Chemokines Mediate Induction Of Endometriosis By The Combined Estrogen And TCDD

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.Current evidence suggests local peritoneal immuno-inflammation might be associated with the origin of endometriosis. The increased and activated pelvic macrophages and lymphocytes and the elevated levels of specific cytokines and growth factors support this hypothesis, but the potential mechanisms for the phenomena is unknown. Chemokine produced by implanting cells is required in the process of recruiting circulating leukocytes into pelvic sites. Chemokine activity is mediated through cell surface receptors that are related to the 7-transmembrane-domain receptor family. The function of chemokine depends on binding to the corresponding receptors. To explore the role of chemokine in the pathogenesis of endometriosis, it will be facilitated to investigate chemokine as well as its receptor because of the complexity of the interaction between the chemokine and its receptor. To pick up chemokine receptors that highly transcribed in ectopic tissues and to explore function of the receptors as well as their ligands may provide some useful insights into the molecular mechanisms of the pathogenesis of endometriosis.The transcription and translation of chemokine and its receptor in endometriotic tissue may be regulated by endocrino-immune network. Endometriosis might be anestrogen-depending disease, and estrogen may be one of the regulatory factors in expression of chemokine and its receptor. Furthermore, the increased prevalence and severity of endometriosis is considered to be associated with exposure to environmental pollutant. Dioxin, an unwanted byproduct in industrial and combustion processes, is reported to be associated with estrogen. Investigating the regulatory mechanism of dioxin and estrogen in chemokine and its receptor expressions will throw a light in elucidating the role of the chemokine in the etiology of endometriosis. Chemokines and their receptors have been implicated as pivotal players in pathology of endometriosis. In addition to recruiting leukocytes, it has become clear that chemokine also play a role in cell proliferation and angiogenesis. Exploring the selected chemokine in the formation of endometriotic tissue will provide valuable evidence in elucidating the pathogenesis of endometriosis.1. The characteristics of chemokine receptor expression in ectopic tissue and eutopic endometrium of patients with endometriosisThe transcription of 18 chemokine receptors in eutopic and ectopic endometrium with endometriosis were first analyzed by semi-quantified RT-PCR. Among 18 chemokine receptors, CCR5, CCR8 and CXCR1 expressed significantly higher in the ectopic compared to that in the eutopic endometrium (p<0.05). Immunohistochemistry was used to identify the translation of CCR5, CCR8 and CXCR1 as well as the corresponding ligands RANTES, MIP-1α, 1-309 and IL-8. The transcription pattern of the chemokine receptors in primary endometrial stromal cells was in accordance with that of the eutopic endometrial tissue. The expression of CCR5, CCR8 and CXCR1 on the surface of ESC was further confirmed by FCM. These data showed that CCR5, CCR8 and CXCR1 were higher expressed in endometriotic tissue than that of the eutopic endometrium, suggesting the hyper-expression of the chemokine receptors may play a role in formation of the ectopic foci.2. Modulating effect of 17β-E2 and TCDD on expression of CCR5/RANTES, MlP-1α by the corresponding ectopic cellsESC, HPMC and U937 cells were treated by a series of concentration of 17β-E₂, TCDD, or 10^-9M17β-E2 plus 1nM TCDD. The expression of CCR5 on the surface of ESC was analyzed by FCM. The levels of RANTES, MIP-1α secreted by HPMC or U937 cells were determined by ELISA. We then established the co-culture system of ESC, HPMC and U937 cells to analyze the influence of the coculture system treated by 17β-E2 in combination with TCDD on RANTES and MIP-1α secretion.E₂ and TCDD alone inhibited respectively expression of CCR5, whereas the combination of E₂ and TCDD up-regulated the expression. E2 had no effect on RANTES secretion by HPMC and U937, but appeared to promoting RANTES secretion by HPMC, but inhibiting that of U937 cells stimulated by TCDD. The coculture of HPMC and U937 significantly stimulated RANTES secretion, and E2 plus TCDD augmented the stimulatory effect, suggesting the reason of high levels of RANTES in peritoneal cavity with endometriosis.TCDD promoted MIP-1α secretion by HPMC and U937, respectively. The MIP-1α level increased further when E2 was added. The data show E2 and TCDD may recruit circulating monocytes into pelvic cavity by inducing MIP-1α secretion of the corresponding cells in the pathogenesis of endometriosis.3. Modulating effect of 17β-E2 and TCDD on expression of CXCR1/IL-8 by the corresponding ectopic cellsIt was showed that CXCR1 expression on the surface of ESC shared the same trend with CCR5. TCDD promoted IL-8 secretion by HPMC, and 17β-E2 magnified the stimulatory effect. Both 17β-E2 and TCDD inhibited respectively IL-8 secretion of U937, but the combination had no further effect.The co-culture of ESC with HPMC promoted IL-8 secretion, and E2 played a synergic stimulatory roles with TCDD. We thus propose that retrograded endometrium can give rise to acute inflammation, and the inflammation appears more serious and persistent in an environment of E2 and TCDD. The interaction of HPMC and the surrounded leukocytes could significantly stimulate IL-8 secretion, suggesting a main resource of IL-8 in peritoneal cavity with endometriosis. TCDD promoted the secretion of IL-8 by HPMC-U937, but exerted a contrary effect when combinationwith E2. This maybe could explain type of the leukocyte recruited into theinflammatory site.4. Role of CCR8/I-309 in the initiation of endometriosisThe effect of I-309 on integrin β1 and αvβ3 expression intensity on the surface of ESC was analyzed by FCM. The chemotactic effects of 1-309 on ESC and U937 cells was explored by a Chemotactic assay. It was shown that the majority of ESC expressed integrin β1, and its expression intensity could be improved by chemokine I-309.I-309 had no effect on integrin αvβ3 expression on ESC surface. Monocyte but not ESC was recruited by I-309. I-309 induced the adhesion by up-regulating the expression of integrinβ1 on the ESC surface, recruited more monocytes to the implanting sites, leading to change of the immune milieu, which accounts for formation of the endometriotic foci.17β-E2 and TCDD treating alone but not in combination up-regulated the CCR5 expression on the ESC surface. E2 magnified the stimulatory effect of I-309 secretion by U937. The interaction of HPMC and U937 cells promoted I-309 secretion, but E2 and TCDD had no effect on it. Therefore, the infiltration of macrophage in pelvic cavity may be due to I-309 action.Collectively, this study first selected the higher expression of CCR5, CCR8 and CXCR1 in ectopic tissue for further research, and found that they as well as their ligands could be regulated by 17β-E2 and TCDD. We also assessed effect of the co-culture on the corresponding chemokine secretion in vitro, and demonstrated the interaction of different cells could stimulate the secretion of the chemokines. Finally, we have explored the role of CCR8/I-309 in the initiation of endometriosis, suggesting a possible mechanistic link between chemokine/chemokine receptor and endometriosis pathogenesis.