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Pregnancy |
a Center for Research on Reproduction and Women's Health, University of Pennsylvania, Philadelphia, Pennsylvania 19104
b Research Direction, Instituto Nacional de Perinatologia, Mexico D.F. 11000, Mexico
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF), interleukin-1ß (IL-1ß), and either lipopolysaccharide (LPS) alone or LPS with anti-TNF or anti-IL-1ß-neutralizing antibodies. Levels of proMMP-9 in culture media were evaluated by zymography. Enzyme-linked immunosorbant assay was performed to measure the quantity of IL-1ß, TNF, and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) after LPS stimulation. ProMMP-9 activity was upregulated after stimulation of the amnion by LPS, TNF, and IL-1ß. The increased activity of proMMP-9 resulting from LPS stimulation in the amnion was blocked by the addition of TNF neutralizing antibody but not with anti-IL-1ß. No significant effect of LPS, TNF, or IL-1ß on proMMP-9 expression was observed in the chorion; however, the chorion produced both cytokines when stimulated with LPS. In contrast, TIMP-1 levels remained unchanged in all cultures incubated in the presence of LPS. Therefore, these data indicate that proMMP-9 is produced by the amnion but not the chorion in response to LPS. Because anti-TNF-neutralizing antibody inhibits proMMP-9 activity in the amnion, TNF appears to upregulate proMMP-9 production by the amnion in an autocrine fashion. Meanwhile, TNF and IL-1ß produced by the chorion may upregulate amnionic proMMP-9 production in a paracrine manner.
cytokines, parturition, pregnancy
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) and interleukin-1ß (IL-1ß) have been identified in the primary wave response. These cytokines have multiple effects on maternal reproductive tissues [8, 9]. TNF and IL-1ß are synthesized in many tissues, including amniotic epithelial cells, chorionic cells, and tissue macrophages [10–13]. Synthesis of these cytokines is not an exclusive feature of infection-mediated activation of the amniochorion because normal labor is characterized by increased release of these cytokines in association with the initial stages of parturition [14, 15]. Information on this signaling pathway is not available. The molecular mechanisms of PROM involve the activation of matrix metalloproteinases (MMPs) in the amniochorion as a central event in fetal membrane weakening [16, 17]. Activation of these enzymes during normal labor or PROM is characterized by the selective expression of the 92-kDa type-IV collagenase (MMP-9), which has been proposed as a key mediator of this process [18–20]. The production of proMMP-9 is regulated at the transcriptional level, while activation is achieved by proteolytic cleavage of the zymogen and some other less well-understood mechanisms. Specific tissue inhibitors of metalloproteinases (TIMPs) can then block the activity of activated MMP-9. Thus, a delicate balance of transcriptional mechanisms, activation steps, and TIMP levels regulates the final proteolytic activity of MMP-9. Four different TIMPs have been defined so far [21–24]. While TIMP-1 preferentially forms complexes with MMP-9 [25], TIMP-2 controls the activity of MMP-2 and membrane type 1 MMP (MT-1 MMP) [26]. The quantitative ratio between MMP-9 and TIMP-1 expression finally determines the in vivo activities of the protease.
Amnion epithelial cells, trophoblasts, and decidual cells are the source of MMP-9 in normal labor and with choriodecidual infection [18, 27, 28]. TNF and IL-1ß induce the expression of MMP-9 in amnion [29] and MMP-1 and MMP-3 in chorion cells [30, 31], but paracrine/autocrine regulatory mechanisms connecting stimulation of amniochorion with bacterial products that result in cytokine secretion and consecutive MMP-9 expression have not been described. This information is relevant to the understanding of interactions between intrauterine infection-related microorganisms and fetal membrane rupture.
In this study, we determined that the secretion of proMMP-9 in human amnion in response to lipopolysaccharide is mediated by TNF and IL-ß acting in an autocrine/paracrine loop.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Internal Review Board of Instituto Nacional de Perinatologia approved this project. Chorioamnion ex vivo culture was performed using fresh fetal membranes obtained from women with normal term pregnancies with indication for cesarean section without evidence of active labor. Repeat cesarean section or suspected cephalic-pelvic disproportion were the indications for surgical deliveries. General microbiologic analyses, including Ureaplasma urealyticum were conducted on the placenta and membranes upon extraction. Membranes were immediately placed in ice-cold PBS and transported to the laboratory within 20 min after extraction. Membranes were washed with PBS in order to eliminate blood clots. Entire membranes or manually separated amnion and chorion were cut into 10-mm-diameter pieces using a punch cutter. Two pieces of tissue were placed into each well of a 24-well tissue culture dish (Costar, New York, NY) in 2 ml Dulbecco Modified Eagle Medium (DMEM; Gibco, BRL, Bethesda, MD) supplemented with 10% fetal bovine serum (Gibco), 1 mM sodium pyruvate (Gibco), and antibiotic-antimycotic solution (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B; Gibco). Tissues were incubated in 5% CO2 at 37°C. Explants were cultured in DMEM with lactalbumin hydrolysate (Gibco) before each experiment and all studies were carried out in this medium. Viability of ex vivo tissues was followed by the measurement of lactate dehydrogenase (LDH) leakage into the medium using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega Corp., Madison, WI). When incubation was finished, the explants were treated with 1% Triton X-100 to evaluate maximal LDH release. The percentage of viability was calculated as %viability = 100 x (1 - (spontaneous release of LDH/maximum release of LDH)).
Entire membranes or separated amnion or chorion were independently stimulated with different concentrations of bacterial lipopolysaccharide (LPS) from Escherichia coli serotype 055:B5 (Sigma, St. Louis, MO), IL-1ß, and TNF (Gibco) for 24 h. For neutralization assays, amnion explants were stimulated with 1.0 ng/ml of LPS in the presence of different concentrations of a neutralizing anti-human TNF antibody or human anti-IL-1ß (R & D Systems, Minneapolis, MN). At the end of stimulation, media samples and tissues were collected and stored at -70°C until assayed.
Zymography
SDS-polyacrylamide gel electrophoresis was performed according to Laemmli [32] using a minigel apparatus (Bio-Rad, Richmond, CA). Gels were prepared according to standard techniques except that porcine gelatin (1.0 mg/ml) was copolymerized in the 8% running gel and samples were loaded under nondenaturing conditions. Five hundred nanograms of protein from culture media were applied in each lane and run under constant current (10 mA). Gels were washed in 2.5% Triton X-100 for 30 min in order to eliminate the SDS and were incubated for 24 h at 37°C in 50 mM Tris buffer, pH 7.4, containing 0.15 M NaCl, 20 mM CaCl2, and 0.02% sodium azide. Gels were stained with Coomasie blue R-250. Prestained molecular weight standards (Bio-Rad) were included in each gel. A standard for MMP-2 and MMP-9 activity obtained from U-937 promyelocyte cell media (ATCC, Rockville, MD) was included in each gel for location of MMPs. Gelatinolytic activity of MMP-9 was quantitated by scanning of the wet gels using a densitometer and analyzed by Quantity One software, version 4.3.0 (Bio-Rad). After background subtraction, the amount of lytic activity was estimated as the density of the band.
TIMP-1 ELISA
TIMP-1 protein level in culture media was determined by using hTIMP-1 assay kit (R & D Systems), based on a two-step sandwich enzyme-linked immunosorbant assay, according to the manufacturer's protocol. Briefly, a 50-µl volume of the standard or a 1:100 dilution of the samples was transferred to a 96-well microtiter plate previously coated with mouse anti-human TIMP-1 monoclonal antibody having a different epitope specificity. The plate was incubated for 2 h at room temperature (RT) on a shaker. After washing three times, the wells were incubated for 1 h at RT with 200 µl of an anti-human TIMP-1 polyclonal antibody conjugated with peroxidase. The wells were washed again three times and 200 µl of the substrate solution was added and the reaction allowed to proceed for 30 min. The resulting color intensity was measured at 450 nm in an ELISA reader.
Cytokines ELISA
TNF and IL-1ß levels in culture media were quantified using ELISA. The assay procedure involved a multiple-site, two-step sandwich immunoassay using monoclonal antibody according to Harlow and Lane [33]. Solid phase monoclonal anti-IL-1ß (R & D Systems) or monoclonal anti-TNF (Pharmingen, San Diego, CA) were used as the cytokine capture antibody and biotinylated polyclonal anti-IL-1ß (R & D Systems) or biotinylated polyclonal anti-TNF (Pharmingen) as the cytokine detection. Briefly, 96-well microtiter plates (Nunc Maxisorp, Roskilde, Denmark) were coated overnight at 4°C with 50 µl/well, 4 µg/ml of monoclonal antibody in sodium bicarbonate buffer, pH 8.2. Unbound antibody was removed by three washes with PBS containing 0.05% Tween (this buffer was used for all subsequent washing steps), and the wells were incubated for 2 h at 37°C with 200 µl 10% fetal bovine serum in PBS (blocking buffer). Wells were then washed three times and incubated overnight at 4°C with 100 µl standard recombinant IL-1ß (R & D Systems), standard recombinant TNF (Pharmingen), or samples. In each instance, DMEM (Gibco BRL) was used as the diluent. After washing three times, the wells were incubated for 2 h at room temperature with 4 µg/ml biotinylated antibodies diluted in blocking buffer to a final volume of 100 µl. The wells were washed again three times and incubated for 1 h at room temperature with 100 µl streptavidin-alkaline phosphatase conjugate diluted 1:5000 in blocking buffer. After washing the wells another four times, 100 µl of p-nitrophenylphosphate was added and the reaction allowed to proceed for 45 min, and the resulting color intensity was measured at 405 nm in an ELISA reader.
Statistical Analysis
Results are presented as means ± SEM of relative activity values or cytokine concentration. Multiple comparisons between groups were performed by one-way analysis of variance followed by a Dunnet test. A P value <0.05 was considered as the limit for statistical significance. All experiments were repeated at least three times for evidence of reproducibility.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Viability and functionality of the amniochorion, chorion, and amnion explants during incubation were assessed by the measurement of LDH leakage into the medium and proMMP-9 production as a biomarker. Viability of the chorion was 83% ± 1.8%, the chorioamnion 85% ± 2%, and the amnion 89% ± 1.6 %, and no effects were observed after incubation with FBS (10%) versus lactalbumin hydrolysate (0.2%), time in culture, or LPS treatment (data not shown). Secretion of MMPs was evaluated by gelatin zymography from the culture medium of the fetal membrane explants. Lysis bands corresponding in size to the 72-kDa (proMMP-2), 62-kDa (activated MMP-2), and 92-kDa (proMMP-9) type IV collagenases were observed after 24 h of culture. The proMMP-9 lytic band was detected in all tissues under experimental culture conditions. ProMMP-9 secretion was slightly increased in the first 24 h of culture in association with manipulation of the tissue, and thereafter declined over the next day, reaching basal levels that remained constant for 8 days (Fig. 1).
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ProMMP-9 and TIMP-1 Expression in Lipopolysaccharide-Stimulated Amniochorion, Chorion, and Amnion
Zymography was performed on media from lipopolysaccharide-stimulated amniochorion, amnion, and chorion. LPS treatment resulted in a significant dose-dependent increase in the release of proMMP-9 by the amniochorion and amnion explants after 72 h in culture once the tissue had returned to baseline levels of enzyme expression. No significant effect of LPS on proMMP-9 activity was observed in the chorion (Fig. 2). The levels of TIMP-1 were determined by ELISA in the culture media from the lipopolysaccharide-stimulated amnion, chorion, and amniochorion. In contrast with the proMMP-9, the expression of TIMP-1 was unaffected by LPS in all tissues (Table 1).
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Effect of Cytokines in proMMP-9 Expression by Amnion and Chorion
TNF and IL-1ß treatment significantly increased proMMP-9 activity in the medium of amnion explants (Fig. 3). Both cytokines stimulated proMMP-9 in a dose-dependent manner, with the maximal effect noticed at a concentration of 10 ng/ml. The activity of the proMMP-9 in the amnion explants was 3.8 (range, 2.88–5.84; P < 0.05) times greater and 2.5 (range, 1.44–4.28; P < 0.05) times greater, respectively, when explants were stimulated with TNF or IL-1ß at 10 ng/ml. No significant effect of TNF or IL-1ß on proMMP-9 activity was shown in the chorion alone.
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TNF and IL-1ß Have an Additive Effect Stimulating the Production of proMMP-9 by Amnion Explants
The stimulation of amnion explants with TNF (10 ng/ml) or IL-1ß (10 ng/ml) resulted in a significantly increased proMMP-9 activity of 1.8- and 3.2-fold, respectively. The combination of 10 ng/ml TNF and 10 ng/ml IL-1ß resulted in an additive effect, showing a total of 4.6-fold (P < 0.05) on the proMMP-9 production (Fig. 4).
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Role of an Autocrine Loop in proMMP-9 Induction by TNF
Amnion explants were treated with LPS in the presence of neutralizing antibodies against human TNF or IL-1ß. The stimulatory effect of LPS on proMMP-9 production was blocked by addition of the anti-TNF antibody. Densitometric analysis revealed that proMMP-9 activity was substantially diminished (twofold) in LPS-treated amnion explants exposed to TNF antibody at 60 ng/ml. No effect was observed with the anti-IL-1ß (Fig. 5).
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Proinflammatory Cytokine Production by the Chorion after Stimulation with LPS
A wide standard deviation was observed in the total amount of protein released by the chorion explants under the culture conditions. Therefore, ELISA results were normalized against the protein concentration in the medium. IL-1ß and TNF levels in the medium from chorion explants were significantly increased after treatment with LPS in a dose-dependent manner, with a maximum at 1000 pg/ml (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Matrix metalloproteinases are the physiologic mediators of extracellular matrix catabolism, and it has been proposed that overexpression and activation of some MMPs before labor results in a progressive loss in amniochorion tissue strength resulting from matrix breakdown and clinical presentation as premature rupture of membranes [2]. The 92-kDa type IV collagenase (MMP-9) is selectively expressed in human amniochorion during labor [18, 19]. Using an animal model, MMP-9 was also found to be induced in amnion immediately before the onset of labor [34]. Finally, increased activity of this enzyme has been demonstrated in association with PROM [20].
The collagenolytic process associated with PROM may be due to an imbalance between matrix metalloproteinases and their inhibitors under the influence of physiologic signals that are thought to be involved in the initiation of labor (platelet-activating factor, endothelins, prostaglandins, etc.) and infectious (lipopolysaccharide) or immune signals (cytokines) involved in the PROM process. Although amniotic fluid inflammatory cytokine levels have been noted to increase toward term and in normal labor, there is a much more dramatic increase in amniotic fluid cytokine levels in women with PROM caused by either subclinical infection or microbial invasion of the intraamniotic cavity [1, 35, 36].
Matrix metalloproteinases are secreted by human amnion and chorion cells in response to IL-1, IL-1ß, and TNF, indicating that cytokines can control the expression of matrix metalloproteinases in the fetal membranes [30, 31]. TNF may represent an important link between an infectious process and extracellular matrix degradation in the human amnion. Specifically, we showed that LPS acts via an autocrine TNF-mediated mechanism to stimulate amnionic proMMP-9 production. IL-1ß is synthesized by the chorion and can stimulate the proMMP-9 production in the human amnion. Therefore, the chorion may regulate the proMMP-9 expression by a paracrine system. Furthermore, costimulation of the amnion with TNF and IL-1ß clearly demonstrate an additive effect in the proMMP-9 production. These findings support the concept that the amniochorion responds as a unit to infection by secreting cytokines, leading to extracellular matrix catabolism. In addition, the fetal membranes respond to LPS with increased secretion of IL-8 [11], which induces the recruitment of leukocytes into the amniotic cavity during the course of intrauterine infection. The presence of white blood cell subpopulations in the amniotic fluid increases the production of cytokines, including TNF and IL-1ß, that can potentiate the amnion's MMP production.
Previous reports have shown that LPS and TNF are capable of increasing the level of proMMP-9 produced by the amniochorionic membranes; however, to our knowledge, our experiments represent the first study that demonstrates the link between LPS, TNF, and proMMP-9 expression in the human fetal membranes. Moreover, the results obtained in the present study show clearly that the amnion is responsible for the proMMP-9 production. Fetal membrane production of MMP-9 is considered to be of major importance in the pathology of PROM. An understanding of the mechanism by which the MMP-9 is produced may lead to novel therapies to modulate its expression during an intraamniotic infection.
The activity of MMP-9 is restrained by TIMP-1 [25]. It is notable that, although LPS treatment increased expression of MMP-9, TIMP-1 expression was unaffected, shifting the ratio of enzyme to inhibitor. This imbalance in human fetal membranes during infection favors increased gelatinase activity and could lead to increased membrane degradation. In vivo observations have shown that TIMP-1 levels are decreased during labor, making more pronounced the imbalance between protease and inhibitor [20, 37, 38]. This means that TIMP-1 expression is downregulated by another mechanism that was not identified by our experimental approach.
Intrauterine infection is associated with PROM and preterm birth. The molecular mechanisms linking infection with the changes in fetal membranes that lead to PROM remain to be elucidated. It has been proposed that there is a four-stage process leading to intrauterine infection [39]. The first stage consists of an overgrowth of microorganisms in the vagina and cervix. Once microorganisms gain access to the intrauterine cavity, they reside in the decidua (stage II). The infection may proceed through the fetal membrane into the amniotic cavity (stage III), and into the fetus (stage IV). Our data support this model because the presence of microorganisms in the decidua during the second stage could activate the synthesis of TNF and IL-1ß by the chorion, which triggers the amnionic proMMP-9 expression in a paracrine manner. Once the infection reaches the amniotic cavity, the presence of microorganisms can have a direct action on the amnion, causing the production of proMMP-9 by the TNF-dependent autocrine system.
Because degradation of the extracellular matrix is an exquisitely controlled process, proMMP-9 activation is a critical point of regulation [40]. Activation of MMP-9 in the extracellular space is proposed to occur via proteinases, including other MMPs and the plasminogen-activator/plasmin system [41, 42]. Although the proMMP-9 activation has not been elucidated in this study, we speculate that the presence of MMP-2, MMP-3, and plasminogen-activator in amniotic fluid samples from pregnancies complicated by PROM [43–45] may convert the proMMP-9 into the active MMP-9.
In conclusion, our results allow us to propose that LPS stimulates the amnion and chorion to produce TNF or IL-1ß, which in turn induces proMMP-9 secretion and activation in the amnion. This could lead to extracellular matrix degradation that predisposes the fetal membranes to rupture prematurely. However, the contribution of other cytokines or metalloproteinases to this autocrine/paracrine system remains to be established (Fig. 7).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Felipe Vadillo-Ortega, Instituto Nacional de Perinatologia, Direccion de Investigacion, Montes Urales 800, Mexico, D.F., 11000, Mexico. FAX: 5255 5520 0034; felipe.vadillo{at}uia.mx
Received: 18 February 2002.
First decision: 13 March 2002.
Accepted: 3 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the female reproductive tract. Proc Soc Exp Biol Med 1995 209:325-342[Abstract] and TNFR p55 in cultured amniochorion. Am J Reprod Immunol 1994 32:188-193) release from cultured human fetal membranes in response to endotoxic lipopolysaccharide mirrors amniotic fluid concentrations. Am J Obstet Gynecol 1996 174:1855-1861[CrossRef][Medline] produced by human placental cytotrophoblastic cells. J Immunol 1993 150:5614-5624[Abstract] increases biosynthesis of collagenase and hyaluronic acid in cultured human chorionic cells. FEBS Lett 1989 244:315-318[CrossRef][Medline] stimulates the biosynthesis of matrix metalloproteinases and plasminogen activator in cultured human chorionic cells. Biol Reprod 1992 46:772-778[Abstract]This article has been cited by other articles:
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  G. Garcia-Lopez, F. Vadillo-Ortega, H. Merchant-Larios, R. Maida-Claros, M. Osorio, D. Soriano-Becerril, H. Flores-Herrera, J. Beltran-Montoya, Y. Garfias-Becerra, and V. Zaga-Clavellina Evidence of in vitro differential secretion of 72 and 92 kDa type IV collagenases after selective exposure to lipopolysaccharide in human fetal membranes Mol. Hum. Reprod., June 1, 2007; 13(6): 409 - 418. [Abstract] [Full Text] [PDF]
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 L. Farina and C. Winkelman A Review of the Role of Proinflammatory Cytokines in Labor and Noninfectious Preterm Labor Biol Res Nurs, January 1, 2005; 6(3): 230 - 238. [Abstract] [PDF]
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 R. Menon and S. J. Fortunato The Role of Matrix Degrading Enzymes and Apoptosis in Repture of Membranes Reproductive Sciences, October 1, 2004; 11(7): 427 - 437. [Abstract] [PDF]
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V. Zaga, G. Estrada-Gutierrez, J. Beltran-Montoya, R. Maida-Claros, R. Lopez-Vancell, and F. Vadillo-Ortega Secretions of Interleukin-1{beta} and Tumor Necrosis Factor {alpha} by Whole Fetal Membranes Depend on Initial Interactions of Amnion or Choriodecidua with Lipopolysaccharides or Group B Streptococci Biol Reprod, October 1, 2004; 71(4): 1296 - 1302. [Abstract] [Full Text] [PDF]
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C. Hernandez-Guerrero, F. Monzon-Bordonaba, L. Jimenez-Zamudio, R. Ahued-Ahued, F. Arechavaleta-Velasco, J.F. Strauss III, and F. Vadillo-Ortega In-vitro secretion of proinflammatory cytokines by human amniochorion carrying hyper-responsive gene polymorphisms of tumour necrosis factor-{alpha} and interleukin-1{beta} Mol. Hum. Reprod., October 1, 2003; 9(10): 625 - 629. [Abstract] [Full Text] [PDF]
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