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Department of Gynecological Endocrinology and Reproductive Medicine (R.M.P., M.S.K., J.J., A.G., T.S., M.v.W.), University of Heidelberg, 69115 Heidelberg, Germany; Departamento Fisiologia (I.S.B.), Universidade Federal de Rio Grande do Sul, Porto Alegre, RS-90050-170, Brazil; and Research Laboratory in Immunology and Inflamation (C.G.), Istituto Clinico Humanitas, 20089 Rozzano, Milan, Italy
Address all correspondence and requests for reprints to: Dr. R. M. Popovici, Department of Gynecological Endocrinology and Reproductive Medicine, University of Heidelberg, Voss Strasse 9, 69115 Heidelberg, Germany. E-mail: roxana.popovici{at}med.uni-heidelberg.de.
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Abstract |
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Introduction |
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This very complex process of trophoblast invasion in the human is necessarily regulated by a widespread paracrine network with multiple factors on both fetal and maternal sides. In our initial study (8), we analyzed the expression of various genes in human endometrial stromal cells cocultured with trophoblast by microarray experiments and found in particular a striking increase in the number of genes involved in immune response and modulation of inflammatory reaction by trophoblast influence.
The long pentraxin PTX3, also known as tumor necrosis factor stimulated gene-14 (12), belongs to a family of evolutionarily conserved multifunctional pattern-recognition proteins that are characterized by a cyclic multimeric structure. The pentraxin family consists of the short pentraxins, C-reactive protein and serum amyloid P-component, and the long pentraxin PTX3, which is produced by various tissues in response to proinflammatory signals (13, 14) as well as in tissues undergoing excessive cell death (15). PTX3 interacts with several ligands including growth factors, extracellular matrix components, and selected pathogens; it plays a role in complement activation and facilitates pathogen recognition by phagocytes exposed to inflammatory signals but interestingly only by cells exposed to lipopolysaccaride, IL-1β (16), and TNF
(12) and not exposed to IL-6 or interferon-
(17). The inducibility by IL-1β and TNF is mediated by a nuclear factor-
B binding site (18).
PTX3 is expressed in amniotic epithelium, chorionic mesoderm, trophoblast terminal villi, and perivascular stroma of placentas and increases throughout pregnancy with a peak at delivery (15). PTX3 knockout mice are virtually sterile due to several causes: PTX3 functions in forming the cumulus oophorus in ovarian follicles (19) as well as implantation and decidualization (20). In humans it was found to be higher in patients with preeclampsia (15, 21) and intrauterine growth restriction (21), both pathological conditions caused by faulty implantation.
The known functions of PTX3 makes it an intriguing factor for taking part in human implantation; therefore, we systematically analyzed its expression in the human endometrium and its regulation in endometrial stromal cells by various factors, including trophoblast and its secreted substances.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endometrial biopsies, cell cultures, coculture, and hormonal stimulation
Total endometrial biopsies from 12 patients (n = 6 from the proliferative and n = 6 from the secretory phase) were snap frozen in liquid nitrogen in 1 ml TRIzol.
Further endometrial biopsies from six patients (n = 3 from the proliferative and n = 3 from the secretory phase) were enzymatically digested and separated into stromal and epithelial cells as described previously (22). Briefly, the endometrial tissue was digested for 1 h with collagenase IV (Life Technologies, Inc., Carlsbad, CA), hyaluronidase IV (Sigma-Aldrich, St. Louis, MO), and DNase I (Roche Diagnostics, Indianapolis, IN). Stromal and epithelial cells were separated by sieving through a 40-µm filter, which was followed by centrifugation. Stromal cells were cultured for 1 h at 37 C and 5% CO2 to allow adherence to culture flask. Stromal cells were trypsinized, washed with 1x PBS and snap frozen in liquid nitrogen in TRIzol. Epithelial cells were snap frozen in liquid nitrogen in TRIzol after sieving.
For cell cultures, endometrial stromal cells from an additional four patients were isolated as described above and cultured until confluent and then split onto 32 plates. Approximately 4 x 105 cells were plated into 60-mm plates and cultured again until confluence with 3 ml of standard medium [DMEM/MCDB-105 plus 10% charcoal-stripped fetal bovine serum (Perbio Science, Bonn, Germany)]. Serum medium was changed to serum-free medium 48 h before the experimental treatment started. Trophoblast tissue was mechanically dissected and thoroughly washed with warm PBS. Confluent stromal cells of the same patient were cultured for the time periods shown in Table 1
and treated with serum-free DMEM/MCDB-105 medium plus epithelial growth factor (20 ng/ml; Sigma-Aldrich) and the following hormonal stimulations: progesterone (1 µM; Sigma-Aldrich), 17β-estrogen (10 nM; Sigma-Aldrich), and progesterone (1 µM; Sigma-Aldrich), IL-1β (10 ng/ml; R&D Systems, Minneapolis, MN), human chorionic gonadotropin (HCG; 50 U/ml, Sigma-Aldrich), or supernatant of cultured trophoblast (see below). Stromal cell cultures of the same patient were cocultured with trophoblast as previously described (8). Briefly, confluent stromal cells were cultured with approximately 25 trophoblast explants from one patient in a culture dish, with most of the explants adhering to the stromal cells after 2–3 h. Stromal cell monocultures, again of the same patient, served as negative control. After 4 h, 24 h, and 10 d of different treatments (see Table 1), with medium changed every 2 d in the 10-d group, supernatant was snap frozen in liquid nitrogen, and cells were trypsinized, washed with 1x PBS, and snap frozen in liquid nitrogen in 1 ml of TRIzol.
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). Concentrations of IL-1β and HCG were chosen according to measured levels in previous cocultures. All endometrial stromal cells had been passaged once before starting the hormonal stimulation. Purity of cultures was determined to be greater than 99%, as published previously (22), and dating of the last menstrual period was performed by patient history and serum levels of estradiol, progesterone, and LH of each patient. Decidualization of the stromal cells was verified by measuring prolactin after 10 d. Progesterone levels were measured in the coculture as well as in the trophoblast supernatant and proved to be similar.
RNA isolation
RNA from 22 patients was isolated using TRIzol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA), and 2 µg of total RNA from each sample were then transcribed into cDNA using the first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics, Basel, Switzerland).
Real-time PCR
Quantitative real-time PCR was carried out in a fluorescent temperature cycler (LightCycler; Roche Diagnostics) using the following primer sequences and annealing temperatures: PTX3, 5'-CAT CCA GTG AGA CCA ATG AG-3' and 5'-GTA GCC GCC AGT TCA CCA TT-3' (56 C), accession no. NM_002852, and RPL-19, 5'-GTA AGC GGA AGG GTA CAG CCA-3' and 5'-TTG TCT GCC TTC AGC TTG TG-3' (58 C), accession no. NM_000981. Total RNA (n = 22) was isolated with the RNeasy minikit (QIAGEN, Valencia, CA), and 1 µg total RNA was reverse transcribed using the first-strand cDNA synthesis kit for RT-PCR (Roche Diagnostics). One tenth of each reverse transcriptase reaction was amplified in a PCR tube containing 0.5 µM of each primer and 1 x LightCycler FastStart DNA Master PLUS SYBR Green I (Roche Diagnostics). After the initial preincubation step of 10 min at 95 C, amplification process with 40 cycles at the corresponding annealing temperature and extension followed. SYBR Green I fluorescence was monitored after each cycle. Quantification of the levels of mRNA expression was carried out with the second-derivative maximum method of the LightCycler software (Roche Diagnostics) and normalized to RPL-19. Amplification of specific transcripts was confirmed by melting curve profiles at the end of each PCR.
Western blot analysis
Cell lysates were obtained by adding a lysis buffer [5% Triton X-100; Roth, Karlsruhe, Germany; 1 mM EDTA (pH 8.0); Merck, Darmstadt, Germany; 100 mM Tris HCl (pH 7.6); Roth; and 100 mM NaCl; Merck] including freshly added phonylmethylsulfonyl fluoride, protease inhibitor cocktail, and sodium orthovanadate (all from Santa Cruz Biotechnology, Santa Cruz, CA) to cultured stromal cells from four patients. After placing the samples on ice for 30 min, they were centrifuged (10,000 x g, 4 C, 10 min), and pellets were discarded. Samples were mixed with an equal volume of 2 x electrophoresis sample buffer (Santa Cruz Biotechnologies), boiled for 5 min, and then run on an 8% acrylamide-sodium dodecyl sulfate gel and transferred onto an nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ). Nonspecific binding was blocked for 1 h by 5% skim milk in Tris-buffered saline with 0.05% Tween 20 (TBST), and then membranes were incubated for 14 h with a polyclonal mouse antihuman PTX3 antibody (kindly provided by C.G.) and diluted 1:1000 in 5% skim milk in Tris-buffered saline at 4 C. After washing the membranes in TBST, they were incubated with horseradish peroxidase-conjugated donkey antimouse antibody (Santa Cruz Biotechnologies) diluted 1:3000 in TBST for 1 h. Finally, membranes were washed in TBST and immersed into Luminol (Santa Cruz Biotechnologies) for 1 min, with exposing the membrane to a BioMax MR-1 film (Kodak, Rochester, NY) for 1–2 min. Membranes were stripped for equal loading with polyclonal goat anti-β-actin (clone Ac-15; Sigma-Aldrich, St. Louis, MO) 1:100 in TBST with 5% skim milk.
Statistical analysis
Duplicate cultures were used for all experiments, and all experiments were tested in at least three different patients. In RT-PCR, OD of PTX3 was normalized to the OD of the housekeeping gene RPL-19 of the same sample and run on the same agarose gel to calculate the mean values ± SEM. In real-time PCR, results are shown as arbitrary units of total PTX3 expression normalized to RPL-19 ± SEM.
Densitometry was performed of Western blots after scanning them on a desk scanner (Hewlett Packard, Palo Alto, CA) and analyzing them on the Bio-Rad Quantity One program (Bio-Rad, Hercules, CA). The integrated areas under the absorbance curves were measured for each band and used to determine the relative amounts of specific protein. Levels of PTX3 protein normalized to β-actin equal loading were used to calculate mean values ± SEM.
ANOVA of the Stat View software (Abacus Concepts, Berkeley, CA) was used to analyze the data, and significance between the different groups was determined by using Fisher’s protected least-significant difference post hoc test, with P < 0.05 taken as significant.
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Results |
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. No statistical significant difference of PTX3 expression could be detected in whole endometrial samples between the proliferative and secretory phase (see Fig. 1A). Further biopsies were separated into stromal and epithelial cells by enzymatic digestion immediately after receiving the biopsy as described in Materials and Methods. mRNA was isolated and real-time PCR performed. Figure 1B shows the expression of PTX3 mRNA normalized to RPL-19 in isolated stromal cells of proliferative and secretory phase endometrium, and Fig. 1C shows the expression of PTX3 mRNA normalized to RPL-19 in isolated epithelial cells of proliferative and secretory phase endometrium. In isolated stromal cells of secretory phase endometrium, a tendency of higher PTX3 levels was seen in comparison with stromal cells of proliferative endometrium. These levels, however, did not reach significance (Fig. 1B). In the proliferative phase, PTX3 mRNA is expressed equally in endometrial stromal cells and endometrial epithelial cells. A significantly larger difference is seen in the secretory phase, compared with the proliferative phase, i.e. PTX3 expression is significantly lower in epithelial cells of the secretory phase than in epithelial cells of the proliferative phase (Fig. 1C). Term placenta has previously been shown to express PTX3 mRNA and was used as an internal control. We also analyzed trophoblast explants of early pregnancy terminations of 6–8 wk gestation and detected significantly lower PTX3 mRNA expression in early pregnancy trophoblast vs. full-term placenta (data not shown), which is in accordance to existing published data (15).
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Endometrial stromal cells were treated as shown in Table 1. The increase of PTX3 mRNA normalized to RPL-19 in treatment groups vs. controls are shown in Fig. 2. Both progesterone and the combination of progesterone and estradiol induce PTX3 mRNA expression in endometrial stromal cells with a maximum that reaches significance at 24 h of treatment. Progesterone increases PTX3 mRNA expression by only 3-fold after 4 h. Progesterone with estradiol treatment increase PTX3 by 3.5-fold after 4 h (Fig. 2A). Both progesterone and the combination treatment of progesterone with estradiol increase PTX3 expression to 14-fold after 24 h of treatment (Fig. 2A). There is no significant difference in PTX3 expression between progesterone alone and the combination treatment at either time point. HCG alone does not have an effect on PTX3 mRNA expression after 4 and 24 h of treatment. However, treatment for 10 d elicits a reaction to HCG from stromal cells, which even reaches significance (Fig. 2B).
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depicts PTX3 expression with treatment of trophoblast-conditioned medium for 4 and 24 h and PTX3 expression after stromal cells have been cocultured with trophoblast for 4 and 24 h, respectively. A striking difference between the very high induction of PTX3 with conditioned medium and the significantly lower reaction with trophoblast coculture can be seen. Trophoblast coculture increases PTX3 expression by 14-fold after 4 h and 15-fold after 24 h, whereas trophoblast-conditioned medium elicits a much stronger reaction after 4 h of up to 200-fold, which then drops rapidly to 8-fold after 24 h (Fig. 2C). In quantitative analysis IL-1β alone is the strongest inducer of PTX3 expression in endometrial stromal cells, with the highest peak at 4 h of treatment (Fig. 2D). IL-1β increases PTX3 mRNA expression significantly after 24 h and 10 d (Fig. 2D).
Identification and regulation of PTX3 protein in endometrial stromal cells
PTX3 has previously been shown only as mRNA transcript in human endometrial stromal cells. We analyzed PTX3 protein using Western blot and found PTX3 protein to be produced by endometrial stromal cells. PTX3 was up-regulated by progesterone, estradiol, and progesterone and IL-1β on the protein level by 24 h (see Fig. 4). Both trophoblast-conditioned medium and trophoblast explants increase PTX3 protein production in endometrial stromal cells, whereas HCG alone does not have an effect. Figures 3-5 show representative Western blots of PTX3 protein production induced by different treatments after 4 h, 24 h, and 10 d, respectively. After 4 h of treatment with IL-1β, a thick PTX3 protein band can be detected. This is equaled only by treatment with trophoblast-conditioned medium. Trophoblast explant culture on endometrial stromal cells has less impact on PTX3 protein production after 4 h (Fig. 3). After 24 h, however, trophoblast cocultured with stromal cells has a significant effect in increasing PTX3 protein production (Fig. 4). Also, progesterone and estradiol combination as well as progesterone-only treatment show an increase in PTX3 protein production after 24 h (Fig. 4) but not after 4 h (Fig. 3). IL-1 and trophoblast-conditioned medium also significantly increase PTX3 after 24 h (Fig. 4). In accordance with the RNA data, HCG treatment also has no significant effect on PTX3 protein production after both 4 and 24 h. Ten days of treatment were performed only on the progesterone (P4), estrogen/progesterone (E2P4), IL-1β, and HCG groups, whereas both trophoblast treatments have previously shown that they are technically not feasible over the duration of 10 d. Decidualization achieved by 10 d of progesterone only as well as by 10 d of estrogen and progesterone treatment was confirmed by prolactin measurement in the conditioned medium. PTX3 protein production at 10 d of treatment is significantly lower in all four treatment groups, compared with 24 h of treatment (Fig. 5). An increase of PTX3, compared with control, is seen with decidualization and HCG treatment; the increase, however, does not reach significance. IL-1β treatment increases PTX3 protein production significantly even after 10 d; its peak reaction is, however, seen after 4 h.
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Discussion |
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In the current study, we have shown for the first time that normal nontransformed human endometrium contains mRNA transcripts encoding PTX3 (Fig. 1). Even though a significant regulation of PTX3 mRNA could not be detected in whole endometrial biopsies during the menstrual cycle, we detected a significant down-regulation of PTX3 in endometrial epithelial cells during the secretory phase, compared with endometrial stromal cells showing a compartmental regulation within the endometrium. Further analysis of nonpregnancy transformed endometrium is currently under investigation in our laboratories. Because decidualization by progesterone is crucial for implantation in humans and decidulization by blastocyst attachment is also faulty in PTX3–/– mice (20), we analyzed PTX3 mRNA regulation in human endometrial stromal cells exposed to progesterone as well as a combination of estradiol and progesterone in vitro. Despite decidualization taking place over several days, we surprisingly detected an effect of progesterone on PTX3 mRNA expression after only 4 h of treatment; this effect of progesterone on PTX3 mRNA expression continued to increase after 24 h. Similar results were found with the progesterone and estradiol combination treatment. Whether estradiol alone elicits the same response remains to be investigated. In view of PTX3 mRNA expression in proliferative endometrium samples, in which progesterone does not play a role, an estradiol effect is to be expected.
We confirmed our gene array observations, showing that trophoblast explants induce PTX3 mRNA expression in untreated endometrial stromal cells and could furthermore show that the PTX3 mRNAs are translated into proteins. Considering that trophoblast CM further increases PTX3 expression in decidualized endometrial cells (9), the question arises whether there is an influence of direct trophoblast contact, compared with the paracrine effects of trophoblast CM on the quantity of PTX3 expression. Trophoblast CM has a significantly higher effect on PTX3 expression after 4 h, compared with trophoblast explant coculture, even when trophoblast and trophoblast CM are used from the same pregnancy (i.e. when confounding factors like different gestational weeks are eliminated) (data not shown). This effect is probably due to a higher starting concentration of trophoblast derived factors in the CM, compared with the trophoblast explant coculture. After 24 h of treatment, this difference can no longer be detected.
A question that remains to be answered is which trophoblast factors inhibit PTX3 overproduction. Factors that inhibit PTX3 production (and possibly other immunomodulator) are thought to play a role in implantation because PTX3 is increased in serum of patients with pathological pregnancies, which can be attributed to faulty implantation like intrauterine growth restriction and preeclampsia (21). Considering that paracrine factors play a role in regulating PTX3 in endometrial stromal cells, further factors studied in the current experiments were IL-1β and HCG. Both are secreted by early pregnancy trophoblast. IL-1β significantly increases PTX3 mRNA as well as protein in endometrial stromal cells. Analysis of the PTX3 proximal promoter region accounts for induction by IL-1 through the nuclear factor-B site (18, 25). HCG treatment showed no significant effect on PTX3 regulation in human endometrial stromal cells after 4 and 24 h, whereas it does seem to have an effect in mouse cumulus oopherus cells within 24 h (23). In endometrial stromal cells, a significant increase in PTX3 by HCG can be seen on mRNA levels after 10 d of treatment, which is, however, not as clear on the protein level in our experiments. These differences could be due to different signal pathways in endometrial stromal cells, compared with cumulus oopherus cells, but also to the fact that HCG/LH has a direct modulatory effect on endometrial stromal cells both in vivo and in vitro (10).
The precise role of PTX3 in human endometrium continues to be intriguing. PTX3 is likely to form pentamers, decamers, and even 20-mers due to its molecular structure. Likewise, due to its molecular structure, PTX3 might facilitate attachment and invasion of the blastocyst to the endometrium (26, 27), explaining its inducibility in the human endometrium at the time of implantation. Because PTX3 was shown to bind spermatozoa (27) and we could show that it is produced by endometrial cells, it could also help to attract the sperms and promote fertilization of the oocyte.
Garlanda et al. (28) conducted studies on PTX3 null mice, with all PTX3–/– mice infected with Aspergillus fumigatus succumbing rapidly to the infection, with the wild type surviving. The lungs from the infected null mice showed a massive inflammatory response, whereas the lungs of wild type showed only a modest reaction. In a likewise manner, PTX3 also bound to Pseudomonas aeruginosa and Salmonella typhimurium but not Candida albicans or Escherichia coli, indicating that it interacts only with selected pathogens. Because the PTX3 null mice showed a massive inflammatory response and PTX3 null mice as well seem to be sterile, we think that PTX3 helps to regulate the maternal inflammatory response at the site of implantation, allowing the trophoblast to invade. PTX3 binds with high affinity to the complement component C1q, and, depending on the nature of the interaction, PTX3 can either activate or inhibit the classical complement pathways (29). However, PTX3 can mediate resistance independently of C1q because exogenous PTX3 protected the null mice in the study by Garlanda et al. (28). It would be intriguing to explore further the expression of the complement components in the human endometrium, possibly for a better understanding of the manner of PTX3 action in the human endometrium. Removal of apoptotic cells by uterine NK cells, monocytes, and macrophages remodels the decidua to facilitate trophoblast invasion (30). PTX3 seems to bind to membrane domains of these apoptotic cells (31), possibly helping to prepare endometrium for implantation.
The most recent paper on PTX3 in mice showing clearly reduced implantation rate of wild-type embryos in PTX3–/– mice (20) in combination with the current in vitro and in vivo data in humans makes PTX3 a new candidate for in vivo studies related to both sterile patients with implantation failure and implantation-related pathologies during pregnancy.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: R.M.P., M.S.K., J.J., A.G., I.S.B., C.G., M.v.W. have nothing to declare. T.S. received lecture fees from Serono.
First Published Online November 29, 2007
1 R.M.P. and M.S.K. contributed equally to this paper. 
Abbreviations: CM, Conditioned medium; HCG, human chorionic gonadotropin; NK, natural killer; PTX3, long pentraxin; TBST, Tris-buffered saline with Tween 20.
Received September 9, 2007.
Accepted for publication November 11, 2007.
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References |
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B in tumor necrosis factor- and interleukin-1β regulation. J Biol Chem 272:8172–8178
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