in vivo 22: 55-62 (2008)
Osteopontin Expression in Human Decidua is Associated with Decidual Natural Killer Cells Recruitment and Regulated by Progesterone XUN QU1,2*, MEIXIANG YANG2*, WEIDONG ZHANG3, LU LIANG3, YONGMEI YANG2, YAN ZHANG3, BIPING DENG2, WENJUAN GAO2, JIA LIU2, QIFENG YANG2, BEIHUA KONG2 and FEILI GONG1 1Department
of Immunology, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan; 2Institute of Basic Medical Science and the Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, 250012 Jinan; 3Department of Pathology, Institute of Basic Medicine, Shandong Academy of Medical Science, 250069 Jinan, People's Republic of China
Abstract. The involvement of the chemokine osteopontin has been proposed for maintaining successful pregnancy in mice and ruminants; however, little information of its function in human pregnancy is available. Osteopontin expression was assessed in decidua by RT-PCR and immunohistochemical staining in early pregnant women and RPL (recurrent pregnancy loss) patients. Osteopontin was expressed both in human decidual stromal cells and decidual natural killer (dNK) cells, and higher expression was detected in the later gestational phase compared to the early gestational phase. The osteopontin expression increased with pregnancy progression and higher osteopontin expression was correlated with a larger number of dNK cells. Compared with normal pregnancy, osteopontin expression and dNK cells accumulation were reduced significantly in RPL patients. Osteopontin expression was regulated by progesterone via an in vitro culture model. Our results indicated that osteopontin may play an important role in dNK recruitment and is an essential factor for successful pregnancy.
*Both authors contributed equally to this work. Correspondence to: Feili Gong, MD, Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan, People's Republic of China. Tel: +86 0 27 83693325, Fax: +86 0 27 83693325, e-mail:
[email protected]; and Beihua Kong, Institute of Basic Medical Science and the Department of Obstetrics and Gynecology, Qilu Hospital, Shandong University, 250012 Jinan, People's Republic of China. Tel: +86 0 531 82169008, Fax: +86 0 531 82169251, e-mail:
[email protected] Key Words: Osteopontin, pregnancy, natural killer cells, progesterone, abortion.
0258-851X/2008 $2.00+.40
The interaction of the immune system and the endocrine system enables embryo implantation and pregnancy success (1). One of the most striking events is a dramatic accumulation of decidual natural killer (dNK) cells especially the CD56+ subset during early pregnancy (2, 3). These cells are almost absent during the preovulatory phase of the endometrium in non-pregnant women, but highly increase after pregnancy and constitute around 70% of the lymphocytes in the deciduas (4). Decidual NK cells are specifically recruited to the site around spiral arterioles in the decidualized stroma, and are present in the decidua until the second trimester of pregnancy (2). Several studies have demonstrated that the number of dNK cells is notably decreased in the stroma of patients with unexplained recurrent pregnancy loss (RPL) and in vitro fertilization-embryo transfer failure (5, 6) which suggests that dNK cell recruitment is essential for a successful pregnancy (4). Some chemokines, such as interleukin (IL)-8 and macrophage inflammatory protein1‚ (MIP-‚), have been associated with dNK cell recruitment (7, 8). Osteopontin, a phosphorylated glycoprotein and the ligand of ·Ó‚3 integrin with pleiotropic properties, is a component of the extracellular matrix (ECM) protein and is expressed by a broad range of tissues and cells including the kidney, uterus, placenta, tumors, lymphocytes and vascular smooth muscle cells (9-14). It was originally characterized as a bone matrix protein (15), which was also known as early T lymphocyte activation-1 (Eta-1) and cell transformation-associated protein (Craig). It has been demonstrated that osteopontin mRNA expression was increased in the maternal-fetal interface microenvironment, including the cytotrophoblasts, decidual stromal cells and glandular cells of the decidua in mice
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in vivo 22: 55-62 (2008) and ruminants (16). Previous studies have also revealed that osteopontin mRNA expression was up-regulated in secretory-phase endometrium and in human endometrial stromal cells during decidulization in vitro (16, 17). Osteopontin plays a pivotal role and is associated with the successful implantation in the first-trimester of gestation in pregnant mice and ruminants (18). Osteopontin could induce signaling events that promote cell adhesion, migration and differentiation after binding to the receptors on the cell surface, such as ·v‚3 integrin and CD44 (19). It has been proved that osteopontin may act as a chemokine in the recruitment of T cells and macrophages to the site of inflammation (20, 21). However, little information is available regarding osteopontin expression pattern and its association with the number of dNK cells in human early pregnant decidua and in RPL patients. Whether or not the production of osteopontin in decidua is influenced by female sex hormores remains unknown. To address these questions and to better understand osteopontin expression and regulation in decidua, osteopontin expression was examined in human firsttrimester decidua collected from elective termination by curettage and RPL patients. The relationship between osteopontin expression and the number of decidual NK cells recruited in the decidual tissues was analyzed. In addition, the influence of progesterone on the osteopontin expression in decidual stromal cells was also investigated.
Materials and ªethods Sample collection and tissue preparation. This study was approved by the Ethics Committee of Qilu Hospital in Shandong University. Informed consent was obtained from all of the patients before sample collection. First-trimester decidual samples were collected from 25 women who had undergone elective termination of normal pregnancy, and from 22 patients who had undergone evacuation of the uterus for RPL. None of them had shown any pathological findings, such as uterine abnormalities, infections or pre-existing disease. The decidual samples were collected within 15 min after curettage and the samples were divided into 3 phases according to the gestational weeks, including early phase (week 4-5), middle phase (week 6-7) and later phase (week 8-9). There were 7 samples of early phase (Ec), 8 samples of middle phase (Mc) and 10 samples of later phase (Lc) in the elective pregnancy termination group. The RPL group included 5 samples of early phase (Et), 8 samples of middle phase (Mt) and 9 samples of later phase (Lt). After washing with PBS, part of the samples were routinely fixed in formalin and embedded in paraffin for immunohistochemical processing. The remainder was used for RNA extraction and cell culture. The RNA precipitate was preserved in diethyl pyrocarbonate-treated water and stored at –80ÆC until use.
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Figure 1. Expression of osteopontin gene transcripts in human decidual tissues. A) Total RNA was isolated from human first trimester decidua obtained from pregnacy termination (Ec, Mc and Lc) and RPL patients (Et, Mt and Lt), for semiquantitative RT-PCR. M, Molecular size marker and Et, Ec, 4-5 weeks, Mc and Mt 6-7 weeks, Lc and Lt 8-9 weeks. B) Densitometrical osteopontin (OPN) /‚-actin transcripts ratio in human decidua at each phase. *p
