Abnormal Regulation Of Chemokine TECK And Its Receptor CCR9 In The Endometriotic Milieu Leads To Endometriosis

The pathogenesis of endometriosis remains to be elucidated.According to Sampson's theory of ectopic transplantation of endometrium,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 a larger part of fertile women,other factors must be involved in the disease.Alteration/dysfunction of the serous immunity in the peritoneal cavity may lead to the occurrence and development of endometriosisCurrent evidence suggests the peritoneal immune and inflammatory milieu might be associated with the pathogenesis of endometriosis.During the occurrence of endometriosis,the immune cells are recruited into the peritoneal cavity,and among them regulatory T cells may be critical in immune tolerance.The Tregs appear traffic to these tissues by depending on chemokine-chemokine receptor interactions.CCR9 was transcribed and translated significantly higher in the ectopic tissue than in the eutopic endometrium,and the latter is higher than in the normal fertile endometrium.CCR9/TECK interaction on the CD4~+T leads to activation of several intracellular signaling pathway, such as PI-3/Akt,MAPK/ERK,GSK-3β,and FKHR,and resistance to death stimuli from cycloheximide and Fas,which plays important roles in promoting the proliferation of T cells and escaping the cell apoptosis mediated by Fas.Chemokine is required in the process of recruiting circulating leukocytes into the pelvic and endometriotic sites.The functions of chemokines depend on binding to the corresponding receptors.To explore the role of chemokine TECK and its receptor CCR9 in the pathogenesis of endometriosis,we proposed the present study so as to understand pathogenesis of endometriosis.Endometriosis is an estrogen-associate disease,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),an environmental contaminant,is associated with an increased prevalence and severity of endometriosis.Regulation of dioxin and estrogen in chemokines TECK and their receptors CCR9 expression and in the recruiting of the CD4~+CD25~+T cells into the endometriotic milieu will throw a light in elucidating the involvement of the chemokines in the etiology and pathogenesis of endometriosis.1.The expression of chemokine TECK and its receptor CCR9 in the endometriotic milieuCCR9 expression was analyzed by semi-quantified RT-PCR,and flow cytometry, respectively.CCR9 expression in the ectopic sites is significantly higher than that of the eutopic endometrium.TECK concentration in the peritoneal fluid and different contact and non-contact co-culture model of the endometriotic-associated cells were analyzed by ELISA.It has been found that TECK concentration in the peritoneal fluid from endometriosis is significantly higher than that of the normal fertile women. TECK secretion by ESC is higher than that of HPMC,a serous membrane cell line, and U937,a monocyte line.The co-culture of ESC-U937 secrets more TECK than that of ESC-HPMC and HPMC-U937.The co-culture of three cells further promotes secretion of TECK.Among the three kinds of contact co-culture of three cells,the co-culture of HPMC/ESC-U937 secrets more TECK than that of the ESC/HPMC-U937 and U937/ESC-HPMC.The three kinds of non-contact co-culture of the three cells secret less TECK than that of the contact co-culture.It is suggested that interaction of the endometriotic-associated cells significantly promotes the chemokine TECK secretion,which may play a role in the pathogenesis of endometriosis.2.The regulatory effect of 17β-estradiol and TCDD on chemokine TECK and its receptor CCR9 expression in the endometriotic-associated cellsThe different direct and indirect co-culture models of the endometriotic-associated cells such as ESC,HPMC and U937 cells were used to explore the effect of 17β-estradiol or/and TCDD on expression of chemokine TECK and its receptor CCR9 by using ELISA or FCM,respectively.We have found low dose of TCDD inhibits CCR9 expression by ESC in vitro,but high dose of TCDD promotes CCR9 expression in vitro in ESC.The combination of 17β-estradiol with TCDD increases CCR9 expression in ESC,and the increase is related positively to the concentration of 17β-estradiol.The 17β-estradiol or TCDD has no effect on TECK secretion by HPMC. U937 cells secret higher TECK than HPMC.Among the treatment with different concentration of 17β-estradiol,0.01nM promotes the TECK secretion by U937,but 0.1-100 nM significantly inhibit the TECK secretion by U937,and TCDD inhibits TECK secretion by U937.Combination of 17-estradiol with TCDD can significantly decrease TECK secretion by U937.17-estradiol has no effect on TECK secretion by the co-culture of ESC-HPMC,but TCDD promotes TECK secretion by the co-culture, and combination of 17-estradiol with TCDD further increases TECK secretion in the co-culture.Neither 17β-estradiol nor TCDD has any effect on TECK secretion by the co-culture of HPMC-U937,and combination of 17-estradiol with TCDD counteracts to the promotion of TECK in the co-culture.TCDD promotes TECK secretion in the contact co-culture of ESC-HPMC-U937,and combination of 17-estradiol with TCDD further increases TECK secretion in the contact co-culture.It is suggested that combination 17-βestradiol with TCDD promotes TECK secretion and CCR9 expression in the endometriotic-associated cells owing to the interaction of these cells, which is involved in the pathogenesis of endometriosis.3.The endometrial stromal cells recruit CD4~+CD25~+FoxP3~+ regulatory T cells into the endometriotic milieu via secreting TECK and regulate their functionsThe co-culture of ESC-HPMC promotes chemotaxis of CD4~+CD25~+FoxP3~+ regulatory T cells.The combination of 17-estradiol with TCDD promotes the chemotaxis of CD4~+CD25~+FoxP3~+ regulatory T cells into ectopic milieu containing ESCs.The CD4~+FoxP3~+ regulatory T cells in the peritoneal fluid of the advanced endometriosis(γ-AFSⅢ,Ⅳ)is higher than that of the early endometriosis (γ-AFSⅠ,Ⅱ)and the control without endometriosis.It is suggested ESCs mediate recruitment of the CD4~+CD25~+ regulatory T cells through secreting chemokine TECK after implanted on the HPMC,and combination of 17-estradiol with TCDD promotes chemotaxis of CD4~+CD25~+ regulatory T cells through up-regulating chemokine TECK secretion.After co-cultured with ESC and HPMC respectively,CD4~+CD25~+ T cells are significantly expanded,and 17-estradiol or/and TCDD amplify the effect,but anti-TECK neutralizing antibody only partly inhibits the effect.After co-cultured with ESC or HPMC,CTLA-4 and FoxP3 expression in the CD4~+CD25~+ T cells increases significantly,and 17-estradiol or/and TCDD amplify the effect,but anti-TECK neutralizing antibody only partly inhibits the effect.Meanwhile in the presence of ESC,the CD25~-T cell subset decreases,and the CD25~+T cell subset increases. Concentration of IL-10 and TGF-β1 in the advanced endometriosis are also significantly higher than that of the early endometriosis and the control.ESCs only partly inhibit the apoptosis of CD4~+CD25~+ regulatory T cells through secreting chemokine TECK.It is suggested that ESCs aider implanted on HPMC promote the differentiation,expansion and function of CD4~+CD25~+ regulatory T cells,which maintain the local immunotolerance in the ectopic milieu.4.The CD4~+CD25~+FoxP3~+ regulatory T cells enhance invasiveness of ESCs in the endometriotic milieuInvasion assay shows that the co-culture of ESC/HPMC-U937-CD4~+CD25~+T cells or ESC/HPMC-U937-CD4~+CD25~-T cells increases invasiveness,activity and protein of MMP2,9 of ESCs that is promoted further by TECK,and inhibited by anti-TECK neutralizing antibody.Moreover,combination of 17-estradiol with TCDD significantly increases the expression of MMP2 and MMP9 in ESC in the co-culture. Therefore,the CD4~+CD25~+ regulatory T cells promote invasiveness of ESC in the ectopic milieu through interaction with the endometriotic-associated cells.In conclusion,it has been demonstrated in the present study that the co-culture of the endometriotic-associated cells promotes TECK secretion,and combination of 17-estradiol with TCDD further promotes TECK secretion and CCR9 expression in the endometriotic-associated cells,which in turn recruits CD4~+CD25~+ regulatory T cells into the ectopic milieu,induces differentiation,and improves suppressive function of the regulatory T cells.The CD4~+CD25~+ regulatory T cells then promote invasiveness of ESC in the ectopic milieu,which is involved in the pathogenesis of endometriosis.