Kv1.3, KCa3.1 And The Phagocytic Function Of Carbon Nanotubes And Macrophages

Background and Objective:The effects of nanomaterials on cell function have gained extensive attention at the moment.One of the first-order events after the nanomaterials entering into an organism is the phagocytosis of the nanomaterials by macrophases and other cells and thus inducesbiological effects.However,the potential influences of nanomaterials on immune cell function including ion channel activities are not well elucidated.We used the RAW264.7 macrophages to observe the the effects of Kv1.3,KCa3.1 potassium channels and multiwalled carbon nanotubes on the phagocytosis.Methods:Microscopy and flow cytometry were used to semi-quantitatively and quantitatively estimate the phagocytotic activities of RAW264.7 cells on chicken red blood cells and FITC-tagged E.coli k-12,and designed to determine the roles of Kv1.3 and KCa3.1 channels in the phagocytosis,with or without stimulation with LPS,using ShK(a Kv1.3 selective blocker)and TRAM-34(a KCa3.1 selective blocker)and multi-walled carbon nanotubes(MWCNT),respectively.We also evaluated the roles of Kvl.3 and KCa3.1 channels on the production of nitric oxide(NO)secretion in RAW264.7 cells.The CCK-8 method was used to examine the roles of Kv1.3 and KCa3.1 channels on cell proliferation,ELISA method,was used to measure the effects of blockers of Kv1.3 and KCa3.1 channels and MWCNT on the secretion of cytokines IL-6,TNF-alpha and IL-1beta.Western blot was used to detect the expression of Kv1.3 and KCa3.1.Results:Under a light microscope,resting(not stimulated with LPS)RAW264.7 cells showed a weak phagocytotic activity on chicken red blood cells.However,pre-treatment of the RAW264.7 cells with ShK and TRAM-34 significantly increased the PR.In the flow cytometry assay,blocking KCa3.1with TRAM-34 also both significantly increased the phagocytotic activities of RAW264.7 cells on FITC-tagged E.coli k-12 compared with the control.Blocking Kv1.3 with ShK efficiently increased the untake of FITC-tagged E.coli k-12 both on the resting and LPS-stimulated macrophages.LPS stimulation inhibited Kv1.3 expression,but increased the expression of KCa3.1.Blocking endocytosis with both Cytochalasin D and filipin Ⅲ could upregulate the expression of Kv1.3 but not KCa3.1.MWCNT inhibited the phagocytosis but promoted the expression of Kv1.3 in a concentration-and time-dependent manner.Conclusion:The activity of Kv1.3 exerts an inhibitory role on the phagocytotic function both in the resting and LPS-stimulated RAW264.7 macrophages,while the activity of KCa3.1 only exerts an inhibitory role on the phagocytotic function of the resting RAW264.7 macrophages.MWCNT especially at low concentration can promote the expression of Kv1.3,while MWCNT did not significantly affect the expression of KCa3.1.Theses results may provide experimental evidence for comprehewnsive understanding on the roles and mechanisms of Kvl.3,KCa3.1 and MWCNT in the phagocytotic function of macrophages.