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a Prince Henry's Institute of Medical Research, Clayton, Victoria 3168, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The extravasation of leukocytes from the circulation into tissue is a complex process requiring the expression of adhesion molecules and their receptors by leukocytes and endothelial cells, respectively, and the production of chemokines and cytokines by resident tissue cells, leukocytes, and endothelial cells (for review, see [13]). Chemokines are small proteins that induce cell migration and activation by binding to G-protein-coupled surface receptors on leukocytes. They can be divided into 4 families, based on the relative position of their cysteine residues. In vitro studies have identified a number of chemokines that act on eosinophils, including eotaxin, monocyte chemoattractant protein (MCP)-3, MCP-4, RANTES (regulated and normal T-lymphocyte expressed and secreted protein), and macrophage inflammatory protein (MIP)1-
(for review, see [13]). Of these chemokines, eotaxin is the most selective for eosinophils. Eotaxin is an 8.3-kDa protein belonging to the CC group of chemokines, and it acts through the CCR3 receptor on eosinophils [14–16]. Eotaxin mRNA is expressed at high levels in the small intestine, colon, heart, kidney, and pancreas, and at lower levels in other tissues including the lung, liver, ovary, and placenta [14,15]. The accumulation of eosinophils within human tissue has been correlated with increased eotaxin expression in a number of inflammatory conditions, including atopic asthma [17], nasal polyps [15], and ulcerative colitis [14]. Furthermore, an influx of eosinophils into the injection site has been observed in response to the administration of this protein in vivo [15,18,19]. Therefore, we hypothesized that the perimenstrual accumulation of eosinophils that occurs within the endometrium may be regulated by eotaxin produced at this site. Eotaxin and CCR3 were identified in endometrial tissue by immunohistochemical analyses, and the functionality of the receptor was investigated using purified, cultured endometrial cells. The results obtained suggest that although eotaxin may act as an eosinophil chemoattractant during the perimenstrual phase, it is likely to have additional important functions within the endometrium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Endometrial tissue was obtained by curettage from women with regular menstrual cycles and no obvious endometrial dysfunction. Informed consent was obtained from patients before tissue collection, and protocols were approved by the Human Ethics Committee at Monash Medical Centre. Tissue samples were divided between 10% (v:v) buffered formalin (for histochemical analysis) and culture medium (for preparation of purified stromal and epithelial cells), and fixed tissue was processed to paraffin wax blocks. Some tissues were divided and incubated for 16 h at 37°C in tissue culture medium without or with monensin (3 mM) before formalin fixation. After histological dating of the menstrual cycle by a gynecological pathologist, samples were grouped into early, mid, and late proliferative phases; early, mid, and late secretory phases; and menstrual phase.
Immunohistochemistry
Sections (5 µm) of endometrial tissues were dewaxed, hydrated, and subjected to immunohistochemistry for eotaxin and CCR3. To provide quality control, one slide from a single endometrial tissue block was included in every staining run. As a negative control for eotaxin, a second section of every tissue was incubated with an irrelevant murine monoclonal antibody diluted to the same final protein concentration as the mouse anti-human eotaxin monoclonal antibody (m6H9-1-1), a generous gift from Dr. C. MacKay (Leukosite Inc., Cambridge, MA). Eotaxin expression was investigated for a total of 45 tissues, with between 3 and 9 samples for each phase.
The staining protocol for eotaxin was performed at room temperature. Tissues were washed with 0.6% Tween 20 in Tris-buffered saline (pH 7.6; TBS) for 20 min and then incubated in 10% normal horse serum (NHS) in TBS for 30 min. Tissue sections were incubated for 1 h in either anti-eotaxin monoclonal antibody or control antibody diluted appropriately in TBS supplemented with 10% NHS. After washing, tissue sections were incubated in biotinylated horse anti-mouse IgG (Vector Laboratories Inc., Burlingame, CA) for 30 min. The Dako (Glostrup, Denmark) Strept-ABC system was used to detect eotaxin-expressing cells, with New Fuchsin (Dako) as the substrate. Levamisole (final concentration of 1 mM) was added to the substrate immediately before use to inhibit endogenous alkaline phosphatase activity. Sections were counterstained with Harris' hematoxylin, dehydrated, and mounted. Immunostaining in individual cellular compartments within each section was scored blind by two independent observers from zero (negative) to four (maximal staining intensity), relative to the positive and negative controls. Results are presented as the mean ± SEM. Staining of monensin-treated and their corresponding control tissues was performed in the same way except that a peroxidase-anti-peroxidase detection system with 3,3'-diaminobenzidine as chromogen was used.
Endometrial expression of CCR3 was investigated in a total of 30 tissues, with between 3 and 6 samples for each phase. CCR3 was detected using a rabbit polyclonal antibody kindly provided by Dr. B. Daugherty (Merck Research Lab., Rahway, NJ). The protocol was identical to that used for the detection of eotaxin with the following exceptions: normal goat serum was used to block nonspecific binding, rabbit immunoglobulin (Dako) was used as a negative control, and biotinylated goat anti-rabbit IgG (Vector Laboratories Inc.) was used as the secondary antibody. The carboxyl terminal decapeptide (TAEPELSIVF) of the human CCR3 molecule that was used to generate the polyclonal antibody was provided by Dr. B. Daugherty. Specificity of the CCR3 antibody was determined by incubating the antibody with peptide (1.7 mg/ml) before addition to tissue sections.
Cell Isolation and Culture
Endometrial stromal and glandular epithelial cells were purified as described previously [20]. Minced tissue was digested for 45 min at 37°C with 45 U/ml bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ) in the presence of 3.5 µg/ml deoxyribonuclease (Boehringer Mannheim Biochimica, Mannheim, Germany) in calcium- and magnesium-free PBS. The cell suspension was filtered sequentially through 45- and 10-µm nylon filters, epithelial glands were recovered from the filters by back-washing, and erythrocytes were removed from the stromal cells by centrifugation over Ficoll-Paque (Pharmacia, Uppsala, Sweden). After washing, the stromal cells and glands were resuspended in a 1:1 mixture of DMEM and Ham's F-12 medium (Trace Biosciences, Sydney, Australia) supplemented with 10% charcoal-treated fetal calf serum (CT-FCS) and antibiotics (penicillin, streptomycin, and fungizone), and cultured in 24-well trays (2 x 105 stromal cells or 1000 glands per well). After 2 days, cells were washed and changed to serum-free medium containing insulin (10 µg/ml, human Actraprid; Novo-Nordisk Pharmaceuticals, Sydney, Australia), sodium selenite (25 ng/ml; Sigma), epidermal growth factor (50 ng/ml; Sigma), linoleic acid (10 nmol/ml; Sigma), and BSA (0.1%; Sigma). Where indicated, estradiol-17ß (10 nM; Sigma) and the synthetic progestin ORG2058 (100 nM; Organon Laboratories Ltd., Oss, Holland) were added to the culture medium. Culture medium was harvested 2 days after the change to serum-free conditions, centrifuged, and stored at -20°C. Some stromal cell cultures were maintained for 10 days with estradiol and synthetic progestin (to induce decidualization), with medium renewal every 2 days and serum-free conditions for the final 2 days. Cells for immunohistochemical analysis were cultured for 2 days on glass coverslips in DMEM/F12 supplemented with 10% CT-FCS, antibiotics, estradiol-17ß, and synthetic progestin, before fixation in 70% ethanol and analysis. Cells for flow cytometric analysis were cultured under conditions identical to those just described for 4 days before analysis.
Western Blot Analysis
Samples were concentrated 10-fold using Microsep microconcentrators (3K molecular weight cut-off; Filtron Technology Corp., Northborough, MA) and electrophoresed on 20% SDS-polyacrylamide gels in Tris-tricine buffer under reducing conditions. The proteins were transferred to Hybond-P membrane (Amersham International, Buckinghamshire, UK), blocked with TBS supplemented with 0.1% Tween 20 and 10% skim milk powder, and incubated overnight with the anti-eotaxin monoclonal antibody diluted 1:1000 in TBS containing 0.1% Tween 20 and 5% skim milk. After they were washed, the membranes were incubated with horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham) and developed by chemiluminescence (ECL system; Amersham). Recombinant eotaxin (1–100 ng; Peprotech, Rocky Hill, NJ) was used as a positive control.
Eosinophil Preparation
Mononuclear cells and granulocytes were isolated from peripheral blood collected from healthy donors by centrifugation over Ficoll Paque. Erythrocytes were removed from the cell pellet by dextran sedimentation (0.6% in normal saline) and hypotonic water lysis. Eosinophils were enriched from the granulocyte population by negative selection with anti-CD16-coated immunomagnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany).
Ca2+ Flux Assay
Peripheral blood eosinophils or cultured endometrial epithelial cells were loaded with 1 µM Fluo-4, AM (Molecular Probes, Eugene, OR) in calcium- and magnesium-free PBS containing 0.02% Pluronic F-127 (Molecular Probes) for 30 min at room temperature (RT). After two washes in PBS, the cells were incubated in PBS for an additional 30 min at RT. Epithelial cells were detached using cell dissociation reagent (Sigma), and both cell types were resuspended in PBS supplemented with 1 mM CaCl2 and 1 mM MgCl2 for analysis using a Mo-Flow cytometer (Cytomation, Fort Collins, CO). The samples were excited by an argon laser at 488 nm, emission was measured at 530 nm using the linear mode, and events were continuously acquired using Cyclops software (Cytomation). Recombinant eotaxin (100 nM) and ionomycin (Sigma; 270 nM) were added to each cell type to stimulate Ca2+ uptake. These experiments were repeated on two separate occasions using epithelial cells from two separate donors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our aim was to determine whether eotaxin could be involved in recruiting eosinophils into the human endometrium before menstruation. Therefore, immunohistochemical analysis of eotaxin expression in tissue sections spanning the entire menstrual cycle was performed. The results of the analysis are summarized in Figure 1, and representative photographs are presented in Figure 2.
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Low levels of eotaxin were detected in the cytoplasm of a proportion of the eosinophils that were identified morphologically in menstrual phase tissue (Fig. 2A). However, much stronger staining was observed in glandular and luminal epithelial cells in tissue throughout the menstrual cycle. In fact, these cells expressed the highest levels of eotaxin in all stages of the cycle apart from the menstrual and early proliferative phases. Since the protein was located in the apical cytoplasm of both luminal and glandular epithelial cells (Fig. 2B), it is possible that eotaxin may be secreted into the uterine cavity.
Endometrial stromal cell expression of eotaxin was at its lowest in menstrual and early proliferative phase tissue, and it increased slightly in tissue from the remainder of the cycle. Tissue sections from the mid-late secretory and menstrual phases contained decidualized stromal cells, and a proportion of these cells also stained positively for eotaxin (Fig. 2C).
Eotaxin was detected in the cytoplasm of endothelial and smooth muscle cells in a proportion of the blood vessels present in each of the tissue sections examined (Fig. 2D). The intensity of staining in both of these cell types was low in comparison to that observed for epithelial cells, and eotaxin expression by these cells was minimal in menstrual and early proliferative phase tissue. Although eotaxin was detected in perivascular cells in tissue from other stages of the cycle, the level of expression did not vary greatly.
Immunostaining was also performed on paired tissues that had been incubated either without or with the ionophore monensin, which blocks transport of proteins destined for export through the Golgi apparatus [21]. Substantially increased staining was observed in both epithelial cells (Fig. 2, J and K) and endothelial cells (Fig. 2, L and M) in treated compared with untreated tissue. The results from this investigation demonstrate that eotaxin is synthesized by many different cell types within the endometrium. As this chemokine was expressed by perivascular cells in the mid-late secretory and menstrual phases of the cycle, at a stage when eosinophils are being recruited from the circulation into the tissue, it is likely that eotaxin is involved in this process. However, the reason why peak levels of the protein were expressed by epithelial cells at earlier stages of the menstrual cycle, when only low numbers of eosinophils are present within the vasculature and none within the tissue, remains to be established.
Analysis of Cultured Stromal and Epithelial Cells
To determine whether eotaxin was secreted by endometrial cells, purified populations of stromal and epithelial cells were prepared and cultured for 2–10 days in the presence of estradiol and synthetic progestin. Concentrated supernatants from these cells were analyzed by Western blot for the presence of eotaxin. The protein could not be detected in supernatants from either short-term cultured epithelial or stromal cells, or from stromal fibroblasts that had been induced to decidualize in vitro (data not shown). Since the detection limit of the assay was 10 ng, a more sensitive technique would be required to determine whether low levels of eotaxin were present in the supernatants analyzed, and hence whether eotaxin was secreted by these cells.
Immunohistochemical analysis of the cultured cells provided results similar to those obtained from the analysis of tissue sections. Epithelial cells demonstrated strong cytoplasmic expression of eotaxin (Fig. 2E), whereas stromal cells cultured for 2 days and decidualized stromal cells were only weakly positive (data not shown).
Expression of the CCR3 Receptor by Endometrial Cells
As eotaxin was strongly expressed within the endometrium throughout the menstrual cycle, it was important to identify cells with the ability to respond to this chemokine. Eotaxin signals only through the CCR3 receptor [16,22], and therefore immunohistochemical analysis was performed on tissue sections from across the menstrual cycle using a polyclonal antibody against this molecule. The results are summarized in Figure 3, and representative photographs are presented in Figure 2.
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As expected, CCR3 was strongly expressed by eosinophils located both intravascularly in all tissue sections and extravascularly in menstrual phase tissue (Fig. 2F). The receptor appeared to be located within the cytoplasm of these cells, which is in accord with the fact that the polyclonal antibody was generated using a peptide from the intracellular carboxyl terminus of the receptor.
To our surprise, most other endometrial cell types also expressed CCR3, albeit to varying levels depending on cell type and menstrual cycle stage. Luminal and glandular epithelial cells exhibited diffuse cytoplasmic staining with the anti-CCR3 antibody, with expression levels peaking in the late proliferative phase of the cycle (Fig. 2G). Perivascular cells in a portion of the blood vessels examined also contained cytoplasmic CCR3 (Fig. 2H), with an increase in expression in the mid proliferative phase. Endometrial stromal cells demonstrated variable reactivity with the anti-CCR3 antibody, and decidual cells present in mid-late secretory and menstrual phase tissue also expressed this receptor.
To establish the specificity of the immunostaining for CCR3, the anti-CCR3 antibody was preadsorbed with the peptide against which it was generated. Immunostaining was completely inhibited in tissue sections that had been incubated with the preadsorbed antibody (Fig. 2, I and J). Furthermore, the intensity of staining was markedly reduced in tissue sections that were incubated with recombinant eotaxin before treatment with the anti-CCR3 polyclonal antibody, thus masking the antigenic epitopes (data not shown). Together, these results strongly suggest that CCR3 is indeed expressed by the majority of endometrial cells, and that the antibody does not bind to other cellular components that contain a similar peptide sequence.
To our knowledge, this is the first report of CCR3 expression by cells other than leukocytes. The results suggest that eotaxin and its specific receptor may have functions in the endometrium, particularly in the epithelium, other than eosinophil chemoattraction.
Analysis of Endometrial Epithelial Cell CCR3 Function
Leukocyte migration and activation are induced when chemokines bind to their G-protein-coupled receptors on the cell surface. Signaling through these receptors generally results in a transient calcium flux within responding cells [22]. To determine whether the CCR3 receptors detected on endometrial epithelial cells were functional, a Ca2+ flux assay with eotaxin as the stimulus was performed on cultured cells. The assay was initially established using the response of peripheral blood eosinophils to stimulation with this chemokine. Response to ionomycin was used as the positive control. The results of the assay are presented in Figure 4.
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When either eotaxin or the nonspecific ionophore, ionomycin, was added to peripheral blood eosinophils, a transient increase in intracellular calcium levels was recorded (data not shown). A similar result was obtained when endometrial epithelial cells were stimulated with either of these reagents (Fig. 4). Therefore, the CCR3 receptor expressed by endometrial epithelial cells is functional. Further analyses are required to identify the physiological role of this receptor on these cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eosinophils are found outside blood vessels in the normal endometrium only perimenstrually and during the menstrual phase of the cycle [6,8]. Therefore, it is likely that eosinophils are recruited into the tissue during the late secretory phase, and in support of this hypothesis, eotaxin was detected in perivascular cells at this stage (Figs. 1 and 2D). Interleukin (IL)-8 and MCP-1, important neutrophil and monocyte chemoattractants, respectively, have also been detected in perivascular cells in the late secretory phase of the cycle [24,25], while numbers of these leukocyte subsets peak at a stage similar to the peak stage of eosinophils [5,8,10]. In situ binding assays using endometrial tissue would be required to identify chemokines bound to the surface of endothelial cells that may be directly involved in recruiting these leukocytes from the circulation. This technique has recently been used to identify the binding sites of IL-8, RANTES, MCP-1, MCP-3, and MIP-1 within normal skin [26,27], but the difficulty in obtaining sufficient human endometrial tissue at the appropriate time of the cycle limits the possibility of such studies in this tissue.
It has been suggested that human endometrium undergoes inflammatory-type reactions in association with both embryo implantation and menstruation [28]. Our results suggest that eotaxin may play an important role in regulating the movement of leukocytes into healthy nonhematopoietic tissues including the endometrium. In support of this argument, the analysis of mice lacking the eotaxin gene has revealed that this chemokine is required for the physiological trafficking of eosinophils into normal tissues such as the thymus and jejunum [29].
Eotaxin was strongly expressed by endometrial luminal and glandular epithelial cells (Figs. 1 and 2B), which is in agreement with previous reports describing the synthesis and expression of this chemokine by epithelial cells in both normal and inflamed tissue [15,17,30]. Maximal levels of eotaxin were detected in these cells during the mid-late proliferative phase of the cycle. In contrast, the lowest levels of the chemokine IL-8 were found in endometrial epithelial cells at this stage of the cycle [24]. These results suggest that endometrial epithelial cell expression of IL-8, but not of eotaxin, may be regulated by progesterone.
The results obtained in this study suggest that eotaxin may play a role in regulating the activity of endometrial epithelial cells. In support of this notion, biological functions apart from leukocyte chemoattraction have been described for other chemokines. First, IL-8 has a variety of other effects, including the ability to induce endometrial stromal cell proliferation [31], to promote tumor metastasis and angiogenesis (for reviews, see [13,32]), and to stimulate the mitogenesis of epidermal [33], melanoma [34], and vascular smooth muscle cells [35]. Second, the chemokines interferon-inducible protein of 10 kDa and platelet factor 4 have been shown to inhibit the growth, metastasis, and neovascularization of tumors (for reviews, see [13,32]). Third, stromal cell-derived factor 1 is required for B-cell lymphopoiesis, the recruitment of hematopoietic progenitors from the fetal liver into the bone marrow [36], and vascularization of the gastrointestinal tract [37]. Further studies are required to determine whether eotaxin is involved in endometrial remodeling by regulating the proliferation and differentiation of epithelial cells or by directing the formation of glands by these cells.
Although eotaxin was detected by immunohistochemistry in cultured endometrial epithelial (Fig. 2E) and stromal cells, secreted protein was not identified in culture supernatants from these cells. One explanation for this result is that the amount of protein secreted by these cells was below the limit of detection of the Western blot assay utilized. Alternatively, cultured endometrial cells may need to be stimulated with exogenous cytokines before they will secrete substantial levels of eotaxin. In support of this notion, pulmonary epithelial cell lines did not secrete significant levels of eotaxin unless treated with tumor necrosis factor (TNF)- or IL-1ß [14,38]. Similarly, preincubation with IL-4 [39] or TNF- [40] was required to induce eotaxin synthesis and secretion by dermal fibroblasts. All of these cytokines are widely expressed within endometrial tissue and would be available to endometrial cells in situ [41–43].
Previous studies have identified CCR3 on eosinophils [16,22], basophils [44], a subset of peripheral blood CD4+ T cells [45], and T cells that co-localize with eosinophils in inflamed tissue from contact dermatitis, nasal polyps, and ulcerative colitis [46]. The results presented here describe the expression of this receptor by additional cell types within the endometrium including epithelial cells, endothelial cells, fibroblasts, and vascular smooth muscle cells (Fig. 2, F–H; Fig. 3). The immunostaining of these cells cannot simply be explained by cross-reactivity of the anti-CCR3 antibody with other cellular constituents containing a similar peptide sequence. First, the immunostaining for CCR3 was substantially inhibited by pretreatment of tissues with eotaxin. Second, the stimulation of cultured endometrial epithelial cells with eotaxin resulted in a transient increase in intracellular Ca2+ levels (Fig. 4). Finally, the expression of CCR3 by cells other than leukocytes was not reported when bronchial mucosal biopsies were analyzed immunohistochemically with this antibody, although epithelial cells in these biopsies stained for eotaxin [30]. The finding that CCR3 was expressed by various endometrial cell types is not without precedent, since the expression of other chemokine receptors is not restricted to leukocytes, and indeed each of the cell types mentioned above must express the appropriate receptors for the various chemokines to exert their diverse effects.
The localization of CCR3 to diverse endometrial cell types supports the hypothesis that chemokines at this site may have important functions other than the attraction of leukocytes from the vasculature into the tissue. As CCR3 and eotaxin were both strongly expressed by epithelial cells, this chemokine may act as a paracrine growth factor or chemoattractant for these cells in the constant remodeling that occurs within the endometrium over the course of the menstrual cycle. The chemokines regulated upon activation, normal T cell expressed and secreted (RANTES), MCP-3, and MCP-4, also bind to CCR3, and thus may be involved in the regulation of endometrial cell function. Although RANTES has been localized to endometrial stromal cells [47], the presence of MCP-3 and MCP-4 within the endometrium has not been investigated.
In conclusion, our results suggest that the chemokine eotaxin is involved in regulating eosinophil entry into the endometrium before menstruation. Although the role of these cells at this site has not been defined, it is likely that eosinophils are involved in both tissue degradation and regeneration by their release of enzymes and cytokines (for review, see [1]). Finally, the identification of functions other than leukocyte chemoattraction that chemokines may have in the endometrium will greatly enhance our understanding of the action of these proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 NH & MRC of Australia (grant 971292), NIH (grant HD33233), WHO Human Reproduction Programme (grant 96908). 
2 Correspondence: L.A. Salamonsen, Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, 3168, Victoria, Australia. FAX: 61 3 9594 6125; lois.salamonsen{at}med.monash.edu.au
Accepted: September 13, 1999.
Received: May 11, 1999.
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