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Medical Research Council Human Reproductive Sciences Unit (A.E.K., R.W.K.) and Department of Reproductive and Developmental Sciences (H.O.D.C.), University of Edinburgh, Centre for Reproductive Biology, Edinburgh, United Kingdom EH16 4SB; and Centre for Inflammation Research (J.-M.S.), Edinburgh University Medical School, Edinburgh, United Kingdom EH8 9AG
Address all correspondence and requests for reprints to: Dr. Anne King, Medical Research Council Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, United Kingdom EH16 4SB. E-mail: a.king{at}hrsu.mrc.ac.uk.
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Abstract |
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, increased elafin mRNA levels by 4.6-fold. These results show that endometrium expresses elafin in a menstruation-dependent manner. This is attributable to the presence of infiltrating leukocytes and increased inflammatory signaling. Elafin will regulate proteolytic enzymes during menstruation and will contribute to the innate defense against uterine infection. |
Introduction |
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Human endometrium is an important site of cyclical tissue degradation and remodeling during reproductive events such as implantation and menstruation (13, 14). These processes involve the activity of proteases [e.g. matrix metalloproteinases (MMPs)] and are regulated by proteinase inhibitors. Previously, we have described expression of SLPI by human endometrium and first-trimester decidua (15). SLPI is expressed in the endometrial epithelium and is present mainly during the mid-late secretory phase and in decidua. It is likely to have a role in control of the tissue remodeling necessary for implantation and early pregnancy to occur. The endometrium is also a key site of natural antimicrobial production (16, 17), and SLPI will contribute to the innate defenses that prevent genital tract infection, which can compromise both implantation and pregnancy.
In contrast to SLPI, elafin expression in human endometrium has not been documented, although it is present in vagina and cervix, suggesting that it has a role in the female reproductive tract (18). The aim of the current study was to examine elafin mRNA and protein expression in the endometrium during the menstrual cycle and in early pregnancy.
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Materials and Methods |
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Endometrial biopsies (n = 55) were collected from women undergoing gynecological procedures for benign conditions. All women had regular menstrual cycles (25–35 d) and had not received any hormonal treatments for 3 months preceding biopsy. Endometrium was collected throughout the menstrual cycle (menstrual n = 7, proliferative n = 17, secretory n = 31) and was histologically dated according to the criteria of Noyes et al. (19). In addition, serum was collected for the measurement of circulating estradiol and progesterone concentrations, which were consistent with the histological assessment of cycle stage. Elafin concentrations were also measured in serum samples (n = 21).
Decidual biopsies (n = 6) were collected from women undergoing surgical termination of pregnancy in the first trimester. Decidua parietalis was collected by curettage of the uterine wall away from the site of implantation. Serum was also collected from women in the first trimester of pregnancy (n = 4) for inclusion in elafin ELISAs.
Written informed consent was obtained from all patients before sample collection, and ethical approval was received from Lothian Research Ethics Committee.
Endometrial and decidual biopsies were collected in RPMI 1640 medium (Sigma, Poole, Dorset, UK) and were: 1) fixed in 10% neutral buffered formalin overnight at 4 C, stored in 70% ethanol, and wax embedded for histological examination and immunohistochemistry; 2) immersed in Tri reagent (Sigma) for RNA extraction and RT-PCR analysis; and 3) digested with collagenase so that the glandular component could be separated from endometrium for cell culture experiments.
Separation and culture of endometrial glandular epithelial cells
The protocol for separation of glandular epithelial cells from whole endometrial biopsies has been described previously (17). In summary, biopsies were chopped into small fragments and incubated with collagenase/deoxynuclease (1 and 0.1 mg/ml; both Sigma) for 80 min at 37 C. Endometrial glands were isolated by density sedimentation and then incubated as above, for a further 2 h. Epithelial cells were then mixed with 50% Matrigel (BD Biosciences, Bedford, MA) and grown on 12-well culture plates (Nunc, Life Technologies, Inc., Paisley, UK) in RPMI 1640 medium supplemented with 10% fetal calf serum (Mycoplex; PAA Laboratories, Teddington, UK), penicillin (100 IU/ml; Sigma), streptomycin (100 µg/ml; Sigma), gentamycin (20 µg/ml; Sigma), epidermal growth factor (25 ng/ml; Peprotech Ltd., London, UK), vascular endothelial growth factor (1 ng/ml; Peprotech), basic fibroblast growth factor (5 ng/ml, Peprotech), and estradiol (10-7M). Epithelial cell purity was determined by immunohistochemical staining of representative samples for cytokeratin (mouse antihuman cytokeratin antibody, clone MNF116; Dako Ltd., Cambridge, UK) and was more than 90% at the time of treatment.
Cells were grown to near confluence (7–10 d) and then treated for 24 h with the following stimuli: IL-1ß (1 ng/ml, Peprotech), TNF (2 ng/ml) and IL-1ß+TNF (1 ng/ml and 2 ng/ml). During the treatment period, cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics as described above. Growth factors were not present during treatment. Estradiol (10-8 M) was maintained throughout, and controls (estradiol alone) were included in each experiment. Each treatment was tested in triplicate wells, and cells were pooled for RNA extraction. Treatments were tested at least four times using epithelial cells from separate endometrial biopsies. After 24 h, cells were harvested in Tri reagent for RNA extraction, and culture supernatants were collected for subsequent ELISA.
RT-quantitative PCR
RNA was extracted from biopsies/epithelial cells as detailed in the manufacturer’s protocol (Sigma). Elafin mRNA levels were determined by real-time quantitative PCR. This technique relates the amount of elafin mRNA present to levels of ribosomal 18S, controlling for the amount of RNA present. Details of the RT and PCR method used have been detailed fully elsewhere (15). Briefly, RNA samples were reverse transcribed using random primers. PCR reaction mixtures included Sure Start Taq DNA polymerase (0.025 IU/ml; Stratagene, Amsterdam, The Netherlands), MgCl2 (3.6 mM), deoxynucleotide triphosphates (all at 200 µM), and specific forward and reverse primers (300 nM; Biosource, Nivelles, Belgium) and probe (50 nM; Biosource) for elafin. In addition, ribosomal 18S primers and probe (all at 50 nM; PE Biosystems, Warrington, UK) were added. Samples were measured in triplicate, and no template controls (containing water in place of RNA) were included throughout. PCR reactions were run on ABI Prism 7700 (PE Biosystems). A Taqman-specified protocol was used for the PCR reaction. The conditions of the protocol were as follows. The protocol began with an incubation for two min at 50 C, followed by an incubation for 10 min at 95 C. After this, there were 40 cycles of the denaturing stage (95 C for 15 sec) and the annealing/extension phase (60 C for 1 min; data were acquired during this phase). For further details, see ABI Prism 7700 Sequence Detection System User’s Manual.
Elafin primers and probe were designed using PRIMER express software (PE Biosystems), and sequences were as follows: forward primer 5'-TGGCTCCTGCCCCATTATC-3', reverse primer 5'-CAGTATCTTTCAAGCAGCGGTTAG-3', and probe 5'-ATCCGGTGCGCCATGTTGAATCC-3'. Product length was 71 bp, and the product melting temperature was 81 C. Primers and probe were validated at the above concentrations, and linearity of response was confirmed by serial dilution of cDNA. Within-assay variation of PCR measurements was calculated from six replicates and was 1.9%. Variability of the reverse transcription step was determined by reverse transcribing one RNA sample on eight separate occasions. The eight cDNA samples were then included within one PCR run, and variability (relative to SD) was calculated to be 3.65%.
The primer and probe set amplified a sequence present in exon 2 of the elafin gene (20) and did not span an intron. The possibility of genomic DNA contamination was excluded by measurement of ß-actin levels in RNA samples (which had not been reverse transcribed). All measurements were above a previously defined arbitrary level of 27 cycles (21), indicating that there were not high concentrations of ß-actin genomic DNA present in any RNA sample included in this study.
Elafin immunohistochemistry
Tissue sections were dewaxed in Histoclear (National Diagnostics, Atlanta, GA) and rehydrated in descending grades of alcohol. Nonspecific endogenous peroxidase activity was blocked with 3% hydrogen peroxide (BDH Laboratory Supplies, Poole, UK) in distilled water for 10 min at room temperature. Diluted normal goat serum (NGS, 20% vol/vol in PBS; Diagnostics Scotland, Edinburgh, UK) was applied to all tissue sections for 20 min at room temperature. Sections were then incubated overnight at 4 C with 50 µl rabbit antielafin antibody (6, 22), which was diluted 1:400 in NGS. In negative control sections, the primary antibody was substituted with an approximately equivalent Ig concentration of rabbit IgG (RIgG; Vector Laboratories, Inc., Peterborough, UK). Sections were incubated with biotinylated goat antirabbit Ig (Vector) and were then subjected to an avidin-biotin peroxidase detection system (both for 60 min at room temperature; Elite ABC, Vector). The peroxidase substrate, diaminobenzidine, was used to identify positive staining. Sections were counterstained with Harris’s hematoxylin (Pioneer Research Chemicals Ltd., Colchester, UK), dehydrated in ascending grades of alcohol, and mounted from xylene in Pertex (Cellpath, Hemel Hempsted, UK).
SLPI immunohistochemistry
The method used has been described previously (23) and was identical to the elafin immunohistochemistry protocol, with the following exceptions. The nonimmune block was performed with diluted horse serum (Vector) for 20 min at room temperature. The primary antibody was mouse anti-SLPI (diluted 1:200 in horse serum; Hycult Biotechnology, Uden, The Netherlands), negative controls were incubated with an equimolar concentration of mouse IgG (MIgG), and the secondary antibody was biotinylated horse antimouse Ig (Vector).
Immunofluorescent colocalization of elafin and neutrophil elastase
Tissue sections were treated as detailed in the elafin immunohistochemistry protocol until application of the nonimmune block. At this stage, sections were incubated with NGS (20% vol/vol) + BSA (5% (wt/vol) (Sigma) for 20 min at room temperature. Sections were then incubated with rabbit antielafin (1:80) and mouse antielastase antibodies (1:10) (both in NGS + BSA) overnight at 4 C. Negative controls were incubated with: 1) rabbit antielafin and MIgG (Vector); 2) mouse antielastase and RIgG; and 3) RIgG and MIgG. After overnight incubation, all sections were washed in PBS containing Tween 20 (PBST, 100 µl/liter) and incubated with fluorescein isothiocyanate-labeled goat antimouse (1:20; Sigma) and peroxidase-labeled goat antirabbit antibodies (1:100, Dako) (both in NGS + BSA) for 60 min at room temperature. Sections were washed in PBST, followed by PBS, and then incubated for a further 10 min with tyramide-cyanine 5 fluorescent complex (TSA Plus Cyanine 5 System, Tyramide Signal Amplification; PerkinElmer Life Sciences, Inc., Boston, MA) at a dilution of 1:50 for 10 min. After further washing (1x PBST, 1x PBS), sections were counterstained with propidium iodide (1:2000 in PBS; Sigma) for 1 min, washed in PBS, and then mounted in Permafluor.
Fluorescent images were taken on a Zeiss LSM 510 confocal laser scanning microscope (Zeiss, Welwyn Garden City, Herts, UK). Fluorescein isothiocyanate (for elastase visualization) was visualized using an argon laser with an excitation beam of 488 nm and was detected using a band pass filter from 505–550 nm. Cyanine 5 (for elafin visualization) was visualized using a helium/neon 2 laser with an excitation beam of 633 nm and was detected using a long-pass filter at 650 nm. Propidium iodide was visualized using a helium/neon 1 laser with an excitation beam of 543 nm and detected using a band pass filter from 560–615 nm.
Elafin ELISA
Elafin protein concentrations in cell culture supernatants and serum samples were determined by ELISA. The method used was adapted from one described previously (6) and has been detailed elsewhere (24). In brief, 96-well plates (Linbro, Flow Labs, McLean, VA) were coated overnight with antielafin IgG (1:500 in carbonate buffer, pH 9.6) at 4 C. The elafin antibody used was a RIgG fraction that has been described in previous studies (6, 22). Plates were then blocked, and samples/standards were diluted in PBS (with 1% BSA and 0.05% Tween 20) and incubated in plates for 2 h at room temperature. Plates were then incubated with biotinylated antielafin IgG (2 µg/ml; RIgG fraction as detailed above), followed by streptavidin horseradish peroxidase (Sigma), both for 2 h at room temperature. Plates were washed and 100 µl chromogenic substrate (2,2'-azino-bis-3-ethyl benz-thiazoline-6-sulfonic acid) with 0.001% H2O2 was added. Absorbance was measured at 490 nm on a microplate reader (Dynatech, Guernsey, UK). Within- and between-assay precision measurements were 8.12 and 12.74%, respectively.
Statistical analysis
ANOVA (StatView 3.0) was used to determine significant differences. Fisher’s protected least-squares differences test was used to assign individual differences. P < 0.05 was considered significant.
Primary endometrial epithelial cells were treated as one group for statistical analysis, because we have previously shown that these cells do not exhibit characteristics of the cycle stage during which they were derived (17).
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Results |
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Elafin mRNA was detected in endometrial biopsies (n = 24) throughout the menstrual cycle and in first-trimester decidua (n = 4). Elafin mRNA showed a dramatic increase at the time of menstruation. Expression was 4-fold higher than in the midsecretory phase and was at least 62-fold higher than during the other cycle stages (Fig. 1
; P < 0.004). Decidual elafin expression was also significantly lower than during menstruation (P < 0.001).
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Elafin protein was localized in endometrium (n = 16) and decidua (n = 4) by immunohistochemistry. This showed positive elafin immunoreactivity in cells scattered throughout the endometrial stroma. These cells were most abundant in biopsies from the late secretory and menstrual phases (Fig. 2A), with only occasional cells present at other times of the menstrual cycle (Fig. 2B). This pattern of expression is consistent with expression of elafin by endometrial neutrophils, which are present in highest numbers in the late secretory and menstrual phases (25). Faint and variable immunoreactivity was also noted in the glandular epithelium and secretions of some biopsies from the late secretory and menstrual phases. In first-trimester decidua, elafin immunoreactivity was present only in occasional cells that had the bilobular nuclei characteristic of neutrophils. This is consistent with the paucity of neutrophils in decidua in early pregnancy (26)
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). Negative controls were included in immunohistochemical experiments. These are shown in insets in Fig. 2, A (elafin) and C (SLPI). Immunofluorescent colocalization of elafin and neutrophil elastase in endometrium
The expression of elafin and neutrophil elastase was examined in endometrial biopsies (n = 1 proliferative; n = 1 late secretory; n = 2 menstrual) using immunofluorescence (Fig. 2D). The two proteins colocalized in cells scattered in the endometrial stroma, confirming that elafin is expressed by endometrial neutrophils during the late secretory and menstrual phases. In addition, propidium iodide was used to counterstain sections, revealing bilobular nuclei, a characteristic of neutrophils, in the cells that expressed elafin and elastase.
Elafin mRNA and protein expression in primary endometrial epithelial cells
Elafin mRNA expression in primary endometrial epithelial cells was examined in 16 separate experiments. Elafin mRNA levels were increased 4.6-fold above control levels by treatment with a combination of IL-1ß and TNF (Fig. 3; P < 0.01). Significance was determined by comparison with 8 controls (the maximum number of samples in any treatment group) using ANOVA.
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, in six separate experiments. Elafin concentrations in these supernatants were 1.75-fold higher than in controls, although this was not significant (data not shown). Elafin concentrations in serum during the menstrual cycle and in early pregnancy
Elafin was present in serum from nonpregnant women (n = 21) at concentrations ranging from 6.4–37.5 ng/ml (mean, 20.65 ng/ml). There were no differences in elafin concentrations in serum collected during different phases of the menstrual cycle, and no increase was observed around the time of menstruation. In addition, concentrations found during early pregnancy (range, 8.1–35.8 ng/ml; mean, 19.48 ng/ml; n = 4) were comparable with those detected in nonpregnant women.
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Discussion |
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In addition to expression by endometrial neutrophils, faint and variable elafin immunoreactivity is present in the glandular epithelium in some samples from the late secretory and menstrual phases. A primary endometrial epithelial cell model was used to examine regulation of elafin expression at this site. Cells were grown in the presence of growth factors (detailed in Materials and Methods) to promote epithelial cell growth. These growth factors were not present during treatment with inflammatory stimuli. Elafin mRNA expression was measured, and elafin protein was detected by ELISA in culture supernatants from our cell model, confirming that the endometrial epithelium can secrete elafin. Elafin mRNA was increased by the proinflammatory cytokines, IL-1ß and TNF. This is consistent with previous studies reporting the presence of an NF
B site in the elafin promoter (30) and showing elafin expression to be induced by inflammatory stimuli in other systems (6). Both of these cytokines are present in endometrium during the menstrual cycle (with TNF expression peaking during menstruation) (31, 32) and are likely to be increased during uterine infection; and hence, they may contribute to the regulation of epithelial elafin expression in vivo.
Measurement of elafin present in serum samples from women at different stages of the menstrual cycle and in early pregnancy showed that there is no systemic increase in elafin production during menstruation. Serum elafin concentrations are constant throughout the menstrual cycle and in early pregnancy. This suggests that the combination of infiltrating neutrophils and increased proinflammatory signaling in endometrium at the time of menstruation both contribute to a specific up-regulation in local elafin expression as detailed above. Neutrophils circulating in the peripheral circulation do not increase their release of elafin during menstruation, and the elafin released in endometrium during menstruation does not reach the blood. The role of elafin in endometrium is likely to involve both its antimicrobial and antiproteinase activities.
Cyclical endometrial degradation and repair occurs during each menstrual cycle. Progesterone withdrawal in the late secretory phase increases expression of inflammatory mediators (e.g. IL-8), encouraging neutrophil entry into the endometrium, and also up-regulates expression of several MMPs (33). MMPs are key molecules involved in the tissue degradation that occurs at menstruation. Several of these proteases (MMP-2, 3, and 9) are activated by neutrophil elastase (27). In addition, the endogenous inhibitors of MMPs, tissue inhibitors of MMPs (TIMPs), can be degraded by neutrophil elastase (TIMP-1 and TIMP-2) (27). The presence of elafin, a neutrophil elastase inhibitor, is likely to be important in the limitation of MMP activation and, hence, tissue breakdown. These antiproteinase actions will ultimately contribute to successful endometrial repair.
In addition to the above role during the normal menstrual cycle, the natural antimicrobial properties of elafin suggest that it is likely to be involved in the prevention of uterine infection. The human endometrial epithelium produces several antimicrobial molecules, such as ß-defensins and SLPI, that contribute to mucosal defense (15, 16, 17). The breakdown of the surface epithelial barrier during menstruation will leave the endometrium particularly vulnerable to any infection ascending from the cervix. Elafin has been shown to have direct antimicrobial activity (independent of antiproteinase activity) (9), and this will strengthen the innate defenses at this time. Also, host antiproteinases have been suggested to have a role in the inhibition of the proteolytic enzymes that bacteria use as virulence factors (34), and this may be another mechanism by which elafin contributes to mucosal defense.
Interestingly, the spatial and temporal localization of elafin in human endometrium differs from that of SLPI (a second proteinase inhibitor that shares 40% homology with elafin). Previously, we have reported that SLPI is expressed in the endometrial glandular epithelium, with no evidence of expression in leukocytes (15). Protein expression peaks during the mid-late secretory phase. In addition, the regulation of elafin and SLPI in a primary endometrial epithelial cell model differs. As described above, elafin mRNA expression is up-regulated by treatment with IL-1ß and TNF. In contrast, we have previously shown that SLPI mRNA expression was resistant to these mediators, whereas SLPI protein concentrations decreased after treatment with the two cytokines (17). These contrasting expression profiles suggest that elafin and SLPI will have differential roles in endometrium, and these may be related to menstruation and implantation, respectively.
In summary, the antiproteinase and antimicrobial molecule, elafin, is present in endometrium during menstruation. Elafin is expressed predominantly by endometrial neutrophils and will likely contribute to the antiproteolytic mechanisms that regulate menstruation and aid tissue repair and will also be involved in the innate mucosal defenses that prevent uterine infection.
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Acknowledgments |
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Footnotes |
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Received February 12, 2003.
Accepted May 27, 2003.
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