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Canadian Institute of Health Research (M.P., W.L., N.A., J.R.G.C.), Departments of Physiology, Obstetrics, Gynecology and Medicine, University of Toronto, Toronto, Ontario, Canada M5S 1A8; Department of Obstetrics and Gynecology and Physiology (A.D.B., K.C.), University of Western Ontario, London, Ontario, Canada N6A 5B8; and Departments of Obstetrics and Gynecology (W.G.), University of Ottawa, Ottawa, Ontario, Canada K1N 6N5
Address all correspondence and requests for reprints to: Marina Premyslova, Department of Physiology, Obstetrics, and Gynecology, Medical Science Building, University of Toronto, 1 King’s College Circle, Toronto Ontario, Canada M5S 1A8. E-mail: marina.premyslova{at}utoronto.ca.
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Abstract |
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on mPGES, cPGES, and cyclooxygenase (COX)-2 expression in villous trophoblast (VT) and chorion trophoblast (CT) cell cultures. mPGES localized to the syncytiotrophoblast and vascular endothelium in PL and to the amnion epithelium, CT, and decidual cells in FM. cPGES protein was localized only to the syncytiotrophoblast in PL and had the same profile of expression as mPGES in FM. With infection, there was an increase in mPGES expression in PL and a decrease in the expression in FM. cPGES protein did not change in either PL or FM with infection. In VT cells in culture, IL-1ß up-regulated COX-2 protein expression but did not affect mPGES. However, TNF- increased both mPGES and COX-2 protein expression in these cells. In CT cells in culture, IL-1ß and TNF- increased both mPGES and COX-2 protein levels. Neither IL-1ß nor TNF- affected cPGES in either VT or CT cells. We conclude that protein levels of mPGES, as well as COX-2, can be stimulated by cytokines, potentially contributing to the increased prostaglandin production at the time of infection-driven preterm labor. However, multiple mechanisms, which apparently are inductor- and cell-type-specific, exist for the regulation of these enzymes. |
Introduction |
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The concentrations of inflammatory cytokines such as TNF- and IL-1ß are increased in amniotic fluid of women in preterm labor in the presence of chorioamnionitis (an inflammatory process involving the chorion, its fetal blood vessels, the umbilical cord, and the amnion) (9, 10, 11, 12). These cytokines activate the PG biosynthetic pathway primarily via induction of cyclooxygenase-2 (COX-2) and increased synthesis of PGE2 by human amnion (13), chorion, and decidual (14) cells. Furthermore, IL-1ß can stimulate PG output not only by increasing expression of COX-2 in human villous and chorion trophoblast (VT and CT) cells but also by decreasing expression of prostaglandin dehydrogenase (PGDH) in VTs (15, 16). However, the importance of PGES, which acts downstream of COX-2, in regulating PG synthesis during preterm labor of infectious etiology in human placenta (PL) and fetal membranes (FM) is unknown. Previously, no changes in mPGES mRNA distribution with term labor or with infection-driven preterm labor were observed in amnion, chorio-decidual, or villous tissues (17). However, we found increased mPGES protein expression in chorion (18), although not in myometrium (19) with labor, and others have reported increased mPGES mRNA expression in myometrial cells after treatment with IL-1ß (20).
In the present study, we examined whether PGES protein expression changed with infection-driven preterm labor, whether PGES enzyme in human PL and FM was regulated by proinflammatory cytokines, and whether there was a coordinate regulation of mPGES, cPGES, and COX-2 by cytokines. We examined mPGES and cPGES immunohistochemical localization and protein expression in preterm human membranes and PL with chorioamnionitis, and we determined the regulatory effect of IL-1ß and TNF- on mPGES, cPGES, and COX-2 protein expression using purified cultures of VT and CT cells.
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Materials and Methods |
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PL and FM from preterm (24.5–36 wk gestation) vaginal delivery with (n = 16) or without (n = 16) chorioamnionitis were collected at St. Josephs Health Centre (London, Ontario, Canada). FM were separated from PL. Rolls of FM and PL tissue were immediately snap frozen in liquid nitrogen for Western blotting analysis or fixed in 4% paraformaldehyde and paraffin embedded for immunohistochemistry (IHC). Diagnosis of chorioamnionitis was confirmed by histopathology. Leukocyte infiltration was used as a criterion for chorioamnionitis. Term PL (n = 8) and membranes were also collected from normal uncomplicated pregnancies, at term, after elective cesarean section in the absence of labor (Mount Sinai Hospital, Toronto, Canada). Patient consent and ethical approval were obtained in accordance with the guidelines of Research Ethics Committees of Mount Sinai Hospital and the University of Toronto, as well as the Review Board for Research Involving Human Subjects of the University of Western Ontario (London, Ontario, Canada). Term tissues were used as a source of PL and CT cells, not affected by inflammation, labor or any complication of pregnancy that could change the expression of prostaglandin synthesizing enzymes.
IHC for mPGES and cPGES
The PL and FM tissues with and without chorioamnionitis (10-µm sections) were deparaffinized with xylene (EMD Chemicals, Inc., Gibbstown, NJ) and rehydrated in graded ethanol washes (six sections for each of four tissue groups were processed). Endogenous peroxidase activity was inhibited by treatment with 0.3% hydrogen peroxide in PBS for 30 min. The sections were then washed in 0.01 M PBS and incubated for 2 h at room temperature (RT) with normal goat serum to block nonspecific binding. Sections were incubated overnight, at +4 C, with primary polyclonal antibodies against a peptide corresponding to amino acids 59–75 (CRSDPDVERSLRAHRND) of human mPGES (Cayman Chemical, Ann Arbor, MI) or with primary polyclonal antibodies against a peptide corresponding to amino acids 58–67 (CIDPNDSKHK) of human cPGES (Cayman Chemical), both used at a dilution of 1:200 in antibody dilution buffer (1% BSA in 0.01 M PBS). Primary antibody binding was visualized using the Vectastain ABC Kit (Vector Laboratories, Burlingame, CA): sections were incubated for 2 h with antirabbit biotinylated secondary antibodies, at RT, followed by a PBS wash and 2-h incubation, at RT, with avidin biotin-peroxidase complex. 3,3' diaminobenzidine (Sigma St. Louis, MO) was used as chromagen. The sections were counterstained with Carazzi’s hematoxylin, dehydrated in graded ethanol, and cover slips were applied. For negative controls, the primary mPGES and cPGES antibodies were pre-absorbed overnight with 10 µg/ml mPGES or cPGES blocking peptides (Cayman Chemical), respectively.
Western blot analysis of mPGES and cPGES protein expression in human preterm PL and FM with and without chorioamnionitis
Levels of mPGES and cPGES protein in PL and FM tissues were assessed by Western blotting. Proteins were extracted from frozen tissues by homogenization on ice in T-PER protein extraction buffer (Pierce, Rockford, IL) containing Mini EDTA-free protease inhibitors (Boehringer Mannheim Biochemicals, Mannheim, Germany) and 100 µM sodium orthovanadate (Na2VO2, Sigma). Insoluble material was pelleted by centrifugation at 14,000 x g for 10 min at +4 C, and the clarified lysates were collected. Protein concentrations in the supernatants were determined by the Bradford assay (21). Proteins samples (10 µg/lane for mPGES and 20 µg/lane for cPGES) were diluted and boiled for 3 min in sodium dodecyl sulfate-containing sample buffer (22) and then resolved on a 14% (vol/vol) sodium dodecyl sulfate-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane (23). Transfer was confirmed by protein visualization with Ponceau S (Sigma). Membranes were blocked for 2 h at 25 C in 5% skimmed milk powder diluted in Tris-buffered saline (TBS)-Tween, pH 7.6 [50 mM Tris-HCl, 150 mM NaCl, and 0.1% (vol/vol) Tween 20]. After blocking, membranes were incubated for 1 h with mPGES (1:1000 in blocking solution) or cPGES (1:1000) antibodies. Blots were washed 5 times (10 min each) with TBS-Tween and subsequently incubated for 1 h with horseradish peroxidase-conjugated goat antirabbit IgG (1:2000 in blocking solution) (Amersham Life Science, Quebec, Canada). Then, membranes were washed 5 times (10 min each) with TBS-Tween, and proteins bands were visualized with enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK). The membranes were exposed to X-OMAT blue film (Kodak Scientific Imaging Product, Rochester, NY). The relative intensities of immunoreactive bands were measured by scanning and analysis with Scion Image software (Scion Image Corporation, Frederick, MD). Protein bands were digitized, and the mean pixel density for each band was analyzed to obtain relative optical density units for each protein. Sample loading was standardized to the expression of ß-globulin using specific antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; 1:2000) (24). To compare measurements between different blots, a reference PL or FM sample was included in each gel.
VT and CT cell culture and treatment
VT and CT cells were isolated and cultured using a modification of the technique described by Kliman et al. (25, 26). Briefly, approximately 60 g of villous tissue was digested with 0.125% trypsin (Life Technologies Inc., Grand Island, NY) and 0.02% deoxyribonuclease I (Sigma) in DMEM, three times for 30 min. The chorion, after the removal of decidua and blood clots, was digested three times for 60 min with the same digestion medium supplemented with 0.2% collagenase (Sigma). The dispersed villous or chorio-decidual cells were filtered through a 200-µm-pore-size nylon gauze and loaded onto a 42-ml preformed discontinuous Percoll (Sigma) gradient of 5–70%, in 5% steps of 3 ml each, then centrifuged (1,200 x g) at RT for 20 min. Cells between densities of 1.049 and 1.062 g/ml were collected and plated in 100-mm-diameter Petri dishes at 2 x 106 cells/ml in 15 ml DMEM containing 10% fetal bovine serum (Life Technology, Inc.) and 1% (vol/vol) of antibiotic-antimycotic solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin sulfate, and 25 mg/ml amphotericin B in 0.9% sodium chloride) (Sigma). Cells were then cultured for 3 d at 37 C in 5% CO2-95% air. After 3 d, PL trophoblast cells aggregated to form a syncytium, and CT cells formed clumps. Chorion and PL cells were then washed twice with Hanks’ solution, equilibrated at 37 C, and cultured for 16 h in DMEM free of FCS and antibiotics. Cells viability was assessed by using trypan blue exclusion and was higher then 80% in all experiments.
Effect of cytokines
After 16 h incubation in DMEM free of fetal bovine serum and antibiotics, the medium was changed, and the cells were treated with: 1) medium alone; and 2) IL-1ß (recombinant human IL-1ß, 0.1 ng/ml; 1 ng/ml and 10 ng/ml) (Sigma) for 4 h, or with TNF- (recombinant human TNF-, 0.1 ng/ml; 1 ng/ml and 10 ng/ml) (Sigma) for different periods of time (2, 4, 6, and 24 h). CT cells were treated with IL-1ß (1 ng/ml) or TNF- (10 ng/ml) for 4 h. We chose 4 h for IL-1ß treatment, based on our preliminary experiments when we failed to observe any effect of IL-1ß on mPGES expression after 24 h incubation. Cells were characterized by cytokeratin (an epithelial cell marker) or vimentin (a fibroblast cell marker) immunostaining as published previously (15). About 90% of PL and chorionic cells were cytokeratin positive. All experiments were performed in duplicate for PL cells and in singleton or duplicate for CTs (depending on the cell yield after extraction).
Western blotting for mPGES, cPGES, and COX-2 expression in cultured VT and CT cells
Cultured cells were collected, proteins extracted, and Western blotting carried out as described for tissue samples. The membranes were sequentially probed with mPGES, cPGES, and COX-2 antibodies (1:1000 in blocking solution, Cayman Chemical).
Data analysis
Student’s paired and unpaired t tests were used to compare the results for infected and noninfected tissue samples and to determine the effects of IL-1ß and TNF- on CT cells. One-way ANOVA, followed by Student-Newman-Keuls test, was used to compare dose effects of the cytokines on VT cells. Data are expressed as the mean ± SEM, with P = 0.05 as the limit of significance (Sigma Stat; Jandel Scientific Software, San Rafael, CA).
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Results |
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In PL, mPGES protein was localized to the syncytiotrophoblast (ST) and vascular endothelium (VE) in noninfected (Fig. 1A
) as well as in infected with CA tissues (Fig. 1B). In noninfected FM, mPGES was localized to amnion epithelium (AE), CT, and decidual cells (Fig. 1C). With infection, there was a loss of normal structure of the membrane, loss of the chorion layer, and diminished mPGES immunostaining (Fig. 1D). cPGES was localized to ST in normal (Fig. 1E) and in infected (Fig. 1F) PL tissues. In FM, cPGES had the same pattern of expression as mPGES (Fig. 1, G and H, show cPGES localization in noninfected and infected FM, respectively). mPGES and cPGES immunostaining in FM and PL was entirely abolished after preabsorption of the antibodies with the corresponding blocking peptides (Fig. 1; I, J, K, and L).
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By Western blotting, immunoreactive mPGES protein was present as a single band with the expected molecular weight of 16 kDa; immunoreactive cPGES was present as a 23-kDa band. In PL samples, the level of mPGES was increased (P < 0.05) with infection (Fig. 2, A and C). However, there was a decrease in the expression of mPGES within FM with infection (P < 0.05, Fig. 2, B and C), which is likely attributable to the loss of cells from the CT layer (see Fig. 1
). There was no significant effect of chorioamnionitis on cPGES expression within these tissues (Fig. 3, A–C).
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on mPGES, COX-2, and cPGES expression in primary VT and CT cell cultures
The increase in mPGES expression in infected PL tissue might be attributable to the elevated level of proinflammatory cytokines, mainly IL-1 ß and TNF-, known to be secondary to chorioamnionitis. To test this hypothesis and to show whether the changes in mPGES expression correlated with any changes in COX-2 and cPGES expression, we determined the effect of IL-1ß and TNF- on mPGES, COX-2, and cPGES protein expression in primary cultures of VT and CT cells, isolated from PL, at term, after elective cesarean section.
In cultured VT cells, IL-1ß (0–10 ng/ml) did not affect mPGES or cPGES expression during 4 h of incubation (Fig. 4, A and C). In contrast, COX-2 protein expression was up-regulated by IL-1ß at 10 ng/ml (Fig. 4B). TNF- caused a 2-fold increase in mPGES expression at 10 ng/ml (Fig. 5A). COX-2 was also up-regulated by TNF- (Fig. 5B). The increase was dose-dependent for both enzymes, with maximum effect at the concentration of 10 ng/ml TNF- for mPGES and 1.0 ng/ml for COX-2. cPGES was unaffected by the range of TNF- concentrations (Fig. 5C). The time-dependent character of TNF- stimulation of mPGES and COX-2 is shown in Fig. 6, A and B. The increase in mPGES and COX-2 expression occurred within the first 2–4 h of treatment. After 4 h, there was a decline in mPGES expression, whereas up-regulation of COX-2 lasted at least 24 h. cPGES remained unchanged under the same conditions of TNF- treatment (Fig. 6C). In CT cells, IL-1ß (1 ng/ml) and TNF- (10 ng/ml) up-regulated both mPGES (Fig. 7, A and B) and COX-2 enzymes (Fig. 7, C and D) but did not affect cPGES expression (Fig. 7, E and F).
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Discussion |
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on mPGES and COX-2 protein expression in cultured VT and CT cells prepared from term PL without infection. IL-1ß, though not affecting mPGES expression in VT cells, up-regulated COX-2 in these cells and caused an increase in mPGES as well as in COX-2 protein expression in CTs. Regulation of cPGES was clearly different from that of mPGES or COX-2 and was unaltered by the amount of proinflammatory cytokines used in the present experiments. IHC showed a loss of the normal structure of the amnio-chorion and especially of the chorion layer in tissues from preterm labor patients with infection. There was diminished mPGES as well as cPGES immunostaining in these tissues. We have reported previously similar observations showing that reduction of mRNA, protein, and enzyme activity of 15-OH PGDH in chorion was accounted for by the loss of the trophoblast cells that accompany the infective process (27, 28, 29). Our finding of expression of mPGES, rather then cPGES, in VE implies that mPGES may be important for regulation of PL vascularity (and thus, PL blood flow) and that it may be acutely regulated. In the FM, cPGES had the same pattern of staining as mPGES, localizing to the amnion epithelial, CT, and decidual cell. Based on our culture experiments, its appearance is more constitutive and not amenable to short-term regulation at least by two pro-inflammatory cytokines.
By Western blotting, we have shown increased mPGES expression in PL and decreased mPGES expression in FM with infection. The decrease may reflect the mechanical loss of CT cells in infected FM. However, there was no change in cPGES expression in these tissues, despite the same profile of immunolocalization for both isoforms. Our use of full-thickness membranes for these experiments might explain this difference. We suggest that mPGES, but not cPGES, has a relatively higher protein expression in chorion, compared with amnion epithelium and decidua. Thus, we measured a detectable decrease in mPGES, but not in cPGES protein expression, in the infected full-thickness FM, which exhibits loss of chorion layer with infection.
The increase in mPGES in PL could result from stimulatory effects of proinflammatory cytokines. Indeed, we speculated that cytokines might also increase initially mPGES in chorion at an earlier time point in the infective process. The decrease seen in intact tissues would reflect then a later loss of chorion cells.
Levels of proinflammatory cytokines are known to be elevated with chorioamnionitis (9, 10, 11, 12). Previous studies have shown the induction of COX-2 enzyme by proinflammatory cytokines (30, 31, 32, 33), and coordinate induction of COX-2 and mPGES (4, 5, 7). Therefore, we investigated whether mPGES was coordinately regulated with COX-2 in VT and CT cells. In VT cells, IL-1ß had no effect on mPGES expression, but it induced the expression of COX-2 in a dose-dependent manner. In CTs, also cultured for 4 h, IL-1ß up-regulated both mPGES and COX-2 enzymes. Although we did not measure PGE2 output in the present study, these results are consistent with increased PGE2 output from VTs (starting already after 4 h of incubation) and from CTs with Il-1 ß treatment, reported from our laboratory previously (15).
It is not clear why VT and CT cells exhibited different responses to IL-1ß, although this could be related to the tissue-specific balance of pro- and anti-inflammatory stimuli. The up-regulation of COX-2, accompanied with unchanged mPGES expression in VTs under IL-1ß treatment, may suggest also that there are different mechanisms involved in the regulation of the two enzymes. In contrast, TNF- stimulated mPGES as well as COX-2 protein in VTs in a dose- and time-dependent manner. The increase in COX-2 expression at 0–1 ng/ml TNF- was followed by a decrease at 10 ng/ml (Fig. 5B). We speculate that this decrease might be the result of COX-2 negative self-regulation (34). CT cells also responded to TNF- treatment by an increased level of mPGES and COX-2 proteins. These data suggest that the mechanisms of mPGES and COX-2 regulation are inductor specific. The shapes of dose- and time-response plots, which are not the same for mPGES and COX-2, support the possibility of the existence of different mechanisms responsible for their regulation. Further studies are required to examine whether there are differences in cellular and/or subcellular colocalization of these enzymes, and to determine changes that occur with acute stimulation. cPGES was unchanged in both cell types under either IL-1ß or TNF- action. These data are in agreement with studies using other cell and tissue types (2). The current findings regarding the stimulatory effect of inflammatory cytokines on mPGES are consistent with previous reports on the ability of cytokines to up-regulate mPGES in other cell systems and tissues (1, 3, 4, 5, 6, 7, 8). Consistent with this, in human gestational tissues, there is increased expression of mPGES mRNA after IL-1ß treatment in myometrium (20), and there is increased mPGES protein expression in chorion with labor (18). However, others did not find differences in mPGES mRNA expression with term labor in amnion, chorio-decidua, or villous tissues (17) or in human myometrium with labor (19). The reasons for these conflicting results are not apparent at the present time. In conclusion, our results suggest that chorioamnionitis up-regulates mPGES protein expression in human preterm PL and that proinflammatory cytokines, produced in response to chorioamnionitis, might underlie the effect. Thus, mPGES seems to be involved in the process of inflammation in the human PL and FM, and may contribute to an overall increase in PGE2 production at the time of preterm labor with infectious etiology. However, the mechanisms of mPGES and COX-2 induction do not seem to be coordinate in the trophoblast cells and may be both cell-type- and inductor-specific.
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Footnotes |
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Received April 9, 2003.
Accepted September 8, 2003.
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References |
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. Placenta 143:2936–2943
13, 14-dihydro-15-keto-prostaglandin F2 (PGFM) and 11-deoxy-13, 14-dihydro-15-keto-11, 16-cyclo-prostaglandin E2 (PGEM-LL) in preterm labor. Prostaglandins 37:149–161[CrossRef][Medline]
, and epidermal growth factor on prostaglandin production in human chorion cells. Am J Reprod Immunol 30:146–153
on the activity and expression of prostaglandin H synthase-2 and the NAD-dependent 15-hydroxyprostaglandin dehydrogenase in cultured term human villous trophoblast and chorion trophoblast cells. J Clin Endocrinol Metab 84:4665–4651 in human myometrium in term and preterm labor: a mechanism for parturition. J Clin Endocrinol Metab 79:1835–1839[Abstract]
and 1ß on prostaglandin production by human trophoblast cells from first and third trimesters. J Clin Endocrinol Metab 80:1641–1646
induces cyclooxygenase-2 gene expression in first trimester trophoblasts: suppression by glucocorticoids and NSAIDs. Placenta 18:521–526[CrossRef][Medline]
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