Store-operated Ca²⁺ Entry （SOCE） Mediated Regulation Of Polarization In Differentiated Human Neutrophil-like HL-60Cells Under Hypoxia

Background:Polymorphonuclear neutrophils (PMNs) are the fist-line defense againstmicroorgnism invasion in the innate immunity system, as is tightly related with theirsencing and direct polarization and migration toward a variety of chamoattractantstimulis. Upon exposure to chemoattracant, neutrophils quickly charactirized as adistinct asymmetric shape with oriented leading edge and trailing tail throughactivating various of signaling cascades. Thus, neutrophil polarization is a crucialconstituent of the innate immune response during bacterial infection. Failure togovern 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, ViralMyocarditis, rheumatoid arthritis, anaphylaxis, some inflammatory dermatoses, andeven tumorigenesis and tumor metastasis. Defects in the mobility and polarizationof neutrophils in response to inflammatory stimuli have been reported in humans,such as lazy leucocyte syndrome (LLS), Shwachman-Diamond syndrome, anddysgammaglobulinemia type I. However, the mechanisms for these defects arepoorly understood. Thus, it’s a very importment and meaningful study for themeachanism of neutrophil porization and migration, in order to povide potentialtherapeutic strategies for counteracting chrnic activation or defection of neutrophilswhich leads to tissue damage or neutrophil dysfunction.Elevation of [Ca2+]iis very crucial for a number of responses of the neutrophilto a variety of stimuli. Although the modulation of Ca2+influx in neutrophils hasbeen studied for many years, there still exist a lot of conflicting reports. Resently, ithas been reported levels of this Ca2+signal might be probably regulated through atleast two mechanisms:(1) the state of filling of Ca2+stores in the endoplasmicreticulum (ER), termed store-operated Ca2+entry (SOCE), and (2) receptoroccupation, which is named receptor-operated Ca2+entry (ROCE). The molecular components of ROCE are still unknown, and efforts made on establishingpharmacological approaches for modulating ROCE have not come to a solution.SOCE, however, has been characterized more completely. SOCE was first describedas the predominant mechanism for Ca2+influx in non-excitable cells, and so far twomajor molecular components of SOCE have been identified:(1) stromal interactionmolecule1(STIM1), which serves as a Ca2+sensor that oligomerizes andtranslocates to ER/PM (plasma membrane) junctional domains to aggregate intopuncta in response to Ca2+store depletion, and (2) Orai1(also named CRACM1),which is localized diffusely in the plasma membrane of resting cells and is recruitedby STIM1into the puncta through an interaction with its C-terminal region. Thisestablished knowledge about the characterizations of SOCE is favorable for furtherstudy on its roles in various cell functions. And at the present, many reports haveshown that Ca2+influx via SOCE plays a vital role in various cellular processes, suchas cell polarization, survival, migration and metastasis of non-excitable cellsfollowing exposure to a variety of stimuli. In fact, in vitro/in vivo experimets haveindicated that SOCE is involved in cell polarization, migration and metastasis, byregulating a variety of cytosolic Ca2+signals. A clinical study by Hauser et al. havefurther suggested that human neutrophil dysfunction secondary to injury andinflammation involves an enhancement in SOCE. Despite of the above findings onthe neutrophil, the mechanisms by which SOCE mediate cell polarization and otherfunctions remain unclear.Neutrophils are able to feel a small gradient concentration of chemoattractantwith phase difference of even only2%, which is enough to induce neutrophilpolarization and migration. And nertrophils can be activated by a variety ofchemoattractants, 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 stimulis inducevarious cell responses through G-protein coupled receptor, each chemoattractantworks differently along with their influenced subfamily of G-protein and theirdownstream signaling pathways.Hypoxia lead nucleus granulocyte aggregation in organizations, at the same time in the inflammatory response it plays an important role, because it inhibitedapoptosis of neutrophil through the HIF–1αdependent regulations. Meanwhile, thepolarity of neutrophils is the initiation of cell migration, and is a part of thecongenital immune response to bacterial infection. Although some clinical andanimal studies have shown that neutrophils sterilization, consuming activity andwound healing is affected in hypoxic conditions, a handful of the experimentalresearch on the effect of hypoxia on neutrophil function in vitro. Rotstein et al.Found neutrophils resulting in a decrease of minority but significant chemotaxismigration in extreme hypoxic conditions (less than30mmHg). McGovern et al.Scholars showed hypoxia damage ability to generate ROS of peripheral bloodneutrophils and kill Staphylococcus bacteria aureusbut ability in human, but hypoxiadoes not affect their sports, migration, receptor regulation and degranulation. SOCEplays an important role in cell polarization, the SOCE change in neutrophils has notbeen reported, and the role of SOCE in neutrophils polarity is unknown underhypoxic conditions.Objective:In the present study, differentiated HL-60(dHL-60) cells, as a validated modelsystem for the analysis of neutrophil polarization, were used to investigate themechanisms whereby SOCE regulates neutrophil polarization. Through treatment byhypoxia, the polarizaiton of differentiation HL-60cell with DMSO was observed,and research on the SOCE basic composition and the change of the SOCE. Throughgenetic manipulation techniques, clarify the role of SOCE in neutrophils polarizationunder hypoxic conditions.Methods:HL-60cells were differentiated with1.3%DMSO for0-6days, without changemedium. Cell density, vability were tested at every treatment day. The differentiatedHL-60cells, which could be used for polarization or plasmid overexpressionexperiments.1. The experimental groups were divided into two groups: hypoxia treatment andnormoxia groups of cells. Normoxia groups: DMSO induced differentiation of4,5,and6days to dHL-60cells, respectively named N4, N5and N6group. Hypoxia groups: after DMSO induced differentiation of four days of dHL-60cells,respectively give3%O2treatment1-2days, respectively named group N4+H1andN4+H2group.2. Through zigmond chamber establish concentration gradient of fMLP (chemotaxismaterial) as (0-100-nM), observe orientation polarization changes of hypoxic celland normoxic group dependent on fMLP concentration gradient.3. By Western blot method, extraction of hypoxic and normoxic group cell protein,electrophoresis analyze expression of two important SOCE related molecular Orai1and STIM1under hypoxia or normoxia conditions, to observe the effects of hypoxiaon Orai1and STIM1expression.4. By using laser confocal microscope and the method of determination of calciumion concentration in cells, detect the effects of hypoxia on calcium release and SOCEcalcium entry of TG stimulation.5. The extraction and purification of plasmid (STIM1-mOrange、Orai1-mKo andpcDNA3.1), through cell electroporation experiment, overexpression Orai1andSTIM1molecules in hypoxic2days group cells, and then observed after plasmidoverexpression, orientation polarization changes dependent on fMLP concentrationgradient in hypoxic group of cell.6. The results of experimental data with mean±standard deviation (x±SD), usingSPSS17.0statistical packages for statistical anylysis. Group compared with one-wayANOVA and inter-group comparision was significant using LSD when thehomogeneity of variances was meet, while, Group compared with Welch andinter-group comparision was significant using Dunnett T3when the homogeneity ofvariances was not meet. P <0.05means the significance.Result:1. Compared N6group’s cells with N4+H2group’s cells, polarization responsedependence on fMLP concentration gradient in hypoxia2days dHL-60cellsincreased significantly (P <0.01).2. Compared N5group’s cells with N6group’s cells, the polarization by fMLPstimulate in N6group’s dHL–60cells dependence on fMLP concentration gradientweakened. 3. Compared N6group’s cells with N4+H2group’s cells, Orai1and STIM1expression level decreased in hypoxia2days group dHL-60cells obviously.4. Compared N5and N6group’s cells with N4group’s cells, Orai1and STIM1expression levels in dHL-60cells of N6group rose.5. Compared N4+H2group’s cells with N6group’s cells, SOCE calcium entrysignificantly reduced in dHL-60cells of hypoxia2days group.6. Compared N5group’s cells with N6group’s cells, SOCE calcium entry withoutsignificant difference in dHL-60cells.7. Compared N4+H2group’s cells with N6group’s cells, calcium release by TGsignificantly reduced in dHL-60cells of hypoxia2days group.8. Plasmid electroporation was used for overexpression of STIM1and Orai1, asshown in Fig.4A. STIM1-mOrange plasmid seemed to be more effective thanOrai1-mKO, and the expression was enhanced by80%and20%as compared withthat treated with pcNDA3.1, respectively.9. STIM1and Orai1overexpression by plasmid resulted in a substantial suppressionof polarization in the direction of fMLP gradient at day2of hypoxia, with thepercentage of polarized cells in the presence of STIM1-mOrange, Orai1-mko, andN4+H2being10%,13%, and50%, respectively. Then plasmid overexpression assayshowed that the response of polarization was returned to the control level.Conclusion:1. The regulation of neutrophil polarization by calcium entry is critical formaintaining an effective host response. Hypoxia has a major effect on the apoptosisof neutrophils, but the role of SOCE in neutrophil polarization under hypoxiaremains to be elucidated. In this study, we examined the polarization ofdifferentiated human neutrophil-like HL-60(dHL-60) cells exposed to hypoxia(3%O2). It was found that hypoxia enhances the polarization of dHL-60cells.2. STM1and Orai1expression in dHL-60cell of hypoxia2days group showedobvious drop, prompt hypoxia maybe has influence on the SOCE.3. With a laser confocal microscope observation the change of intracellular calciumion concentration, we use the TG lead calcium release, by adding calcium, causes theSOCE, results show that the SOCE declined in dHL-60cell of hypoxia2days group, and consistent with Western blot results, showed hypoxia has influence onthe SOCE.4. In order to prove the role of the SOCE in polarization of dHL-60cell underhypoxia, we using plasmid overexpressing experiment, through the electroporation,overexpressing the Orai1and STIM1molecules in dHL-60cells, polarizationexperiment found that the response of polarization was returned to the control level,suggests that a protective effect of SOCE on cell polarization.5. We demonstrated the inhibitory role of SOCE in the polarization of dHL-60cellsunder hypoxic conditions, which may be the mechanism for the adaption ofneutrophils to hypoxia. SOCE is also suggested to be a key modulator of immunedeficiency under hypoxic conditions, potentially as a therapy target.