In Vitro Morphology, Viability And Cytokine Secretion Of Uterine Telocyte-activated Mouse Peritoneal Macrophages

Objective:Telocytes(TCs) is distinct interstitial cell population that was present in a wide variety of human and mammalian reproductive organs/tissues. Recently, more and more studies gradually revealed that TCs might modulate activities of immunocytes, through direct intercellular junctional complexes or possibly indirect paracrine messages. We here evaluated in vitro paracrine effect of uterine telocytes on morphology, viability and cytokines/enzyme production in mouse peritoneal macrophages. Aimed to get in vitro evidence of immunoregulation/immunosurveillance roles for uterine TCs.Methods:Adult female BALB/c mice were selected to obtain primary uterine TCs and p MACs.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 methylene blue vital staining, mitochondrial labelling and immunofluorescence cytochemistry(ICC)(vimentin, CD34 and c-kit).3. TCs behaviour was monitored by time-lapse videomicroscopy and images were captured every 5 min.4. TCs content in our culture was analyzed in a BD FACSCanto II cytometer by doublelabeling with Per CP-labeled anti-vimentin monoclonal antibodies and FITC-conjugated anti-CD34 monoclonal antibodie.5. TCs entered into the logarithmic growth phase and cultured with serum-free DMEM/F12 for 24 hrs, then harvested the media(TCM).6. Thioglycollate was injected into the mouse abdominal cavity. 3 days later, p MACs were obtained by collection of peritoneal lavage fluids.7. PMACs were cultured with TCM, or serum-free DMEM/F12 as negative control, or serum-free DMEM/F12 with lipopolysaccharide(LPS) as positive control.8. Morphology of p MACs were observed and cell viability were determined by using CCK-8 kits, immunofluorescence intensity of mitochondrial labelling was measured, the quantitative determination of fluorescence intensity was calculated by multiplying the average number of pixels/area and p MACs-related cytokines/enzyme, including i NOS, IL-6, TNF-α, IL1R1, IL-10, TGF-β1, IL-1β, IL-23α and IL-18, were determined by using and ELISA kits. To study the ability of TCs to activate p MACs and potentially trigger subsequent immunoresponse through indirect paracrine effect.Results:1. TCs demonstrated with small bipolar or multipolar cellular body with one or more extremely long, thin, very sinuous cellular prolongations(Tps) which was composed of an alteration of thin segments and thick segments.2. Double positive vimentin/CD34 was found for TCs. However, no fluorescence for c-Kit could be observed.3. TCs is a kind of active cell, time-lapse images showed that TCs scouting the area and planting marks out of its cell body and extending Tps.4. Flow cytometry revealed the presence in primary culture of double-positive cells(vimentin+CD34+) in an average proportion of 89.2 ± 7.5 %.5. After co-cultured with TCM, p MACs presented with abundant pseudopodia and secretory granules within cytoplasm, with no obvious cell death. However, no sign of activation/immunoresponse can be observed in negative control group. For LPS-treated group, more dramatic morphological changes were observed, accompanied by obvious cell death.6. By using CCK-8 assay, significant difference between TCM and DMEM/F12. However, no significant tendency for slightly higher viability was found for TCM-treated p MACs, as compared with LPS(P > 0.05).7. Quantitative analyze showed that the expression of fluorescence intensity in DMEM/F12-group was respectively: 9732.27?1996.87, the LPS-treated group was respectively: 21939.16?2159.58, and the TCM-treated group was respectively: 29478.73?3652.01, which significantly different from DMEM/F12-group and LPS-treated group(P > 0.05).8. By using Elisa kits, i NOS and IL-6 were significantly increased in TCM-treated p MACs than in DMEM/F12 both at 24 and/or 48 hrs(all P < 0.05). Meanwhile, slightly but significantly increased levels of TNF-α, IL1-R1, IL-10 were observed(all P < 0.05). However, no obvious fluctuations of TGF-β1, IL-1β, IL-23α and IL-18 were observed throughout studies(all P > 0.05).Conclusions:After indirected co-cultured with p MACs, TCs demonstrated the ability to activate p MACs and potentially trigger subsequent immunoresponse. We believed that, the panel of elevated cytokines/enzyme play an important role in reproductive physiology. Based on the change of TCM-treated p MACs, we suggested that TCs might active players in induction and maintenance of inflammatory process.