Molecular Human Reproduction, Vol. 7, No. 9, 881-886,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Co-localization of oestrogen receptor ß and leukocyte markers in the human cervix
1 Division for Reproductive Endocrinology and 2 Division for Obstetrics and Gynecology, Department of Woman and Child Health, Karolinska Institutet, Stockholm, Sweden
Abstract
Cervical ripening during parturition is associated with rapid production of catabolic enzymes by invading leukocytes and increased collagen metabolism. The recruitment of leukocytes is regulated by various factors including inflammatory mediators, prostaglandins and matrix metalloproteinases. Sex steroids may be indirectly or directly involved in this process. This study aimed to evaluate the expression of oestrogen receptor ß (ERß) in blood cells infiltrating the cervix during pregnancy and parturition. Cervical biopsies were obtained from term pregnant, post-partal and non-pregnant women. The ERß protein and leukocyte markers CD45 and CD68 were evaluated by single and double labelling immunohistochemistry. Quantitative values were assessed using a microscope and a high-resolution camera connected to a computer with image analysis program. The number of CD45+ and CD68+ cells in the cervix increased in term pregnancy and post-partum compared with the non-pregnant state. The ERß antigen was co-localized with CD45 leukocyte common antigen and CD68 macrophage specific antigen in blood leukocytes infiltrating the cervical tissue. The presence of ERß in the cervical leukocytes suggests that oestrogen may directly regulate leukocyte functions in the cervix.
cervix/immunochemistry/leukocyte/oestrogen receptor/parturition
Introduction
During pregnancy the human cervix undergoes consecutive biochemical changes (Uldbjerg et al., 1983a
). A final rapid remodelling of the cervical connective tissue is an important and obligatory event for a normal delivery. The cervix consists mainly of connective tissue with a high amount of collagen and a small proportion of smooth muscle tissue (Danforth and Evanston, 1954). Cervical ripening is associated with increased collagen metabolism and rapid production and activation of catabolic enzymes including collagenases (Uldbjerg et al., 1983b). The two main sources of collagenase enzymes in the cervix are resident fibroblasts and invading leukocytes (Junqueira et al., 1980; Osmers et al., 1992). Collagenases of the granulocytes have recently been referred to as matrix metalloproteinases (i.e. MMP-8, MMP-9). Invading leukocytes have been shown to be responsible for an increase of collagenase and elastase activity (Uldbjerg et al., 1983b; Kanayama and Terao, 1991; Osmers et al., 1992) and, more recently, for increased MMP-8 concentrations during parturition (Rath et al., 1998; Winkler et al., 1999; Maymon et al., 2000). Cervical ripening has been described as an inflammatory reaction (Liggins et al., 1981). Compared to the non-pregnant state, a dramatic increase in the density of neutrophils and macrophages in the cervix has been observed at term pregnancy and parturition, indicating a role for these cells during cervical ripening (Bokström et al., 1997). The recruitment of leukocytes is regulated by inflammatory mediators such as interleukin (IL)-8, prostaglandin (PG) E and F2
, monocyte chemotactic protein (MCP)-1 and MMP (Chwalisz et al., 1994; Bokström et al., 1997; Sennström et al., 2000). Gonadal steroids may also be indirectly or directly involved in this process.
Two subtypes of oestrogen receptor (ER) have been described: the `classical' ER, now named ER, and the more recently discovered ERß (Kuiper et al., 1996). The human ERß is a protein of 530 amino acids with 47% identity to the larger ER, which consists of 595 amino acids (Enmark and Gustafsson, 1999).
In humans, there is no abrupt change of serum oestrogen or progesterone levels immediately before parturition (Csapo et al., 1971). However, the level of gonadal steroid receptors in the human cervix changes during pregnancy. Studies of ERß may reveal further sites of potential oestrogen action and lead to reconsideration of oestrogen function in many clinically important events including cervical ripening. In contrast to ER and progesterone receptor (PR) subtypes A and B, the level of ERß protein increases in the human cervix significantly at term pregnancy compared with the non-pregnant state (Wang et al., 2001; Y.Stjernholm Vladic et al., unpublished data).
Although the distribution of ERß is often related to that of ER, some tissues lack ER but express ERß, and vice versa (Taylor and Al-Azzawi, 2000). Endothelial cells of cervical blood vessels are negative for ER and PR, but express ERß (Wang et al., 2001; Y.Stjernholm Vladic et al., unpublished data). Previous studies have indicated the presence of oestrogen binding sites in macrophages, but a monoclonal antibody specific for the classical ER does not react with those binding sites (Gulshan et al., 1990). Other studies have shown no evidence of expression of either PR or ER in lymphocytes, macrophages and other leukocyte populations in the human endometrium (King et al., 1996; Stewart et al., 1998). It is noteworthy though that the antibodies used in those studies recognize ER and have an unknown specificity to ERß. In a previous study, we found specific immunostaining of ERß in morphologically recognized granulocytes in cervical tissue (Wang et al., 2001). Our finding led us perform this study aimed at evaluating the expression of ERß in blood cells infiltrating the cervix during pregnancy and parturition.
Materials and methods
Patients
Twenty-four women previously described in detail (Wang et al., 2001) were included in the study. The patients were divided into three groups: non-pregnant (NP, n = 6), term pregnant (TP, n = 8) and post-partal (PP, n = 10). The study was approved by the local ethical committee of the Karolinska Hospital (Ref. no. 99-099). Informed consent was obtained from all patients.
Tissue collection
Cervical biopsies were obtained transvaginally from the anterior cervical lip at the 12 o'clock position from 10–20 mm depth. The biopsies were fixed in 4% formaldehyde at 4°C overnight and stored in 70% ethanol before being embedded in paraffin. Paraffin sections were cut at 5 µm, mounted on positively charged slides (SuperFrost Plus; Menzel-Glaser, Braunschweig, Germany) and dried at 50°C overnight before use.
Antibodies
Commercially available monoclonal antibodies were used for detection of the CD68 macrophage specific antigen (M0876; Dako, Glostrup, Denmark) and CD45 RB leukocyte common antigen (M0833; Dako). CD45 RB antibody reacts with B cells, T cell subsets, monocytes, macrophages and weakly with granulocytes. A polyclonal chicken antibody was used for detection of the ERß. The preparation of this antibody has been described (Saji et al., 2000). Specifications of the antibodies are listed in Table I.
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Immunohistochemistry
Samples from all women were dewaxed with Bio-Clear (Bio-Optica, Milan, Italy), rehydrated in graded ethanol and washed consecutively in double-distilled water and 0.01 mol/l phosphate-buffered saline (PBS) (pH 7.4). After washing, the sections to be labelled with CD68 antibody were transferred to plastic jars containing 0.01 mol/l citrate buffer (pH 6.0) and heated in a microwave oven for 3x5 min at 700 W. If necessary, more citrate buffer was added after 5 and 10 min of heating to avoid drying of the sections. Sections were allowed to cool for 20 min before washing in PBS. No microwave pre-treatment was needed for labelling with the CD45 antibody. All sections were blocked for 30 min with normal horse serum. Excess liquid was removed and slides were incubated with the respective primary antibody (CD45 and CD68, both diluted 1:200 in PBS) for 60 min at room temperature. A normal mouse IgG (Santa Cruz Biotechnology, Santa Cruz, USA) was used to obtain negative controls. After washing in PBS, sections were incubated for 60 min at room temperature with horse anti-mouse biotinylated secondary antibody (Vector Laboratories, Burlingame, USA) diluted 1:200 in PBS. After an additional wash in PBS, the sections were incubated for 30 min at room temperature with alkaline phosphatase avidin–biotin complex (Vector Laboratories) diluted 1:100 in PBS. After further washing in PBS, the sections were developed with Vector Red (Vector Laboratories) for 8–9 min, washed with tap water for 5 min and counterstained with haematoxylin. The sections were then rewashed in water, dehydrated by graded alcohol, cleared with `Bio-Clear' and permanently mounted using Pertex mounting medium (Histolab Products, Göteborg, Sweden)
Immunostaining for ERß was performed as described elsewhere (Wang et al., 2000).
Immunochemical double labelling
Five patients from each group were included in a double immunostaining study.
Samples were dewaxed, rehydrated and microwaved (3x5 min) as described above. Endogenous peroxidase activity was blocked using hydrogen peroxide (3% v/v). After washing in PBS (2x5 min), sections were blocked for 30 min with normal rabbit serum. Excess liquid was removed and slides were incubated for 18 h at 4°C with the first primary antibody (ERß) diluted 1:500 in 1% BSA in PBS. The primary antibody was substituted by a normal rabbit IgG (Santa Cruz Biotechnology) to obtain negative controls. After washing in PBS, sections were incubated for 60 min at room temperature with rabbit anti-chicken peroxidase-conjugated secondary antibody (Sigma Chemicals, St Louis, USA) diluted 1:1000 in PBS. After an additional wash in PBS, sections were developed with 3,3-diaminobenzidine (Dako Corp., Carpinteria, USA) for 1.5–2.5 min and washed in double-distilled water.
After the ERß immunostaining procedure was finished, sections were heated in the microwave oven twice for 2x5 min and allowed to cool as described above. Sections were washed in PBS and incubated sequentially with normal horse serum for 30 min and the second primary antibody (CD45 1:200, CD68 1:200) for 60 min at room temperature. After washing in PBS, sections were incubated with a biotinylated secondary horse anti-mouse antibody (1:200) for 60 min, thereafter washed in PBS and incubated with alkaline phosphatase avidin–biotin complex (1:100) for 30 min at room temperature. After further washing in PBS, sections were developed with Vector Red (Vector) for 6–8 min, washed with tap water for 5 min and slightly counterstained with haematoxylin. Sections were then rewashed in water, dehydrated by graded alcohol, cleared with Bio-Clear and permanently mounted using Pertex mounting medium.
Image analysis
A microscope and a video camera connected to a computer with image analysis software (Leica Imaging System Ltd, Cambridge, UK) were used to assess quantitative values from immunohistochemistry. Immunostaining was quantified as described before (Wang et al., 1999). In brief, the total area of positively stained cells in the sub-epithelial stroma was measured using colour discrimination software and presented as the ratio between the positively stained area and the total cell nuclei area.
Statistics
Data from the image analysis were subjected to analysis of variance on ranks (Kruskal–Wallis) test for statistical evaluation. Significances were calculated using Dunn's test. P < 0.05 was considered to be significant.
Results
The samples had a typical morphology of cervical tissue, consisting mainly of cervical stroma, smooth muscles, blood vessels and stratified squamous epithelium. In most of the samples, there were also areas of cervical glands and some samples contained the mucosa of the cervical canal covered with columnar epithelium.
Immunohistochemistry
ERß expression
ERß immunostaining was found in the cervical epithelium and stroma in all groups (Figure 1a). Intensity of staining and the number of ERß+ cells in epithelium and stroma appeared to be increased in the term pregnant compared with the non-pregnant and the post-partal groups. Immunostaining of ERß mainly followed the pattern reported previously (Wang et al., 2001). Some smooth muscle cells and endothelial cells identified by morphology showed positive staining for ERß. Morphologically recognized granulocytes both within and outside the vessels were positive for ERß (Figure 1b). A negative control for ERß immunostaining is shown in Figure 1c.
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CD45 and CD68 expression
CD45+ and CD68+ staining was observed as a bright purple cytoplasmic staining around the nuclei, which were blue due to counterstaining with haematoxylin.
CD45+ cells were detected in all samples (Figure 1d, e, f
). Leukocytes were distributed around the tissue and tended to accumulate around blood vessels and in the glandular and sub-epithelial areas. CD45+ cell density was increased both at term pregnancy (Figures 1e, 2a) and in the post-partum period (Figures 1f and 2a), compared with the non-pregnant state (Figures 1d and 2a). There was no positive staining in the negative control (Figure 1j). CD45 immunostaining image analysis results are shown in Figure 2a.
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CD68 antigen was expressed in all three groups (Figure 1g, h, i
). The distribution of macrophages in the tissue was similar to that of CD45+ leukocytes. A low number of positively stained macrophages (arrow) was observed in the non-pregnant group (Figure 1g). The number of CD68+ cells tended to increase in the term pregnant group (Figures 1h and 2b) and there was a 3-fold increase in the post-partal group (Figures 1i and 2b) compared with the non-pregnant group (Figures 1g and 2b). Negative controls showed no positive staining (Figure 1j). Figure 2b illustrates CD68 immunostaining image analysis results.
Immunochemical double labelling
Using double immunohistochemical labelling, the ERß antigen was co-localized with the CD45 leukocyte common antigen (Figure 1k, m) and the CD68 macrophage specific antigen (Figure 1l, n) in blood cells infiltrating the cervical tissue. Co-localization was observed in all groups. CD45 and CD68 monoclonal antibodies showed a bright purple cytoplasm staining in the cells with phenotypical characteristics of leukocytes and macrophages. ERß was detected in the nuclei of the same cells with a dark brown colour. However, some CD45 and CD68+ cells did not show any specific ERß staining and there were numerous ERß+ cells that were not leukocytes. The general pattern of ERß immunostaining was similar to the one obtained with the single labelling procedure (Figure 1a).
Discussion
Cervical ripening at parturition is characterized by an extensive connective tissue remodelling with a significant decrease in collagen and proteoglycan levels. Leukocyte invasion of the cervix at term pregnancy has been described in several species including the human (Liggins et al., 1981; Owiny et al., 1995; Lugue et al., 1996; Bokström et al., 1997). Leukocytes are recognized as a main source of the catabolic enzymes involved in the remodelling of the cervix during parturition (Osmers et al., 1992). There is also a resident population of leukocytes in the cervix (Stern et al., 1998).
In a recent study, we observed positive ERß immunostaining in granulocytes infiltrating the human cervix at term pregnancy (Wang et al., 2001). In addition, ERß immunostaining was also reported for leukocytes infiltrating the human vagina (Taylor and Al-Azzawi, 2000). In the present study, the expression of ERß was found in several leukocyte subsets within the cervical tissue at term pregnancy, after parturition and in the non-pregnant state. CD45+ and CD68+ leukocyte populations include macrophages, monocytes, B cells, T cells and some granulocytes. The ERß antigen and leukocyte markers CD45 and CD68 were co-localized in leukocytes using a double immunostaining technique. Neutrophilic granulocytes identified by morphology showed positive nuclear staining for ERß.
The number of leukocytes in the cervix can be modulated by different cytokines and inflammatory mediators such as IL-8, prostaglandins and MMP (Chwalisz et al., 1994; Bokström et al., 1997; Luo et al., 2000), indicating possible roles for these compounds in the recruitment of leukocytes. Gonadal steroids have also been shown to influence leukocyte invasion (Critchley et al., 1996; Ramos et al., 2000). It has been implied that sex steroids may influence leukocyte traffic and activation indirectly by initiating a local cascade of events involving inflammatory mediators and cytokines (Critchley et al., 1996; Robertson et al., 1996).
The results from the current study suggest a possibility for a direct effect of oestrogens on leukocytes via ERß.
Previously published studies have not shown any evidence for ER expression in leukocytes (King et al., 1996; Stewart et al., 1998). However, the presence of oestrogen binding sites in a human macrophage cell line has been reported (Gulshan et al., 1990). The protein had a relatively high affinity to diethylstilboestrol but was not recognized by a monoclonal antibody for ER. The type of binding site found in macrophages could have been what was later described as ER subtype ß (Kuiper et al., 1996).
In vascular endothelium, positive ERß, but not ER, immunostaining has been found in the rat uterus (Wang et al., 1999), cervix and oviduct (Wang et al., 2000), in the human cervix (Wang et al., 2001) and in the human and non-human primate endometrium (Critchley et al., 2001).
The presence of ERß in vascular endothelium and cervical leukocytes suggests that oestrogen may directly regulate leukocyte functions in the cervix, possibly including the production of MMP-8, one of the enzymes involved in cervical remodelling in humans. The inflammatory reaction in the cervix, particularly the release of inflammatory mediators and catabolic enzymes from the leukocytes during cervical ripening, could potentially be mediated via ERß. The up-regulation of ERß in the human cervix at term pregnancy (Wang et al., 2001) indicates an important role for this ER subtype at parturition and supports the hypothesis of a receptor-mediated functional regulation of the response to oestrogens.
Acknowledgements
We wish to acknowledge Jan-Åke Gustafsson from the Department of Medical Nutrition, Karolinska Institutet, Huddinge, Sweden for providing the ERß 503 antibody. The present study received financial support from the Magn. Bergvalls foundation and the Swedish Medical Research Council (grants 3972 and 9508). Denis Stygar was supported by a scholarship from the Swedish Institute.
Notes
3 To whom correspondence should be addressed at: Division for Reproductive Endocrinology, Karolinska Hospital, L05:01, S-171 76 Stockholm, Sweden. E-mail: Lena.Sahlin{at}kbh.ki.se 
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Submitted on March 29, 2001; accepted on June 27, 2001.
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