Calcium Signal In The Process Of Cell Polarity In Neutrophils

BackgroundPolymorphonuclear neutrophils are phagocytic cells which have the function of directional locomotion.They are play an important role of host response in resisting to bacterium and eumycete,which due to their chemiotaxis.Neutrophils can recept signaling molecule and follow the concentration gradient of chemiotaxin to the location of inflamation.And the foundation of this migratory function is the process of neutrophils polarity according to the chemiotaxin.Calcium signal plays an important role not only in signal conduction in cells but also in executing function of cells and it participates the whole vital process from fertilization of ovum to apoptosis of cells.Calcium ion can be not only the first messenger but also the second messenger.It can regulate the vital process and itself as well.The concentration of calcium ion in extracellular fluid is more than 10^-3M, while it in intracellular fluid is around 10^-7M.To maintain the homeostatic of Ca²⁺,it depends on some important region of accommodation to controlling and accommodation.Some proteins can be the ion channels in cell membrane or organelle plasmalemma to permeation Ca²⁺,and they have three groups according to their gating mechanisms:voltage-gating channel,ligand-gating channel and store-operated channel.. James Putney introduced the concept of a store-operated ca2+ entry(SOCE) in 1986.A select group of stimuli initiate cellular responses by acting on cell-surface receptors to increase the cytosolic Ca²⁺ concentration.The stimulation of these receptors induces the hydrolysis of membrane phosphoinositides by phospholipase C (PLC) enzymes yielding the diffusible Ca²⁺-mobilizing messenger inositol 1,4,5-trisphosphate[Ins(1,4,5)P3],to release Ca²⁺ from non-mitochondrial Ca²⁺ stores.However,Ca²⁺ release is also followed by a stimulated Ca²⁺ entry in such cells and the ultimate reason for the Ca²⁺ influx is a decrease in the Ca²⁺ content of the endoplasmic reticulum(ER).The mechanism by which the ER Ca²⁺ stores communicate with the plasma membrane(PM) and the molecules participating in the Ca²⁺-influx process remained elusive until a series of key discoveries identified the stromal interaction molecule(STIM) and Orai(also known as calcium-release-activated calcium-modulator[CRACM]) proteins that serve as ER calcium sensors and calcium channels,respectively.Following with the mechanism of SOCE was gradually recognized,the pharmacological means are also highlight to manipulate the functions of SOCE,and SK&F96365 and 2-APB were used widespread.Neutrophils can be polarity when stimulated by chemotaxin,and in that process the concentration of Ca²⁺ in introcelluar fluid can developed a series of changes as well as its distribution.The chemotaxin fMLP can active the receptor of IP3 in ER and induce releasing Ca²⁺ from Ca²⁺ storage of ER.The releasing Ca²⁺ from Ca²⁺ storage of ER can active SOCE and promote Ca²⁺ inflow from extrocellar fluid.It is still unknown about whether SOCE participates in the process of cell polarity in neutrophils and what SOCE plays in this process.It also is not reported about what influence in the process of cell polarity and chemotaxis in neutrophils by block SOCE. SOCE may play an important role in the process of cell polarity,and it may has well relationship with lipid raft in membrane.The key function of lipid raft is to be a platform for cell signal.To understand more about the relationship of SOCE with lipid raft,we have studied the influence in cell polarity by mβCD,mβCD can clear the cholesterol of membrane to destroy the integrality of lipid raft.It may influence the process of polarity.ObjectiveThe experiment is to investigate the role of SOCE in the process of cell polarity in neutrophils by observation the changes of calcium ion in introcellular fluid and the changes in cell polarity and chemotaxis after using the pharmacological means to manipulate the functions of SOCE.Methods1.Human peripheral blood neutrophils were obtained from healthy adult volunteers and separated on a discontinuous gradient consisting of percoll solution.The cells were preserved in 4℃.2.Confocal microscopy was used to observe the changes of the concentration of calcium ion in intracellular fluid of neutrophils after stimulated by chemotaxis fMLP in different groups.According to different disposal,four groups were set: no calcium group(there was not any calcium ions in extracellular fluid),normal calcium group(there was 1mM calcium ions in extracellular fluid),high calcium group(there was 2mM calcium ions in extracellular fluid),and 2-APB group (there was 1mM calcium ions in extracellular fluid and 100nM 2-APB was used to block SOCE).3.Patch clamp was used to observe the changes of the whole-cell current after stimulated by chemotaxis fMLP in different groups by using different protocol in the whole-cell and voltage clamp mode.According to different disposal,five groups were set:Control group(no drug was used to pretreat the neutrophils and after that fMLP was not used either),fMLP group(no drug was used to pretreat the neutrophils and after that fMLP was used as other experimental groups), SKF96365 group(10μM SKF96365 was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),2-APB group(100nM 2-APB was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed),TG group(100nM TG was used to pretreat the neutrophils in 15 minutes and after that 100nM fMLP was followed).4.Flow cytometry was used to observe the changs of immunofluorescence of F-actin,the skeleton protein related to polarity,after stimulated by chemotaxis fMLP in different groups of neutrophils.According to reported,six time points (0 second,10 seconds,30 seconds,60 seconds,120 seconds and 240 seconds) after using fMLP was taken to observe the difference among the different groups in each time point.And in all these time points,the group settings are all similar to the setting in the experiment of patch clamp.5.Transwell was used to observe the changes of chemotaxis of neutrophils in different groups.According to different disposal,six groups were set:Control group(RMPI1640 300μL with PBS was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room,the dosage of PBS and DMSO was just as the dosage which dissolve the drugs using in experimental groups),fMLP group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with DMSO was put in upper-room),SKF96365 group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with SKF96365 10μM pretreatment for 15 minutes in 37℃and 5%CO₂ was put in upper-room),2-APB group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with 2-APB 100nM pretreatment for 15 minutes in 37℃and 5%CO₂ was put in upper-room),TG group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with TG 100nM pretreatment for 15 minutes in 37℃and 5%CO₂ was put in upper-room),and mβCD group(RMPI1640 300μL with fMLP 100nM was dropped in infra-room and the cell suspension of neutrophils 100μL with mβCD 10mM pretreatment for 15 minutes in 37℃and 5%CO₂ was put in upper-room).Results1.The standard ratio of the change of fluorescence of calcium ion in intracellular fluid of neutrophils in no calcium group is significant minor to which in normal calcium group(n=9,P=0.027),and in 2-APB group it is also significant minor to which in normal calcium group(n=9,P=0.005).2.The whole cell current of neutrophils began to augment quickly after stimulated by 100 nM fMLP and last about 1 minute around the peak.After that,the current began to decrease slowly but last a few minutes above the base current(more than 5 minutes).In the command potential of-80,the augmentation of the current was significantly inhibited if SKF96365 10μM or 2-APB 100nM was used to pretreat the cells(P=0.004 and 0.022 respectively in step protocol and P=0.030 and 0.043 respectively in ramp protocol).The whole cell current augmented significantly after the cells pretreatment by TG 100nM(P=0.002 in step protocol and P=0.002 in ramp protocol),while after that,the current did not augment significantly any more when stimulated by fMLP.3.In the time point of 240 seconds,the fluoresence of F-actin in 2-APB group is significant smaller than fMLP group(P=0.036).In all of these time points,the fluoresence of F-actin in SKF96365 group is smaller than fMLP group according to their means.4.The migration capability of neutrophils in 2-APB group is significant less than which in fMLP group(P=0.034).And The capability in SKF96365 and mβCD groups is both less than which in fMLP group just according to their means.Conclusion1.After neutrophils stimulated by fMLP,the augment of calcium ion concentration in cells has two resources:the release of ca²⁺ from ca²⁺ storage in cells and the inflow of ca²⁺ from outside.And the inflow of ca²⁺ from outside due to SOCC. So SOCE plays an important role in the process of cell polarity in neutrophils.2.According to the experiment of patch clamp,it can be concluded that SOCC play an important role in the process of neutrophils polarity,and SOCE is most important to polarity.At the same time,other ca²⁺ channels may participate this process.3.According to the experiment of flow cytometry,it suggested that SOCE participates in the process of neutrophils polarity and may play the key role in it. Blocking SOCE can inhibite the process of polarity.4.SOCE plays an important role in the process of migration of neutrophils,and blocking it can inhibit the process.The process of neutrophils migration also has well relationship with lipid raft.Destroying the integrality of lipid raft can also inhibit the process of migration.Whether SOCE is correlated with lipid raft in this process still needs more experiments.