| BackgroundCell polarity is a fundamental feature of diverse cell type, it is essential for thefunction of many cells. Cell polarity refers to the asymmetry in cell shape resultingfrom asymmetrical signaling molecules asymmetrical distribution within a cell inorder to serve a specialized cell function or directional cell division. A highlypolarized cell is characterized by elongated shape and many intracellular signalingmolecules including cytoskeletal proteins and lipids showing asymmetric distributionand cyto-architecture reconstruction. The development of cell polarity is mediated byactin cytoskeleton and cortical extensions. First, cells develop ruffles all over thebody, accompanied with a two to three-fold transient rise in polymeri7ed actin whichaccumulates in the ruffles. A few minutes later the cells develop a polarizedmorphology with formation of a lamellipodium at the leading edge and a uropod atthe tailing edge. During cell polarization, the rapid nucleation and polymerization ofactin at the leading edge drive the protrusion of the cell membrane, forming theleading lamellipodium, whereas cell retraction, driven by myosinⅡ-generated forces,results in detachment of the uropod from the substratum. The temporal and spatialcoordination of lamellar protrusion and uropod retraction is essential for cell polarityin response to chemotactic stimulation. The polarity play central role in basic cellfuctions such as cell migration, axonal growth, wound repair, vasculogenesis, the organization of the embryo in metazoan.Cell polarity of neutrophils plays a key role in physiological function. Thepolarity of neutrophils is responsible for initiation and progression of many humandiseases, including septic shock, asthma, atherosclerosis, ischemia-reperfusion injury,virus-induced myocarditis, rheumatoid arthritis, and even tumorigenesis. Neutrophilsemerge as the first line of defense against invading bacteria and pathogens. Thisfunction depends on cell polarity and chemotaxis, cell polarity is an essential featureof neutrophil chemotaxis. Neutrophils can sense chemoattractant gradients releasedby bacteria and pathogens, even it is as little as 2%, and develop polarity toward sitesof infection and finally migrate toward these sites which is based on the optimizedand amplified polarized signal mediated by intracellular signalling molecules.Recent studies on the mechanisms of neutrophils polarization focused on thefollowing three aspects: firstly, investigate the signal transduction pathway involvedin the formation of cell polarity; secondly, explore the effective mechanism of SOCE(Store-operated Ca2+ entry); lastly, elucidate the role of lipid rafts on cell polarity.However, the correlation among the mechanisms of neutrophils polarity, especiallySOCE mediated by TRP ion channel and the influence of lipid rafts on TRP duringcell polarization remain to be further clarified.Thus, a better understanding how to regulate the polarity of neutrophils mayprovide novel therapeutic targets or strategies to prevent tissue damage induced bychronic activation of neutrophils.Objective1. Investigate the effects of 4℃pretreatment on the membrane current and cellpolarity of human neutrophils.2. Investigate the formation of human neutrophils polarity in response to uniformconcentration of fMLP. 3. Elucidate the change of [Ca2+]i in human neutrophils and the cell polarizationduring different period induced by different Concentration of fMLP.4. Elucidate the relationship between the change of cytosolic Ca2+ induced by fMLPand store-operated Ca2+ entry (SOCE).5. Explore the effect of TRPC1 and lipid raft on cell polarization and the correlationbetween them.Methods1. Human peripheral blood neutrophils were obtained from healthy adult volunteersand separated on a discontinuous gradient consisting of percoll solution. Confirmisolated neutrophils purity by Wright's stain and cell viability by Trypan blue.Observe the change of human neutrophils in response to fMLP after 4℃pretreatment through inverted microscope. K+ currents were recorded in singlechannel and whole-cell voltage-clamp mode.2. The cytosolic free Ca2+ concentration ([Ca2+]i) of neutrophils were measuredusing laser scanning confocal microscope (LSCM) and highly sensitive Ca2+fluorescent dye(Fluo—3/AM).3. Local expression of TRPC1 in three groups (control, fMLP and fMLP+mβCD)was detected by immunofluorescence using confocal microscopy.4. Lipid raft was separated using sucrose gradient ultracentrifugation and theexpressions of TRPC1 in different groups was analyzed by Western-blotting.Results1. The purity of isolated neutrophils using Wright's stain was found to be＞95%.The viability of the cells as determined by Trypan blue dye exclusion was＞94%.After 4℃pretreatment the isolated neutrophils, the results showed that the timeof lamellipod formation was respectively (45.93±7.87msec, n=35) and(33.75±6.02msec, n=28) in the 4℃pretreatment group and the control group, there were significant difference (P＜0.01).The time of the polarized periodbetween the 4℃pretreatment group (538.26±27.74sec, n=23) and the controlgroup (260.25±25.87sec, n=23) was significantly different (P＜0.01). However,voltage-dependent K+ currents (Kv) were recorded in whole-cell voltage-clampmode, the current-voltage curve of two groups showed no significant difference(n=5, P＞0.05). Moreover, large-conductance Ca2+-activated K+ currents (BKCa)were recorded using inside-out patches,the conductance and NPo of two groupswere also no significant difference (P＞0.05).2. Under the stimulation of three uniform concentrations fMLP (10nM, 50nM,100nM), The time of 50% neutrophils development polarizability wasrespectively(51.16±6.49sec,45.83±4.95sec and 37.33±5.16sec),there weresignificant difference among three concentration groups (n=6, P＜0.01)Meanwhile, the lengths of cell polarization (1.62±0.050μm, 1.73±0.036μm,1.81±0.027μm; n=30) and the frequency of polarization oscillation period(209.37±17.70sec, n=52; 234.00±21.42sec, n=50; 286.15±19.44sec, n=52)were also obviously different in the three concentrations (P＜0.01). Moreover, theextension rate of pseudopod (0.067±0.0073μm/sec, 0.107±0.0086μm/sec,0.120±0.0132μm/sec),contraction rate of pseudopod (0.051±0.0050μm/sec,0.091±0.0051μm/sec, 0.110±0.0142μm/sec) and change rate of pseudopod(0.057±0.0071μm/sec, 0.099±0.0059μm/sec, 0.114±0.0124μm/sec)under thethree concentration groups were significant difference(n=30, P＜0.01).3. The change of[Ca2+]i was mainly classified into the following five phases: restingphase (0sec),rapid rise phase (10sec),rapid descent phase (150sec),slowdescent phase (250sec) and final phase after adding 100nM fMLP. During thefive phases, neutrophils developed ruffles all over the cell body at the time of10sec which further elicited the cell polarization of lamellar protrusion and uropod retraction. After this, polarization and depolarization were repeatedlyshown.4. 10μM SKF96365 and 10mM mβCD can inhibit the formation of neutrophilspolarity and the increasing of [Ca2+]i induced by fMLP.5. TRPC1 were distributed uniformly in the plasma membranes of controlneutrophils, and its were concentrated obviously on the lamellipodium of polarityneutrophils induced by uniform fMLP, however, there were no aggregation whenneutrophils were treated with mβCD before stimulated with uniform fMLP (100nM). TRPC1 levels were monitored by Western blotting in DRMs and N-DRMsof control group, fMLP group and mβCD+fMLP group. In N-DRMs,differences between control group (289.00±8.00) and mβCD+fMLP group(283.33±6.02) versus fMLP group (76.00±7.00) were statistically significant(n=3,P＜0.01). However, in DRMs, there were significant differences (n=3, P＜0.01)between fMLP group (215.00±6.55) and the other two groups (0.00±0.00,21.33±5.68).Conclusions1. 4℃pretreatment can inhibit neutrophils polarity, but has no effects on membranecurrents. Therefore, 4℃pretreatment decreased the rate of spontaneouspolarization and provided favorable cells for the further experiments.2. We established a set of indexes to evaluate the change of neutrophils polarity.According to the experiment data, we discovered that the development ofneutrophils polarity induced by fMLP depended on the time and concentrationsof fMLP.3. The results suggested that there were no significant difference among the changesof [Ca2+]i induce by different concentrations fMLP. Although the increasing[Ca2+]i elicited the polarization of neutrophils, it had no obvious effects on the following polarization periods. This experiment further revealed that inceased[Ca2+]i was related to the initiation mechanism of neutrophils polarity.4. Research identified that the mechanism of SOCE and lipid raft involved in thethe development of neutrophils polarity and the change of [Ca2+]i induced byfMLP. This experiment further elucidate a role for some key intracellularsignaling molecules (Ca2+ and TRPC1 ) in neutrophils polarized signaltransduction.5. We firstly discorvered that TRPC1 located in lipid raft during the polarization ofneutrophils. |
