pathways are only partially understood. Anionic phospholipids, phosphatidylserine (PS) and phosphoinositides (PPI) provide an important attachment site for numerous proteins, including several oxidase subunits. RESULTS: We investigated the kinetics of p47phox and Rac2 phagosomal membrane recruitment. Both subunits are known to interact with anionic phospholipids; we therefore addressed the role of PS in this recruitment. Phagosomal accumulation of p47phox and Rac2 tagged with fluorescent proteins was analysed by videomicroscopy. We used the C2 domain of lactadherin (lactC2) that interacts strongly and specifically with PS to monitor intracellular PS localisation and to decrease PS accessibility. During phagocytosis of opsonised zymosan, p47phox and constitutively active Rac2G12V briefly translocated to the phagosomal membrane, whereas ROS production continued for a longer period. However, in the presence of lactC2, Rac2G12V recruitment was inhibited and the kinetics of p47phox recruitment and detachment were delayed. A reduced phagosomal ROS production was also observed during the first 7 min following the phagosome closure. CONCLUSIONS: These results suggest that p47phox and Rac2 accumulate only transiently at the phagosome at the onset of NADPH activity and detach from the phagosome before the end of ROS production. Furthermore, lactC2, by masking PS, interfered with the phagosomal recruitment of p47phox and Rac2 and disturbed NADPH oxidase activity. Thus, PS appears as a modulator of NADPH oxidase activation. [less ▲]

An essential role of STIM1, Orai1, and S100A8-A9 proteins for Ca2+ signaling and FcγR-mediated phagosomal oxidative activity

in Journal of Immunology (2011), 186(4), 2182-2191

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS ... [more ▼]

Phagocytosis is a process of innate immunity that allows for the enclosure of pathogens within the phagosome and their subsequent destruction through the production of reactive oxygen species (ROS). Although these processes have been associated with increases of intracellular Ca(2+) concentrations, the mechanisms by which Ca(2+) could regulate the different phases of phagocytosis remain unknown. The aim of this study was to investigate the Ca(2+) signaling pathways involved in the regulation of FcγRs-induced phagocytosis. Our work focuses on IgG-opsonized zymosan internalization and phagosomal ROS production in DMSO-differentiated HL-60 cells and neutrophils. We found that chelation of intracellular Ca(2+) by BAPTA or emptying of the intracellular Ca(2+) store by thapsigargin reduced the efficiency of zymosan internalization. Using an small interfering RNA strategy, our data establish that the observed Ca(2+) release occurs through two isoforms of inositol 1,4,5-triphosphate receptors, ITPR1 and ITPR3. In addition, we provide evidence that phagosomal ROS production is dependent on extracellular Ca(2+) entry. We demonstrate that the observed Ca(2+) influx is supported by ORAI calcium release-activated calcium modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1). This result suggests that extracellular Ca(2+) entry, which is required for ROS production, is mediated by a store-operated Ca(2+) mechanism. Finally, our data identify the complex formed by S100A8 and S100A9 (S100 calcium-binding protein A8 and A9 complex), two Ca(2+)-binding proteins, as the site of interplay between extracellular Ca(2+) entry and intraphagosomal ROS production. Thus, we demonstrate that FcγR-mediated phagocytosis requires intracellular Ca(2+) store depletion for the internalization phase. Then phagosomal ROS production requires extracellular Ca(2+) entry mediated by Orai1/STIM1 and relayed by S100A8-A9 as Ca(2+) sensor. [less ▲]

STIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells

in Biochemical Pharmacology (2009), 78(5), 504-513

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has ... [more ▼]

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development. [less ▲]

Cell passaging rapidly affects expression, secretion and activity of MMP9 as well as mobility of HL60 leukemia cells

in Journal of Cell and Animal Biology (2008), 2(9), 160-165

The HL60 cell line, derived from acute promyelocytic leukemia cells, can differentiate into neutrophil-like cell following DMSO treatment. Mobility of HL60, or DMSO-differentiated HL60 cells (≠HL60 ... [more ▼]

The HL60 cell line, derived from acute promyelocytic leukemia cells, can differentiate into neutrophil-like cell following DMSO treatment. Mobility of HL60, or DMSO-differentiated HL60 cells (≠HL60), requires surface expression of adhesion molecules and production of matrix metalloproteinases (MMPs). The aim of this study was to investigate in HL60 and ≠HL60 the effects of cell passaging (over 5 passages after delivery (P and P+5)) on i) surface expression of adhesion molecule CD11b, which is considered a neutrophil differentiation marker ii) MMP9 mRNA expression, protein release and zymographic activity and iii) cellular mobility. As expected, CD11b expression at both cell passages increased in ≠HL60 relative to undifferentiated HL60, but expression levels of this neutrophils marker did not change over 5 passages. MMP9 mRNA expression however, in basal conditions was increased in HL60 at P+5. At P+5 versus P, MMP9 protein levels, MMP9 zymographic activity and cellular mobility in HL60 and ≠HL60 were elevated. Stimulation by N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine had no effects on HL60, but raised MMP9 protein concentration and zymographic activity in ≠HL60. Since passage history is likely to also influence cellular functions other than MMP-related effects, it is important to carefully consider passage numbers when designing experiments [less ▲]

Co-cultures of Human Coronary Smooth Muscle Cells and Dimethyl Sulfoxide-differentiated HL60 Cells Upregulate ProMMP9 Activity and Promote Mobility"”Modulation By Reactive Oxygen Species

in Inflammation (2008)

Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We ... [more ▼]

Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We investigated by co-culture experiments the effects of human coronary smooth muscle cells (HCSMC) on MMPs characteristics and mobility of neutrophil-like dimethyl sulfoxide-differentiated HL60 cells (≠HL60). The effects of superoxide dismutase (SOD) and catalase were also analyzed. All the studied MMP2 characteristics remained unchanged. HCSMC stimulated MMP9 protein level, activity and mobility of ≠HL60 cells and expressed and secreted a variety of cytokines implicated in atherosclerosis. SOD and catalase increased MMP9 expression, protein level and activity of ≠HL60, but migration of ≠HL60 cells was only decreased by catalase, demonstrating that ROS are more efficient in modulating MMP9 activity of ≠HL60 than their mobility. Finally, HCSMC being able to stimulate ≠HL60, their co-cultures may represent an in vitro approach to study cellular interactions occurring in vivo during atherosclerosis. [less ▲]