| The pathogenesis of endometriosis remains to be elucidated.According to Sampson's transplantation theory,the pelvic endometriosis is initiated by the retrograde menstruation when menstrual fragments flow out of the fimbriated end of the fallopian tubes,and become established on the ovarian surface or other sites in the peritoneal cavity.Since the retrograde menstruation is a physiological process occurring in fertile women,other factors must be involved in the disease.Alteration/ dysfunction of the immune system may lead to the development of endometriosisCurrent evidence suggests the peritoneal immuno-inflammation might be associated with the origin of endometriosis.During the development of endometriosis, the immune cells are recruited into the peritoneal cavity,and among them macrophages are the dominant cell type that are involved in phagocytosis and inflammation,especially in cleaning the retrograded endometrial debris.The peritoneal macrophages isolated from patients with endometriosis have been found to have phenotypic and functional alterations leading to poor phagocytotic capacity, which is highly associated with severity of endometriosis.The peritoneal macrophages may improve pathogenesis of endometriosis by secreting many sorts of cytokines,and thereby play a very important role in endometriosis.Chemokine is required in the process of recruiting circulating leukocytes into pelvic sites. Chemokine activity is mediated through cell surface chemokine receptors,and the functions of chemokine depend on binding to the corresponding receptors.To explore the role of chemokine in the pathogenesis of endometriosis,it is facilitated to investigate chemokine as well as its receptor because of the complexity of the interaction between the chemokines and their receptors.To pick up chemokine receptors as well as their ligands may provide some useful insights into the molecular mechanisms of the pathogenesis of endometriosis.So we evaluated 18 chemokine receptors of U937 cells,which would help us to explore pathogenesis of endometriosis.Endometriosis is an estrogen-depending disease.Recently,environmental contaminants have been also suggested to play a role in the pathogenesis of endometriosis.Research on nonhuman primates has shown that exposure to the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin(TCDD) is associated with an increased prevalence and severity of endometriosis.Investigating the regulatory mechanism of dioxin and estrogen in chemokines and their receptors expression will throw a light in elucidating the role of the chemokines in the etiology of endometriosis.1.The expression characteristics of chemokine receptors in U937 cells and the modulating effect of 17β-estradiol and TCDD on the expressed chemokine receptorsThe transcriptions of 18 chemokine receptors in U937 cells were first analyzed by semi-quantified RT-PCR.Among 18 chemokine receptors,CCR5,CCR9,XCR1 and CX3CR1 were highly expressed in U937 cells,while CCR4,CCR7 and CXCR4 were moderately expressed.Other chemokine receptors were lowly expressed.Both 17β-estradiol and TCDD increased the transcription and translation of CCR1.The combination of 17β-estradiol with TCDD further promoted the transcription and translation of CCR1.The 17β-estradiol or/and TCDD had no significant effect on transcription of CCR2,CCR3,CCR4,CCR5,CCR7,CCR8,CCR9,CXCR1, CXCR2,CXCR3,CXCR5,CXCR6 and CX3CR1 mRNA.The 17β-estradiol had no significant effect on transcription of CCR6,while TCDD significantly decreased expression of CCR6 mRNA.The 17β-estradiol or/and TCDD significantly decreased transcription of CCR10 mRNA.The 17β-estradiol of 10-7M decreased expression of CXCR4 mRNA,but TCDD or the combination of 17β-estradiol with TCDD had no effect on expression of CXCR4 mRNA.The results above suggest that the effect of 17β-estradiol on chemokine receptors expression in U937 cells is one of mechanisms involved in pathogenesis of endometriosis.TCDD not only influences development of the disease as a pro-inflammatory factor but also produces a marked effect through the signal transduction of estrogen.The interaction of 17β-estradiol with TCDD were very complicated.Women with endometriosis were influenced by the internal and external environment,and their immune system were changed,which may be one important mechanism of onset of this disease.Chemokine receptor CCR1 of U937 cells may play an important role in pathogenesis of endometriosis.2.The modulating effect of 17β-estradioi and TCDD on expression of CCR1, CCR5/RANTES,MIP-1αby the endometriosis-associated cellsWe established different contact and non-contact co-culture model of the corresponding ectopic cells such as ESC,HPMC and U937 cells,and explored the effect of 17β-estradiol or/and TCDD on expression of CCR1,CCR5/RANTES, MIP-1αby using ELISA and Western blot.The co-culture of ESC and HPMC promoted secretion of RANTES.The RANTES secretion in co-culture of HPMC-U937 were significantly higher than the co-culture of ESC-U937 or ESC-HPMC,and the co-culture of three cells further promoted secretion of RANTES. The contact co-culture of U-E-H secreted highest RANTES.Among the three non-contact co-culture of three cells,secretion of RANTES in co-culture of E/H-U was significantly higher than the other two non-contact co-culture.Moreover,we found that the non-contact co-culture the three cells of U/H-E,H/E-U,E/H-E secreted more RANTES than the co-culture of two cells,H-E,E-U,H-E,respectively.The combination of 17β-estradiol with TCDD significantly increased secretion of RANTES of co-culture of E/H-U and H/E-U.The MIP-1αsecretion in the co-culture of HPMC-U937 was significantly higher than the co-culture of ESC-U937 or ESC-HPMC.The co-culture of three cells further promoted secretion of MIP-1α.The contact co-culture of U-E-H secreted highest MIP-1α.Among the three non-contact co-culture of three cells,secretion of MIP-1αof co-culture of E/H-U was significantly higher than the other two non-contact co-culture.It was found that the non-contact co-culture the three cells of U/H-E,H/E-U,E/H-E secreted more MIP-1αthan co-culture of two cells of H-E,E-U,H-E respectively.The combination of 17β-estradiol with TCDD increased the secretion of RANTES in the co-culture of E/H-U.Among all the co-culture units,the combination of 17β-estradiol with TCDD increased the expression of CCR1 in U937 cells.While the combination of 17β-estradiol with TCDD only increased the expression of CCR5 in U937 cells of E/H-U co-culture unit.The results above suggest that the combination of 17β-estradiol with TCDD promotes recruitment of mono-macrophages in peritoneal cavity by up-regulating expression of CCR1,CCR5/RANTES,MIP-1β,leading to the development of endometriosis. 3.Combination of 17β-estradiol with TCDD promoted recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteractionThe co-culture of ESC-HPMC promoted chemotaxis of U937 cells,which could be partly inhibited by anti-RANTES or anti-MIP-1αneutralizing antibody.The combination of 17β-estradiol with TCDD promoted the chemotaxis of U937 cells to ESC,which could be partly inhibited by anti-RANTES,anti-MIP-1α,anti-CCR1 or anti-CCR5 neutralizing antibody,which suggests the combination of 17β-estradiol with TCDD promotes chemotaxis of U937 cells via RANTES,MIP-1α/CCR1,CCR5 interaction.Invasion assay showed that the co-culture unit ESC/HPMC-U937 or the combination of 17β-estradiol with TCDD promoted ESC invasiveness and increased activity and protein expression of MMP 2,9 which could be partly inhibited by anti-RANTES,anti-MIP-1α,anti-CCR1 or anti-CCR5 neutralizing antibody. Meanwhile,both hrRANTES and hrMIP-1αcould promote invasiveness of ESC.The results above suggest that the combination of 17β-estradiol with TCDD promotes recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteraction.Collectively,it has been demonstrated in the present study that all the 18 chemokine receptors were expressed in U937 cells.The combination of 17β-estradiol with TCDD promotes expression of CCR1.The different co-culture units increase secretion of RANTES and MIP-1α,and the combination of 17β-estradiol with TCDD significantly promotes secretion of RANTES and MIP-1αof the co-culture unit of ESC/HPMC-U937.The combination of 17β-estradiol with TCDD promotes recruitment of monocyte and invasion of ESC via CCR1,CCR5/RANTES,MIP-1αinteraction.Our research suggests a possible mechanistic link between chemokine/chemokine receptor and endometriosis pathogenesis. |
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