Requirement For Both ROCE-and SOCE-mediated Calcium Influx In FMLP-induced Neutrophil Polarization

BackgroudNeutrophils constitute a key component of the innate immune system and represent50-60%of the total leukocytes in circulation. Accumulation of neutrophils in damaged tissues is a pathogenic signature of many inflammatory diseases. Polarization and migration of neutrophils toward the chemotactic gradient produced by an inflammatory stimulus is an essential part of this process. In the resting state, neutrophils adhere poorly and exhibit a spherical shape. However, in response to an inflammatory stimulus, neutrophils first become polarized, exhibiting a leading edge (pseudopod) at one end and a tail (uropod) at the other end, and then migrate towards the source of the stimulus. Failure to govern any molecules of this event may lead to neutrophil inappropriate activation, which develop such several infammatory pathologies as ARDS, COPD, asthma, ischemia-reperfusion, rejection in organ transplantation, atherosclerosis, Viral Myocarditis, rheumatoid arthritis, anaphylaxis, some inflammatory dermatoses, and even tumorigenesis and tumor metastasis. Defects in the mobility and polarization of neutrophils in response to inflammatory stimuli have been reported in humans, such as lazy leucocyte syndrome (LLS), Shwachman-Diamond syndrome, and dysgammaglobulinemia type I. However, the mechanisms for these defects are poorly understood. Thus, it’s a very importment and meaningful study for the mechanism of neutrophil polarization and migration, in order to provide potential therapeutic strategies for counteracting activation or defection of neutrophils which leads to tissue damage or neutrophil dysfunction.Neutrophils are able to feel a small gradient concentration of chemoattractant with phase difference of even only2%, which is enough to induce neutrophil polarization and migration. And neutrophils can be activated by a variety of chemoattractants, including N-formyl-Met-Leu-Pue (fMLP), leukotriene B4(LTB4), platelet-activating factor (PAF), IL-8, complement factor5a (C5a). Among of them, fMLP represents the prototypic chemotactic factor. Chemoattractant stimuli induce various cell responses through G-protein coupled receptor, each chemoattractant works differently along with their influenced subfamily of G-protein and their downstream signaling pathways.Elevated cytoplasmic Ca2+concentrations in neutrophils play a critical role in mediating the pro-polarizing and pro-motility effects induced by the inflammatory stimulus. In neutrophils, fMLP stimulates activation of phospholipase C (PLC) via the fMLP receptor, leading to breakdown of phosphatidylinositol4,5-bisphosphate into inositol1,4,5-trisphosphate (IP3) and1,2-diacylglycerol (DAG). IP3binds to its receptor on the endoplasmic reticulum (ER) and causes rapid Ca2+release from ER stores. When the Ca2+stored in the ER is depleted, it then activates the extracellular Ca2+influx across the plasma membrane by store-operated calcium entry (SOCE) mechanisms. SOCE was originally proposed three decades ago by Putney and colleagues. And at the present, many reports have shown that Ca2+influx via SOCE plays a vital role in various cellular processes, such as cell polarization, survival, migration and metastasis of non-excitable cells following exposure to a variety of stimuli. However, despite significant progresses, it is still not clear if SOCE is the sole mechanism responsible for the sustained phase of Ca2+influx in neutrophils following inflammatory-mediator stimulation. And in vitro/in vivo experiments have indicated that SOCE is involved in cell polarization, migration and metastasis, by regulating a variety of cytosolic Ca2+signals. A clinical study by Hauser et al. have further suggested that human neutrophil dysfunction secondary to injury and inflammation involves an enhancement in SOCE. Indeed, recent studies in a number of cell types have suggested the existence of a non-store operated or non-capacitative Ca2+-entry pathway, referred to as receptor-operated calcium entry (ROCE), which may involve the DAG-activated protein kinase C (PKC) pathway.Intracellular Ca2+mobilization is known to play an important role in the control of cell polarization. Because two routes of Ca2+influx appear to be activated following fMLP stimulation, we propose that both routes are required for fMLP-induced neutrophil polarization; indeed, fMLP-induced cell polarization was not observed if either SOCE or ROCE was inhibited. Cell polarization is defined by the development of morphological changes to create pseudopod and retraction of uropod.It is well known that PI3K-dependent pathway plays a pivotal role in cell polarization and migration, However, recent studies have identified PI3K-independent pathway that acts in parallel with the PI3K-dependent pathway, and these two pathways work together to activate Rac and Cdc42and thereby regulate cell polarization and migration. Although the function of Rho family GTPases (Rac1, Rac2and Cdc42) in cell polarization and chemotaxis process was widely described, it is still a subject of controversy and remains unclear. However, what is their up regulation pathways? It has been reported that pretreatment fMLP-stimulated neutrophils with wortmannin or LY294002, two PI3K inhibitors, could not completely inhibit activation of Rac or Cdc42, which also was blocked by pretreatment of fMLP-stimulated neutrophils with genistein, a tyrosine kinase inhibitor. Furthermore, other studies pointed out that activation of both PI3K and Src kinase pathways induced the activation of Rac1and Cdc42.Members of the small Rho-GTPase family, including Rac2and Cdc42, localize to the leading edge. Accordingly, Rac and Cdc42are essential for polarization and directional migration. We therefore also used these GTPases as markers to determine the involvement of Rho-GTPase family members in SOCE and ROCE signaling during fMLP-induced neutrophil polarization. However, the mechanisms whereby different Ca2+-signaling routes communicate with Rho-GTPase family members, leading to subsequent cell polarization, remain unclear.ObjectivesWe hypothesized that fMLP stimulation caused not only store-operated calcium entry (SOCE), but also receptor-operated calcium entry (ROCE) in neutrophils. In this study, both pharmacological inhibitors, i.e.PKC inhibitor calphostin C and PLC inhibitor U73122, and neutralizing monoclonal antibody approaches were used to observe their effects on the fMLP-induced influx of Ca2+entry. We also investigated whether Rac2or Cdc42activation could take place if either SOCE or ROCE was inhibited. The SOCE and ROCE inhibitors and STIM1antibody approaches were used to investigate the effect of fMLP-induced polarization.Methods1. Human neutrophils were isolated from heparin anti-coagulated venous blood from healthy consenting adults, erythrocytes were removed by Dextran sedimentation (6%dextran/0.9%NaCl) followed by two rounds of hypotonic lysis using sterile ddH2O. With the exception of dextran sedimentation, the entire isolation procedure was performed at4℃. Purified neutrophils were re-suspended in D-Hanks without CaCl2at a concentration of1.0×106cells/ml prior to use.2. Intracellular Ca2+was monitored using the Ca2+-sensitive fluorescent indicator, Fluo-4/AM under an inverted laser scanning confocal microscope. Cells were loaded with2μM Fluo-4/AM at37℃for30min in the dark in Hanks’buffered salt solution (pH7.4). Ca2+-free buffer solution was prepared by omitting CaCl2and adding0.3mM EGTA. In certain experiments, neutrophils were maintained in Ca2+-free buffer for30min before Ca2+image recording. The green fluorescence of Fluo-4was excited by a10-mW multi-tune argon laser at488nm.3. The neutrophil suspension was transferred to an electroporation chamber containing2.5μg/ml anti-STIM1antibody and the electrotransjection efficacy was determined by immunoprecipitation and western blotting.4. Cell polarization toward an fMLP gradient (0-100nM) was observed by using Zigmond chamber. A total of1×106neutrophils were allowed to adhere to coverslips for5min at room temperature. The coverslips were then immediately inverted onto the chamber, which was assembled loosely. One channel of the chamber was filled with HBSS (vehicle) and the other with100nM fMLP. Digital images of the cells were taken after exposure to the chemoattractant for15min, using an inverted microscope with a×20objective. Polarized cells were quantified by counting the total number of cells, the cells that were polarized in any direction, and the cells polarized towards the direction of the gradient[5]. Cells with a leading edge and a trailing tail were scored as polarized. The effects of PKC inhibitor calphostin C, PLC inhibitor U73122and GdCl3on cell polarization were observed by using Zigmond chamber.5. The effects of PKC inhibitor calphostin C, PLC inhibitor U73122and GdCl3on Rac2and Cdc42were determined with GST pull-down assay. Freshly isolated neutrophils (1×107/ml) were treated with pharmacological inhibitors, Then cell lysates were centrifuged at12,000rpm for15min at4℃, then5%of each supernatant was removed for detection of total (both GTP-and GDP-bound) Rho-GTPases. The remaining supernatants were incubated with20μg PAK-GST Protein Beads for1h at4℃. The beads were eluted in1×sample buffer (2%SDS,10%glycerol,5%β-mercaptoethanol, and0.01%bromophenol blue dye in50mM Tris-HCL, pH6.8). Active Rac2or Cdc42were detected by western blot analysis using anti-Rac2or anti-Cdc42antibodies.6. Statistical analysis was performed using SPSS13.0software. Results were expressed as mean±SEM. Data were analyzed by t-tests or one-way analysis of variance. Differences Group compared with one-way ANOVA and inter-group comparision was significant using LSD when the homogeneity of variances was meet, while, Group compared with Welch and inter-group comparision was significant using Dunnett T3when the homogeneity of variances was not meet. P<0.05means the significance.Results1. A low GdCl3concentration (2μM) had little effect on the Ca2+release induced by fMLP, but it partially inhibited the subsequent Ca2+influx by approximately85%(T=9.168, P=0.000) following the addition of CaCl2.2. STIM1was clearly detected in electropermeabilized cells, whereas much lower levels were detected in non-permeabilized cells. And anti-STIM1antibody had no effect on fMLP-induced Ca2+store release, and it was unable to entirely abolish the Ca2+influx(F=8.430, P=0.009)3. Sr2+influx in response to fMLP stimulation produced an obvious fluorescence ratio[△F/F0] that was approximately20%of the fMLP-induced Ca2+influx (F=50.320, P=0.000).4. Inhibition of PLC by U73122diminished the ability of fMLP stimulation to induce neutrophil polarization(T=44.274, P=0.000).5. In the absence of Gd3+, approximately80%of neutrophils exhibited a spread and polarized morphology after fMLP stimulation. However, only about5%of neutrophils showed a similar polarized morphology in the presence of Gd3+(T=21.166,P=0.000).6. Anti-STIM1neutralizing antibody introduced into the cells by electroinjection reduced fMLP-induced neutrophil polarization to about6%(F=421.560, P=0.000).7. Calphostin C had no effect on fMLP-induced Ca2+store release (AF of the first peaks of fluorescence signal intensity curves were similar), but it almost completely blocked Sr2+influx (T=2.827, P=0.012).8. In the presence of calphostin C, the ratio of fMLP-induced neutrophil polarization was reduced from80%to only13%(T=14.056,P=0.000).9. The fMLP-triggered Rac2and Cdc42activities were significantly inhibited when neutrophils were pretreated with either Gd3+or the PKC inhibitor calphostin C, However, when the cells were pretreated with the PLC inhibitor U73122, no further inhibition of Rac2or Cdc42activity was observed. ConclusionfMLP triggers not only SOCE, but also ROCE, in human primary neutrophils. Each of these two routes of Ca2+influx appears to play a critical role in regulating downstream small GTPase Rac2and Cdc42activity.