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Royal Veterinary College, Departments of Veterinary Clinical Sciences (S.H., D.P.F., E.J.W., S.T.L., I.M.S.) and Pathology and Infectious Diseases (D.W.), University of London, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom; Department of Veterinary Clinical Science and Animal Husbandry (H.D.), Faculty of Veterinary Science, University of Liverpool, Leahurst, Neston CH64 7TE, United Kingdom; and Centre for Veterinary Science (C.E.B.), Department of Clinical Veterinary Medicine, University of Cambridge, Cambridge CB3 0ES, United Kingdom
Address all correspondence and requests for reprints to Prof. Martin Sheldon, Royal Veterinary College, Department of Veterinary Clinical Sciences, University of London, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom. E-mail: sheldon{at}rvc.ac.uk.
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Abstract |
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(PGF) and prostaglandin E2 (PGE), which are secreted by the uterine endometrium. However, the uterus is readily infected with bacteria such as Escherichia coli, which disrupt luteolysis. Immune cells detect E. coli by Toll-like receptor 4 (TLR4) binding its pathogenic ligand, lipopolysaccharide (LPS), although signaling requires accessory molecules such as CD14. The objective of this study was to determine the effect of E. coli or LPS on the function of bovine endometrial cells, and whether purified populations of epithelial and stromal cells express the molecules involved in LPS recognition. In addition, because the female sex hormones estradiol and progesterone modify the risk of uterine infection, their effect on the LPS response was investigated. Endometrial explants produced prostaglandins in response to LPS, with an increased ratio of PGE to PGF. Addition of LPS or E. coli to stromal and epithelial cells stimulated production of PGE and PGF and increased their cyclooxygenase 2 mRNA expression. The production of prostaglandins was abrogated by an LPS antagonist. In addition, estradiol and progesterone inhibited the production of PGE and PGF in response to LPS, indicating a role for steroid hormones in the response to bacterial infection. For the first time, Toll-like receptor 4 mRNA and CD14 mRNA and protein were detected in bovine endometrial stromal and epithelial cells by RT-PCR and flow cytometry. In conclusion, epithelial and stromal cells detect and respond to bacteria, which modulate their endocrine function. |
Introduction |
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(PGF) and prostaglandin E2 (PGE) from the uterine endometrium (1). Both PGF and PGE are synthesized from arachadonic acid (AA) under the regulation of cyclooxygenase 2 (COX-2) isoenzymes in the endometrium (2, 3). The uterine epithelial cells predominantly secrete PGF, whereas the stromal cells produce PGE (4). The secretion of PGF, in response to endometrial oxytocin receptor activation, induces regression of the corpus luteum (luteolysis) and initiates the follicular phase of the ovarian cycle (1). In contrast, PGE has a luteotrophic role to help maintain the corpus luteum, particularly during pregnancy. However, infection of the uterus with bacteria disrupts the process of luteolysis, causing either premature regression of the corpus luteum or failure of luteolysis and an extended luteal phase (5). For most of the reproductive cycle in humans and animals, the uterus is thought to be sterile or at least clear of pathogenic bacteria, but it is readily contaminated with bacteria during sexual intercourse and around the time of parturition. Sexually transmitted infections are widespread in the human population with an estimated 350 million new, mainly bacterial, cases each year and a prevalence of 5% in US women of reproductive age (www.who.int/topics/sexually_transmitted_infections). The majority of these infections are initially asymptomatic, but the consequences range from subfertility to severe pelvic inflammatory disease (PID) (6, 7, 8). Mammalian fertility is also compromised by PID associated with bacterial infections after parturition. Bos taurus is a particularly extreme species, where bacterial contamination of the uterus is ubiquitous after parturition in dairy cattle (9, 10, 11). In 15% of these animals, uterine bacterial infections persist for more than 3 wk after parturition, causing clinical diseases ranging from acute PID to chronic endometritis (12). These infections perturb normal ovarian cycles by suppressing follicular growth and disrupting luteolysis (5, 10). The well-characterized uterine disease, the ready availability of tissues, and the similarity of uterine bacterial pathogens among mammals, make cattle a good model for studying the effects of infection on the endocrine and immune functions of the uterus.
Escherichia coli are the most commonly isolated pathogenic bacteria from cases of clinical uterine disease in cattle (9, 10, 11). In the uterine lumen, there are high concentrations of the main pathogenic ligand of E. coli, lipopolysaccharide (LPS) (9). The endometrium provides a barrier against infection and an opportunity to detect these bacteria by innate immune receptors recognizing conserved sequences on pathogens, known as pathogen-associated molecular patterns (13). A key group of receptors that recognize pathogen-associated molecular patterns are the Toll-like receptors (TLRs), which were first identified on immune cells, but have since been identified on other cell types (14, 15, 16, 17, 18). Engagement of these receptors initiates a signaling cascade stimulating the production of immune mediators such as TNF and nitric oxide (NO), which orchestrate the immune response to clear the infection (14, 19). The TLR pathways can also stimulate the production of prostaglandins by immune cells (20). In the human endometrium, nine TLRs are expressed at the mRNA level including TLR4 (16, 18, 21, 22). It is the principal role of TLR4 to detect LPS, although signaling through TLR4 also requires accessory molecules such as CD14, LPS binding protein, and MD2 (14, 15).
The endometrium is regulated by changing concentrations of the female sex hormones estradiol and progesterone during the ovarian cycle, and these steroids also have a profound effect on the establishment of infections (23, 24). For example, in humans, rodents, and cattle, progesterone suppresses uterine immune function by decreasing the proliferative capacity of lymphocytes, thereby increasing the susceptibility to bacterial infection (23, 24). Furthermore, increased concentrations of progesterone are associated with decreased production of PGF (4, 25). Conversely, estradiol may play a role in the recruitment of immune cells as more macrophages are present in the endometrium when estradiol concentrations are high in rodents (24). However, the effect of steroids on the immune response to infection in the bovine uterus is less clearly defined.
The objective of the present study was to determine the effect of E. coli or LPS on the endocrine function of bovine endometrial cells, and whether purified populations of epithelial and stromal cells express the mRNA for molecules involved in LPS recognition and binding. In addition, the effect of steroids on the stromal and epithelial cell response to LPS was investigated.
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Materials and Methods |
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For tissue explants, the endometrium was cut into strips and placed into serum-free RPMI 1640 (Sigma, Poole, UK) supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, and 2.5 µg/ml amphotericin B (Sigma), working under sterile conditions. The strips were then chopped into 1-mm3 pieces using a mechanical tissue chopper (McIlwain Laboratory Engineering, Gomshall, UK) and placed into HBSS (Sigma) (27). For each experimental group within the study, 50 mg of tissue were weighed in triplicate and then transferred onto sterile tissue-lined metal grids in six-well plates (Nunc, Nottingham, UK) with 4.25 ml serum-free RPMI 1640 per well (28). The tissue explants were incubated at 37 C, 5% CO2 in air, in a humidified incubator overnight, and then supernatants were removed and replaced with fresh media.
For cell isolation, endometrial tissue was used as previously described (29, 30) with the following modifications. Briefly, tissue was digested in 25 ml sterile filtered digestive solution, which was made by dissolving 50 mg trypsin III (Roche, Lewes, UK), 50 mg collagenase II (Sigma), 100 mg BSA (Sigma), and 10 mg DNase I (Sigma) in 100 ml phenol-red-free HBSS. After a 1.5-h incubation in a shaking water bath at 37 C, the cell suspension was filtered through a 40-µm mesh (Fisher Scientific, Loughborough, UK) to remove undigested material, and the filtrate was resuspended in phenol-red-free HBSS containing 10% fetal bovine serum (FBS; Sigma) and 3 µg/ml trypsin inhibitor (Sigma) (washing medium). The suspension was centrifuged at 100 x g for 10 min and, after two further washes in washing medium, the cells were resuspended in RPMI 1640 containing 10% FBS, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. The cells were plated at a density of 1 x 105 cells in 2 ml per well using 24-well plates (Nunc). To obtain separate stromal and epithelial cell populations, the cell suspension was removed 18 h after plating, which allowed selective attachment of stromal cells (29). The removed cell suspension was then replated and incubated allowing epithelial cells to adhere (31). Stromal and epithelial cell populations were distinguished by cell morphology as previously described (29) and the purity was greater than 95% as determined by microscopy and the production of prostaglandins (4). The culture media was changed every 48 h until the cells reached confluence. All cultures were maintained at 37 C, 5% CO2 in air, in a humidified incubator.
Tissue explant and cell culture challenge
Tissue explants were challenged after 24 h culture with different concentrations of oxytocin (OT, 3–300 nM; Bachem, St. Helens, UK), LPS (0.03–1 µg/ml; Sigma; E. coli serotype 055.B5) and polymyxin B (2 µg/ml; Sigma) individually or in combination as indicated in Results.
Stromal and epithelial cells were challenged once confluence had been reached with different concentrations of AA (10–300 µM; Sigma), OT (100 nM), LPS (0.1–3 µg/ml), heat-killed E. coli (102–105 CFU/ml, isolated from a case of clinical bovine endometritis associated with pyrexia) (10), polymyxin B (2 µg/ml), 17-ß estradiol (3 pg/ml; Sigma), or progesterone (5 ng/ml; Sigma) individually or in combination and for the period of time indicated in Results.
For both tissue explants and cell cultures, the supernatants were harvested and frozen at –20 C until used for cytokine and prostaglandin determination. Endometrial cells were collected immediately for RNA isolation or flow cytometry.
Prostaglandin RIAs
Culture supernatants were analyzed for PGE and PGF by RIA as previously described (32). Samples were diluted in 0.05 M Tris buffer containing 0.1% gelatin and 0.01% sodium azide. Standards and tritiated tracers for the PGs were purchased from Sigma and Amersham International PLC (Amersham, Little Chalfont, Buckinghamshire, UK), respectively. The antisera were a generous gift from Prof. N. L. Poyser (University of Edinburgh, Edinburgh, Scotland, UK) and their cross-reactivities were: PGF antiserum, 0.54% with PGE; PGE antiserum, 0.47% with PGF (33). The limits of detection for PGE and PGF were 2 pg/tube and 1 pg/tube, respectively. The intraassay and interassay coefficients of variation were 4.4 and 7.8% for PGE, and 5.1 and 9.7% for PGF, respectively.
RT-PCR
Total RNA was isolated from cell cultures using the RNeasy Mini kit (Qiagen, Crawley, UK) and first strand cDNA was prepared using SuperScript II RNase H– Reverse Transcriptase (Invitrogen Life Technologies, Paisley, Scotland, UK) according to the manufacturers’ protocols. Amplification of cDNA used the following conditions: denaturation for 5 min at 94 C; followed by 30 cycles of 94 C for 30 sec, 54–56 C (depending on primer Tm) for 30 sec, and 72 C for 30 sec; followed by a final extension of 5 min at 72 C. Primer combinations were designed using the MacVector software package and were purchased from MWG (https://ecom.mwgdna.com). Primer sequences are presented in Table 1
. All PCR products were sent for sequencing (MRC Geneservice, Cambridge, UK) and products showed >98% homology to published sequences on the NCBI database (http://www.ncbi.nlm.nih.gov/blast).
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Determination of TNF- and NO concentrations
Levels of bioactive TNF- were determined using L929 cells as previously described (35), with the following modifications. The L929 cells were cultured in DMEM (Sigma) supplemented with 12.5% FBS, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 20 mM HEPES buffer (Sigma). Cells were plated at a density of 2.5 x 104 cells per well in a 96-well plate (Nunc) in 100 µl medium. Cytotoxicity was determined by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay involving the addition of 0.1 µg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye (Sigma) to each well. The cells were then lysed using 100 µl DMSO (Sigma) per well, and color development was read at 560 nm on a Spectra Max 250 (Molecular Devices). The limit of detection was 10 pg/ml; standards were made using recombinant human TNF- (Sigma) and cross-reactivity was determined using recombinant bovine TNF- (kindly provided by Prof. C. Howard, Institute for Animal Health, Compton, UK).
The concentrations of NO were measured using the Greiss Reagent System (Promega) according to the manufacturer’s instructions. The limit of detection was 2.5 µM nitrite.
Statistical analysis
Data were analyzed using mixed model ANOVA in SAS version 9.1. Results are quoted as mean ± SEM, and significance attributed when P < 0.05.
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Results |
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). However, the media required supplementation with AA, the substrate for prostaglandin synthesis. Stromal cells produced little PGF and epithelial cells produced no PGE (stromal cells, 0.5 ± 0.1 ng/ml PGF; and epithelial cells, PGE levels below limits of detection at 100 nM OT stimulation). As expected, more PGF was produced in response to OT than PGE (172 ± 4 vs. 13 + 1 ng/ml at 100 nM OT stimulation).
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, A and C). Moreover, this response was dependent on LPS because the LPS-stimulated production of PGE or PGF could be abrogated by addition of polymyxin B (Fig. 2, B and D), which neutralizes LPS (36).
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). This observation was further confirmed using flow cytometric analysis, which showed an absence of CD45+ cells (Fig. 3B).
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). The prostaglandin response was abrogated by the addition of polymyxin B to the cultures (Fig. 4). Because LPS is considered the active component of E. coli, the production of prostaglandin by stromal and epithelial cells was analyzed after incubation of the cells with LPS alone. Both stromal and epithelial cells produced increasing amounts of PGE and PGF, respectively, in a dose-dependent manner (Fig. 5). Stromal cells produced little PGF in response to LPS (2.31 + 0.33 vs. 1.09 + 0.27 ng/ml at 1 µg/ml LPS). Interestingly, epithelial cells did produce PGE (81.8 + 17.3 vs. 24.3 + 5.0 ng/ml at 1 µg/ml LPS). However, as our aim was to focus on the effect of LPS on the endocrine function of uterine cells, subsequent results focused on the polarized production of PGE and PGF from stromal and epithelial cells, respectively. The PGE and PGF response to LPS was significantly abrogated by neutralizing LPS with polymyxin B (Fig. 5). Additionally, endometrial epithelial and stromal cells stimulated with LPS, up-regulated COX-2 mRNA expression (see Fig. 7).
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). Flow cytometric analysis confirmed the expression of CD14 on stromal and epithelial cells (Fig. 3B).
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mRNA (Fig. 7
). However, the concentrations of NO or TNF- in culture supernatants were below the limits of detection of the assays.
Progesterone and estradiol partially inhibit LPS-induced prostaglandin production
Preincubation of stromal and epithelial cells with either estradiol or progesterone for 48 h resulted in a significantly reduced production of prostaglandins in response to LPS challenge (Fig. 8). Additionally, the perturbation of prostaglandin secretion was greater following progesterone than estradiol treatment (P < 0.05).
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Discussion |
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Both PGE and PGF are synthesized from AA in many cell types, under the regulation of COX-2 isoenzymes and these prostaglandins have numerous endocrine and immune functions (1, 2, 38). The ability of endometrial explants to produce both PGE and PGF after OT stimulation was confirmed in the present study. However, these explants consisted of a variety of cells, so the individual cell phenotypes were isolated and two methods used to confirm that the cultures were not contaminated with immune cells. First, RT-PCR determined the absence of mRNA transcripts for the pan-leukocyte marker CD45 in epithelial or stromal cells. Second, flow cytometric analysis using a bovine CD45 antibody confirmed the absence of contamination with CD45+ cells. In response to OT, the stromal cells produced predominantly PGE, whereas epithelial cells secreted PGF, confirming previous observations (4). Because research on the interactions of uterine cells with invading pathogens is often hampered by the lack of cell accessibility or established models, these bovine tissue cultures provide a biologically relevant model to study the immune function of the endometrium.
The main pathogenic ligand of E. coli, LPS, was added to tissue explants at concentrations spanning the range found in the uterus of infected animals (9). There was a dose-dependent secretion of PGE and PGF in response to LPS, confirming data where a single concentration of 0.1 µg/ml LPS was used to challenge endometrial explants (27). These observations were extended in the present study by demonstrating the specificity of the tissue response using an LPS antagonist, which abrogated the increased production of prostaglandins, even for the maximal LPS challenge. The explants in both studies secreted more PGE than PGF in response to LPS. This greater ratio of PGE to PGF production is the reverse of that when endometrial cells or explants are stimulated with OT. After uterine infection, the altered prostaglandin secretion toward PGE may disrupt luteolysis and ovarian cycles, and provide unfavorable conditions for the establishment of pregnancy. The increased prostaglandin secretion by the endometrial explants could reflect the presence of immune cells within the explants, as they also respond to LPS challenge by secreting PGE and, to a lesser extent, PGF (20, 38). So, to examine further the uterine response to bacterial challenge, it was necessary to isolate and challenge pure populations of epithelial and stromal cells that were not contaminated by leukocytes.
Endometrial stromal and epithelial cells challenged with heat killed E. coli secreted PGE and/or PGF. To ensure the bacteria were biologically relevant, E. coli was isolated from the uterus of a cow with persistent uterine disease. Although it is difficult to reconcile the numbers of bacteria added to the cultures with those in the uterine lumen, they are likely to be of a similar magnitude as swabs collected from the uterus of infected cows often contain several hundred E. coli (10). Interestingly, epithelial cells responded to the microbial challenge at lower concentrations of bacteria than the stromal cells, and this differential sensitivity may reflect their spatial roles in the endometrium. Epithelial cells are in contact with the uterine lumen, so that sensitivity to fewer bacteria than stromal cells should ensure a rapid immune response to remove the pathogen. Indeed, direct contact of epithelial cells with the uterine lumen plays an important role in rats, where E. coli stimulates secretion of an anti-bacterial product (39). The effect of E. coli on secretion of prostaglandins by the present cell cultures was, in part, mediated by LPS as polymyxin B abrogated the response. Therefore, the next step was to challenge the endometrial cells with purified LPS.
Addition of appropriate concentrations of E. coli LPS to stromal and epithelial cells stimulated a dose-dependent secretion of PGE and PGF, respectively, and there was increased expression of COX-2 mRNA. Similar to the explant cultures, there was more PGE than PGF produced as a consequence of LPS challenge. In addition, both epithelial and stromal cells produced PGE. This is in contrast to the greater PGF to PGE ratio after OT stimulation and further confirms that uterine infections disrupt ovarian cycles. The production of PGE by the epithelial cells may be of considerable significance, and this modulation of the prostaglandin pathways in these cells warrants further investigation.
Work in humans and rats has focused on the increased secretion of inflammatory molecules such as IL-8 and TNF after LPS challenge of endometrial cells, and prostaglandin production does not appear to have been investigated (21, 40). The specificity of the LPS response in the present study was demonstrated by addition of polymixin B, which abrogated the secretion of prostaglandins. However, as TLR4 is the principal receptor for the recognition of LPS, it was important to determine whether the bovine endometrium expresses this receptor (14, 15).
The present study is the first to report mRNA transcript expression of the TLR4-CD14 complex in pure populations of bovine endometrial epithelial and stromal cells. In the human, TLR4 mRNA has been identified in the endometrium and in endometrial epithelial cell lines (18, 22). Recently, TLR4 mRNA and protein expression were reported in primary cultures of epithelial and stromal cells from the human endometrium (21). However, the absence of a suitable antibody to bovine TLR4 hampers the detection of TLR4 protein on bovine endometrial cells. The TLR4 signaling pathway requires accessory molecules such as CD14 (14, 15). In the present study, a bovine CD14 antibody was used to demonstrate that epithelial and stromal cells express CD14 protein, in addition to expressing CD14 at the mRNA level. In the human, CD14 was detected by flow cytometry only on stromal cells and not on epithelial cells, which may reflect a species difference (21).
The innate immune response is governed by the ability of immune cells to recognize pathogens and produce immune mediators, such as TNF- and NO (14). Because the endometrial cells expressed TLR4 and responded to LPS with an enhanced production of prostaglandins, the capacity of these cells to produce TNF- and NO was determined. Transcripts for iNOS, the enzyme required for the production of NO, and TNF- were up-regulated by endometrial cells after LPS challenge, but NO and TNF- were not detected in culture supernatants. These observations are in contrast to findings in the rat, where endometrial epithelial cells secrete detectable concentrations of TNF in response to LPS (40). In contrast, NO and TNF in the bovine endometrium may act in an autocrine or paracrine manner or may produce concentrations that are below the limits of detection of the assays in the present study. Indeed, the concentrations of TNF- and NO that stimulate the production of prostaglandins by bovine stromal and epithelial cells are around the limits of detection of the assays in the present study (41, 42).
Progesterone suppresses uterine immune responses resulting in increased susceptibility of the uterus to infection, at least in rodents and cattle (23, 43, 44, 45). The role of estradiol in infection is dependent on the species, tissue, and concentration of the sex hormone, but is generally thought to confer protection against uterine infections (24). In the present study, basal secretion of PGE by stromal cells was unaffected by steroids, as previously described (4), although the suppression of PGF secretion by estradiol and stimulation by progesterone was less marked than in the earlier study. However, the endometrial cells both secreted less prostaglandin after LPS challenge, when they were preincubated with physiological concentrations of progesterone or estradiol. The effect of estradiol was not as marked as that after progesterone treatment and this observation concords with the more potent suppression of immunity in the endometrium by progesterone (23, 43, 44, 45).
Taken together, the results showed that bovine endometrial stromal and epithelial cells express TLR4 and CD14, and produced a functional response to Escherichia coli and its pathogenic ligand, LPS. There was increased secretion of PGE and PGF, and greater expression of mRNA for inflammatory mediators. The increased secretion of prostaglandins may explain why luteolysis is often disrupted during uterine infection. However, the response to LPS was dependent on the steroid hormone milieu, which has important implications for the establishment of uterine infections and treatment of disease. In conclusion, epithelial and stromal cells have an important immune function to detect and respond to bacteria, as well as fulfilling endocrine roles in the endometrium.
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Footnotes |
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S.H., D.P.F., D.W., E.J.W., S.T.L., H.D., C.E.B., and I.M.S. have nothing to declare.
First Published Online October 13, 2005
Abbreviations: AA, Arachadonic acid; COX, cyclooxygenase; FBS, fetal bovine serum; iNOS, inducible NO synthase; LPS, lipopolysaccharide; NO, nitric oxide; OT, oxytocin; PGE, prostaglandin E2; PGF, prostaglandin F2; PID, pelvic inflammatory disease; TLR, Toll-like receptor.
Received August 31, 2005.
Accepted for publication September 30, 2005.
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and E2 and response to oxytocin in cultured epithelial and stromal cells of the bovine endometrium. Biol. Reprod 54:371–379 metabolite concentrations in postpartum beef cows. J Anim Sci 70:3158–3162[Abstract], -2 and -6 on oxytocin receptor expression and prostaglandin production in bovine endometrium. J Endocrinol 168:497–508[Abstract] production and oxytocin receptor concentrations in bovine endometrium in explant culture. Domest Anim Endocrinol 20:217–226[CrossRef][Medline] production by oxytocin and platelet-activating factor in bovine endometrial cells. J Reprod Fertil 103:239–247 and tumor necrosis factor production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys. Infect Immun 73:476–484 on production of prostaglandin F2 and E2 in bovine endometrial cells. J Reprod Dev 50:333–340[CrossRef][Medline] by cultured bovine endometrial cells in response to tumor necrosis factor : cell type specificity and intracellular mechanisms. Biol Reprod 62:1116–1120This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
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) or absence (
) of polymyxin B (2 µg/ml). After 24 h in culture, supernatants were harvested and prostaglandin production was measured by RIA. *, Differences were statistically different at P < 0.05 compared with control. 
, Differences were statistically different at P < 0.05 compared with corresponding LPS stimulation. Numerical values are presented as the mean + SEM of three experiments.
