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Prince Henry’s Institute of Medical Research (N.J.H., R.L.J., L.A.S.), Clayton, Victoria 3168, Australia; Department of Reproductive and Developmental Sciences, University of Edinburgh (H.O.D.C.), Edinburgh, Scotland EH1645B; Department of Obstetrics and Gynecology, Monash University (G.J.K., P.A.W.R.), Clayton, Victoria 3168, Australia; and Department of Obstetrics and Gynecology, Faculty of Medicine, University of Indonesia (B.A.), Jakarta, Indonesia 1033
Address all correspondence and requests for reprints to: Dr. Natalie J. Hannan, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: natalie.hannan{at}phimr.monash.edu.au.
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Abstract |
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Introduction |
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Abnormal leukocyte infiltration, particularly of neutrophils, eosinophils, macrophages, and uNK, is seen in women using progestin-only contraceptives, where break-through bleeding is common (3, 4). Which leukocyte subpopulations are present depends on the particular contraceptive method used. Intrauterine levonorgestrel (Lng-IUS) and injectable progesterone, depo-medroxyprogesterone acetate (DMPA; Depo-Provera; Pharmacia & Upjohn, Kalamazoo, MI) induce decidualization of the endometrium (5), and this is associated with elevated numbers of uNK and macrophages (4). Conversely, subdermal levonorgestrel (Norplant; Wyeth Laboratories, Philadelphia, PA) has varying effects on endometrial morphology (atrophic, shedding, and decidualized), and the latter is associated with increased numbers of neutrophils, eosinophils, and macrophages (3). The abnormal leukocyte infiltration in these endometria also supports the importance of leukocytes in endometrial remodeling in both decidualization and endometrial breakdown. Thus, it is critical to understand the mechanisms involved in the selective recruitment of leukocytes to the endometrium and their activation.
Leukocyte migration into tissues is regulated by specific chemokines that mediate the adhesion of leukocytes to endothelial cells and initiate transendothelial migration and homing to specific regions of the tissue (6). To date, the endometrium has been demonstrated to produce IL-8, monocyte chemotactic protein-1 and -2, macrophage inflammatory protein-1
and -3, eotaxin, and RANTES (regulated upon activation normal T cell expressed and secreted) (7, 8, 9, 10, 11). Because IL-8 and monocyte chemotactic protein-1 are negatively regulated by progesterone (7, 12), its withdrawal premenstrually is postulated to up-regulate the expression of these chemokines (13), thus indirectly stimulating the recruitment of leukocytes that lack genomic progesterone receptors.
Fractalkine (also known as neurotactin) is the sole member of a novel subclass of chemokines designated the CX3C family. It is known to attract monocytes, neutrophils, T cells, and NK cells (14, 15, 16). Due to the novel arrangement of its cysteine residues, it is structurally distinct from other chemokines and can exist in two active forms, a transmembrane protein and soluble fractalkine, which is released by proteolytic cleavage at a membrane-proximal dibasic cleavage site (17). Fractalkine’s unique structure enables dual functions; it acts not only as a potent chemoattractant, but also as an adhesion molecule by capturing leukocytes in an integrin- or G protein-independent manner (14), facilitating their migration into tissue.
In an expression screen for chemokine family members on normal human endometrium (18), fractalkine was found to be one of the most abundant chemokines. Given its chemotactic action toward leukocyte subsets important during menstruation and its high expression in menstrual phase endometrium, fractalkine was an attractive candidate for a pivotal role in leukocyte recruitment to the endometrium during menstruation and breakthrough bleeding.
To investigate the potential role of fractalkine in the human endometrium, we examined the production and localization of fractalkine protein by immunohistochemistry in endometrial samples at the different stages of the menstrual cycle corresponding to known leukocyte accumulation and in early pregnancy. Endometrium from women using progestin-only (P-only) contraceptives (Lng-IUS, Depo-Provera, and Norplant) was compared with normal endometrium to establish whether fractalkine was dysregulated by long-term progestin administration and was related to abnormal leukocyte recruitment. The distribution of the fractalkine receptor CX3CR1 was also assessed to establish the potential cellular sites of action for fractalkine during endometrial remodeling.
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Patients and Methods |
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Ethical approval was obtained from appropriate institutional ethics committees for all tissue collections. Written informed consent was obtained before tissue collection from all subjects.
Nonpregnant and pregnant human endometrium
Human endometrial biopsies were obtained by curettage at all stages of the menstrual cycle from normal fertile women undergoing curettage after laparoscopic sterilization or assessment of tubal patency (n = 30). Patients with uterine abnormalities, such as leiomyomas, endometrial polyps, or endometriosis, or those who had received steroid hormone therapy in the last 6 months were excluded. Samples were collected from all stages of the menstrual cycle, and cycle stage was confirmed by histological dating according to the criteria of Noyes et al. (19) (Table 1
). First trimester decidua parietalis (n = 10) was collected away from the implantation site by curettage before termination of pregnancy (8–12 wk gestation) by vacuum aspiration.
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Lng-IUS (Mirena, Leiras Oy, Finland) contraception comprises a T-shaped plastic intrauterine device with a cylinder of synthetic progestin (levonorgestrel) enclosed in a SILASTIC sleeve (Dow Corning, Midland, MI), which allows controlled release of 20 µg levonorgestrel daily directly into the uterine cavity. This results in high local concentration and low serum progestin levels. Women were recruited to the study, and endometrial biopsies were collected by pipette suction curette before insertion of the device either during the proliferative or secretory phase and at 3, 6, and 12 months after insertion (Table 1
). Therefore, this study was longitudinal, where each patient acted as her own control (4). After insertion of the device, endometrial cyclic activity was completely abolished; therefore, postinsertional endometrial samples at each of the time points were examined independently of stage of menstrual cycle.
Depo-Provera
Depo-Provera contraception is the administration of 150 mg DMPA injected im every 90 d (20). Women presenting to Family Planning Victoria for administration of Depo-Provera volunteered for endometrial biopsy (by curette) 3 wk to 12 months after commencing Depo-Provera contraception (Table 1).
Norplant
Norplant uses the same synthetic progestin (levonorgestrel) as Lng-IUS but has a different route of administration. Six SILASTIC rods containing the hormone are subdermally implanted, allowing systemic release of 0.05 and 0.08 mg/d in the first 12 months of use (21), resulting in lower concentrations of progestin in the uterus compared with Lng-IUS. Endometrial samples from women using Norplant were obtained from Klinik Raden Saleh (Jakarta, Indonesia). Between 3 wk and 12 months after Norplant insertion, an endometrial sample was collected by Pipelle suction curette (Laboratoire CCD, Paris, France). These samples were histologically assessed and divided into three groups by their distinct morphological appearance; atrophic (n = 6), shedding (n = 6), and progesterone modified (decidualized stroma; n = 6; see Table 1) (21).
Tissue fixation
Endometrial biopsies were immersion fixed overnight (17 ± 1 h) in 10% buffered formalin at 4 C, then washed three times in Tris-buffered saline (TBS; pH 7.6) and stored at 4 C until embedding in paraffin wax.
Immunohistochemistry
Fractalkine. Localization of fractalkine used a goat polyclonal antihuman fractalkine antibody (SSC-7226 C18, Santa Cruz Biotechnology, Inc., Santa Cruz, CA). A number of positive control tissues (kidney, brain, and heart) with established expression patterns for fractalkine protein were included for validation of the specificity of immunostaining. Negative controls were also included, where the primary antibody was preabsorbed with 1 µg/ml fractalkine blocking peptide (SC-7226, Santa Cruz Biotechnology) at 4 C for 48 h. A section from a single block of endometrial tissue was included in each staining run for quality control. Antibody specificity has previously been confirmed by Western blot analysis (15).
Endometrial sections from all stages of the menstrual cycle, from early pregnancy, and from women using the three types of P-only contraceptives and respective controls (described in Table 1) were immunostained for fractalkine. In the staining runs for Depo-Provera and Norplant tissues, normal tissues (proliferative and secretory, n = 3 for each) were also included to ensure the validity of the conclusions.
Paraffin sections (5 µm) were dewaxed in Histosol (Sigma-Aldrich Corp., St. Louis, MO) and rehydrated through descending grades of alcohol to dH2O. Sections were microwaved at high power (1000 watts) in 0.01 mol/liter sodium citrate buffer (pH 6.0) twice for 5 min each time, then incubated in the hot buffer for an additional 20 min. Endogenous peroxidase activity was quenched with 3% H2O2 in dH2O for 10 min at room temperature. Nonspecific binding was blocked with a nonimmune blocking solution [10% normal horse serum (H0146, Sigma-Aldrich Corp.), 2% normal human serum (in-house) in 0.1% Tween 20 (Bio-Rad Laboratories, North Ryde, Australia) and TBS] in a humidified chamber for 30 min at room temperature. Antifractalkine antibody was applied at 1 µg/ml in nonimmune blocking solution to sections in a humidified chamber overnight (16–18 h) at 4 C. Antibody localization was detected using an avidin-biotin peroxidase detection system after stringent washing in TBS-Tween (0.6%). Biotinylated horse antigoat immunoglobulin G (IgG; Vector Laboratories, Inc., Burlingame, CA) diluted in nonimmune blocking solution was applied for 30 min, followed by avidin-biotin conjugated with horseradish peroxidase (DakoCytomation, Botany, Australia). Positive localization of fractalkine protein was identified by the application of the peroxidase substrate 3,3'-diaminobenzidine (DakoCytomation), which produces a brown precipitate. Tissue sections were counterstained with Harris hematoxylin, dehydrated through ascending grades of ethanol, and mounted.
Fractalkine receptor (CX3CR1). Fractalkine receptor (CX3CR1) was localized using rabbit polyclonal antihuman CX3CR1 (ab7201, Abcam Ltd., Cambridge, UK). A number of positive control tissues (kidney, brain, and heart) with established expression patterns for CX3CR1 were included for validation of the specificity of the immunostaining. Negative controls were included for each section where rabbit IgG (in-house) was substituted at matching concentration to the primary antibody. The immunostaining protocol was the same as that used for fractalkine, except the primary antibody was applied at 2 µg/ml, the nonimmune block used 10% normal swine serum (in-house) instead of horse serum, and the secondary antibody used was biotinylated swine antirabbit IgG (E353, DakoCytomation).
Identification of leukocyte subpopulations positive for fractalkine and CX3CR1. It was not possible to perform dual immunostaining to colocalize fractalkine or CX3CR1 with the various leukocyte subpopulation markers, because the microwave antigen retrieval necessary for fractalkine and CX3CR1 detection is not compatible with the leukocyte marker staining protocols. Instead, immunostaining was conducted for fractalkine/CX3CR1 and leukocyte subpopulation markers on 2-µm serial sections cut from representative endometrial samples. Immunostaining for fractalkine and CX3CR1 was conducted as described above. Leukocyte subpopulations were identified using antibodies directed against various leukocyte cell markers: total leukocytes (CD45, leukocyte common antigen, DakoCytomation), T cells (CD3, DakoCytomation), uNK (CD56, Zymed Laboratories, South San Francisco, CA), macrophages (CD68, DakoCytomation), neutrophils (neutrophil elastase, DakoCytomation), eosinophils (EG1, DakoCytomation), and mast cells (mast cell tryptase, DakoCytomation). Immunostaining protocols were similar to that described for fractalkine. After deparaffinization, sections were subjected to antigen microwave retrieval before staining for CD3 and CD56. Trypsin (Sigma-Aldrich Corp.) digestion (0.2% in 0.2% calcium chloride/TBS) was used for CD68 detection, for 10 min at 37 C. None of the other antibodies required antigen retrieval. After application of nonimmune blocking solution, primary antibodies were applied to each section overnight (16–18 h) at 4 C at the following concentrations: CD45, 1 µg/ml; CD3, 1 µg/ml; CD56, 1.1 µg/ml; CD68, 2.4 µg/ml; neutrophil elastase, 1.3 µg/ml; EG1, 1 µg/ml; and mast cell tryptase, 0.5 µg/ml. Negative controls were included for each tissue section with substitution of the primary antibody at matching concentrations of rabbit IgG and mouse IgG (in-house) as appropriate.
Positive immunostaining was revealed by the sequential application of biotinylated horse antimouse or swine antirabbit IgG (both from Vector Laboratories, Inc.; 1:200 dilution) and avidin-biotinylated alkaline-phosphatase conjugate (DakoCytomation) for 30 min. The chromogen substrate New Fuchsin (DakoCytomation), containing 0.1 M levamisole to quench endogenous alkaline phosphatase activity, was applied to each section, producing a pink precipitate representing the positive labeling of leukocytes. Sections were then counterstained and mounted as described previously.
Analysis of immunostaining
Staining was examined with an Olympus CH30 microscope (Melville, NY) and high resolution images were captured with an Hc-2000 digital camera (Fujix, Tokyo, Japan). The positive immunostaining was scored semiquantitatively by two independent observers blind to the nature of the tissue. The amount of staining within the different cell types in the endometrium (glandular and luminal epithelium, stroma, decidualized stroma, and vessels) was analyzed and allocated a score from 0–3 with 0.5 increments, where 0 is no staining, and 3 is intense staining. In addition, the number of leukocytes staining positively within the tissue stroma was semiquantitatively scored, also using a seven-point scoring system where 0 is no positive cells, and 3 is very abundant positive cells. Statistical analysis was performed using ANOVA, followed by Newman-Keuls test (P < 0.05 was taken as significant) after testing for normal distribution using PRISM version 3.00 for Windows (GraphPad, San Diego, CA).
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The specificity of the fractalkine antibody was verified by the cellular location of specific immunostaining in positive control tissue. Immunoreactive protein was localized in epithelial cells of the collecting tubules in the kidney, in glial cells of the brain, and in endothelial cells of the heart, consistent with previous reports (22, 23, 24, 25). No staining was observed in the negative control, where the fractalkine antibody was preadsorbed with the fractalkine peptide (Fig. 1E, inset).
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Immunoreactive fractalkine protein was detected in endometrium from nonpregnant women at all stages of the menstrual cycle and in decidua from early pregnancy. Immunostaining was predominantly localized to glandular and luminal epithelia, with additional immunostaining in decidualized stromal cells and leukocytes. The intensity and localization of immunostaining varied at different stages of the menstrual cycle (Fig. 1).
During the proliferative phase, fractalkine immunostaining in the glandular epithelium was relatively faint and diffuse (Fig. 1A). As the cycle progressed, immunoreactivity was observed in intensely staining vesicles in glandular epithelial cells (Fig. 1, B, D, and inset). The subcellular localization of the vesicles changed with progression of the menstrual cycle. In the early secretory phase, the immunostaining was predominantly localized basally within glandular epithelial cells (B), whereas during the midsecretory phase, fractalkine immunoreactivity was dispersed throughout these cells (C). In the late secretory phase, glandular immunostaining was mainly localized in the apical region of the glandular epithelium of actively secreting glands (D). Fractalkine immunoreactivity was also present in glandular epithelium of early pregnancy tissue (Fig. 1F, inset). Immunoreactive fractalkine was also detected in the luminal epithelium during most of the cycle, with a marked increase in late secretory phase endometrium, where the staining pattern was similar to that in glandular epithelium (data not shown).
Fractalkine was not detected in endometrial stromal cells throughout most of the menstrual cycle (Fig. 1, A–D, and Fig. 2A). However, with the onset of progesterone-induced decidualization in the mid- to late secretory phase, fractalkine expression was observed in the decidualizing stromal cells and increased with cycle progression (Figs. 1E and 2A). Immunoreactive fractalkine was also observed in the decidual cells of early pregnancy tissue (Figs. 1F and 2A). Fractalkine immunoreactivity was absent from vessels throughout the cycle, except in immediately premenstrual tissue, where strong expression was seen in endothelial and the perivascular cells (Fig. 1E).
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and 2B) and in early pregnancy decidua (Figs. 1F and 2B). Identification of fractalkine-producing leukocytes
Leukocyte common antigen was used as a marker to determine that the cells producing fractalkine were leukocytes (data not shown) before detailed analysis of individual subpopulations (macrophages, uNK, T cells, mast cells, neutrophils, and eosinophils). To determine which endometrial leukocytes were producing the fractalkine protein, very thin (2-µm) serial sections were stained for fractalkine (brown stain) and markers of individual leukocyte subpopulations (pink stain).
Analysis of serially stained sections revealed that fractalkine immunoexpression was present in subsets of uterine NK cells (CD56+; Fig. 1, G and g) and macrophages (CD68+; Fig. 1, H and h) within the human endometrium. Fractalkine was not detectable in T cells, mast cells, neutrophils, and eosinophils (data not shown).
Immunolocalization of CX3CR1 in the endometrium
The specificity of the CX3CR1 antibody was verified by specific immunostaining in positive control tissue. Immunoreactive protein was localized in proximal tubular epithelial cells of the kidney, resting microglial cells of rat brain, and endothelial cells of the heart, which was consistent with previous reports (24, 25, 26). No positive immunostaining was observed in the negative control (Fig. 1O, inset).
CX3CR1 immunostaining in the normal human endometrium
CX3CR1 immunostaining was detected in the endometrium from all stages of the menstrual cycle and in decidua from early pregnancy. CX3CR1 immunostaining in the glandular epithelium was low during the early stages of the menstrual cycle (Figs. 1J and 3A), with an increase in staining intensity during the secretory phase (Figs. 1N and 3A). In contrast, luminal epithelial cells were consistently negative for CX3CR1 expression at all stages of the cycle (data not shown).
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, J and N, and Fig. 3A), but some immunostaining was evident with the onset of decidualization in the mid- to late secretory phase (Fig. 3A). Some CX3CR1 expression was also observed in early pregnancy decidua (Figs. 1O and 3A). Endometrial vessels were weakly immunoreactive for CX3CR1 throughout the cycle and in early pregnancy without a significant change (Fig. 1, N and inset).
As anticipated, CX3CR1 was identified on peripheral blood leukocytes in the endometrial vessels and on a subset of endometrial leukocytes within the stroma (Fig. 1, I–L and N, and Fig. 3B). Increased numbers of leukocytes positive for CX3CR1 were detected in the menstrual phase (Fig. 1I), with consistent numbers thereafter across the cycle (Fig. 3B). Many leukocytes positive for CX3CR1 appeared to be tethered to vessel walls or were localized in the tissue surrounding blood vessels (Fig. 1, K, L, N, and inset). These leukocytes possess the morphological characteristics of neutrophils (Fig. 1Q).
Identification of CX3CR1-positive leukocytes
CX3CR1 was present on a large subset of endometrial leukocytes (Fig. 3B) throughout the cycle, particularly in the menstrual phase. To determine which cells expressed CX3CR1, CX3CR1 and markers of individual leukocyte subpopulations (macrophages, uterine natural killer cells, T cells, mast cells, neutrophils, and eosinophils), were colocalized on serially stained sections. The expression of CX3CR1 was detected on subpopulations of macrophages (CD68; Fig. 1, M and m) and neutrophils (Fig. 1, P and p). CX3CR1 was not detected on eosinophils, mast cells, T cells, or NK cells (data not shown).
Localization of fractalkine in endometrium from women using P-only contraceptives
To determine whether fractalkine protein production was altered in progestin-exposed endometrium, immunohistochemical analysis of fractalkine expression was performed on endometrium exposed to LNG-IUS, Depo-Provera, and Norplant.
Lng-IUS
After insertion of the Lng-IUS, the endometrium becomes thin and atrophic, with highly decidualized stroma (27). In particular, glandular activity ceases, and epithelial glands become very narrow and atrophic. After insertion of Lng-IUS, immunostaining in glandular epithelial cells declined compared with that in the preinsertion control (proliferative and secretory) endometrium (Figs. 1R and 4A). Strong immunostaining for fractalkine was observed in the extensively decidualized stroma, correlating to the extent of decidualization (Figs. 1R and 4A). The number of leukocytes immunoreactive for fractalkine was increased in women using Lng-IUS compared with preinsertion endometrium (Fig. 4A), consistent with the numbers seen in early pregnant decidua.
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Administration of injectable progestin DMPA causes extensive decidualization of endometrial stromal cells. Glandular epithelium exposed to Depo-Provera had much reduced fractalkine immunostaining compared with control (proliferative and secretory) endometrium (Figs. 1S and 4B). Whereas fractalkine production was significantly increased in the decidualized stromal cells and in leukocyte subpopulations (Figs. 1S and 4B) compared with control endometrium (Fig. 4B).
Norplant
Immunoreactive fractalkine was significantly reduced in glandular epithelial cells in all classes of Norplant tissue (Fig. 4B), but increased immunoreactive fractalkine was observed in the decidualized stroma of Norplant tissue that had been classified as progesterone modified (decidualized; Fig. 1T) compared with control endometrium. However, no staining was observed in the stroma of tissue classified as atrophic or shedding (Fig. 4B). Fractalkine-positive leukocyte numbers were not increased in Norplant tissues compared with control tissues (Fig. 4B).
Localization of CX3CR1 in endometrium from women using P-only contraceptives
CX3CR1 was not assessed in tissue from women using LNG-IUS or Depo-Provera because insufficient tissue was available.
Norplant
After Norplant insertion, expression of CX3CR1 was not notably altered in endometrial glands compared with control endometrium regardless of tissue morphology (atrophic, shedding, and progesterone modified; Fig. 4C). Strong immunostaining for CX3CR1 was observed in the extensively decidualized stroma of Norplant tissue that was classified as having a progesterone-modified appearance (Figs. 1U and 4C). No stromal staining was observed in the stroma of the other two Norplant classifications, in which there was no decidualized stroma. Similar levels of leukocytes immunoreactive for CX3CR1 were observed for the three different Norplant classes (Fig. 4C), significantly lower than in control endometrium.
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Discussion |
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Fractalkine is a potent chemoattractant for a wide range of relevant leukocyte subtypes, making it an attractive candidate for the endometrial recruitment of leukocytes, particularly monocytes and neutrophils, whose numbers rise dramatically in the perimenstrual phase. Fractalkine is the first chemokine identified that is able to mediate leukocyte attraction, capture, firm adhesion, and activation in the absence of integrins (15), a property resulting from its unique membrane-anchored structure. It may therefore act independently, in the absence of other proinflammatory mediators, during the initial stages of leukocyte recruitment before and during menstruation. Previously, fractalkine expression has been observed in a number of tissues (17, 22, 28, 29, 30, 31, 32), including uterine decidual cells at the implantation site (33). However, this is the first description of fractalkine protein production in nonpregnant endometrium during the menstrual cycle or under the influence of P-only contraceptives. Furthermore, fractalkine production by endometrial leukocytes was previously unknown. Although immunoreactive fractalkine is present in endometrium throughout the menstrual cycle, maximal production was noted in the secretory phase, and importantly, its production by endothelial cells in vessel walls was only detected immediately premenstrually.
Increased fractalkine in the early to midsecretory phase of the menstrual cycle with increasing levels continuing into early pregnancy corresponds temporally to the infiltration of pregnancy-associated leukocytes, uNK and macrophages. However, a recent study has reported that the putative precursors of the uNK cells (34), a subset of circulating NK cells identified as CD56bright and CD16–, do not possess CX3CR1 and do not migrate in vitro in response to fractalkine (35). We have confirmed that the receptor is not expressed by uNKs, whereas it is strongly expressed by a subpopulation of endometrial macrophages. Therefore, fractalkine may be involved in the recruitment, differentiation, activation, or homing of pregnancy-associated monocytes.
The increased production of fractalkine seen premenstrually by glands and decidualized stroma and particularly the up-regulation in vascular endothelium suggest a role in the initial recruitment of leukocytes, particularly neutrophils and macrophages, from the periphery during the early stages of menstruation. Fractalkine is a potent neutrophil chemoattractant (16), and the identification in this study of CX3CR1 on endometrial neutrophils and macrophages, especially those in or immediately surrounding vessels, also supports such a role for fractalkine. This data are summarized diagrammatically in Fig. 5.
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Subpopulations of macrophages and uNK were immunoreactive for fractalkine, with elevated numbers of stained cells in the early proliferative phase, where they may be involved in regeneration of the endometrium. During early pregnancy, uNK probably have important roles in regulating decidualization, spiral arteriole remodeling, and trophoblast invasion (42). The production of fractalkine by endometrial leukocytes has the potential to amplify focal leukocyte accumulation and activation in specific areas, such as those of tissue breakdown (late secretory/menstrual phases), regeneration (proliferative phase), or decidualization (late secretory phase).
CX3CR1 was expressed by subpopulations of neutrophils and macrophages. Some were observed to be tethered to vessel walls or localized in the stroma surrounding vessels as described previously for neutrophils (43). It appeared that these cells were migrating through the endothelium and invading the tissue. Their numbers were elevated in the menstrual phase. CX3CR1 expression by macrophages highlights a probably direct role for fractalkine in their recruitment into the endometrium, and the time of high expression coincides with macrophage accumulation. CX3CR1 was not present on T cells within the endometrium. Fractalkine is a potent chemoattractant of T cells in other inflammatory situations; this suggests that fractalkine is not involved in the recruitment of T cells to the endometrium, or that CX3CR1 may be down-regulated after ligand binding or recruitment into the endometrium. Chemokine receptor expression can be altered on leukocytes depending on their activation status (44, 45); once recruited to the endometrium, the leukocytes may switch on or off the expression of CX3CR1. Phenotypically distinct populations of macrophages can be found within the human endometrium (2), and these may be differentiating or changing activation status. It is likely that multiple chemokines, including fractalkine, acting sequentially or in combination, are involved in the initial recruitment, extravasation, homing, and activation of leukocytes within the endometrium.
Fractalkine is predominantly detected in the glandular epithelium, where it appeared to be contained within secretory vesicles that progressed from the basal to apical region of the actively secreting glands as the cycle progressed, suggesting that fractalkine is being secreted into the uterine lumen. Glandular secretions are believed to be important for endometrial receptivity and may be envisaged to interact with the preimplantation embryo. Indeed, human blastocysts have recently been shown to express chemokine receptors (46, 47). Should CX3CR1 be expressed by human blastocysts, fractalkine could act on the periimplantation phase blastocyst and stimulate cell activation, up-regulate adhesion molecules, and hence assist in attracting the blastocyst to the uterine epithelium or homing the invading trophoblast cells to maternal blood vessels. The processes involved in leukocyte migration (i.e. rolling, attachment, and migration from the endothelium into the tissue) are remarkably similar to those events occurring during blastocyst implantation and trophoblast invasion. Recently, a ligand-receptor system that allows leukocyte capture from the endothelium and migration into tissues has been shown to be functional between the endometrium and the blastocyst (48). A system involving fractalkine may likewise prove important at implantation.
Glandular epithelial cells, but not luminal epithelial cells, coexpress CX3CR1 during the secretory phase, and thus, it is possible that fractalkine has autocrine/paracrine actions on the glandular epithelium due to the high expression of ligand and receptor during the secretory phase. The high expression of fractalkine and CX3CR1 coincides with the time of maximal glandular activity; a potential role therefore exists for fractalkine in glandular remodeling and activity.
The increase in fractalkine production during the secretory phase is suggestive of positive progesterone regulation. This is likely to be an indirect action or via a membrane-bound progesterone receptor, because secretory phase glandular epithelium, decidualized stromal cells, and leukocytes do not possess nuclear progesterone receptor. The apparently biphasic pattern of fractalkine production by the epithelium across the cycle (i.e. a decrease in the midsecretory phase) is difficult to explain given that progesterone levels are high at this time. However, it is consistent with our findings for fractalkine production by the epithelium in women using P-only contraceptives, in whom epithelial fractalkine production decreased after P-only use. However, the reduction seen in the midsecretory phase was much less pronounced than that seen in P-only contraceptive users.
Fractalkine was overall increased in the endometrium of women using P-only contraceptives, but only those in which decidualization is a feature (Lng-IUS, Depo-Provera, and some Norplant tissues). By contrast, production was reduced in glandular epithelium after Lng-IUS insertion and Depo-Provera use, particularly with long-term use, consistent with the decreased secretory activity of glands in such women (5, 27, 49). Increased numbers of leukocytes producing fractalkine were also observed in the endometrium from women using Lng-IUS and Depo-Provera, consistent with increased distribution of uNK and macrophages in the endometrium in these women (5, 20, 50).
Differences in the morphological appearance of Norplant-exposed endometrium have been noted in the past and have made it difficult to compare with Lng-IUS- and Depo-Provera-exposed endometria. This sc delivery system results in lower uterine progestin levels. The morphology of Norplant-exposed endometrium can be described as hypoplastic, with poorly secretory glands, progestin-modified or shedding (21, 51). In the absence of decidualization, fractalkine was absent from stromal cells, and only low numbers of fractalkine positive leukocytes were observed, whereas in extensively decidualized tissue, fractalkine was elevated in the decidualized stroma and in the leukocyte subpopulations. However, glandular production of fractalkine was reduced in all three morphologies, consistent with the two other types of P-only contraceptives.
In conclusion, this paper describes the temporal and cellular location of fractalkine and its receptor within the human endometrium, supporting a role for this chemokine in recruiting leukocytes in the secretory phase, during menstruation, and during early pregnancy. A direct role can be predicted for fractalkine in recruiting macrophages and neutrophils into the endometrium due to the detection of CX3CR1 on subsets of these. This is an important finding, because the mechanisms of their appearance in the uterus are poorly understood. The production of fractalkine by glandular and luminal epithelium and decidualized stroma along with the presence of CX3CR1 on glandular epithelium and decidualized stroma suggest important additional roles for fractalkine, possibly embryo implantation and blastocyst homing. Additional studies investigating the presence of CX3CR1 on the human blastocyst and trophoblast cells are needed. The presence of both fractalkine and CX3CR1 on glands and decidua also suggests roles in endometrial remodeling.
Fractalkine expression was altered in the endometria of women using P-only contraceptives. Its elevated levels in progestin-induced decidualized endometrium support its involvement in the abnormal recruitment of leukocytes in such tissue. Fractalkine may contribute to the altered endometrial microenvironment and thus to the endometrial fragility observed with the use of these contraceptives.
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Acknowledgments |
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Footnotes |
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Abbreviations: DMPA, Depo-medroxyprogesterone acetate; Lng-IUS, levonorgestrel-intrauterine system; P-only, progestin-only; TBS, Tris-buffered saline; uNK, uterine-specific natural killer cell.
Received August 7, 2004.
Accepted September 14, 2004.
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 W. V. Ingman and R. L. Jones Cytokine knockouts in reproduction: the use of gene ablation to dissect roles of cytokines in reproductive biology Hum. Reprod. Update, March 1, 2008; 14(2): 179 - 192. [Abstract] [Full Text] [PDF]
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 R.D. Catalano, H.O. Critchley, O. Heikinheimo, D.T. Baird, D. Hapangama, J.R.A. Sherwin, D.S. Charnock-Jones, S.K. Smith, and A.M. Sharkey Mifepristone induced progesterone withdrawal reveals novel regulatory pathways in human endometrium Mol. Hum. Reprod., September 1, 2007; 13(9): 641 - 654. [Abstract] [Full Text] [PDF]
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 M.-P. Belot, L. Abdennebi-Najar, F. Gaudin, M. Lieberherr, V. Godot, J. Taieb, D. Emilie, and V. Machelon Progesterone reduces the migration of mast cells toward the chemokine stromal cell-derived factor-1/CXCL12 with an accompanying decrease in CXCR4 receptors Am J Physiol Endocrinol Metab, May 1, 2007; 292(5): E1410 - E1417. [Abstract] [Full Text] [PDF]
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N. J. Hannan, R. L. Jones, C. A. White, and L. A. Salamonsen The Chemokines, CX3CL1, CCL14, and CCL4, Promote Human Trophoblast Migration at the Feto-Maternal Interface Biol Reprod, May 1, 2006; 74(5): 896 - 904. [Abstract] [Full Text] [PDF]
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E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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 R. L. Jones, N. B. Morison, N. J. Hannan, H. O.D. Critchley, and L. A. Salamonsen Chemokine expression is dysregulated in the endometrium of women using progestin-only contraceptives and correlates to elevated recruitment of distinct leukocyte populations Hum. Reprod., October 1, 2005; 20(10): 2724 - 2735. [Abstract] [Full Text] [PDF]
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 R. L. Jones, N. J. Hannan, T. J. Kaitu'u, J. Zhang, and L. A. Salamonsen Identification of Chemokines Important for Leukocyte Recruitment to the Human Endometrium at the Times of Embryo Implantation and Menstruation J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6155 - 6167. [Abstract] [Full Text] [PDF]
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). More intense staining was seen in the early secretory (B), midsecretory (C), and late secretory (D and E) phases. Staining appeared to be in vesicles (V) in the basal (ba) region in GE in the early secretory phase (B), more diffuse in the midsecretory phase (C), and apically located (ap) in the late secretory phase (D and inset), when strong staining was also observed in blood vessels (bv) and decidualizing stroma (Dec) surrounding the spiral arterioles (E). Fractalkine was also identified in decidua (Dec) in early pregnancy (F). No staining was observed after preadsorption of antibody with fractalkine peptide (E and inset). Fractalkine-staining leukocytes were identified by colocalization studies on adjacent 2-µm sections. Staining pairs are fractalkine (G) and CD68+ (g) macrophages, and fractalkine (H) and CD56+ (h) uNK cells. CX3CR1 was expressed in leukocytes (
