An in vitro investigation of telocytes-educated macrophages: morphology, heterocellular junctions, apoptosis and invasion analysis

Abstract

Background: Telocytes (TCs), a recently discovered novel type of interstitial cells, were also found in a wide variety of human and mammalian reproductive organs/tissues, including uterus, oviduct and placenta. Previously, we demonstrated that TCs-conditioned media was capable of activating peritoneal macrophages (pMACs) through paracrine effects. This study investigates the hypothesis that direct interaction of TCs with pMACs will also play a significant role in immunoregulation of pMACs.

Methods: TCs and pMACs were derived from the uterus and intraperitoneal cavity of female BALB/c mice, respectively. TCs were identified by immunofluorescence and then co-cultured directly with pMACs for 24 h without added cytokines, to observe the in vitro biological behavior of pMACs. We used histochemical staining to study morphology and mitochondrial metabolism of pMACs, scanning electron microscopy to study heterocellular junctions, flow cytometry to investigate mitochondrial membrane potential (ΔΨm) and apoptosis, and transwell chambers to study invasion ability. Student-t test was used accordingly.

Results: Presently, TCs with typical structure and immunophenotype of double CD-34-positive/vimentin-positive were successfully isolated. pMACs co-cultured with TCs showed obviously morphological activation, with enhanced energy metabolism (P < 0.05). Meanwhile, direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs. Furthermore, TCs treatment markedly reduced the depletion of ΔΨm in co-cultured pMACs (all P < 0.05), and inhibited their apoptosis (P < 0.05). Functionally, pMACs co-cultured with TCs showed enhanced invasion ability (P < 0.05).

Conclusions: Direct physical cell-to-cell interaction promoted the development of heterocellular junctions between TCs and pMACs, presumably responsible for the observed novel efficient way of pMACs activation via mitochondrial signaling pathway. TCs-educated pMACs might be a promising way to restore the defective immunosurveillance in endometriosis (EMs), led to the enhanced treatment efficacy of EMs in a simple and clinically feasible fashion.

Keywords: Apoptosis; Endometriosis (EMs); Heterocellular junctions; Immunoregulation; Interstitial cells; Macrophage activation; Mitochondrial membrane potential (ΔΨm); Peritoneal macrophages (pMACs); Telocytes (TCs).

Fig. 1

Representative double-labeled immunofluorescence identification of TCs. Images of c-kit negative staining were not shown; Scale bar = 50 μm. a Positive FITC labelling for Vimentin (green). b Positive CY3 labelling for CD34 (red). Nuclei were counterstained with DAPI (blue). c In the merged image, Vimentin and CD34 were overlapped, both in the cellular body and full length of Tp, with characteristic podomer (thin segments) and podom (thick segments) arrayed alternatively along Tp

Fig. 2

Real-time dynamic observation of primary directly co-cultured TCs and pMACs at 12 h intervals under phase-contrast microscopy; mice uterus. TCs have small oval cell bodies and long Tp branched from the cell body, appearing as an alteration of podom and podomer. a At 0 h, pMACs showed its normal regular round shape and no signs of activation. b At 12 h, pMACs showed moderate activation with irregular polyhedron shape and pseudopodia, but without any intercellular contacts to TCs. c At 24 h, pMACs exhibited intensive morphological changes and continuous activation, with obvious polyhedron, abundant pseudopodia; more importantly, direct heterocellular junctions can be observed between the activated pMACs and TCs (black arrowhead). d At 24 h, vital staining with crystal violet demonstrated heterocellular junction between the pseudopodia of activated pMACs and TCs (black arrowhead)

Fig. 3

Fluorescence microscopy of Mito Tracker green staining. Error bars = SD. The MFI of each group was repeatedly detected three times under the same conditions. a–c The observed fluorescent intensity for TCs and pMACs in the co-cultured system at two time points. MFI at 24 h was significantly higher than that of 0 h (*P < 0.05), indicating active energy metabolism of pMACs in the co-cultured system. d–f No significant difference for background MFI of cultured-alone pMACs was found in two time points

Fig. 4

a–c Scanning electron microscopy of the direct co-cultured system at 24 h. TCs displayed oval cell body with long TP. The activated pMACs with irregular shape, abundant fluff-like structures on the surface of the cell membrane was observed. The complex direct heterocellular junction between the activated pMACs and Tp was presumably a certain type of immunological synapse (IS)

Fig. 5

Flow cytometry analysis of apoptosis of pMACs (cultured-alone, co-cultured with TCs) under three conditions (DMEM/F12 for control conditions, UV irradiation and DXM for positive control). a Annexin V-FITC/PI double staining method was used to quantitatively analysis the percentage of apoptotic cells. b The considerably lower percentage of apoptosis was detected for the co-cultured pMACs, compared with that of cultured-alone pMACs in all of three conditions (all *P < 0.05 versus cultured-alone pMACs). Error bars = SD. The mean and SD were calculated from three independent experiments

Fig. 6

Changes of ∆Ψm in pMACs (cultured-alone, co-cultured with TCs) were evaluated by flow cytometry analysis of JC-1 and Rhodamine fluorescence, respectively, under three conditions (DMEM/F12 for control conditions, UV irradiation and DXM for positive control). Results showed that the depletion of ∆Ψm in pMACs can be reduced by TCs, further prevent their apoptosis. Error bars = SD. The mean and SD were calculated from three independent experiments. a For JC-1, value of ∆Ψm was represented as the green/red fluorescence ratio by flow cytometry. b For both groups of pMACs, higher values of ∆Ψm were detected in cultured-alone pMACs compared with that of co-cultured pMACs, in all of three conditions (all *P < 0.05 versus cultured-alone pMACs). c For Rhodamine, value of ∆Ψm was represented as Rhodamine fluorescence intensity by flow cytometry. d For both groups of pMACs, higher values of ∆Ψm were detected in the co-cultured pMACs compared with that of cultured-alone pMACs, in all of three conditions (all *P < 0.05 versus cultured-alone pMACs)

Fig. 7

Invasion assay of pMACs (blue dye cells) after 24 h by Transwell chambers assay. a When cultured alone, very few pMACs invasion can be observed on the lower surface of the membrane. b When co-cultured with TCs, cellular invasion of activated pMACs was significantly higher than that of cultured-alone pMACs. c The average number of pMACs invasion significantly increased. (*P < 0.05 versus DMEM/F12). Error bars = SD. The mean and SD were calculated from three independent experiments