Cellular Specificity of Interleukin-1ß-Stimulated Expression of Type-2 Prostaglandin H Synthase in Human Amnion Cell Cultures¹

^a Department of Obstetrics and Gynecology and Physiology, University of Ottawa, Ottawa General Hospital, Ottawa, Ontario, Canada K1H 8L6

	ABSTRACT

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Interleukin-1ß (IL-1ß) has been shown in numerous studies to increase prostaglandin output by cultures of human amnion cells. This is due to an increase in the expression of type-2 prostaglandin H synthase (PGHS-2), the inducible form of the enzyme, in these cultures. Amnion consists of an epithelial layer of cells and a subepithelial mesenchymal layer of cells. The purpose of the present study was to determine the cell-type(s) responsible for the IL-1ß-induced PGHS-2 expression in amnion cultures. Amnion was obtained at term after elective Cesarean section or vaginal delivery. Tissues were dispersed with collagenase, and cells were plated in multichamber culture slides and cultured for 7 days in media supplemented with 10% fetal bovine serum. Cell types were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Cultures contained both cell types, and the proportion of these varied considerably from one culture to another. Cells were treated with various concentrations of IL-1ß for 6 or 24 h and were then fixed in 4% paraformaldehyde. The fixed cells were permeabilized with Triton and examined by immunohistochemistry for PGHS-2 protein using specific antisera, and PGHS-2 mRNA was localized by in situ hybridization using a specific oligonucleotide probe. The cell type(s) expressing PGHS-2 was characterized using double labeling with antisera to keratin (epithelial cell marker) and vimentin (mesenchymal cell marker). IL-1ß was found to increase expression of immunoreactive PGHS-2 and PGHS-2 mRNA. This increased expression was found to occur only in the vimentin-positive cells and not the epithelial cells. These results highlight the potential importance of the subepithelial cells in the mesenchymal layer of amnion in the formation of prostaglandins during pregnancy and possibly in preterm labor with infection.

	INTRODUCTION

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The human fetal membranes, amnion and chorion laeve, form the amniotic sac in human pregnancy and have been proposed to be intimately involved in the onset and/or progression of labor [1]. These tissues produce various substances that may be involved in labor including prostaglandins. Extensive studies have investigated the regulation of prostaglandin formation in the fetal membranes; and various substances, including glucocorticoids [2–4], peptides [5], and cytokines [6–9], have been found to stimulate prostaglandin production in these tissues. Since these tissues are derived from human products of conception, the approaches to studying the regulation of prostaglandin formation by them are severely limited. Primary cultures of cells from both chorion laeve and amnion have been extensively used as models for investigating prostaglandin formation by these tissues.

Primary cultures of amnion cells respond to various cytokines including interleukin-1ß (IL-1ß) by increasing prostaglandin output [6], and it has been shown that IL-1ß-stimulated output results from increased expression of type-2 prostaglandin H synthase (PGHS-2), the inducible form of PGHS [10]. These studies have supported the view that infection-driven preterm labor may result from cytokine-induced prostaglandin formation in the fetal membranes.

Amnion is composed of an epithelial layer of cells and a mesenchymal layer. Recently, we have shown that glucocorticoid stimulation of PGHS-2, which occurs in amnion cultures, is specific for the cells derived from the mesenchymal layer rather than the epithelial cell layer. This indicated that the cells in the mesenchymal layer, heretofore largely ignored, may have an important physiological role in the onset and progression of labor in humans. With respect to interleukin action, it is not known which cell type or types respond by increasing PGHS-2 expression and prostaglandin output. The purpose of the present study, therefore, was to determine which cell type(s) in amnion cultures responded to IL-1ß by increasing PGHS-2 expression. We present evidence that the mesenchymal cells and not the epithelial cells are the major cell type that up-regulates PGHS-2 mRNA and protein expression in response to IL-1ß.

	MATERIALS AND METHODS

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Tissues

Term (38–40 wk gestation) placentae with adherent membranes were obtained immediately after spontaneous vaginal delivery or elective Cesarean section from patients with no clinical evidence of infection. Membranes were cut, rolled, fixed in 4% paraformaldehyde (PFA), and embedded in paraffin. Six- to 7-µm sections were mounted on Superfrost Plus slides (Fisher Scientific, Nepean, ON, Canada) and deparaffinized by standard procedures before immunohistochemistry. Tissues were also set aside for culture.

Isolation of Amnion Cells and Primary Culture

All manipulation was carried out under sterile conditions. Amnion was peeled from chorion laeve and was cut about 25 mm above the placental disc. It was washed several times in PBS (150 mM NaCl, 10 mM Na₂HPO₄, 1.5 mM NaH₂PO₄, pH 7.5) and finely minced with scissors. Tissue was transferred into a sterile tube containing 40 ml PBS and 2.5 mg/ml collagenase A (Boehringer Mannheim Canada, Dorval, PQ, Canada) and was incubated at 37°C with gentle shaking for 2 h. After digestion, 3 µg/ml of trypsin inhibitor (Boehringer) was added, and the suspension was filtered through nylon screens of 100 and 35 µm. The cells were pelleted by centrifugation at 2000 x g for 10 min, washed once in PBS, counted on a hemocytometer, and suspended in culture medium. Culture medium consisted of a 1:1 (v:v) mixture of Ham's F-12 (Gibco/BRL, Burlington, ON, Canada) and Dulbecco's Modified Eagle's Medium (Gibco/BRL), 10% fetal calf serum (ICN Canada, Montréal, PQ, Canada), 1 g/L BSA, 29 mM NaHCO₃, 45 mM 4-(2-hydroxymethyl)-1-piperazine-ethanesulfonic acid at pH 7.4, 10⁵ U/L penicillin, 16 mg/ml gentamycin, and 8 µg/ml fungizone. Cells were plated in 8-well slides (Nunc, Naperville, IL) at 1.25 x 10⁵ cells per 0.64 cm² well/0.4 ml. They were maintained in culture at 37°C with a water-saturated atmosphere and 5% CO₂ and air. Media were replaced at least every second day. On Day 6, cells were treated for 6 h or 24 h with Il-1ß (0.1–1 ng/ml). Control cells received media alone. After treatment, cells were fixed in 4% PFA and stored in 95% ethanol at 4°C.

Immunohistochemistry

PGHS-2 was localized with a polyclonal rabbit antiserum (Oxford Biomedical Research Inc., Oxford, MI; product #PG27), keratin with a polyclonal rabbit antiserum (Dako, Glostrup, Denmark), and vimentin with a monoclonal mouse antibody (Dako). Cells were permeabilized in 0.2% Triton X-100 for 1 h before staining. Double labeling of antigens was carried out as follows. Label 1 was detected with a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA). All antibody dilutions and washes for this kit were in PBS (150 mM NaCl, 10 mM Na₂HPO₄, 1.5 mM NaH₂PO₄, pH 7.5). Endogenous peroxidase activity was quenched by 30 min of treatment with 0.3% H₂O₂ in PBS. Slides were washed twice for 10 min. Blocking serum was applied for 20 min, and the slides were incubated with primary antibody in 0.1% BSA overnight at 4°C. The primary antibody dilutions found to be appropriate for this kit were 1:4000 anti-keratin, 1:3000 anti-vimentin, and 1:200 anti-PGHS-2. Slides were washed twice for 5 min, incubated with biotinylated secondary antibody for 30 min, washed twice for 5 min, incubated with avidin-biotin-peroxidase complex (ABC) for 30 min, and washed twice more for 5 min. Immunoreactive proteins were visualized by treatment with diaminobenzidine (Fast DAB tablets; Sigma) for 5 min. Slides were then washed once in water and once in PBS for 5 min each. Label 2 was detected with an Immunopure ABC Alkaline Phosphatase kit (Pierce, Rockford, IL). Antibody dilutions for this kit were in Tris-buffered saline (TBS: 150 mM NaCl, 10 mM Tris, pH 7.5), and washes were in PBS. Beginning with the blocking serum and up to the ABC treatment, slides received the same treatment as for label 1, except that the primary antibody incubation was for 30 min at room temperature. The primary antibody dilutions found to be appropriate for this kit were 1:4000 anti-keratin and 1:3000 anti-vimentin. Immunoreactive proteins were visualized by 10-min treatment with a solution of reagents from Substrate Kit I (TR SALT/NABP; Pierce) in Substrate TBS (100 mM Tris, pH 8.2) to yield a red product. Slides were counterstained for 1 min in Harris' hematoxylin, dehydrated in graded ethanol, cleared, and mounted. In single labeling, the Vectastain Elite ABC kit was used. Controls included replacement of antibody with buffer and with preimmune serum.

Quantification of Cell Types

At least 300 cells were counted in a field of 1 mm² at a magnification of x100. The percentage of epithelial cells in each culture was determined from the percentage of cells that were keratin-immunopositive as calculated from the sum of all the cell counts from each treatment group. (Within a culture, no difference in epithelial cell content between groups was found.) Percentage of fibroblasts was calculated as the percentage of epithelial cells subtracted from 100%. The percentages of PGHS-2-immunopositive fibroblasts were determined in control and IL-1ß-treated cells in a similar fashion. Values are expressed as the mean ± SD of determinations by three independent observers.

In Situ Hybridization

The 50-mer antisense oligonucleotide probe for PGHS-2 (complementary to bases 1734–1783 of the human PGHS-2 gene [11]) was synthesized by the molecular biology facility at the University of Ottawa using an Oligo 1000 DNA synthesizer (Beckman Instruments, Inc., Palo Alto, CA), and was purified by HPLC. The uniqueness of the sequence for PGHS-2 was verified with a nucleotide sequence database using Blastn 1.4.6 MP [12]. Hybridization with the corresponding sense probes, prepared in a similar fashion, served as a control.

Hybridization procedures have been described previously [13]. The probes were labeled using terminal deoxynucleotidyl transferase (Gibco/BRL) and ³⁵S-labeled deoxyadenosine 5'--thio triphosphate (1300 Ci-mmol; NEN, Dupont Canada Inc., Mississauga, ON, Canada). The labeled probe was purified using a Nensorb 20 column (NEN; Dupont Canada Inc.). Slides were allowed to air-dry at room temperature and were then incubated overnight in a moist chamber at 45°C with 5000 cpm/µl of radiolabeled probe in hybridization buffer. Hybridization buffer contained 4-strength SSC (single-strength SSC is 150 mM sodium chloride, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.0), 1 mM sodium pyrophosphate (pH 7.0), 50% deionized formamide, 0.02% BSA, 200 µg/ml hydrolysed salmon sperm DNA, 0.02% polyvinylpyrrolidone, 10% dextran sulphate, and 40 mM dithiothreitol (DTT). After being washed for 20 min at room temperature and for 45 min at 55°C in single-strength SSC containing 10 mM DTT, slides were rinsed in single-strength SSC containing 10 mM DTT and in 0.1-strength SSC (10 sec each). Slides were then dehydrated in graded ethanol and air-dried. Emulsion autoradiography was carried out using standard procedures with Ilford (Mobberly, England) K5 liquid emulsion. The slides were developed after 14 days using standard procedures and were counterstained with 0.1% cresyl violet to permit identification of nuclei.

	RESULTS

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The results shown are typical of results obtained from at least 6 different cultures. The specificity of the antisera for epithelial and mesenchymal cells is shown in Figure 1. Clearly, the keratin antiserum labeled the amnion epithelial cells and did not interact with the mesenchymal cells (Fig. 1:1), and the vimentin antiserum labeled the mesenchymal cells and did not interact with the epithelial cells (Fig. 1:2). In culture, both epithelial and mesenchymal cells were found (Fig. 1:3 and 1:4). However, the proportions of these cell types varied considerably from one preparation to another, and the results obtained from 6 separate cultures are shown in Table 1.

View larger version (133K): [in this window] [in a new window]   FIG. 1. Influence of IL-1ß on PGHS-2 expression in primary cultures of human amnion cells. 1) Immunohistochemical localization of keratin to the epithelial cells. 2) Immunohistochemical localization of vimentin to the mesenchymal cells. 3 and 4) Typical amnion culture with keratin-positive cells (red) and vimentin-positive cells (brown). 5, 7, 11, and 13) Control (untreated) cultures, keratin-positive cells (red); PGHS-2-positive cells (brown). 6, 8, 12, and 14) IL-1ß-Treated cultures, cytokeratin-positive cells (red); PGHS-2-positive cells (brown). IL-1ß increased PGHS-2 staining in the nonepithelial cells. 9, 10, 15, and 16) In situ hybridization for PGHS-2 mRNA in control (untreated) cells (9 and 15) and IL-1ß-treated cells (10 and 16). Magnification bars: 1–10, 13–16 = 10 µm; 11 and 12 = 20 µm.

IL-1ß was found to stimulate expression of PGHS-2 protein and mRNA only in the nonepithelial cells. The results of immunohistochemistry and in situ hybridization from 2 typical cultures are shown in Figure 1:5–16. The two cultures shown were chosen because one had a high proportion of epithelial cells (Fig. 1:5–10) and the other a high proportion of fibroblasts (Fig. 1:11–16). Clearly, in both instances, only the nonepithelial cells responded to IL-1ß and increased expression of PGHS-2 protein and mRNA. When the percentages of PGHS-2-immunopositive fibroblast cells were calculated, using three different cultures, there was a significant increase (p < 0.05) from control cultures (11 ± 2%) to IL-1ß-treated cultures (37 ± 5%). Also, it should be emphasized that keratin staining did not mask any changes in PGHS-2 protein in the keratin-positive cells: in the absence of antibodies to keratin, no increases in PGHS-2 expression was found in the epithelial cells (data not shown).

	DISCUSSION

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The present study indicates that routine cultures of amnion cells can result in considerable variation in the proportion of epithelial and mesenchymal cells and that up-regulation of PGHS-2 in human amnion cell cultures in response to IL-1ß occurs principally in the mesenchymal cells and not in the epithelial cells, indicating the potential importance of cells in the mesenchymal layer in infection-induced prostaglandin production and hence infection-induced labor.

The variation in proportions of cell types found in the present study highlights the importance of continually monitoring the purity of amnion cell cultures, even when the procedure used is not altered. It is possible that the variation seen in proportion of epithelial and mesenchymal cells isolated was due to the genetic diversity of the tissues used in human studies, and the fact that tissues obtained may be at differing states of proteolytic degradation.

The present study demonstrates that the mesenchymal cells respond to IL-1ß by up-regulating PGHS-2 protein expression through increasing PGHS-2 mRNA levels. No evidence was found for IL-1ß increasing PGHS-2 expression in the epithelial cells, although a much lower increase occurring in these cell types cannot be completely ruled out. These findings may also explain why freshly dispersed cells, which might contain a higher proportion of epithelial cells, do not increase prostaglandin output in response to IL-1ß, whereas cultured cells do [14].

Human amnion cultures respond to glucocorticoids by increasing prostaglandin output [2–4] rather than inhibiting prostaglandin output, as occurs in most other systems [15–17]. The increase seen in human amnion is due to increased expression of PGHS-2 [18]. Recently, we have shown that this glucocorticoid-induced up-regulation of PGHS-2 expression also occurs in the cells from the mesenchymal layer and not the epithelial cells [19], further highlighting the potential importance of the cells in the mesenchymal layer in prostaglandin production by another potential effector.

Infection has been shown to be an important determinant of preterm labor in humans, and various interleukins including IL-1ß are increased in amniotic fluid in preterm labor with infection [20]. In vitro studies with amnion cell cultures found that interleukins increased prostaglandin production and, in the case of IL-1ß at least, did so through increased expression of PGHS-2 [6, 10]. The present results clearly demonstrate that this up-regulation of PGHS-2 mRNA and protein occurs principally in the mesenchymal cells and not in the epithelial cells. This would indicate that cells in the mesenchymal layer may respond to infection via IL-1ß stimulation of PGHS-2 and, therefore, have an important role in infection-induced preterm labor. Whether these cell types have increased PGHS-2 expression in vivo, however, will require further studies.

In conclusion, results from the present study together with our previous studies with glucocorticoids in amnion cultures underline the importance of the cells in the mesenchymal layer of amnion in prostaglandin production. This previously ignored population of cells may therefore be important in many other aspects of amnion and fetal membrane metabolism in relation to parturition. Indeed, recent evidence indicates that cells in the mesenchymal layer express the inducible form of nitric oxide synthase [21] and are involved in collagen processing and synthesis [22].

	ACKNOWLEDGMENTS

 
The authors wish to thank the labor and delivery staff at Ottawa General Hospital for their help in obtaining tissues.

	FOOTNOTES

 
¹ These studies were supported by M.R.C. Canada Grant #MT 13413.

² Correspondence: W. Gibb, Department of Obstetrics and Gynecology, Ottawa General Hospital, 501 Smyth Rd., Ottawa, ON, K1H 8L6, Canada. FAX: 613 737 8470; wgibb{at}netcom.ca

Accepted: July 2, 1998.

Received: February 10, 1998.

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