| BackgroundNeutrophils are the most abundant leukocyte and fastest moving cell in the body. Neutrophils play a central role in innate immunity as cellular defense against microorganisms and inflammatory processes. The mature neutrophils are released from bone marrow. They circulate in the peripheral blood. In response to an inflammatory stimulus, neutrophils first become polarized, exhibiting a leading edge (pseudopod) at one end and a tail (uropod) at the other end. Then these leukocytes migrate towards the source of the stimulus and contribute to hyper-inflammatory reactions caused by tissue injury. However, neutrophil inappropriate activation are clinically associated with many diseases, including rheumatoid arthritis, ischemia-reperfusion syndrome, acute respiratory distress, and systemic inflammatory response syndromes. 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.Some chemokines stimulate neutrophils to move toward factor source direction, the phenomenon known as chemotaxis. There are two kinds of Chemokines:one’s own tissue injury released factors, such as collagen and fibrin fragments, complement activation products and immune cytokines; the other is a polypeptide containing N-early acyl methionine residue of microbial origin, e.g. formyl methionyl-leucyl-phenylalanine (fMLP). Heit found fmlp as neutrophil terminal chemokine is not dependent on PI3K pathway through P38 MAPK, and IL-8 as intermediate chemokine places PI3K-dependent signaling pathway. If the terminal and intermediate chemokines exist, non-dependent PI3K will be dominant. Therefore, our study used fmlp and IL-8 chemokines as stimulating neutrophil chemo-attractant.There has been recent progress in understanding how Ca2+signaling that takes part in the activation of neutrophil reactions such as chemotaxis and migration, release of Reactive Oxygen Species (ROS). Neutrophil chemo-attractant bind G-protein coupled receptors on human neutrophils membrane, so that phospholipase C (PLC) hydrolyze into IP3 and DAG. IP3 bind to the IP3 of receptor the endoplasmic reticulum inducing rapid depletion from endoplasmic reticulum (ER). This activates the store-operated calcium entry (SOCE) and an external Ca2+influx. DAG can also activate receptor-operated calcium entry (ROCE) within a external Ca2+influx. Endoplasmic reticulum and mitochondria are calcium store. The endoplasmic reticulum can store about 500μM calcium. Depletion from endoplasmic reticulum (ER) can make the cytoplasmic calcium concentration rise from 0.1 LM to 1μM, causing the cell calcium toxicity to be very high. This need a mitochondrial calcium buffering system. Italy’s Rizzuto measured that the mitochondrial calcium ions can be stored 100-300μM. Calcium ions from IP3 channel streams through the outer mitochondrial membrane VDAC channel, and then flows into a mitochondrial calcium uniporter (MCU).It has been known for almost 50 years that Ca2+ uptake across the ion-impermeable inner mitochondrial membrane (IMM) is mediated by low-affinity mitochondrial Ca2+ uniporter (MCU) that has the properties of a highly selective ion channel. Indeed, it was known that the driving force for Ca2+ accumulation in the mitochondrial matrix is the steep membrane potential (negative inside) (△ψ m) across the inner membrane. Mitochondrial Ca2+ uptake occurs by a uniport mechanism driven by the negative-inside membrane potential without direct coupling to ATP hydroslysis or transport of other ions. However, its molecular identity remains elusive. Most traditional approaches in this molecular search could not be applied because there’s no highly specific inhibitor that can be utilized in biochemical purifications. Although ruthenium red (RuR) and the related compound Ru360 can inhibit the activity of the uniporter, it shows some major drawbacks because it binds a broad array of glycoproteins. Until 2011, researchers of Harvard Medical School and Massachusetts General Hospital found the key protein of mitochondrial calcium uniporter CCDC109A (MCU) by referring to the human genome project database combined with experimental analysis. MCU, localized to the inner membrane, and three components of the MCU complex have been recently identified:MCU, mitochondrial calcium uptake 1, and mitochondria calcium uniporter regulator 1. A characteristic feature of the MCU is its low affinity for Ca2+.but the mitochondrial Ca2+concentrations can increase quickly by cell stimulation, as mitochondria are exposed to micro domains of high [Ca2+]. Mitochondria localized at close proximity to intracellular Ca2+ stores or plasma membrane Ca2+channels sense and respond to the Ca2+transients by taking up Ca2+. The study of MCU function is not very complete, a new study has found the possibility of adjusting the mitochondrial transport. Mitochondria continually change shape through the combined actions of fission, fusion, and movement along cytoskeletal tracks, known as mitochondrial dynamics. In more recent years, the biological relevance of these phenomena has become clear with the discovery of human diseases that are caused by mutations in fission and fusion proteins and the discovery of numerous connections with apoptosis and mitophagy. Mitochondrial fission and fusion are now considered cornerstones for cell survival because of their contributions to health and disease.In mitochondrial fission, cytosolic Drp-1 trans locates to the outer mitochondrial membrane. Drp-1 then self-assembles and form helices that encircle the dividing mitochondria to mediate scission of the mitochondria. The divided mitochondria can then enter the fusion-fission cycle, or if the mitochondria are damaged then these fragmented mitochondria are removed by the process of mitophagy. In mitochondrial fusion, Mfnl and Mfn2 on the OMM tether adjacent mitochondria and mediate fusion of the OMM, which is then followed by IMM fusion mediated, by OPA1.It has been reported that higher mitochondrial [Ca2+] can promote DRP-1 transposition and promote mitochondria fission, while mitochondrial channel inhibitors Ru360 can inhibit the mitochondrial fission. Mirol, a mitochondrial outer membrane protein, has been shown to play a role in mitochondrial transport. Selective binding of mirol to TRAK1 or TRAK2 may regulate interaction with kinesin or dynein to modulate directionality of mitochondrial movement. It has also been reported that mitochondrial calcium intake influence mitochondrial moving speed.ObjectiveIn this study,we used the uniform, low concentration of fMLP and IL- 8 as stimulating neutrophil chemoattractant to set up neutrophil cell polarity model. First we use patients neutrophils polarization experiment and western blot to find the relationship between MCU and neutrophils polarization.And then we explore the effects of MCU’s blockers and agonist in neutrophil polarization and chemotaxis, through the observation of cell morphology, the distribution of intracellular F-actin and cell migration.By observing the distribution of mitochondria and mitochondrial division protein expression to explore whether MCU involved in human neutrophil polarity and chemotaxis formation by mitochondrial fission.Methods1. The classic method of density gradient centrifugation separation neutrophils. It mainly includes three steps:erythrocyte sedimentation; lymphocyte separation medium density gradient centrifugation; and hypotonic lysis of the remaining red blood cells. More than 95% of the cells isolated were neutrophils, as assessed by Wright-Giemsa staining. Viability, determined by trypan blue exclusion, was> 98%.2. Human neutrophils were pretreated with or without Ru360 or spermine pretreatment for 30 min at 37 ℃, Zigmond chambers were used for quantification of neutrophil polarization..3. Human neutrophils were pretreated with or without Ru360 or spermine pretreatment for 30 min at 37 ℃, transwell experiment testing the changes of neutrophil migration.4. Confocal laser-scanning microcopy was used to observe the location of F-actin and the distribution of Mitochondria by fluorescence stain.5. Using the Ca2+-sensitivity indicator fluo4/AM and rhod2 pretreatment neutrophils under an inverted laser confocal microscope monitoring the change of intracellular Ca2+and mitochondrial Ca2+.6. The expressions of MCU, Drp-1, P-Drp-l(616) and P-Drp-1(637) in different groups was analyzed by Western-blotting.7. Statistical analysis was performed using SPSS 13.0 software. Results were expressed as mean ± SEM. Differences Group compared with one-way ANOVA and inter-group comparision was significant using LSD when the homogeneity of variances was meet, while, Group compared with Welch and inter-group comparision was significant using Dunnett T3 when the homogeneity of variances was not meet. p< 0.05 means the significance.Result1. The neutrophils polarization rate of Chronic obstructive pulmonary disease patients was decrease,and MCU protein expression was also decreased..2.Stimulation of MCU by spermine promotes fMLP-induced neutrophil polarization and chemotaxis, whereas inhibition of MCU by Ru360 inhibits both processes.3. Stimulation of MCU by spermine promotes IL-8-induced neutrophil polarization and chemotaxis, whereas inhibition of MCU by Ru360 inhibits both processes.4.1n un stimulated cells, only faint F-actin staining was observed at the periphery with round loop.After 100 nM 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 pre incubated with Ru360.while cells were pre incubated with spermine. This asymmetry became more obvious.5.MCU protein expression, intracellular Ca2+ and μ-calpain activity had no difference.6. Ru360 inhibit mitochondrial calcium uptake, while spermine promotes mitochondrial calcium uptake.7. Resting neutrophils present the linear clusters, distribute in the middle of the cell,. After exposed to fMLP, mitochondria appear scattered point, mainly in the tail, this linear clusters appeared when cells were pre incubated with Ru360, distribution in the middle of the cell. However when the cells were pre incubated with spermine, this scattered point became more obvious.8. Ru360 inhibit P-Drp-1 (Ser616) protein expression, while spermine promotes P-Drp-1 (Ser616) protein expression.9. Mitochondrial fission protein Drp-1 inhibitors Mdivi-1 inhibit neutrophil polarization and chemotaxis.Conclusion1. MCU channel play an important role in neutrophil polarization and chemotaxis.2. MCU involved in human neutrophil polarity and chemotaxis formation by mitochondrial fission. |
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