- Email: [email protected]
Endometrial and placental CRH as regulators of human embryo implantation
Journal of Reproductive Immunology 62 (2004) 53–59
Review
Endometrial and placental CRH as regulators of human embryo implantation A. Makrigiannakis a,∗ , E. Zoumakis b , S. Kalantaridou b , G. Chrousos b a
b
Department of Obstetrics & Gynocology, Medical School, University of Crete, Heraklion 71110, Greece Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Received in revised form 28 November 2003; accepted 28 November 2003
Abstract Epithelial cells of the human endometrium and differentiated endometrial stromal cells express the corticotropin-releasing hormone (CRH) gene. CRH is also produced by human placental cytotrophoblast. Endometrial and placental CRH are under the endocrine control of gonadal steroids as well as under autocrine/paracrine regulation by prostanoids and interleukins. Human endometrium, myometrium and placenta express the relevant receptors. Human trophoblast and decidualized endometrial cells also express Fas ligand (FasL), a pro-apoptotic molecule. These findings suggest that intra-uterine CRH may participate in local inflammatory phenomena associated with blastocyst implantation, while FasL may assist with maternal immune tolerance to the semi-allograft embryo. A nonpeptidic CRH receptor type 1 (CRH-R1)-specific antagonist decreased the expression of FasL by human trophoblasts, suggesting that CRH regulates the pro-apoptotic potential of these cells in an auto-paracrine fashion. Invasive trophoblasts promoted apoptosis of activated Fas-expressing human T lymphocytes, an effect potentiated by CRH and inhibited by the CRH antagonist. Female rats treated with the CRH antagonist in the first 6 days of gestation had a dose-dependent decrease of endometrial implantation sites and live embryos as well as markedly diminished endometrial FasL expression. However, embryos of mothers lacking T cells (nude rats) and embryos of syngeneic matings were not rejected when mothers were treated with antalarmin, suggesting that the effect of antalarmin on embryonic implantation is not due to a nonspecific toxicity of this compound but a specific effect on T cells. Our data suggest important physiological roles of
∗
Corresponding author. Tel.: +30-2810-392333; fax: +30-2810-392759. E-mail address: [email protected] (A. Makrigiannakis).
0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2003.11.006
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endometrial and placental CRH in the regulation of blastocyst implantation and early maternal tolerance. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: CRH; Implantation; FasL; Decidualization; Antalarmin
1. Introduction The hypothalamic neuropeptide corticotropin-releasing hormone (CRH) is produced in several reproductive organs, including endometrial glands and decidualized stroma as well as trophoblast, syncytiotrophoblast and placental decidua (Usui et al., 1998; Suda et al., 1984; Makrigiannakis et al., 1995b). Similarly, the CRH receptor type 1 (CRH-R1) gene is expressed in human endometrial stromal cells (DiBlasio et al., 1997). CRH is secreted at inflammatory sites and possesses potent proinflammatory properties influencing both innate and acquired immune processes. One of the early effects of “immune” CRH is degranulation of mast cells and release of histamine and several inflammatory cytokines, including TNF-␣ and interleukin-6 (IL-6) (Theoharides et al., 1998). During blastocyst implantation, the maternal endometrial response to the invading semi-allograft has characteristics of an aseptic inflammatory response. The mother’s immune system prevents a graft versus host reaction in pregnancy deriving from the immune system of the fetus. Fas receptor (Fas) and its ligand (FasL) play an important role in the regulation of immune tolerance. The major function of the Fas–FasL interaction is induction of apoptosis in activated cells carrying Fas (Runic et al., 1996). FasL is expressed on the surface of fetal cytotrophoblast as well as maternal decidual cells of the placenta, i.e. on cells located in the interface between fetal placenta and maternal endometrium. Abnormalities of maternal immune tolerance to the fetal semi-allograft, and vice versa, have been implicated in several common disease processes of pregnancy, including recurrent early miscarriage, pre-eclampsia and eclampsia.
2. Endometrial CRH The CRH transcript and its peptide product are present in normal and neoplastic human glandular endometrial cells (Makrigiannakis et al., 1995b). The epithelial cells of the rat uterus also express the CRH gene (Makrigiannakis et al., 1995a). The CRH transcript and peptide product are detectable in human decidua and in stromal cells that have been decidualized in vitro by a mixture of progesterone, relaxin and estrogens. The principal receptor for the CRH ligand, the CRH-R1, is also present in both epithelial and stromal cells of human endometrium (DiBlasio et al., 1997) as well as in human myometrium (Hillhouse et al., 1993). Inducers of hypothalamic CRH, 8-bromo-cAMP, forskolin and epidermal growth factor (EGF), stimulate the activity of the CRH promoter (Makrigiannakis et al., 1996). Estrogens suppress the activity of CRH promoter in endometrium, while glucocorticoids decrease the activity of the CRH promoter via activation of cAMP- and EGF-dependent
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pathways. The inhibitory effect of glucocorticoids in endometrium is in agreement with that described for hypothalamus and opposite to what has been found in human placenta, suggesting that the regulation of transcription of CRH gene is cell-specific depending on specific transcription factors, nuclear receptor coregulators, etc. Finally, the cytokines IL-1 and IL-6 stimulate the activity of the CRH promoter (Makrigiannakis et al., 1999b). This effect appears to be mediated by prostaglandins, in accordance to what has been described in hypothalamus and placenta.
3. CRH is involved in endometrial decidualization CRH is present at sites of inflammation in both humans and rodents, and its immunoneutralization appears to drastically attenuate the inflammatory response (Karalis et al., 1991). In human endometrium, a phenomenon with characteristics of an aseptic inflammatory reaction takes place during the differentiation of endometrial stroma to decidua. CRH induces the decidualization of endometrial stroma (Ferrari et al., 1995; Zoumakis et al., 2000) and potentiates the decidualizing effect of progesterone. Furthermore, progestins stimulate the expression of endometrial CRH in a cAMP-dependent manner (Makrigiannakis et al., 1999a). Indeed in stromal cells, CRH may mediate, via the CRH-R1 receptor, the cAMP-dependent part of the decidualizing effect of progesterone, an effect blocked by cAMP inhibitors. In addition to progesterone, several locally produced pro-inflammatory immune factors also exert decidualizing effects. In humans, prostaglandin (PG) E2 enhances, while interleukin-1 inhibits, the decidualizing effect of progesterone. Furthermore, PGE2 and interleukins IL-1 and IL-6 are also major inducers of endometrial CRH. During the decidualizing process CRH interacts with local factors, i.e. CRH inhibits the production of PGE2 and stimulates the production of both IL-1 and IL-6 by human endometrial stromal cells (Zoumakis et al., 2000). IL-1 is a principal modulator of the decidualization process, blocking the differentiation of human endometrial stromal cells induced by ovarian steroids or cAMP (Frank et al., 1995). The stimulatory effect of CRH on stromal IL-1 suggests that the former may exert its decidualizing effect either as a principal regulator or as a modulator of progesterone, the classic decidualizing effector. It is interesting that progesterone per se induces the expression of the CRH gene in the stromal cells of human endometrium (Makrigiannakis et al., 1999a). Thus, it is possible that progesterone-driven CRH may exert an inhibitory effect on endometrial decidualization through induction of a local inhibitor, possibly interleukin-1, establishing a complex feedback system, fine tuning the response of stroma cells to these factors. It appears that a close interaction takes place within human endometrium involving CRH, prostanoids and cytokines.
4. A role for CRH in supporting blastocyst implantation The implanting blastocyst secretes several inflammatory mediators, including IL-1 and prostaglandin PGE2. Blastocyst-derived IL-1 plays an essential role on implantation. In mice, blockade of its effect by the specific antagonist IL-1ra inhibits implantation (Sheth et al., 1991). As mentioned, IL-1 and PGE2 are inducers of CRH expression in human en-
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Fig. 1. Antalarmin decreases fertility in Sprague-Dawley rats. The pregnant uterus of antalarmin-treated rats has decreased size with significantly reduced number of fetuses.
dometrial cells. The blastocyst may modulate the expression of endometrial CRH through IL-1 and/or PGE2 produced by it at the very site of nidation. Subsequently, endometrial CRH, in association with other local factors, may participate in a local inflammatory response at the site of implantation, rendering the endometrial surface “adhesive” for the attachment of the blastocyst, culminating in the formation of the nidus. This hypothesis is supported by several lines of data showing a significantly higher concentration of the CRH transcript and its peptide product at the early implantation sites of pregnant rats compared to the inter-implantation uterine areas (Makrigiannakis et al., 1995a). In vivo experiments in the mouse have shown that intra-peritoneal injections of CRH antibodies at day 2 of pregnancy decrease the number of fetuses within the uterus by 60% (Athanassakis et al., 1999). This observation is further supported by experiments in rats using antalarmin, a specific CRH-R1 antagonist. Administration of antalarmin to early pregnant rats results up to a 70% reduction in the number of implantation sites (Makrigiannakis et al., 2001) (Fig. 1). Thus, blocking of CRH has an anti-nidation effect when it takes place at a very early stage of pregnancy. It is evident that both methods of blocking the effects of uterine CRH (antibodies or antalarmin) do not completely abolish nidation, suggesting the presence of other mechanisms supporting the implanted embryo. This is also compatible with the fact that CRH and CRH-R1-knock-out mice are hypofertile rather than entirely sterile (Muglia et al., 1995; Turnbull et al., 1999). Recent experimental findings show that CRH participates in the nidation of the fertilized egg by inhibiting the local maternal immune response to the implanted embryo. Indeed, CRH stimulates the expression of the pro-apoptotic FasL protein in decidual and trophoblastic cells, thus potentiating their ability to induce apoptosis of surrounding maternal T lymphocytes activated by the presence of the embryo. Expression of Fas ligand (FasL) by fetal extravillous trophoblast cells can induce apoptosis of activated T lymphocytes expressing increasing numbers of the Fas membrane protein. Another role that is attributed to maternal and fetal FasL is that it limits the migration of fetal cytotrophoblast cells into maternal tissue, and vice versa. The intra-uterine presence of CRH, both in maternal (decidua) and fetal (trophoblast) sites, suggests that locally produced CRH regulates FasL production, thus affecting the invasion process through a local auto/paracrine regulatory loop of cytotrophoblast cells regulating their own apoptosis. Therefore, inadequate CRH-mediated self-induction of FasL in extravillous trophoblasts might be involved in the pathophysiology of infertility and recurrent fetal resorption or miscarriage (Fig. 2).
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CRH
57
CRH
CRH-R1
CRH CRH-R1
CRH
FasL Fas
CRH-R1
FasL
Fas Fas
CRH CRH-R1
Fas
Fig. 2. Schematic presentation of the involvement of CRH/Fas system in the early phase of human implantation. CRH is produced by both EVT and decidual cells and acts in an autocrine-paracrine manner to stimulate FasL expression and to cause apoptosis of activated maternal T lymphocytes. CRH: corticotropin-releasing hormone; CRH-R1: CRH receptor type 1; DC: decidual cell; EVT: extravillous trophoblast; Fas: Fas receptor; FasL: Fas ligand; T: T lymphocyte; NK: natural killer cell; m: macrophage; BV: blood vessels.
Abrogation of immune privilege at the placental–uterine interface or unbridled invasion of trophoblast may have deleterious consequences for the developing fetus, as evidenced by the high rates of fetal morbidity and mortality and, for the mother, observed in pregnancies complicated by inflammation at maternal–fetal interfaces or in preeclampsia/eclampsia. CRH stimulates the expression of FasL in extravillous trophoblast cells, thus potentiating their ability to induce apoptosis of the surrounding activated T lymphocytes. This effect of
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CRH appears to be specifically mediated through its type 1 receptor. These findings are in agreement with previously published reports suggesting that expression of Fas ligand by fetal extravillous trophoblast cells can induce apoptosis of activated T lymphocytes expressing increasing amounts of the Fas membrane protein (Itoh et al., 1991; Yamashita et al., 1999). It should be noted here that mice with missense or inactivating mutations of FasL gene (gld) are hypofertile but can reproduce, suggesting that trophoblast FasL expression is not obligatory for maternal immunotolerance. Thus, in the absence of a functional Fas–FasL system, other mechanisms supporting maternal immunotolerance are sufficient to prevent total pregnancy failure but at the cost of reproductive efficiency. The observed effect of CRH in implantation and early pregnancy appears to be mediated by primarily the Fas–FasL system for the following reasons: (i) CRH had no effect on expression of TRAIL protein in trophoblastic JEG3 cells; (ii) neutralizing antibodies raised against FasL inhibited the CRH-induced apoptosis of activated T lymphocytes; (iii) exposure of cells not expressing Fas (WS1 cells) to CRH, and co-cultured with activated T lymphocytes, showed very little evidence of T cell apoptosis, and (iv) nonactivated T lymphocytes, which express neither the Fas protein nor the TRAIL system, when co-cultured with extravillous trophoblasts showed very little evidence of apoptosis. It has been suggested that FasL of maternal and fetal origin limits the migration of fetal cytotrophoblast cells into maternal tissue, and vice versa. Our data strengthen this hypothesis, suggesting that CRH of fetal and maternal origin regulates FasL production and thus affecting the invasion process through a local auto-paracrine regulatory loop of cytotrophoblast cells, regulating their own proapoptotic properties.
References Athanassakis, I., Farmakiotis, V., Aifantis, I., Gravanis, A., Vassiliadis, S., 1999. Expression of corticotrophinreleasing hormone in the mouse uterus: participation in embryo implantation. J. Endocrinol. 163, 221– 227. DiBlasio, A., Peroci-Giraldi, F., Vigano, P., Petraglia, F., Vignall, M., Cavagnini, F., 1997. Expression of corticotropin-releasing hormone and its R1 receptor in human endometrial stromal cells. J. Clin. Endocrinol. Metab. 82, 1594–1597. Ferrari, A., Petraglia, F., Gurpide, E., 1995. Corticotropin releasing factor decidualizes human endometrial stromal cells in vitro. Interaction with progestin. J. Steroid. Biochem. Mol. Biol. 54, 251–259. Frank, G.R., Brar, A.K., Jikihara, H., Cedars, M.I., Handwerger, S., 1995. Interleukin-1 beta and the endometrium: an inhibitor of stromal cell differentiation and possible autoregulator of decidualization in humans. Biol. Reprod. 52, 184–191. Hillhouse, E.W., Grammatopoulos, D., Milton, N.G., Quartero, H.W., 1993. The identification of a human myometrial corticotropin-releasing hormone receptor that increases in affinity during pregnancy. J. Clin. Endocrinol. Metab. 76, 736–741. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y., Nagata, S., 1991. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 66, 233–243. Karalis, K., Sano, H., Redwine, J., Listwark, S., Wilder, R., Chrousos, G., 1991. Autocrine or paracrine inflammatory actions of corticotropin-releasing hormone in vivo. Science 254, 421–424. Makrigiannakis, A., Margioris, A., Chatzaki, E., Zoumakis, E., Chrousos, G., Gravanis, A., 1999a. The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells. Mol. Hum. Reprod. 5, 789–796.
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Makrigiannakis, A., Margioris, A., Zoumakis, E., Stournaras, C., Gravanis, A., 1999b. The transcription of corticotropin-releasing hormone in human endometrial cells is regulated by cytokines. Neuroendocrinology 70, 451–459. Makrigiannakis, A., Margioris, A.N., Le Goascogne, C., Zoumakis, E., Nikas, G., Stournaras, C., Psychoyos, A., Gravanis, A., 1995a. Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus. Life Sci. 57, 1869–1875. Makrigiannakis, A., Zoumakis, E., Margioris, A.N., Theodoropoulos, P., Stournaras, C., Gravanis, A., 1995b. The corticotropin-releasing hormone (CRH) in normal and tumoral epithelial cells of human endometrium. J. Clin. Endocrinol. Metab. 80, 185–193. Makrigiannakis, A., Zoumakis, E., Kalantaridou, S., Coutifaris, C., Margioris, A., Coukos, G., Rice, K.C., Gravanis, A., Chrousos, G.P., 2001. Corticotropin-releasing hormone promotes blastocyst implantation and early maternal tolerance. Nat. Immunol. 2, 1018–1024. Makrigiannakis, A., Zoumakis, E., Margioris, A.N., Stournaras, C., Chrousos, G.P., Gravanis, A., 1996. Regulation of the promoter of the human corticotropin-releasing hormone gene in transfected human endometrial cells. Neuroendocrinology 64, 85–93. Muglia, L., Jacobson, L., Dikkes, P., Majzoub, J.A., 1995. Corticotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 373, 427–432. Runic, R., Lockwood, C.J., Ma, Y., Dipasquale, B., Guller, S., 1996. Expression of Fas ligand by human cytotrophoblasts: implications in placentation and fetal survival. J. Clin. Endocrinol. Metab. 81, 3119–3122. Sheth, K.V., Roca, G.L., Al-Sedairy, S., Parhar, R., Hamilton, G., Al-Abdul Jabbar, F., 1991. Prediction of successful embryo implantation by measuring interleukin-1-alpha and immunosuppressive factor(s) in preimplantation embryo culture fluid. Fertil. Steril. 55, 952–958. Suda, T., Tomori, N., Tozawa, F., Mouri, T., Demoura, H., Shizume, K., 1984. Distribution and characterization of immunoreactive corticotropin-releasing factor in human tissues. J. Clin. Endocrinol. Metab. 59, 861–867. Theoharides, T.C., Singh, L.K., Boucher, W., Pang, X., Letourneau, R., Webster, E., Chrousos, G., 1998. Corticotropin-releasing hormone induces skin mast cell degranulation and increased vascular permeability, a possible explanation for its proinflammatory effects. Endocrinology 139, 403–413. Turnbull, A.V., Smith, G.W., Lee, S., Vale, W.W., Lee, K.F., Rivier, C., 1999. CRF type I receptor-deficient mice exhibit a pronounced pituitary-adrenal response to local inflammation. Endocrinology 140, 1013–1017. Usui, T., Nakai, Y., Tsukada, T., Jingami, H., Takahashi, H., Fucada, J., 1998. Expression of adrenocorticotropin-releasing hormone precursor gene in placenta and other nonhypothalamic tissues in man. Mol. Endocrinol. 2, 871–879. Yamashita, H., Otsuki, Y., Matsumoto, K., Ueki, K., Ueki, M., 1999. Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle. Mol. Hum. Reprod. 5, 358–364. Zoumakis, E., Margioris, A., Stournaras, C., Dermitzaki, I., Angelakis, E., Makrigiannakis, A., Koumantakis, E., Gravanis, A., 2000. Corticotrophin-releasing hormone (CRH) interacts with inflammatory prostaglandins and interleukins and affects the decidualization of human endometrial stroma. Mol. Human Reprod. 6, 344–351.
Review
Endometrial and placental CRH as regulators of human embryo implantation A. Makrigiannakis a,∗ , E. Zoumakis b , S. Kalantaridou b , G. Chrousos b a
b
Department of Obstetrics & Gynocology, Medical School, University of Crete, Heraklion 71110, Greece Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA Received in revised form 28 November 2003; accepted 28 November 2003
Abstract Epithelial cells of the human endometrium and differentiated endometrial stromal cells express the corticotropin-releasing hormone (CRH) gene. CRH is also produced by human placental cytotrophoblast. Endometrial and placental CRH are under the endocrine control of gonadal steroids as well as under autocrine/paracrine regulation by prostanoids and interleukins. Human endometrium, myometrium and placenta express the relevant receptors. Human trophoblast and decidualized endometrial cells also express Fas ligand (FasL), a pro-apoptotic molecule. These findings suggest that intra-uterine CRH may participate in local inflammatory phenomena associated with blastocyst implantation, while FasL may assist with maternal immune tolerance to the semi-allograft embryo. A nonpeptidic CRH receptor type 1 (CRH-R1)-specific antagonist decreased the expression of FasL by human trophoblasts, suggesting that CRH regulates the pro-apoptotic potential of these cells in an auto-paracrine fashion. Invasive trophoblasts promoted apoptosis of activated Fas-expressing human T lymphocytes, an effect potentiated by CRH and inhibited by the CRH antagonist. Female rats treated with the CRH antagonist in the first 6 days of gestation had a dose-dependent decrease of endometrial implantation sites and live embryos as well as markedly diminished endometrial FasL expression. However, embryos of mothers lacking T cells (nude rats) and embryos of syngeneic matings were not rejected when mothers were treated with antalarmin, suggesting that the effect of antalarmin on embryonic implantation is not due to a nonspecific toxicity of this compound but a specific effect on T cells. Our data suggest important physiological roles of
∗
Corresponding author. Tel.: +30-2810-392333; fax: +30-2810-392759. E-mail address: [email protected] (A. Makrigiannakis).
0165-0378/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2003.11.006
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endometrial and placental CRH in the regulation of blastocyst implantation and early maternal tolerance. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: CRH; Implantation; FasL; Decidualization; Antalarmin
1. Introduction The hypothalamic neuropeptide corticotropin-releasing hormone (CRH) is produced in several reproductive organs, including endometrial glands and decidualized stroma as well as trophoblast, syncytiotrophoblast and placental decidua (Usui et al., 1998; Suda et al., 1984; Makrigiannakis et al., 1995b). Similarly, the CRH receptor type 1 (CRH-R1) gene is expressed in human endometrial stromal cells (DiBlasio et al., 1997). CRH is secreted at inflammatory sites and possesses potent proinflammatory properties influencing both innate and acquired immune processes. One of the early effects of “immune” CRH is degranulation of mast cells and release of histamine and several inflammatory cytokines, including TNF-␣ and interleukin-6 (IL-6) (Theoharides et al., 1998). During blastocyst implantation, the maternal endometrial response to the invading semi-allograft has characteristics of an aseptic inflammatory response. The mother’s immune system prevents a graft versus host reaction in pregnancy deriving from the immune system of the fetus. Fas receptor (Fas) and its ligand (FasL) play an important role in the regulation of immune tolerance. The major function of the Fas–FasL interaction is induction of apoptosis in activated cells carrying Fas (Runic et al., 1996). FasL is expressed on the surface of fetal cytotrophoblast as well as maternal decidual cells of the placenta, i.e. on cells located in the interface between fetal placenta and maternal endometrium. Abnormalities of maternal immune tolerance to the fetal semi-allograft, and vice versa, have been implicated in several common disease processes of pregnancy, including recurrent early miscarriage, pre-eclampsia and eclampsia.
2. Endometrial CRH The CRH transcript and its peptide product are present in normal and neoplastic human glandular endometrial cells (Makrigiannakis et al., 1995b). The epithelial cells of the rat uterus also express the CRH gene (Makrigiannakis et al., 1995a). The CRH transcript and peptide product are detectable in human decidua and in stromal cells that have been decidualized in vitro by a mixture of progesterone, relaxin and estrogens. The principal receptor for the CRH ligand, the CRH-R1, is also present in both epithelial and stromal cells of human endometrium (DiBlasio et al., 1997) as well as in human myometrium (Hillhouse et al., 1993). Inducers of hypothalamic CRH, 8-bromo-cAMP, forskolin and epidermal growth factor (EGF), stimulate the activity of the CRH promoter (Makrigiannakis et al., 1996). Estrogens suppress the activity of CRH promoter in endometrium, while glucocorticoids decrease the activity of the CRH promoter via activation of cAMP- and EGF-dependent
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pathways. The inhibitory effect of glucocorticoids in endometrium is in agreement with that described for hypothalamus and opposite to what has been found in human placenta, suggesting that the regulation of transcription of CRH gene is cell-specific depending on specific transcription factors, nuclear receptor coregulators, etc. Finally, the cytokines IL-1 and IL-6 stimulate the activity of the CRH promoter (Makrigiannakis et al., 1999b). This effect appears to be mediated by prostaglandins, in accordance to what has been described in hypothalamus and placenta.
3. CRH is involved in endometrial decidualization CRH is present at sites of inflammation in both humans and rodents, and its immunoneutralization appears to drastically attenuate the inflammatory response (Karalis et al., 1991). In human endometrium, a phenomenon with characteristics of an aseptic inflammatory reaction takes place during the differentiation of endometrial stroma to decidua. CRH induces the decidualization of endometrial stroma (Ferrari et al., 1995; Zoumakis et al., 2000) and potentiates the decidualizing effect of progesterone. Furthermore, progestins stimulate the expression of endometrial CRH in a cAMP-dependent manner (Makrigiannakis et al., 1999a). Indeed in stromal cells, CRH may mediate, via the CRH-R1 receptor, the cAMP-dependent part of the decidualizing effect of progesterone, an effect blocked by cAMP inhibitors. In addition to progesterone, several locally produced pro-inflammatory immune factors also exert decidualizing effects. In humans, prostaglandin (PG) E2 enhances, while interleukin-1 inhibits, the decidualizing effect of progesterone. Furthermore, PGE2 and interleukins IL-1 and IL-6 are also major inducers of endometrial CRH. During the decidualizing process CRH interacts with local factors, i.e. CRH inhibits the production of PGE2 and stimulates the production of both IL-1 and IL-6 by human endometrial stromal cells (Zoumakis et al., 2000). IL-1 is a principal modulator of the decidualization process, blocking the differentiation of human endometrial stromal cells induced by ovarian steroids or cAMP (Frank et al., 1995). The stimulatory effect of CRH on stromal IL-1 suggests that the former may exert its decidualizing effect either as a principal regulator or as a modulator of progesterone, the classic decidualizing effector. It is interesting that progesterone per se induces the expression of the CRH gene in the stromal cells of human endometrium (Makrigiannakis et al., 1999a). Thus, it is possible that progesterone-driven CRH may exert an inhibitory effect on endometrial decidualization through induction of a local inhibitor, possibly interleukin-1, establishing a complex feedback system, fine tuning the response of stroma cells to these factors. It appears that a close interaction takes place within human endometrium involving CRH, prostanoids and cytokines.
4. A role for CRH in supporting blastocyst implantation The implanting blastocyst secretes several inflammatory mediators, including IL-1 and prostaglandin PGE2. Blastocyst-derived IL-1 plays an essential role on implantation. In mice, blockade of its effect by the specific antagonist IL-1ra inhibits implantation (Sheth et al., 1991). As mentioned, IL-1 and PGE2 are inducers of CRH expression in human en-
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Fig. 1. Antalarmin decreases fertility in Sprague-Dawley rats. The pregnant uterus of antalarmin-treated rats has decreased size with significantly reduced number of fetuses.
dometrial cells. The blastocyst may modulate the expression of endometrial CRH through IL-1 and/or PGE2 produced by it at the very site of nidation. Subsequently, endometrial CRH, in association with other local factors, may participate in a local inflammatory response at the site of implantation, rendering the endometrial surface “adhesive” for the attachment of the blastocyst, culminating in the formation of the nidus. This hypothesis is supported by several lines of data showing a significantly higher concentration of the CRH transcript and its peptide product at the early implantation sites of pregnant rats compared to the inter-implantation uterine areas (Makrigiannakis et al., 1995a). In vivo experiments in the mouse have shown that intra-peritoneal injections of CRH antibodies at day 2 of pregnancy decrease the number of fetuses within the uterus by 60% (Athanassakis et al., 1999). This observation is further supported by experiments in rats using antalarmin, a specific CRH-R1 antagonist. Administration of antalarmin to early pregnant rats results up to a 70% reduction in the number of implantation sites (Makrigiannakis et al., 2001) (Fig. 1). Thus, blocking of CRH has an anti-nidation effect when it takes place at a very early stage of pregnancy. It is evident that both methods of blocking the effects of uterine CRH (antibodies or antalarmin) do not completely abolish nidation, suggesting the presence of other mechanisms supporting the implanted embryo. This is also compatible with the fact that CRH and CRH-R1-knock-out mice are hypofertile rather than entirely sterile (Muglia et al., 1995; Turnbull et al., 1999). Recent experimental findings show that CRH participates in the nidation of the fertilized egg by inhibiting the local maternal immune response to the implanted embryo. Indeed, CRH stimulates the expression of the pro-apoptotic FasL protein in decidual and trophoblastic cells, thus potentiating their ability to induce apoptosis of surrounding maternal T lymphocytes activated by the presence of the embryo. Expression of Fas ligand (FasL) by fetal extravillous trophoblast cells can induce apoptosis of activated T lymphocytes expressing increasing numbers of the Fas membrane protein. Another role that is attributed to maternal and fetal FasL is that it limits the migration of fetal cytotrophoblast cells into maternal tissue, and vice versa. The intra-uterine presence of CRH, both in maternal (decidua) and fetal (trophoblast) sites, suggests that locally produced CRH regulates FasL production, thus affecting the invasion process through a local auto/paracrine regulatory loop of cytotrophoblast cells regulating their own apoptosis. Therefore, inadequate CRH-mediated self-induction of FasL in extravillous trophoblasts might be involved in the pathophysiology of infertility and recurrent fetal resorption or miscarriage (Fig. 2).
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CRH
57
CRH
CRH-R1
CRH CRH-R1
CRH
FasL Fas
CRH-R1
FasL
Fas Fas
CRH CRH-R1
Fas
Fig. 2. Schematic presentation of the involvement of CRH/Fas system in the early phase of human implantation. CRH is produced by both EVT and decidual cells and acts in an autocrine-paracrine manner to stimulate FasL expression and to cause apoptosis of activated maternal T lymphocytes. CRH: corticotropin-releasing hormone; CRH-R1: CRH receptor type 1; DC: decidual cell; EVT: extravillous trophoblast; Fas: Fas receptor; FasL: Fas ligand; T: T lymphocyte; NK: natural killer cell; m: macrophage; BV: blood vessels.
Abrogation of immune privilege at the placental–uterine interface or unbridled invasion of trophoblast may have deleterious consequences for the developing fetus, as evidenced by the high rates of fetal morbidity and mortality and, for the mother, observed in pregnancies complicated by inflammation at maternal–fetal interfaces or in preeclampsia/eclampsia. CRH stimulates the expression of FasL in extravillous trophoblast cells, thus potentiating their ability to induce apoptosis of the surrounding activated T lymphocytes. This effect of
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CRH appears to be specifically mediated through its type 1 receptor. These findings are in agreement with previously published reports suggesting that expression of Fas ligand by fetal extravillous trophoblast cells can induce apoptosis of activated T lymphocytes expressing increasing amounts of the Fas membrane protein (Itoh et al., 1991; Yamashita et al., 1999). It should be noted here that mice with missense or inactivating mutations of FasL gene (gld) are hypofertile but can reproduce, suggesting that trophoblast FasL expression is not obligatory for maternal immunotolerance. Thus, in the absence of a functional Fas–FasL system, other mechanisms supporting maternal immunotolerance are sufficient to prevent total pregnancy failure but at the cost of reproductive efficiency. The observed effect of CRH in implantation and early pregnancy appears to be mediated by primarily the Fas–FasL system for the following reasons: (i) CRH had no effect on expression of TRAIL protein in trophoblastic JEG3 cells; (ii) neutralizing antibodies raised against FasL inhibited the CRH-induced apoptosis of activated T lymphocytes; (iii) exposure of cells not expressing Fas (WS1 cells) to CRH, and co-cultured with activated T lymphocytes, showed very little evidence of T cell apoptosis, and (iv) nonactivated T lymphocytes, which express neither the Fas protein nor the TRAIL system, when co-cultured with extravillous trophoblasts showed very little evidence of apoptosis. It has been suggested that FasL of maternal and fetal origin limits the migration of fetal cytotrophoblast cells into maternal tissue, and vice versa. Our data strengthen this hypothesis, suggesting that CRH of fetal and maternal origin regulates FasL production and thus affecting the invasion process through a local auto-paracrine regulatory loop of cytotrophoblast cells, regulating their own proapoptotic properties.
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