Effect Of Lipid Rafts On FMLP-induced Human Neutrohpil Polarization

BackgroundPolymorphonuclear nertrophils(PMNs) is one of important components in innate immune system. As the first line of defense, Main functions of PMNs is leaking to vessel wall, migrating to inflammatory lesions and releasing superoxide and/or proteolytic enzymes to kill bacteria and invading microbes, when the body exposed to external invasion of bacteria and other pathogens. The motile force is generated by the local polymerization of actin at the leading edge and it pushs the membrane forward and generates protrusions (mammalian cells frequently use a specialized subset of flat, sheet-like pseudopods, which named lamellipodia) which is essential for eukaryotic cell migration. Cell migration implicates in nutrition acquirement, proliferation, wound repair, axon growth, angiogenesis and embryo development. However, failure to govern any molecule of this event may lead to inappropriate activation of neutrophil, which would develop several infammatory pathologies such 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. Thus, it’s a very important and meaningful research for exploring the mechanism of neutrophil porization, in order to provide potential therapeutic strategies for counteracting chronic activation or defection of neutrophils which leads to tissue damage or neutrophil dysfunction.Plasma membrane microdomain, so-called lipid rafts, is a recent focus of interest as organizers of signaling molecules. Lipid raft is sphingolipid and cholesterol-rich membrane microdomain, which exist in a highly liquid-ordered phase and is less fluid than glycerophospholipid rich membranes which mainly in a ’liquid-desordered’phase. Depletion of PMNs 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 typically 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. The key function of lipid rafts is to be a platform for cell signal. We have studied the influence to cell polarity by mβCD and CLM. mβCD can clear the cholesterol of membrane to destroy the integrality of lipid raft. CLM can protect the cholesterol of membrane to recover the integrality of lipid raft. It may influence the process of polarity.Calcium signaling is involved in a variety of physiological and pathological processes. Increasement of concentration of cytoplasmic Ca2+depends on two pathways:.the releasing of endoplasmic reticulum calcium stores and plasma membrane calcium influx. SOCE(store-operated calcium entry) is the major forms of calcium influx in non-excitable cells. When the Ca2+stored in the ER is depleted, it then activates the extracellular Ca2+influx across the plasma membrane by SOCE mechanisms. There are good evidences that lipid rafts are organizers of signaling molecules, involved SOCE entity in neutrophil polarization and NADPH products. So, the role of SOCE in neutrophil polarization is important.Rho GTPases are important regulators to the actin cytoskeleton and thereby to control the adhesive and migratory behavior of cells. Rho GTPases cycle between an inactive, GDP-bound form and an active, GTP-bound form. The guanine nucleotide-exchange factors (GEFs) regulate the activation of Rho GTPases by promoting the exchange of GDP for GTP, while GTPase-activating proteins (GAPs) promote the intrinsic GTPase activity and thus the transition back to the GDP-bound state. The family of Rho GTPases contains20members and their splice variants can be divided into various subfamilies, i.e. Cdc42, Rac, Rho, RhoF, RhoU, Rnd, RhoBTB and RhoH. Neutrophils are among the first cells which respond to infection and migrate quickly to the site of infection. Therefore, the high levels of these Rho GTPases in neutrophils could reflect a high demand for Rho GTPase activity which might be required for this fast response.The polarity of human neutrophils is complex. To elucidate the role of lipid rafts on human neutrophil polarization and chemotaxis and their regulatory mechanisms, in this report, we study the role of lipid rafts in human neutrophils.ObjectiveTo investigate effect of lipid rafts on fMLP-induced human polarizationMethods1. Neutrophils were freshly isolated following 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 centrifugation separates mononuclear cells from neutrophils with remainning RBC removed using ddH2O. More than95%of isolated cells were neutrophils, as assessed by Wright-Giemsa staining. Viability, determined by trypan blue exclusion, was>98%.2. Neutrophils polarization and chemotaxis was observed by Zigmond chamber.3. Transwell chamber assay was used to detect the human neutrophil polarization.4. Patch clamp instrument was used to seal and rupture the neutrophils, record changes in membrane capacitance and process factors membrane currents.5. Intracellular Ca2+was monitored using the Ca2+-sensitive fluorescent indicator, Fluo-4/AM, under an inverted laser scanning confocal microscope.6. Activations of Rac2, Racl were observed by GST pull-down assay and Western blotting was used to detect the changes of related signaling molecules in fMLP-induced human neutrophil polarization.7. The results of experimental data were shown as mean±standard deviation (x±SD). SPSS13.0statistical packages was used for statistical analysis. One-way ANOVA was used for group compared and LSD was used for inter-group comparison when the homogeneity of variances was meet. Welch was used for group compared and Dunnett T3was used for inter-group comparison when the homogeneity of variances was not meet. P<0.05means the significance.Result1. SOCE can be caused by fMLP-induced human Neutrohpil polarization, The changes of neutrophil membrane current can be caused by fMLP.2. mβCD and CLM can cause Neutrophil membrane current changes. Compared with the control group and/or fMLP group, current density changes were statistically significant (P<0.05). mβCD can inhibit the formation of fMLP-induced Ca2+store release. CLM can recover the formation of fMLP-induced Ca2+store release.3. mβCD can inhibit the formation of neutrophils polarity, but CLM can recover neutrophils polarization. 4. Transwell chamber assay showed that mβCD can inhibit the formation of neutrophils polarization and migration. CLM can restore the formation of neutrophils polarization and migration.5. Western blot analysis showed activities of Racl and Rac2activation were increased after fMLP stimulation compared with control.Conclusion1. fMLP can regulate neutrophil membrane current and polarization. It also closes contact with SOCE.2. The mβCD can reduce the membrane currents of the fMLP-induced polarity cells and CLM can restore the membrane currents of the fMLP-induced polarity cells partially.3. mβCD can inhibit the formation of SOCE and CLM can restore the formation of SOCE.4. The mβCD can reduce the polarization and migration of the fMLP, CLM can restore the polarization and migration of the fMLP.5. Lipid rafts structure up-regulate the activities of both Rac1and Rac2during fMLP-induced human neutrophil polarization.