Problem. In endometriosis, regurgitating endometrial cells fail to undergo apoptosis and implant themselves outside the uterus, particularly in the peritoneum. During endometriosis a breakdown occurs in endometrial and peritoneal homeostasis caused by cytokine induced cell proliferation and dysregulation of apoptosis. TNFα, a member of the TNF family of death ligands, binds to cell surface TNFR1 and TNFR2 and appears to mediate different biological activities ranging from the proliferation, differentiation and angiogenesis to the activation of apoptosis. Methods. Peritoneal fluid samples of women with endometriosis at different stages were collected at the time of the laparoscopy and centrifuged. The supernatants was used to assay soluble TNFα by ELISA. The cell pellet was suspended in PBS, layered onto Histopaque-1077 and centrifuged. The cells were collected at the interface, washed, suspended, counted and identified as PFMCs. An aliquot of these cells was used to evaluate TNFR1 and TNFR2 cell membrane expression by indirect immunofluorescence. The remaining cells were assayed for TNFR1 and TNFR2 mRNA and protein levels by real-time PCR and western blotting respectively. Results. Firstly we found that soluble TNFa decreased from minimal to severe stage of the disease. In the same way, we observed that TNFR1 mRNA and protein levels as well as the percentages of TNFR1-positive PFMCs decreased from minimal to severe stage of the disease. On the contrary, TNFR2 mRNA and protein levels as well as the percentages of TNFR2-positive PFMCs increased from minimal to severe stage of the disease. Conclusions. The utilization of different signalling mechanism by TNFR1 and TNFR2 is consistent with the ability of each receptor to trigger distinct biological responses after TNFα binding. TNFα/TNFR1 interaction is necessary to induce the endometriotic cell apoptosis. We hypothesize that the decreasing levels of both TNFα and TNFR1 from the minimal to severe stages reduce the possibility of their optimal binding, and so endometriotic cells could escape to this pathway of apoptosis. On the other hand, the TNFR2, increasing throughout the worsening of the disease, could compete with TNFR1 for TNFα binding, promote endometriotic cell proliferation and angiogenesis, and contribute to their survival.

Stage-related changes of peritoneal soluble TNFa and TNFR2 in cells recovered from peritoneal fluid of women with endometriosis

LAGANA', ANTONIO SIMONE;SALMERI, Francesca Maria;RETTO, Giovanni;STURLESE, Emanuele;PIZZO, Alfonsa;DE DOMINICI, ROSANNA;SOFO, Vincenza
2012

Abstract

Problem. In endometriosis, regurgitating endometrial cells fail to undergo apoptosis and implant themselves outside the uterus, particularly in the peritoneum. During endometriosis a breakdown occurs in endometrial and peritoneal homeostasis caused by cytokine induced cell proliferation and dysregulation of apoptosis. TNFα, a member of the TNF family of death ligands, binds to cell surface TNFR1 and TNFR2 and appears to mediate different biological activities ranging from the proliferation, differentiation and angiogenesis to the activation of apoptosis. Methods. Peritoneal fluid samples of women with endometriosis at different stages were collected at the time of the laparoscopy and centrifuged. The supernatants was used to assay soluble TNFα by ELISA. The cell pellet was suspended in PBS, layered onto Histopaque-1077 and centrifuged. The cells were collected at the interface, washed, suspended, counted and identified as PFMCs. An aliquot of these cells was used to evaluate TNFR1 and TNFR2 cell membrane expression by indirect immunofluorescence. The remaining cells were assayed for TNFR1 and TNFR2 mRNA and protein levels by real-time PCR and western blotting respectively. Results. Firstly we found that soluble TNFa decreased from minimal to severe stage of the disease. In the same way, we observed that TNFR1 mRNA and protein levels as well as the percentages of TNFR1-positive PFMCs decreased from minimal to severe stage of the disease. On the contrary, TNFR2 mRNA and protein levels as well as the percentages of TNFR2-positive PFMCs increased from minimal to severe stage of the disease. Conclusions. The utilization of different signalling mechanism by TNFR1 and TNFR2 is consistent with the ability of each receptor to trigger distinct biological responses after TNFα binding. TNFα/TNFR1 interaction is necessary to induce the endometriotic cell apoptosis. We hypothesize that the decreasing levels of both TNFα and TNFR1 from the minimal to severe stages reduce the possibility of their optimal binding, and so endometriotic cells could escape to this pathway of apoptosis. On the other hand, the TNFR2, increasing throughout the worsening of the disease, could compete with TNFR1 for TNFα binding, promote endometriotic cell proliferation and angiogenesis, and contribute to their survival.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11570/1968028
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