Roles Of TRPC1Protein And Lipid Rafts In FMLP-induced Neutrophil Polarization

BackgroundPolymorphonuclear leukocytes (PMNs) are the first-line defense against microorganism invasion in the innate immunity system, as is tightly related with their directed migration known as chemotaxis. In this process, to establish polarized morphology is a vital step for neutrophils to effectively moving. Upon exposure to chemoattractant, PMNs quickly characterized as a distinct asymmetric shape with actin-rich leading edge (pseudopod) and tail (uropod) through activating various of signaling cascades. Failure to govern any molecules of this event may lead to neutrophil inappropriate activation, which develop such several inflammatory pathologies as ARDS, COPD, asthma, ischemia-reperfusion,and so on. In this regard, inhibiting any signaling pathways maybe candidate pharmaceuticals target. Therefore, unraveling of the mechanisms regulating neutrophil polarization could potentially lead to novel therapeutic strategies for counteracting chronic activation of PMNs which leads to tissue damage.Neutrophils can be activated by a variety of chemoattractants,including complement component factor5a (C5a), formylated peptides such as formyl-Met-Leu-Phe (fMLP), leukotriene B4(LTB4), platelet activating factor (PAF),and interleukin8(IL-8). Among of them, fMLP represents the prototypic chemotactic factors.On stimulated with fMLP, neutrophils initiated through the binding of bacterial peptides to G protein-coupled receptors, releasing the Gβγ heterodimer. Gβγ appears to regulate most of the known pathways activated by chemoattractants in neutrophils.These effectors include:phosphatidylinositol3-kinases(PI3Ks),guanosine triphosphatases of the Rho family(Rho GTPases),cytosolictyrosine kinase,and etc.The regulation of PMN polarization by signaling via the enzyme PI3Ks is a conserved polarity module.The resting neutrophil is maintained in a rounded, non-adherent state and, in response to the chemoattractant, adopts a polarized morphology with actin-rich leading edge (pseudopod) and tail (uropod). Striking,as well as in steep or shallow gradient of stimuli, PMNs can even polarize and chemotaxis in very low uniform concentrations of stimuli,indicating that intrinsic signalings must exist in cell mophologic polarization regardless of an exracellular gradient. Neutrophils respond to stimulation for chemoattractants by quickly establishing a leading edge,which requires cells translates extranal gradient cues into intracellular signal gradient. A hallmark of the polarized morphology is the asymmetric recruitment of both membrane and signaling components. There are evidences for local positive feedback in neutrophils,in which the lipid product of PI3K phosphatidylinositol3,4,5-trisphosphate (PIP3) and Rac serve as signals that amplifies th external signal.However, the mechanism by which this is achieved and how the responses may differ to uniform chemoattractants remains poorly understood.There has been recent porogress in understanding how Ca2+signaling takes part in the activition of neutrophil reactions such as chemotaxis and migration, realease of reactive oxygen species(ROS). The precise temporal and spatial regulation of [Ca+]i is required for cell polarization.It is well known that an increase of cytosolic intracellular free calcium results from both a stores rapid depletion from endoplasmic reticulum (ER) and a external Ca2+influx.Because of lack of voltage-gated calcium entry, Ca influx is predominantly mediated by store-operated Ca2+entry (SOCE) in fMLP-stimulated human neutrophils. There are good evidence that Orail and STIM1are two essential molecullars comprising SOCE entity to involved in neutrophil polarization and NADPH products,however,the role of transient receptor potential canonical l(TRPC1),as Ca2+-permeable channel,in neutrophil polarization is not available.Cell polarization requires differential activation and/or localization of signaling enzymes in different areas of the plasm membrane. Plasma membrane microdomains,so-called lipid rafts,are a recent focus of interest as organizers of signaling molecules.Lipid rafts are sphingolipid-and cholesterol-rich membranes microdomain,which exist in a highly liquid-ordered phase and are less fluid than glycerophospholipid-rich membranes with mainly in a’liquid-desordered’phase. Depletion of PMN cholesterol with methyl-β-cyclodextrin(MβCD),which results in raft disruption,prevents chemotactic peptide-induced ruffling and development of polarity.Given that chemoattactant receptors are typicallly distributed uniformly across the cell surface even after polarization,this internal signal occurs somewhere between receptor occupancy and actin polymerization.Lipid rafts are the exact point at which amplification of polarization signals occurs.However,it is not known whether TRPC1protein inserts into lipid rafts in neutrophil polarization.Because human neutrophils are short life-span, terminally differentiated cells, difficult in genetic manipulations, we explored the effects of TRPC1channel and lipid rafts on fMLP-induced neutrophil polarization in the present study by pharmocological approaches. Objectives1. To investigate the expression profiles of TRPC mRNA and the expression ofTRPC1protein in primary human neutrophils.2. To examine the effects of2-APB, SKF96365, GsMTx-4and MβCD on morphological changes and localization of polymerized F-actin of human neutrophils responding to fMLP.3. To explore the effects of2-APB, SKF96365, GsMTx-4and MβCD on activities of Rho small GTPases Rac2and Cdc42in fMLP-induced neutrophil polarization.4. To elucidate the effects of2-APB, SKF96365, GsMTx-4and MβCD on phosphorylation of Akt, a major downstream target of PI3K,in fMLP-induced neutrophil polarization.5. To determine the relationship between TRPC1protein and lipid rafts in fMLP-induced neutrophil polarization. Methods1. The neutrophils were freshly isolated by standard isolation protocol. This protocol comprises three successive steps:dextran sedimentation, Ficol-Histopaque density (1.077g/L) centrifugation and hypotonic lysis.Most of erythrocytes were removed during dextran sedimentation, while Ficol-Histopaque density (1.077g/L) centrifugation separates mononuclear cells from neutophils with remaining RBC removed using ddH2O. More than95%of the cells isolated were neutrophils, as assessed by Wright-Giemsa staining. Viability, determined by trypan blue exclusion, was>98%.2. Total RNA was extracted and real-time RT-PCR was performed to analyze expression profile of TRPC in human neutrophils. Whether TRPC1protein were expressed in resting human neutrophils using immunofluorescence.3. Human neutrophils were pretreated without or with100μM2-APB,10μM SKF96365,5μM GsMTx-4or lOmM MβCD for15min at37℃. Zigmond chambers (Neuroprobe) were used for quantification of neutrophil polarization. Digital images of the cells were taken after the exposure to chemoattactant for15min with a20×objective inverted microscope (Olympus IX-71).4. Human neutrophils were pretreated without or with100μM2-APB,10μM SKF96365,5μM GsMTx-4or lOmM MβCD for15min prior to100nm fMLP stimulation or not for5min at37℃.The reactions were stopped by immediately centrifugation. Proteins were extracted to perform immunoblot analysis.GTP-Rac2and GTP-Cdc42levels were purified using GST Pull down assay, and phosphoate Akt473and308, Rac2, Cdc42were analysised by western blot.5. After human neutrophils were stimulated with100Onm fMLP for0s,30s,60s,120s and300s. The level of Akt phosphorylation on Ser473was examined by immunoblot analysis.6. Confocal laser-scanning microcopy was used to observe the location of actin polymerization, TRPC1protein and lipid raft by immunofluorescence stain.7. Lipid rafts was separated using sucrose gradient ultracentrifugation and the expressions of TRPC1protein in different fractions was analyzed by Western-blotting.Results1. TRPC1, TRPC3, TRPC4and TRPC6mRNA were found in human primary neutrophils (n=5). A specific antibody for TRPC1revealed the expression of TRPC1protein in neutrophils.2. Resting neutrophils looked like round with8.32±2.43%polarized cells. After exposed to fMLP, the majority of primary human neutrophils (85.91±10.44%) adopted polarized shape with a distinct head and tail,among of them68.10±9.40%polarized cells along fMLP gradient.When cells were pretreatment with100μM2-APB,10μM SKF96365,5μM GsMTx-4or lOmM MβCD before fMLP stimulation,the percentages of polarized cells in arbitrary direction are21.91+4.76%,25.53±10.95%,27.37±2.85%and21.59±4.67%, respectively. They are lower than that of fMLP group (P<0.001).Correspondingly the percentages of polarized cells towards fMLP gradient are lower than that of fMLP group (P <0.001),with13.14±3.40%,15.50±6.34%,18.68±1.32%and15.26±4.56%, respectively.3. In unstimulated cells, only faint F-actin staining was observed at the periphery with round loop.After100nM fMLP treatment, the polymerized F-actin became concentrated at the leading edge of cells with a strong fluorescence intensity,however,this asymmetry disappeared when cells were preincubated with2-APB, SKF96365,GsMTx-4or MBCD.4. Both Rac2and Cdc42activities were low in resting neutrophils. On stimulation with100nM fMLP, the activities of both Rac2and Cdc42were increased. However,on inhibition of TRPC1channel or diruption of lipid rafts,the activities of both Rac2and Cdc42were significantly decreased, compared with those of fMLP group(P<0.05).5. The level of Akt phosphorylation on Ser473were similar at all indicated times after fMLP stimulation.The level of Akt phosphorylation on Ser473and Thr308 induced by fMLP were maintained when PMNs were incubated with2-APB, SKF96365,GsMTx-4or MβCD prior to stimulation.6. TRPC1protein and lipid rafts were evenly distributed around the periphery of unstmulated cells. When cells were stimulated with100nM fMLP, lipid rafts aggregated a platform together with the colocalization of TRPC1protein. However, after lipid rafts was disrupted with MβCD followed by stimulation,the localization of TRPC1protein and lipid rafts were similar with those of resting cells.7. Lipid rafts isolation using sucrose gradient ultracentrifugation demonstrated the majority of TRPC1protein in low density fractions in activated cells,while TRPC1protein released from low density fractions to high density fractions in MβCD pretreatment cells.Conclusions1. Human primary neutrophils express TRPC1on the level of both mRNA and protein.2. Both TRPC1protein and lipid rafts are involved in the formation of polarized morphology and cytoskelton reorganization of neutrophils responding to fMLP.3. Both TRPC1protein and lipid rafts up-regulate the activities of both Rac2and Cdc42during fMLP-induced neutrophil polarization.4. Akt, a major downstream target of PI3K, is not involved in fMLP-induced neutrophil polarization. Likely, Akt is not involved in both TRPC1channel protein-and lipid rafts-mediated signaling pathways in neutrophil polarization.5. TRPC1protein translocates into lipid rafts to transmmit signaling during fMLP-induced neutrophil polarization.