Effect Of Electrotransjection With Anti-STIM1 Antibodies On FMLP-induced Human Neutrophpils Polarization And Chemotaxis

BackgroundPolymorphonuclear neutrophils(PMNS) are an important part of innate immune system. When the body subjected to external invasion of bacteria and other pathogens, PMNS, as the first line of defense, their main function is leaking vessel wall and migrate to inflammatory lesions, through phagocytosis and release of superoxide and /or proteolytic enzymes to kill bacteria and invading microbes. The mechanism of extravasation and migration is based on cell polarization and chemotaxis. Some diseases, such as clinical sepsis, asthma, ischemia/reperfusion injury and organ transplant rejection, atherosclerosis, viral myocarditis, rheumatoid arthritis, allergies, inflammatory skin diseases and even the development and metastasis of tumor are associated with function of polarity of neutrophils. Therefore, a better understanding how to regulate the polarity of neutrophils may provide novel therapeutic targets or strategies for preventing tissue damage caused by chronic activation of neutrophils.Calcium signaling is involved in a variety of physiological and pathological processes. Concentration of cytoplasmic Ca2+ depends on two pathways:the release of endoplasmic reticulum calcium stores and plasma membrane calcium influx. Store-operated calcium entry (SOCE) is the major forms of calcium influx in non-excitable cells. In recent years, findings on (stromal interaction molecule, STIM) lead to large progress on regulation mechanism of SOCE. Liou, Roos, etc. have been suggested that STIM1,but not STIM2, regulates SOCE. STIM1, as a key molecule in SOCE, located mainly in the endoplasmic reticulum (Endoplasmic Reticulum, ER), less in plasma membrane (PM), contains an EF-hand motifs in ER. It has been suggested that STIM1 acts as a calcium sensor in the ER lumen. Study shows that SOCE plays an important role in cell polarization and chemotaxis. However, whether STIM1 involves in the neutrophils polarization and chemotaxis and the mechanism in it still remains unclear.There are two signaling pathways of cell polarity:PI3K-dependent and PI3K-independent pathway. PI3K-Akt is considered as a classic polarity signaling pathway, and studies have shown that Src tyrosine kinase and Rho small G proteins are involved in polarity formation, cell migration, tumor invasion and metastasis. It is not clear that whether the existence of such signaling pathways for fMLP-induced human neutrophils polarization and chemotaxis.Human neutrophils are the terminal blood cells, which are short survival time, and they are difficult to carry out gene manipulation or RNA interference. So electrotransjection with antibodies for this short duration of studying is an effective means. To elucidate the role of SOCE in human neutrophils polarization and chemotaxis and regulatory mechanisms, in the present study, electrotransjection with anti-STIM1 is applied for observe its effect on rate of neutrophils polarization and chemotaxis and changes in related signaling proteins such as Akt, Src, Rho small G protein. ObjectiveInvestigate effect of STIM1 on fMLP-induced human neutrophils polarization and chemotaxis and elucidate the roles of SOCE in this and regulatory mechanisms by electrotransjection with anti-STIM1 antibodies.Methods1. The neutrophils were freshly isolated by standard isolation protocol. This protocol comprises three successive steps:Dextran sedimentation, Ficoll-Hypaque density centrifugation and hypotonic lysis.2. Electrotransjection with anti-STIM1 antibodies into human neutrophils by electrotransjection device is in order to study in neutrophils polarization and chemotaxis.3. Immunoprecipitation and Western blotting detected efficiency of electrotransjection with anti-STIM1 antibodies.4. Zigmond chamber observed effect of electrotransjection with anti-STIM1 antibodies on rate of fMLP-induced human neutrophils chemotaxis.5. Application of GST pull-down and Western blotting to detect the changes of related signaling molecules in fMLP-induced human neutrophils polarization and chemotaxis after electrotransjection with anti-STIM1 antibodies.Result1. Immunoprecipitation and Western blotting detection of electrotransjection with anti-STIM1 antibodies showed significantly the efficiency of electrotransjection,electrotransjection with anti-STIM1 antibodies group which detected the protein signal was significantly stronger than non-electrotransjection with anti-STIM1 antibodies group (P<0.001). 2. Each group of control, IgG, anti-STIM1 was significantly different rate of chemotaxis (P<0.001), rate of chemotaxis showed Control> IgG> anti-STIMl.3. Western blotting analysis showed control group p-Akt protein band signal was weak with and without stimulation of fMLP, no significant changes (p=0.505), the level of Src phosphorylation and Racl, Rac2, Cdc42 activation after fMLP stimulation increase significantly than without fMLP (P<0.001); IgG group had a similar trend, p-Akt remained unchanged (P=0.766), p-Src, GTP-Rac1, GTP-Rac2, GTP-Cdc42 after fMLP stimulation increase significantly than without fMLP (P <0.001); anti-STIM1 group demonstrated that p-Akt was no significant difference (P =0.476), p-Src (P=0.315), GTP-Racl (P=0.944), GTP-Rac2 (P=0.386), GTP-Cdc42 (P=0.386) had no significant differences with and without fMLP in each group.Conclusion1. Electrotransjection with anti-STIMl antibodies inhibits fMLP-induced human neutrophils polarization and chemotaxis, suggesting that SOCE might be involved in fMLP-induced human neutrophils polarization and chemotaxis.2. SOCE decreases activity of Src and Rac/Cdc42, indicating that SOCE regulates fMLP-induced human neutrophils polarization and chemotaxis through Src-Rac/Cdc42 signaling pathway.