The expression of fractalkine in the endometrium during the menstrual cycle

International Journal of Gynecology and Obstetrics (2006) 92, 242 — 247

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CLINICAL ARTICLE

The expression of fractalkine in the endometrium during the menstrual cycle M. Watanabe, K. Shimoya *, Q. Zhang, K. Temma-Asano, T. Kimura, Y. Murata Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Suita City, Osaka, Japan Received 18 August 2005; received in revised form 14 December 2005; accepted 15 December 2005

KEYWORDS Fractalkine; Endometrium; CX3CR1; Menstruation

Abstract Objective: The objective of the study was to evaluate the presence of fractalkine in the endometrium of the uterus and the change of fractalkine protein levels during menstrual cycle. Methods: Twelve samples of endometrium of the uterus were obtained from gynecological patients who underwent total hysterectomy. Western blotting, RT-PCR and immunohistochemistry were performed. Results: Fractalkine protein was detected in the endometrium of the uterus. Positive staining was confirmed in the epithelial cells and grandular cells in the endometrium. Expression levels of fractalkine protein and mRNA in the endometrium during secretory phase were significantly higher than those during proliferative phase. Immunohistochemical analysis using an anti-CX3CR1 antibody demonstrated positive staining in the glandular cells of the endometrium of the uterus. Conclusion: Fractalkine was expressed in the endometrium and its production was up-regulated during secretory phase. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.

1. Introduction

* Corresponding author. Tel.: +81 6 6879 3356; fax: +81 6 6879 3359. E-mail address: [email protected] (K. Shimoya).

Chemokines are small proteins which stimulate the migration of leukocytes and mediate inflammation. Fractalkine is the sole member of a novel subclass of chemokines designated the CX3C family [1,2]. In contrast to other chemokines, this chemokine displays potent chemoattractant activity for T cells, NK cells,

0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2005.12.012

Fractalkine in the endometrium of the uterus and monocytes but not neutrophils and is of nonhaemopoietic origin [1]. Fractalkine can exist in two active forms, a transmembrane protein and soluble fractalkine, which is released by proteolytic clevage at a membrane-proximal dibasic cleavage site [3]. This unique structure enables dual function: chemoattractant and adhesion molecule. In the area of reproduction, this molecule is present in the seminal plasma and associated with sperm motility [4]. Fractalkine has already been reported to contribute to the immunodefense mechanism during pregnancy [5]. The expression of fractalkine has been reported to be upregulated by inflammatory signals [6]. Fractalkine may be responsible for the accumulation of lymphocytes in regions of inflammation. The receptor for fractalkine, named as CX3CR1, has also been characterized [7,8]. CX3CR1-mediated signal transduction presumably plays a role in the migration and adhesion of lymphocytes [7,8]. Recently, it was reported that CX3CR1-dependent processes, which controlled host interactions of specialized dendritic cells (DCs) with commensal and pathogenic bacteria, might regulate immunological tolerance and inflammation [9]. The endometrium plays critical role on several important steps, such as implantation, early embryogenesis, placentation, and the maintenance of pregnancy. It follows, therefore, that the environment in the endometrium represents the optimal conditions for these and other important developmental processes. The endometrial cycle in women follows a series of morphologic and physiologic events characterized by proliferation, secretory differentiation, degradation, and regeneration of the uterine lining. These alternations are controlled by cyclically released ovarian estradiol and progesterone. The endometrium produces various kinds of molecules, such as growth factors and cytokines [10]. The larger amounts of inflammatory leukocytes that infiltrate the tissue premenstrually are considered to be critical effecter of endometrium [11]. The lymphocytes in the endometrium have an important role to be capable of initiating focal breakdown of the endometrium and become a decidual change. The aim of this study was to investigate the expression of fractalkine in the endometrium of the uterus and to clarify the functions of fractalkine in the endometrium of the uterus during the process of pregnancy.

2. Materials and methods 2.1. Reagents Goat anti-human fractalkine polyclonal antibodies and recombinant (r-) fractalkine were purchased

243 from R&D Systems (Minneapolis, MN, USA). Normal goat IgG for use as a control in the histological analysis was purchased from Zymed Laboratories (San Francisco, CA). A human CX3CR1 antibody (rat IgG2b) and rat IgG2b were purchased from MBL (Nagoya, Japan).

2.2. Samples Twelve samples of endometrium of the uterus were obtained from gynecological patients who underwent total hysterectomy. The range of ages of patients was 30—45 years old. This study was approved by the local ethics committee of the Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine. Informed consent was obtained from each patient.

2.3. Tissue preparation for Western blot analysis The endometrium of the uterus was homogenized in a 2-ml volume of the homogenizing buffer, as described previously [5]. Homogenates were centrifuged at 4 8C for 30 min at 14,000g to remove debris. Following protein determinations, the samples were aliquoted and subjected to polyacrylamide gel electrophoresis (PAGE).

2.4. Western blot analysis of the endometrium of the uterus To examine fractalkine protein in the endometrium of the uterus, we performed Western blotting analysis using an anti-human fractalkine polyclonal antibody, as described previously [5].

2.5. Protein assay Protein levels were determined with Bio-Rad (Hercules, CA) Protein Determination Reagent, according to the method of Bradford [12].

2.6. Determination of fractalkine levels of the endometrium of the uterus by densitometric analysis of Western blotting To measure titers of fractalkine levels in the endometrium of the uterus during menstruation, the expression of fractalkine protein was quantified and analyzed by NIH image software program (developed and provided by the Research Services Branch (RSB) of the National Institute of Mental Health (NIMH)).

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quantified and analyzed by NIH image software program.

95DKa

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2.9. Immunohistochemical staining of fractalkine in the endometrium of the uterus

P<0.05

To determine the localization of fractalkine in the endometrium of the uterus, we performed immunohistochemical staining by using an avidin—biotin peroxidase complex method kit (OminiTags Universal Streptavidin/Biotin Affinity Immunostaining Systems, Lipshaw, Pittsburgh, PA), as described previously [5,14].

Arbitary unit

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Proliferative phase

Secretory phase

Figure 1 (A) Western blot analysis of fractalkine protein in the endometrium of the uterus. Ten micrograms of uterine endometrial protein were electrophoresed and the fractalkine signal was detected as described in the text. Lanes 1, 2: uterine endometrial protein in the proliferative phase. Lane 3, 4: uterine endometrial protein in the secretory phase. Lane 5; r-fractalkine (10 ng). (B) Fractalkine protein levels in the endometrium in the proliferative phase and in the secretory phase. The expression of fractalkine protein was quantified and analyzed by NIH image software program. Densitometric intensity levels of fractalkine protein in the endometrium of women in the secretory phase (1.00 F 0.20; n = 6) were higher than those in the proliferative phase (0.62 F 0.14; n = 6) ( p b 0.01).

2.10. Immunohistochemical staining of CX3CR1 in the endometrium of the uterus To determine the localization of CX3CR1 in the endometrium of the uterus, immunohistochemical staining of anti-CX3CR1 antibody was performed, as described previously [5,14].

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FRK G3PDH

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2.7. RNA extraction RNA was extracted from the uterine endometrial samples of 0.5 g wet weight by acid guanidine thiocyanate-phenol-chloroform extraction according to the method of Chomczynski and Sacchi [13].

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2.8. RT-PCR amplification RT-PCR was performed using an RT-PCR high kit (TOYOBO Co., Osaka, Japan), as described previously [14]. Amplification using CX3CR1-specific primers yielded a 653-bp DNA product and amplification using the fractalkine-specific primers yielded a 597-bp DNA product [15]. Molecular sizes were estimated using a 100-bp DNA ladder (TOYOBO, Osaka, Japan). All primers were obtained from Life Technologies (Tokyo, Japan). To measure levels of fractalkine mRNA expression in the endometrium of the uterus during menstruation, the expression of fractalkine mRNA was

Proliferative phase

Secretory phase

Figure 2 (A) Fractalkine mRNA expression in the cells of the peritoneal fluid. Agarose gel electrophoresis of RTPCR-amplified fractalkine cDNA. Lane 1: 100-bp DNA ladder. Lane 2, 3: the endometrium in the proliferative phase. Lane 4, 5: the endometrium in the secretory phase. (B) Fractalkine mRNA levels in the endometrium in the proliferative phase and in the secretory phase. The expression of fractalkine mRNA/G3PDH was quantified and analyzed by NIH image software program. The arbitrary units of the ratio in the endometrium of women in the secretory phase (1.00 F 0.25; n = 6) were higher than those in the proliferative phase (0.49 F 0.19; n = 6) ( p b 0.01).

Fractalkine in the endometrium of the uterus

2.11. Statistical analysis The data represent as means F SD. The data were subjected to one-way analysis of variance with the use of the Statview statistic package (Abacus Concepts, Inc., Berkeley, CA). p b 0.05 was considered significant.

3. Results To detect fractalkine protein in the endometrium, Western blot analysis was performed. As shown in Fig. 1A, fractalkine protein was detected as a 95-kDa band in the endometrium. To determine fractalkine levels in the endometrium during menstrual cycle, the densitometric intensity of Western blot analysis was undertaken, using NIH image software. The expression of fractalkine protein in the endometrium of the uterus during secretory phase was significantly higher than that during proliferative phase ( p b 0.05, Fig. 1B). RT-PCR was then performed to examine the expression of the fractalkine mRNA in the endometrium of the uterus. Fig. 2A shows that fractalkine transcripts were present in the endometrium of the uterus. To compare the levels of fractalkine mRNA in the uterine endometrium, we analyzed the densitometric intensity of RT-PCR using the software bNIH imageQ. The expres-

245 sion of fractalkine mRNA in the endometrium of the uterus during secretory phase was significantly higher than that during proliferative phase ( p b 0.01, Fig. 2B). To identify the origin of fractalkine, immunohistochemical staining of sections of the endometrium was performed using an antifractalkine polyclonal antibody. The glandular epithelial cells in the endometrium of the uterus were intensely stained (Fig. 3). To determine whether CX3CR1-positive cells were present in the endometrium, we performed RT-PCR using specific primers for CX3CR1. As shown in Fig. 4A, RT-PCR demonstrated the presence of CX3CR1 mRNA expression in the endometrium. To determine the existence of CX3CR1 molecule in the endometrium of the uterus, immunocytochemical staining was performed. As shown in Fig. 4B, immunohistochemical analysis using an anti-CX3CR1 antibody demonstrated positive staining in the glandular epithelial cells of the endometrium of the uterus.

4. Discussion Fractalkine is one of the chemokines with a CX3C cysteine motif. In the present study, the expression of fractalkine protein and mRNA in the human cycling endometrium of the uterus were demonstrated. The present findings indicated that frac-

Figure 3 Immunohistochemical staining of fractalkine-producing cells in the endometrium. The cells in the endometrium were stained by the avidin—biotin complex method with a goat polyclonal anti-fractalkine antibody. (A), (C): Negative control; (B), (D): stained by anti-fractalkine antibodies. (A), (B): The endometrium of the woman in the proliferative phase; (C), (D): the endometrium of the woman in the secretory phase.

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Figure 4 (A) CX3CR1 mRNA expression in the endometrium. Agarose gel electrophoresis of RT-PCR-amplified CX3CR1 cDNA. Lane 1: 100-bp DNA ladder, Lane 2, 3: the endometrium in the proliferative phase. Lane 4, 5: the endometrium in the secretory phase. (B) Immunohistochemical staining of CX3CR1 positive cells in the endometrium. The cells in the endometrium were stained by the avidin—biotin complex method with a goat polyclonal anti-CX3CR1 antibody. (A), (C): negative control; (B), (D): stained by anti-fractalkine antibodies. (A), (B): The endometrium of the woman in the proliferative phase; (C), (D): the endometrium of the woman in the secretory phase.

talkine was constitutively present in the endometrium. The immunohistochemical analysis using anti-fractalkine polyclonal antibody showed that the glandular epithelial cells of the endometrium were intensely stained, suggesting that these epithelial cells are the main source of fractalkine in the endometrium. The endometrium has bacteriostatic and bactercidal mechanisms to protect against infection of the peritoneal cavity through the uterus. Fractalkine has been reported to have a critical role in the innate immunity [9]. CX3CR1— fractalkine systems may regulate immunological tolerance and inflammation [9]. The mechanism of CX3CR1 dependent processes may have an important role in the immunodefense of the uterine cavity. Fractalkine has an important role in Th1 type cell function and inflammatory response [16,17]. Such lymphocytes might contribute to the immunodefense system in the endometrium of the uterus. This molecule might contribute to homeostasis of the immunodefense system in the endometrium of the uterus in case of endometritis.

Further investigations will be necessary to examine the relationship between the fractalkine level and genital tract infections such as endometritis. The findings of this study were consistent with Hannan’s previous report [18]. Hannan et al. demonstrated that fractalkine was localized predominantly to glandular epithelial and decidual stromal cells, with the highest staining intensity in the secretory phase and early pregnancy [18]. In the present study Western blot analysis clearly demonstrated that the expression of fractalkine in the uterine endometrium of women in the secretory phase was significantly higher that in the proliferative phase. Increased fractalkine has a chemoattractive activity to immunodefense cells, such as T cells and NK cells. These cells produce inflammatory mediators. Several inflammatory mediators enhance the fractalkine mRNA levels and the production of fractalkine [15,19]. The production of fractalkine in the endometrium might be regulated by autocrine and paracrine mechanism. However, the mechanism of the increased fractalkine in the

Fractalkine in the endometrium of the uterus secretory phase is still unclear. Further investigations should be necessary to demonstrate the mechanism of fractalkine production.

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Acknowledgment

[10]

This study was supported by Grants-in-Aid for Scientific Research (nos. 15209054, 15591746, 16390476 and 17791117) from the Ministry of Education, Science, and Culture of Japan (Tokyo, Japan).

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