In Vitro Activation Of Mouse Peritoneal Macrophages By Uterine Telocytes: Morphologic,apoptosis And Invasion Analysis

Objective:Telocytes(TCs),a newly discovered type of interstitial cells,were found in a wide variety of human and mammalian reproductive organs/tissues,including uterus,oviduct and placenta,with multiple proposed potential biological functions.Their unique structure allows them to form junctions with various immunocytes,both in normal and diseased tissues,including macrophage,may influence the activity,apoptosis,invasion of local immunocytes.However,no reliable cytological evidence for this hypothesis is currently available.In this study,we co-cultured primary murine uterine TCs and mouse peritoneal macrophages(pMACs)for 24 h to detect the activity,apoptosis,invasion of pMACs,to provide in vitro evidence of immunoregulatory and immunosurveillance roles for TCs.Methods:Adult female BALB/c mice were selected to obtain primary uterine TCs and pMACs.1.Mice were killed with an overdose of sodium pentobarbital,TCs were obtained from uterine tissue by enzyme digestion.2.Cultured TCs were detected by using immunofluorescence cytochemistry(ICC)(vimentin,CD34).3.Thioglycollate was injected into the mouse abdominal cavity.3 days later,pMACs were obtained by collection of peritoneal lavage fluids.4.After 3–4 days of primary cell culture,the TCs were transferred to 6-well culture plates with 2.5 ml of serum-free DMEM/F12.Peritoneal macrophages were inoculated into the TCs at a ratio of pMACs: TCs of about 10:1,which were isolated from BALB/c mice.At 24 h after seeded,co-cultured TCs and pMACs were fixed and stained in crystal violet solution at room temperature for 10 min.,washed with PBS for three times,and imaged by light microscope..5.The co-cultured cells were incubated in phenol red-free DMEM with 100 nmol/l MitoTracker Green to label mitochondria.Detection under fluorescence microscopy(450–490 nm excitation,520 nm barrier filter;Leica)and analysis by Image-Pro Plus.6.pMACs cultured with DMEM/F12 and co-cultured cells were induced apoptosis under three conditions : serum-free DMEM/F12 as negative control,,irradiated at 15 W ultraviolet lamp 10min(UV,a physical induction of apoptosis stimulus),serum-free DMEM/F12 with 80ug/ml dexamethasone(DXM,a chemical induction of apoptosis stimulus).7.pMACs was observed at 24 hours and apoptosis was measured by Annexion-PI.The changes of mitochondrial membrane potential in p MACs were measured by JC-1 and Rhodamine-123.To investigate the changes of morphology,invasion and apoptosis of pMACs stimulated by TCs.8.For the invasion ability assay,performed in Transwell chambers.Results:1.The TCs had small cell bodies and one or more extremely long,thin telopodes(Tps),with alternation of thin segments(podomers)and thick segments(podoms).Double positive vimentin/CD34 was found for TCs.2.The co-cultured TCs and pMACs were taken picture at 0h,12 h,24h after seeded,the morphology of pMACs changed significantly.At 0hr,pMACs cultured with TCs showed no sign of activation,had normal morphology with a regular round shape.At 12 hr,pMAC occurred phenomenon of activation.At 24 hr,pMACs showed apparent activation phenomenon : they exhibited obvious morphological changes with a polyhedron shape,sufficient pseudopodia,few pMACs formed immunological synapse to contact with TCs.The immunological synapse of TCs and pMACs could be found clearly when co-cultured cells under the condition of crystal violet staining.3.Mito Tracker Green was used to label mitochondria of co-cultured cellls at 0hr and 24 hr.Mitochondrial fluorescent intensity was detected in each group three times under the same condition and 30 photographs of each group were analyzed by Image-Pro Plus to express fluorescence mean density.Results are expressed as means ± SD.Mitochondrial fluorescent intensity had a significant increase at 24 hr(P<0.05).4.Apoptosis of pMACs was examined by Annexin V/PI staining and flow cytometry.Two groups of cells under three conditions.Under both conditions,the considerably low percentage of apoptotic cells was detected in the co-cultured pMACs compared with that of pMACs cultured alone(P<0.05).5.Mitochondrial membrane potential(??m)in co-cultured pMACs was markedly increased than in pMACs cultured alone(P<0.05).??m changes were measured by JC-1 green/red fluorescence ratio.??m in co-cultured pMACs was markedly increased than in pMACs cultured alone(P<0.05).The Rhodamine fluorescence cell ratio of the co-cultured pMACs was higher than pMACs cultured alone(P<0.05).These results showed that TCs were able to prevent the loss of ??m and prevent cell apoptosis.It indicated that TCs reduce cell apoptosis via a mitochondrial pathway in vitro.6.Transwell assay was used to explore whether the TCs promoted the invasion of pMACs.The number of cells was less than that co-cultured with TCs.The data indicate that the TCs can accelerate the invasion of pMACs.Conclusions:After directed co-cultured with pMACs,TCs demonstrated the ability to activate pMACs and potentially trigger subsequent immunoresponse,improve cell viability,increase invasive ability to inhibit macrophage apoptosis.Based on the change of pMACs,we suggested that TCs might active players in induction and maintenance of inflammatory process.