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Prince Henry’s Institute of Medical Research (R.L.J., N.J.H., T.J.K., J.Z., L.A.S.), Clayton, Victoria 3168, Australia; and Department of Immunology (N.J.H.), Monash University, Prahran, Victoria 3181, Australia
Address all correspondence and requests for reprints to: Rebecca Jones, Prince Henry’s Institute of Medical Research, Clayton, Victoria, Australia 3186. E-mail: rebecca.jones{at}phimr.monash.edu.au.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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During every menstrual cycle, the endometrium rapidly regenerates and proliferates after menses; differentiates to provide a specialized environment for embryo implantation; and, in a nonfertile cycle, breaks down and is shed during menstruation. Although these changes are governed by the ovarian steroid hormones, estrogen and progesterone, there is increasing evidence for the local regulation of these endometrial processes. In particular, the differentiation or decidualization of stromal cells before implantation is initiated around spiral arterioles and glands and spreads progressively throughout the endometrium if pregnancy is achieved, suggesting paracrine rather endocrine triggers (4). Similarly, endometrial breakdown and shedding is initiated at focal points throughout the endometrium, with areas of breakdown evident coincident with regeneration of neighboring areas (5).
Infiltrating immune cells are likely candidate effector cells for initiating and facilitating these processes. There is a large accumulation of endometrial leukocytes in the periimplantation phase and during early pregnancy, and a dramatic influx in the immediate premenstrual phase and during menses (4, 5, 6). At these times, leukocytes comprise up to 40% of total endometrial stromal cells. These are present, scattered throughout the stroma, but also seem to be specifically targeted to areas of breakdown or decidualization. Importantly, distinct leukocyte subpopulations are present at the different stages of the menstrual cycle: decidual/pregnancy-associated leukocytes are predominantly a subpopulation of macrophages and uterine-specific natural killer (uNK) cells (4, 7), whereas in the perimenstrual period, there is a dramatic influx of inflammatory-type leukocytes: neutrophils, eosinophils, macrophages, and an activation of mast cells (8, 9, 10).
uNK cells begin to infiltrate the endometrium on d LH+3, specifically accumulate around spiral arterioles and areas of decidualized stroma, and are present in the decidua until the second trimester of pregnancy (11). This highly specialized population of immune cells is a fundamental component of the implantation site, creating a unique immunological environment permissive to, yet regulating, the invasion of fetal cytotrophoblast cells (11, 12). Furthermore, recent evidence from mice lacking natural killer (NK) cells suggests important roles in spiral arteriole remodeling and decidualization (13, 14, 15). Macrophages comprise 20% of endometrial leukocytes and are present during periods of endometrial proliferation, differentiation, and breakdown. There is a marked accumulation of endometrial macrophages specifically in areas of decidualization and trophoblast invasion. These cells are a source of growth factors, cytokines, and proteases, creating local microenvironments permissive to tissue remodeling and have been proposed to participate in fetal-maternal interactions in the implantation site (16, 17, 18). Inflammatory leukocytes (neutrophils, eosinophils, and mast cells) enter the endometrial stroma premenstrually before the first signs of breakdown are evident. These cells produce and locally secrete proteases [matrix metalloproteinases (MMPs), leukocyte enzymes] capable of initiating extracellular matrix breakdown and activating inflammatory mediators and proteases, which together could trigger an inflammatory cascade culminating in endometrial shedding (19). Therefore, the recruitment and activation of these two distinct groups of leukocyte subpopulations must be tightly and specifically regulated at the critical times of embryo implantation and during endometrial destruction.
Leukocyte migration into tissues is regulated by chemokines, an ever-growing family of chemotactic cytokines. More than 50 chemokines have been identified to date, with a large degree of redundancy and overlap of functions (20, 21, 22). Chemokines classically act on specific leukocyte subsets to increase their adhesion to the endothelium, through up-regulation of adhesion molecules, and induce their extravasation and chemotaxis along a concentration gradient. Chemokines also play important roles in both homing of leukocytes to specific regions within a tissue and as potent activators of leukocytes (23, 24). Importantly, the sequential or combinatorial action of multiple chemokines is probably necessary for the recruitment, homing, and activation of a single leukocyte subtype (25). Chemokines act locally and are rapidly and transiently induced in response to an inflammatory stimulus. However, there is recent evidence for the constitutive expression of certain chemokines that are responsible for immunosurveillance and tissue homeostasis (24, 26, 27).
Chemokines are likely to be critical mediators of leukocyte recruitment to the endometrium. There are a number of reports describing the expression and regulation of individual chemokines in the endometrium, including IL-8, monocyte chemotactic proteins (MCPs)-1 and -2, macrophage inflammatory protein (MIP)-1
, eotaxin, and regulated upon activation, normal T cell expressed, and secreted (RANTES) (28, 29, 30, 31, 32). However, given the large number of chemokine family members and the combinatorial actions of chemokines in leukocyte infiltration, homing, and activation, tightly regulated expression of multiple chemokines is likely to be critical in controlling leukocyte recruitment to the endometrium at different stages of the menstrual cycle and creating the unique immunological environment for implantation.
To address this and identify which are the critical chemokines in human endometrium, we used a gene array approach to conduct an unbiased screen of chemokine expression in endometria across the menstrual cycle with focus on the stages corresponding to recruitment of specific subpopulations of leukocytes. Nine chemokines were found to be abundantly expressed in human endometrium, with clear cyclical variability in expression levels. These chemokines were analyzed in depth at the level of mRNA expression and protein localization to produce a clear profile of chemokine expression during endometrial breakdown, repair, and embryo implantation. This study therefore provides a major advance in understanding the integral roles of chemokines in mediating endometrial function, both through their effects on leukocyte recruitment and on remodeling of the endometrium.
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Patients and Methods |
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Endometrial tissue was obtained by dilation and curettage from women with no endometrial abnormalities undergoing minor gynecological surgical procedures, such as laparoscopic sterilization or investigation of tubal patency. All women described regular menstrual cycles and had not used hormonal treatment in the previous 3 months. Endometrial samples were collected at all stages of the menstrual cycle: menstrual (d 1–3), proliferative (d 8–10), early secretory (d 14–17), midsecretory (d 20–23), and premenstrual (d 26–28) phases. Stage of cycle was confirmed by histological assessment according to the criteria of Noyes et al. (33). Decidual tissue was also collected by curettage from women undergoing elective termination of pregnancy (amenorrhea 6–8 wk). Written informed consent was obtained from all participants, and ethical approval was obtained from the appropriate institutional ethics committees.
Endometrial samples were divided: one portion was fixed in 10% buffered formalin for 17 ± 1 h before washing in Tris-buffered saline (TBS) and routine histological processing to paraffin blocks, whereas the remainder was immersed in RNALater solution (Ambion, Austin, TX) before snap freezing and storage at –80C for RNA extraction.
RNA extraction and purification
Total RNA was extracted from endometrial samples by homogenization in Trizol reagent (Qiagen Sciences, Clifton Hill, Victoria, Australia), according to manufacturer’s instructions, with the exception of an additional chloroform extraction step to minimize carryover of phenol into the precipitated RNA. All samples were treated with RNase-free DNase (Ambion) to remove the possibility of genomic DNA contamination. RNA samples were then analyzed by spectrophotometry to determine RNA concentration, yield, and purity. Any samples with ratios of A260/280 less than 1.7 or A230/280 more than 1 were purified through RNeasy spin columns (Qiagen) according to manufacturer’s instructions and thereafter reanalyzed by spectrophotometry. One microgram of purified RNA was then run on a 1% agarose (Roche, Castle Hill, NSW, Australia) gel to ensure integrity of rRNA subunits.
RNA concentrations were also analyzed by Ribogreen fluorescence RNA assay (Molecular Probes, Eugene, OR). RNA samples were diluted to approximately 40 ng/ml based on spectrophotometric readings and analyzed in a 96-well plate in conjunction with a standard curve of serially diluted rRNA (Molecular Probes) between 0 and 80 ng/well, by addition of Ribogreen fluorescent dye at a dilution of 1:500. This assay gave an intraassay coefficient of variation, as defined by the repeated analysis of a single RNA sample, of 3.3% (n = 16) and an interassay variation of 10.3% (n = 14).
Gene array
Screening of chemokine mRNA expression in endometria at times of maximal leukocyte recruitment (menstrual, midsecretory, and premenstrual phases), compared with a time of low leukocyte recruitment (proliferative phase), was conducted using the human common chemokine GeArray nylon membrane (SuperArray Bioscience Corp., Bethesda, MD) containing cDNA probes for 21 chemokine family members in duplicate (for a full gene list, see Table 2
). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin were included as positive controls and PUC18 plasmid DNA as a negative control. RNA samples (n = 5–8) from these four stages of the cycle were pooled, and 5 µg total RNA were reverse transcribed to cDNA using Moloney murine leukemia virus-reverse transcriptase (Promega, Annandale, NSW, Australia) in the presence of biotinylated uridine 5-triphosphate (Roche) and incubated overnight in hybridization buffer (SuperArray) containing 100 µg/ml denatured sheared salmon sperm DNA (Invitrogen Australia, Pty. Ltd., Mount Waverley, VIC, Australia) with prehybridized nylon arrays at 68 C. Posthybridization washes with saline sodium citrate/sodium dodecyl sulfate were conducted at 68 C. Positive cDNA binding was thereafter detected by application of a streptavidin-alkaline phosphatase conjugate in a supplied blocking solution and chemiluminescent substrate CDP-Star (SuperArray). Membranes where exposed to x-ray film for a range of times (between 2 sec and 5 min) to ensure quantitation during the linear phase of the reaction. All arrays were conducted simultaneously, and the entire experiment was repeated. X-ray films were scanned at high resolution and densitometrically analyzed using GelDoc software (Bio-Rad Laboratories, Regents Park, NSW, Australia). Chemokine signals were normalized for the expression levels of GAPDH. Statistical analysis was performed using GBStat (GBStat, Silver Spring, MD).
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Standards for real-time PCR were generated by conventional PCR. A representative endometrial RNA sample (1 µg) was reverse transcribed using AMV-RTase (Promega) and 100 ng random hexanucleotide primers (Amersham Biosciences, Piscataway, NJ), and the cDNA generated was subsequently amplified by PCR for each individual chemokine (Hybaid Express block cycler; Hybaid Ltd., Ashford, UK) using the optimized conditions described in Table 1. Products were electrophoresed on a 2% agarose gel, the bands excised, and cDNA purified using UltraClean GelSpin columns (MoBio Laboratories Inc., Solana Beach, CA). The resultant PCR-generated cDNAs were quantitated by spectrophotometry, sequenced to confirm identify, and thereafter used as standards for real-time RT-PCR.
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). An additional stage at 88 and 84 C for 5 sec was included for fractalkine (FKN) and MIP-1ß, respectively, to exclude quantitation of primer dimers. All samples to be compared were included within the same run, and the entire PCR experiment was repeated. mRNA expression was quantitated by comparison with a 4-point standard curve of serially diluted (10-fold) standards, with top standards of between 50 pg/µl and170 fg/µl, depending on expression level of individual chemokines. Fluorescence from incorporation of SYBR green into double-stranded PCR products was monitored continuously during cycling at the end of each elongation phase, and quantitation of mRNA expression was performed when amplified products were in the log-linear phase and parallel to the standards. A quality control (a single endometrial cDNA sample) was included in every run. Using this method, we achieved an overall 14.9 and 16.1% intra- and interassay variability, respectively. At the end of each program, melting curve analysis was carried out to ensure specificity of the reaction products. The sizes of the products were also confirmed by gel electrophoresis for selected samples.
Mean expression levels were calculated from the triplicate reverse transcriptions (RTs) and duplicate PCRs, and statistical analysis was performed using GBStat. Data were not normalized for the expression of a housekeeping gene due to the regulated expression of all known housekeeping genes examined (18S, cyclophilin, GAPDH, ß-actin) in the endometrium (data not shown). Instead, RNA samples were meticulously purified and quantitated by three distinct methods to ensure confidence in equal RNA loading and RT efficiency and reproducibility (intraassay variability < 15%). Data were statistically analyzed by ANOVA with Tukey’s post hoc test. A value of P < 0.05 was considered statistically significant.
Immunohistochemistry
Immunohistochemistry was performed for six of the nine chemokines [MCP-3, 6Ckine, macrophage-derived chemokine (MDC), hemofiltrate CC chemokine (HCC)-1, HCC-4, MIP-1ß] using polyclonal antibodies raised against human chemokine peptides (MIP-1ß: R&D Systems Inc., Minneapolis, MN; all other antibodies: Santa Cruz Biotechnology, Santa Cruz, CA) on endometrial sections from all stages of the menstrual cycle and early pregnancy (n = 5–8/group). Positive controls (kidney and ovary) were included in every run. Briefly, 5-µm sections of formalin-fixed, paraffin-embedded tissues were dewaxed, rehydrated, and exposed to microwave antigen retrieval for 2 x 5 min on high, followed by 20 min cooling. Endogenous hydrogen peroxidase activity was quenched using 3% H2O2 in dH20 for 5 min at RT. Nonspecific binding was prevented by preincubation of tissue sections with a nonimmune block containing 10% nonimmune horse serum (Sigma-Aldrich, Sydney, Australia), 2% normal human serum (in-house) in TBS and 0.1% Tween 20. Primary antibodies were applied overnight (17 ± 1 h) at 4 C diluted to 1–4 µg/ml in nonimmune block: MCP-3, 6Ckine, MDC, HCC-1, HCC-4, MIP-1ß. Negative controls were included whereby the antibodies were preabsorbed for 48 h with a 5-fold excess of specific chemokine peptide (Santa Cruz Biotechnology). A nonimmune goat IgG (R&D Systems), diluted to a matching concentration as the primary antibody, was also included for each tissue.
After stringent washing with TBS and 0.6% Tween 20, detection of positive binding was performed by the sequential application of biotinylated horse antigoat IgG (1:200 in nonimmune block; Vector Laboratories, Burlingame, CA) and avidin-biotin-peroxidase conjugate (Dako, Glostrup, Denmark), followed by the substrate diaminobenzidine (Dako) for between 2 and 10 min. Wherever possible, samples to be analyzed were included in the same run; otherwise, quality controls were included in each run. Sections were counterstained with Harris’ hematoxylin (Sigma), dehydrated, and mounted from Histosol with DPX mounting medium (BDH Laboratory Supplies, Poole, UK). Immunostaining was analyzed semiquantitatively by two independent observers blind to the stage of the cycle. Staining intensity and heterogeneity in each of the endometrial compartments (epithelium; stroma, including decidualized stromal cells; and vasculature) was assessed and allocated a score between 0 and 3 where 0 = no stain, 1 = faint staining, 2 = strong staining, and 3 = very intense staining. For immunostaining in leukocytes, infiltrating the endometrial stroma, a semiquantitative score between 0 and 2 was allocated based on their relative abundance where 0 = absent, 1 = few positive leukocytes, 2 = abundant positive leukocytes.
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Results |
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The chemokine expression profile of human endometrium during times of leukocyte recruitment was determined by gene array. Endometrium was examined in the perimenstrual phases (premenstrual and menstrual), when there is a dramatic infiltrate of neutrophils, eosinophils, and macrophages; at the time of embryo implantation (midsecretory), when macrophages and uNK cells are increasing in number; and compared with the proliferative phase when there is low leukocyte recruitment (predominantly macrophages).
Of the 21 chemokines on the GeArray (Table 2), nine were found to be highly abundant in the endometrium, with clear variations in mRNA expression levels evident at the different stages of the menstrual cycle (Fig. 1). These chemokines were MCP-3, eotaxin, FKN, MIP-1ß, 6Ckine (also known as secondary lymphoid chemokine), IL-8, HCC-1, HCC-4, and MDC. All other chemokines were expressed below the detection sensitivity level of this technique (Table 2).
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. MCP-3, eotaxin, FKN, and MIP-1ß were found to be the most abundant chemokines in the endometrium at all stages of the menstrual cycle. MCP-3 in particular was very highly expressed in all endometrial samples examined. Five additional chemokines were expressed at lower levels and were not detectable at all stages of the menstrual cycle examined. 6Ckine and HCC-4 were expressed at moderate/low levels in menstrual, proliferative, and midsecretory stages, whereas MDC and HCC-1 were detectable only in menstrual and midsecretory phase endometria. IL-8 was highly expressed only in the menstrual phase. Statistical analysis was not performed on gene array data due to obvious differences in labeling efficiencies using this system, which necessitated normalization with GAPDH. This technique was instead used primarily to select candidate chemokines for subsequent detailed analysis of mRNA expression patterns and protein localization.
Real-time RT-PCR
Real-time quantitative RT-PCR was conducted to verify that the chemokines identified by gene array are expressed in the endometrium and provide a more accurate quantitative analysis of expression pattern for each chemokine during the menstrual cycle and early pregnancy. mRNA expression levels were quantitated by comparison with a purified PCR-generated cDNA standard of known concentration for each chemokine.
All nine chemokines were confirmed to be expressed by endometrium, and the relative abundance of each was generally borne out by the PCR data (Fig. 2). MCP-3 was confirmed the most abundant chemokine (followed by MDC, FKN, 6Ckine, and MIP-1ß), with a peak expression level of 300 fg/µl, between 3 and 100 times higher than other chemokines.
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Outliers from this general pattern were FKN and MDC, which were expressed at relatively constant levels at all stages of the menstrual cycle, and IL-8, which was undetectable at any stage of the menstrual cycle apart from during menstruation when it was dramatically up-regulated, and was also highly expressed in early pregnancy.
Immunohistochemistry results
Immunohistochemistry was performed to verify chemokine protein production and determine the cellular source (and hence potential site of action) of each chemokine. The specificity and selectivity of the chemokine antibodies were confirmed by optimization of immunohistochemical staining conditions on positive control tissues (kidney and ovary) with known localization of the chemokine in question (34). In each case, the positive localization of the chemokine achieved was consistent with previous published data. Furthermore, antibodies were preabsorbed with 1- and 5-fold concentrations of immunogenic peptide, resulting in an absence of staining.
All chemokines examined (HCC-1, HCC-4, MCP-3, MIP-1ß, MDC, 6Ckine) were detectable at the protein level in the endometrium, with differences evident in temporal and spatial immunostaining patterns between chemokines, and for each chemokine at the different stages of the menstrual cycle (see Figs. 3 and 5). We have previously described immunoreactive protein localization of eotaxin, IL-8, and FKN (28, 31, 35).
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and 5). Chemokines could be broadly grouped into three groups according to their epithelial production levels. 6Ckine and MDC were produced at low levels in the menstrual phase and proliferative phases and were dramatically up-regulated in the mid-late secretory phases and in early pregnancy. MIP-1ß and HCC-4 were both produced by menstrual phase glands and were maximal in proliferative phase epithelia, and immunoreactivity declined toward the end of the menstrual cycle. HCC-1 was highly abundant in glands in all but the menstrual phase, whereas MCP-3 was highly abundant throughout the cycle.
Decidual cells first detectable in the mid-late secretory phase and abundant in early pregnancy were a major source of all chemokines (although MCP-3 and MIP-1ß appeared to be produced only by highly decidualized cells) (see Figs. 3 and 5). Overall, chemokine immunostaining was maximal in epithelial and decidual cells in early pregnancy. HCC-1 and HCC-4 were intensely localized to the vasculature (endothelium and supporting perivascular cells) throughout the menstrual cycle and early pregnancy, whereas MDC, 6Ckine, and MIP-1ß were specifically up-regulated in the vessels in the mid-late secretory phase (Figs. 3 and 4), as seen previously for IL-8, eotaxin, and FKN (28, 31, 35). Subpopulations of infiltrating leukocytes were strongly positive for all chemokines, with elevated numbers of leukocyte producing chemokines evident at times of peak leukocyte accumulation (Figs. 3 and 5). Importantly, during the menstrual phase, the highly abundant infiltrating leukocytes were the major source of chemokines.
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), and with the subcellular location changing from basal to apical as the secretory phase progressed (as shown for MCP-3, Fig. 5). In highly decidualized stromal cells (i.e. in early pregnancy but not in the late secretory phase), intense vesicular-type staining for MCP-3 was present in the differentiated stromal cells (data not shown). |
Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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After selection of candidates by gene array, real-time RT-PCR was used to accurately assess mRNA abundance and regulation across the menstrual cycle and early pregnancy. Immunolocalization of chemokine protein provided invaluable information regarding function because chemokines are short lived and very locally acting proteins. Vascular localization suggests roles in initial recruitment into the endometrium, whereas expression in epithelium and decidua indicates involvement in activation and homing of leukocyte subpopulations to specific focal areas. Localization of chemokines is critical, considering the highly heterogeneous nature of endometrium, particularly at the times of menstruation and decidualization. This study demonstrates clearly the limitation of gene expression studies using whole-tissue homogenates, which dilute out localized changes in chemokine production, such as the focal up-regulation of MDC in vessels premenstrually, and HCC-4 and MIP-1ß in epithelial glands in the early proliferative phase. Certain chemokines, e.g. RANTES, MIP-1, and MCP-1, have been described previously in the endometrium (28, 30, 32) but were expressed below the sensitivity limit of this technique. This indicates either low expression levels or tightly restricted spatial expression, but due to their locally acting nature, they may nonetheless be physiologically important.
These data enabled functional grouping of the chemokines into implantation-, menstruation-, and proliferative-associated chemokines (Fig. 6). A large number of chemokines (MDC, MCP-3, FKN, 6Ckine, HCC-1/4, MIP-1ß) were up-regulated in the midsecretory phase and maintained in early pregnancy. These are chemoattractants for macrophages, and all but HCC-1/4 are potent NK cell chemoattractants, corresponding to a major influx of these cells into the implantation site. We did not detect a global elevation of chemokine expression premenstrually, although MDC, 6Ckine, MCP-3, and FKN were maintained at high levels, and localized up-regulation of IL-8, FKN, eotaxin, and MDC protein was detected in vascular endothelium. After the initial premenstrual recruitment of leukocytes, these cells themselves are the predominant source of chemokines, particularly IL-8, FKN, MDC, and HCC-4. Finally, chemokine expression by proliferating endometrium was surprisingly high, with FKN, MCP-3, MDC, eotaxin, MIP-1ß, and HCC-4 being the major chemokines present; all of these are macrophage chemoattractants in keeping with macrophages being the main leukocytes present at during endometrial repair and regeneration.
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, ITAC, IFN-
-inducible protein-10, and stromal cell- derived factor-1) were not detectable in endometrium in the current study, indicating they are not as important for NK cell recruitment to the uterus. Classical NK cells, which would be detrimental to pregnancy (42), lack the receptor for 6Ckine (CCR7) (40), and this may provide selectivity to ensure their exclusion from the endometrium. This highlights a further level of regulation of leukocyte migration by the selective expression of chemokine receptors by subsets of leukocyte subpopulations. We therefore propose that we have identified chemokines that are potentially critical for the recruitment, and possibly activation, of uNK cells. In the present study, we failed to detect any chemokine production by nondecidualized stromal cells, a finding at odds with a body of work in the literature on the production of chemokines by nondecidualized cultured stromal cells (43, 44, 45), probably reflecting changes in cell phenotype with culture. Instead, decidualized stroma are a major source of chemokines, in keeping with the homing and clustering of macrophages and uNKs to areas of decidualization and with their absence in nondecidualized ectopic implantation sites (46). This is fascinating from the perspective of decidual cell function, particularly because chemokines have also been implicated in the specific targeted migration of cytotrophoblast cells to maternal vessels (47, 48). There is intimate spatial contact and communication between cytotrophoblast cells, decidual cells, and uNK cells clustering around the arteries. Decidual cells could therefore, via chemokine production, be playing a much broader role in the process of trophoblast invasion, facilitating the association of maternal and fetal cells in the implantation site. Recruitment of leukocytes by the decidua also supports the concept that the spontaneous initiation of decidualization that occurs in humans, in the absence of pregnancy, is the cause of subsequent menstruation (49). This is reinforced by experimental evidence from a functional model of menstruation in the mouse (50), in which endometrial breakdown and associated leukocyte recruitment occurred only if the uterine horn had been previously stimulated to decidualize.
We found constitutive expression of some chemokines, similar to that seen related to roles in tissue homeostasis, or immune surveillance, e.g. 6Ckine in lymph nodes (51). This relatively high level of chemokine production in the endometrium is similar to that seen in other mucosal tissues (2), in which susceptibility to antigen exposure calls for increased immune surveillance. Many of the endometrial chemokines identified, including pregnancy-associated 6Ckine, MDC, MCP-3, and MIP-1ß, are potent chemokines for dendritic cells and T lymphocytes (52). Whereas T cells are present in the endometrium (comprising up to 10% of decidual leukocytes), they appear to be unresponsive or tolerogenic to trophoblast antigens (18). It has been theorized that successful pregnancy involves a localized Th1-Th2 shift induced by the hormonal milieu (53), although this has never been proven. Alternatively, specific subsets of T lymphocytes may be selectively recruited to the implantation site and governed by decidual chemokine expression and additionally by the expression of distinct chemokine receptors repertoires on circulating T cells (54). A small population of myeloid-derived dendritic cells has recently been identified in the decidua (55), and the up-regulation of dendritic cell chemoattractants may reflect the need for increased tolerance and protection from pathogens during pregnancy (2).
An unexpected finding was that the epithelial compartment of the endometrium is the major source of most chemokines. Several chemokines (FKN (35), MDC, 6Ckine, and MCP-3) were localized to intensely stained vesicles in epithelial cells, which appeared to transit from basal to apical subcellular regions as secretory activity increased, highly indicative of secretion into the uterine lumen. So whereas roles in homing of leukocytes to subepithelial or intraepithelial regions is likely, these data suggest that epithelial chemokines have nonimmune functions, such as has been reported during embryogenesis, tissue remodeling, and tumorigenesis (34, 56, 57, 58), through up-regulation of adhesion molecule expression, cell proliferation, and motility (59). Indeed endometrial cells express a number of chemokine receptors (CXCR1, CXCR2, CXCR4, CCR2B, CCR5, and CX3CR1) (31, 35, 60, 61), indicating that chemokines could facilitate endometrial remodeling (e.g. during glandular morphogenesis and decidualization) or up-regulate cell adhesion molecules in the receptive endometrium. Moreover, the blastocyst expresses CCR2B and invasive trophoblast cells express multiple receptors (CCR1, CCR10, CCR5, CCR7, CXCR4, CXCR6, and XCR) (47, 48, 62, 63), suggesting that endometrial-derived chemokines are involved in cross-talk between endometrium and embryo/trophoblast during the initial attachment and subsequent trophoblast invasion. The latter has recently been demonstrated during caprine implantation with trophoblast migration directed by endometrial IP-10 interacting with trophoblast counterreceptor CXCR3 (64). Furthermore, the presence of chemokines in uterine fluid may impact fertilization because FKN and IL-8 are present in seminal fluid (65, 66), and the presence of the former chemokine is related to sperm motility. These findings therefore indicate that chemokines may have multifactorial roles in the periimplantation endometrium, independent of leukocyte recruitment.
Determining the exact functions of chemokines in endometrium is further complicated by a wealth of new information regarding posttranslational processing of chemokines, which affects their activation status. For example, FKN is rapidly cleaved by proteolytic enzymes including MMP-9 from its transmembrane location and released as a soluble chemokine (67), which appears to have different chemotactic properties to the bound form and additionally can antagonize MCP-1 action (68). Similarly, HCC-1 can be cleaved by serine proteases to release a truncated form of the protein that can interact with additional receptor subtypes (CCR3 and CCR5) (69), and MT1-MMP action produces a superactivated form of IL-8 (70). In contrast, MMP-2 exhibits very high affinity for MCP-3 and produces a truncated MCP-3 that is not only biologically inactive but is also a potent antagonist of chemokines acting through CCR1, CCR2, and CCR3 (70). This is significant, given the very high abundance of MCP-3 in the endometrium and may be evidence for an endogenous antiinflammatory regulatory action within the endometrium. It is also important to note that all these MMPs are found in the endometrium, with cyclical variation in keeping with potential roles in chemokine regulation (5). Whereas chemokines are generally perceived to be inflammatory in nature, a number of the chemokines appear to be associated with the recruitment of antiinflammatory leukocytes, e.g. MDC preferentially recruits Th2 cells or macrophages, and may in fact be important for the resolution of inflammation (71, 72). Furthermore, some chemokines may be antagonists rather than agonists, depending on the receptor subtype to which they bind, e.g. MCP-3 is an antagonist of CCR5 activation, several CXC chemokines can bind to CCR5 and therefore antagonize the natural ligands for that receptor, and eotaxin is an agonist for receptor subtype CCR5 but an antagonist for CCR2 (73, 74, 75, 76).
In conclusion, we report the presence of a highly active and complex chemokine network in the endometrium summarized in Fig. 6, which together would be capable of recruiting, homing, and activating all leukocyte subsets within the endometrium. The majority of these chemokines are macrophage, NK, and T cell chemoattractants, and their up-regulation in the periimplantation phase and early pregnancy supports a role in recruitment of the pregnancy-associated leukocytes. Local up-regulation of IL-8, MDC, and FKN in blood vessels premenstrually suggests that these chemokines may be instrumental in triggering the premenstrual influx of neutrophils. In shedding endometria, chemokine expression was elevated and predominantly localized to infiltrated leukocytes, presumably acting to amplify and regulate the inflammatory reaction. We have therefore broadly identified groups of chemokines whose expression correlates to the recruitment of implantation- and menstruation-associated leukocytes. Leukocyte recruitment to the endometrium is tightly regulated to prevent inappropriate entry of leukocytes, and here we show the regulated expression of nine chemokines that together would be capable of creating a specialized immune environment for pregnancy and in its absence ensuring a tightly controlled and localized inflammatory response that is rapidly resolved. We also propose that chemokines may have paracrine actions on endometrial and embryonic cells during implantation and endometrial remodeling. However, given the complexity of chemokine processing and the variable outcomes of receptor binding, teasing out individual actions of chemokines in the human endometrium and determining how they interact on a cellular level to tightly coordinate leukocyte recruitment presents a major challenge.
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Acknowledgments |
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Footnotes |
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Abbreviations: FKN, Fractalkine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCC, hemofiltrate CC chemokine; MCP, monocyte chemotactic protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; MMP, matrix metalloproteinase; NK, natural killer; RANTES, regulated upon activation, normal T cell expressed, and secreted; RT, reverse transcription; TBS, Tris-buffered saline; uNK, uterine-specific NK.
Received March 16, 2004.
Accepted September 14, 2004.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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A. Germeyer, A. M. Sharkey, M. Prasadajudio, R. Sherwin, A. Moffett, K. Bieback, S. Clausmeyer, L. Masters, R. M. Popovici, A. P. Hess, et al. Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts Mol. Hum. Reprod., January 1, 2009; 15(1): 39 - 48. [Abstract] [Full Text] [PDF]
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 S.-U. Chen, H. Lee, D.-Y. Chang, C.-H. Chou, C.-Y. Chang, K.-H. Chao, C.-W. Lin, and Y.-S. Yang Lysophosphatidic Acid Mediates Interleukin-8 Expression in Human Endometrial Stromal Cells through Its Receptor and Nuclear Factor-{kappa}B-Dependent Pathway: A Possible Role in Angiogenesis of Endometrium and Placenta Endocrinology, November 1, 2008; 149(11): 5888 - 5896. [Abstract] [Full Text] [PDF]
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O. Shynlova, P. Tsui, A. Dorogin, and S. J. Lye Monocyte Chemoattractant Protein-1 (CCL-2) Integrates Mechanical and Endocrine Signals That Mediate Term and Preterm Labor J. Immunol., July 15, 2008; 181(2): 1470 - 1479. [Abstract] [Full Text] [PDF]
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L. Schulke, F. Manconi, R. Markham, and I.S. Fraser Endometrial dendritic cell populations during the normal menstrual cycle Hum. Reprod., July 1, 2008; 23(7): 1574 - 1580. [Abstract] [Full Text] [PDF]
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 N. J Hannan and L. A Salamonsen CX3CL1 and CCL14 Regulate Extracellular Matrix and Adhesion Molecules in the Trophoblast: Potential Roles in Human Embryo Implantation Biol Reprod, July 1, 2008; 79(1): 58 - 65. [Abstract] [Full Text] [PDF]
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 C. Carlino, H. Stabile, S. Morrone, R. Bulla, A. Soriani, C. Agostinis, F. Bossi, C. Mocci, F. Sarazani, F. Tedesco, et al. Recruitment of circulating NK cells through decidual tissues: a possible mechanism controlling NK cell accumulation in the uterus during early pregnancy Blood, March 15, 2008; 111(6): 3108 - 3115. [Abstract] [Full Text] [PDF]
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R. M. Popovici, M. S. Krause, J. Jauckus, A. Germeyer, I. S. Brum, C. Garlanda, T. Strowitzki, and M. von Wolff The Long Pentraxin PTX3 in Human Endometrium: Regulation by Steroids and Trophoblast Products Endocrinology, March 1, 2008; 149(3): 1136 - 1143. [Abstract] [Full Text] [PDF]
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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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T. J. Kaitu'u-Lino, N. B. Morison, and L. A. Salamonsen Estrogen Is Not Essential for Full Endometrial Restoration after Breakdown: Lessons from a Mouse Model Endocrinology, October 1, 2007; 148(10): 5105 - 5111. [Abstract] [Full Text] [PDF]
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Y.-Y. He, M.-R. Du, P.-F. Guo, X.-J. He, W.-h. Zhou, X.-Y. Zhu, and D.-J. Li Regulation of C-C motif chemokine ligand 2 and its receptor in human decidual stromal cells by pregnancy-associated hormones in early gestation Hum. Reprod., October 1, 2007; 22(10): 2733 - 2742. [Abstract] [Full Text] [PDF]
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R. G Lea and O. Sandra Immunoendocrine aspects of endometrial function and implantation Reproduction, September 1, 2007; 134(3): 389 - 404. [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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 B. W. Whitcomb, E. F. Schisterman, M. A. Klebanoff, M. Baumgarten, A. Rhoton-Vlasak, X. Luo, and N. Chegini Circulating Chemokine Levels and Miscarriage Am. J. Epidemiol., August 1, 2007; 166(3): 323 - 331. [Abstract] [Full Text] [PDF]
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C Freyer, L M Kilpatrick, L A Salamonsen, and G Nie Pro-protein convertases (PCs) other than PC6 are not tightly regulated for implantation in the human endometrium Reproduction, June 1, 2007; 133(6): 1189 - 1197. [Abstract] [Full Text] [PDF]
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C.-w. Cheng, H. Bielby, D. Licence, S. K. Smith, C. G. Print, and D. S. Charnock-Jones Quantitative Cellular and Molecular Analysis of the Effect of Progesterone Withdrawal in a Murine Model of Decidualization Biol Reprod, May 1, 2007; 76(5): 871 - 883. [Abstract] [Full Text] [PDF]
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A.P. Hess, A.E. Hamilton, S. Talbi, C. Dosiou, M. Nyegaard, N. Nayak, O. Genbecev-Krtolica, P. Mavrogianis, K. Ferrer, J. Kruessel, et al. Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators Biol Reprod, January 1, 2007; 76(1): 102 - 117. [Abstract] [Full Text] [PDF]
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R. M. Popovici, N. K. Betzler, M. S. Krause, M. Luo, J. Jauckus, A. Germeyer, S. Bloethner, A. Schlotterer, R. Kumar, T. Strowitzki, et al. Gene Expression Profiling of Human Endometrial-Trophoblast Interaction in a Coculture Model Endocrinology, December 1, 2006; 147(12): 5662 - 5675. [Abstract] [Full Text] [PDF]
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S. Yoshioka, H. Fujiwara, T. Nakayama, K. Kosaka, T. Mori, and S. Fujii Intrauterine administration of autologous peripheral blood mononuclear cells promotes implantation rates in patients with repeated failure of IVF-embryo transfer Hum. Reprod., December 1, 2006; 21(12): 3290 - 3294. [Abstract] [Full Text] [PDF]
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R. L Jones, T. J Kaitu'u-Lino, G. Nie, L G. Sanchez-Partida, J. K Findlay, and L. A Salamonsen Complex expression patterns support potential roles for maternally derived activins in the establishment of pregnancy in mouse. Reproduction, November 1, 2006; 132(5): 799 - 810. [Abstract] [Full Text] [PDF]
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P. J. Scaife, J. N. Bulmer, S. C. Robson, B. A. Innes, and R. F. Searle Effector Activity of Decidual CD8+ T Lymphocytes in Early Human Pregnancy Biol Reprod, October 1, 2006; 75(4): 562 - 567. [Abstract] [Full Text] [PDF]
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A. Peloggia, C. A. Petta, L. Bahamondes, M. Oliveira-Ribeiro, J. Zhang, and L. Salamonsen Endometrial chemokines, uterine natural killer cells and mast cells in long-term users of the levonorgestrel-releasing intrauterine system Hum. Reprod., May 1, 2006; 21(5): 1129 - 1134. [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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 X.-H. Ma, S.-J. Hu, H. Ni, Y.-C. Zhao, Z. Tian, J.-L. Liu, G. Ren, X.-H. Liang, H. Yu, P. Wan, et al. Serial Analysis of Gene Expression in Mouse Uterus at the Implantation Site J. Biol. Chem., April 7, 2006; 281(14): 9351 - 9360. [Abstract] [Full Text] [PDF]
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M. K. Tee, J.-L. Vigne, and R. N. Taylor All-Trans Retinoic Acid Inhibits Vascular Endothelial Growth Factor Expression in a Cell Model of Neutrophil Activation Endocrinology, March 1, 2006; 147(3): 1264 - 1270. [Abstract] [Full Text] [PDF]
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R. L. Jones, J. K. Findlay, P. G. Farnworth, D. M. Robertson, E. Wallace, and L. A. Salamonsen Activin A and Inhibin A Differentially Regulate Human Uterine Matrix Metalloproteinases: Potential Interactions during Decidualization and Trophoblast Invasion Endocrinology, February 1, 2006; 147(2): 724 - 732. [Abstract] [Full Text] [PDF]
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 S. Labied, T. Kajihara, P. A. Madureira, L. Fusi, M. C. Jones, J. M. Higham, R. Varshochi, J. M. Francis, G. Zoumpoulidou, A. Essafi, et al. Progestins Regulate the Expression and Activity of the Forkhead Transcription Factor FOXO1 in Differentiating Human Endometrium Mol. Endocrinol., January 1, 2006; 20(1): 35 - 44. [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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 M. Hickey, J. Crewe, J. P. Goodridge, C. S. Witt, I. S. Fraser, D. Doherty, F. T. Christiansen, and L. A. Salamonsen Menopausal Hormone Therapy and Irregular Endometrial Bleeding: A Potential Role for Uterine Natural Killer Cells? J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5528 - 5535. [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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J.V. Fahey, T.M Schaefer, J.Y. Channon, and C.R. Wira Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract Hum. Reprod., June 1, 2005; 20(6): 1439 - 1446. [Abstract] [Full Text] [PDF]
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N. J. Hannan, R. L. Jones, H. O. D. Critchley, G. J. Kovacs, P. A. W. Rogers, B. Affandi, and L. A. Salamonsen Coexpression of Fractalkine and Its Receptor in Normal Human Endometrium and in Endometrium from Users of Progestin-Only Contraception Supports a Role for Fractalkine in Leukocyte Recruitment and Endometrial Remodeling J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6119 - 6129. [Abstract] [Full Text] [PDF]
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, HCC-1 binds CCR3 and -5 only in cleaved state). (Data collated from Refs.