Mol. Hum. Reprod. Advance Access originally published online on December 10, 2008
Molecular Human Reproduction 2009 15(1):49-57; doi:10.1093/molehr/gan071
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Alpha 1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes
1Department of Regenerative Medicine, University of Toyama Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan 2Department of Obstetrics and Gynecology, University of Toyama Graduate School of Medicine and Pharmaceutical Sciences, Toyama, Japan 3Department of Biomolecular Engineering, Tokyo Institute of Technology, Tokyo, Japan 4Department of Obstetrics and Gynecology, Kyoto University Graduate School of Medicine, Kyoto, Japan
5 Correspondence address. Tel: +81-76-434-7210; Fax: +81-76-434-5011; E-mail: tnikaido{at}med.u-toyama.ac.jp
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Preterm premature rupture of the membranes (PPROM) has been considered to be closely associated with chorioamnionitis. However, the detailed mechanism is not well understood. Alpha 1 antitrypsin (AAT) was reported to decrease in concentration in amniotic fluid obtained from patients with PPROM. However, the origin of AAT in amniotic fluid has not been clarified. In this study, we assessed the expression and localization of AAT in human amnion, as well as its biological activity in cases with PROM. Human amniotic epithelial (hAE) cells expressed AAT. After stimulation with oncostatin M (OSM), interleukin-6 (IL-6) or tumor necrotic factor alpha (TNF
), hAE cells increased the expression of AAT, while the expression of MMP9 was reduced by OSM and induced by TNF . Oxidized AAT (inactivated form) was detected in the amnion with PPROM and TPROM, but not in specimens without PROM. Moreover, AAT activity was decreased in amnions from cases with PROM, regardless of gestational age. Thus, the results showed that AAT in the amnion may function as a protective shield at inflammatory sites, and not as it loses it inhibitory activity in cases with PROM, possibly by oxidation, suggesting that its imbalance contributes to PROM. Key words: amnion/alpha 1 antitrypsin/oxidization/MMP9/PPROM
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Introduction |
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Preterm premature rupture of the membrane (PPROM) is defined as spontaneous fetal membrane rupture earlier than the 37th week of gestation. It causes preterm delivery leading to perinatal morbidity. PPROM has been reported to be associated with infection (Mazor et al., 1994), cigarette smoking (Williams et al., 1992) and second trimester bleeding (Ekwo et al., 1993), and the main etiology is considered to be chorioamnionitis (CAM). Therefore, understanding the inflammatory mechanisms in CAM will lead to prevention of PPROM.
The amnion is the inner layer of the fetal membrane which lines the amniotic cavity. It is composed of a single layer of epithelial cells on a thicker basement membrane and a spongy collagen layer containing mesenchymal cells. The amnion is of fetal origin and is considered to protect the fetus from mechanical injury by enveloping it in the amniotic fluid. The amnion derives its strength from collagen, especially type IV collagen, in the basement membrane. Collagen in the basement membrane and the degradation of these collagens within the chorioamnion is controlled by matrix metalloproteinases (MMPs). Matrix metalloproteinase-1 (MMP-1) degrades type I, II and III collagens, while MMP-2 and MMP-9 (gelatinase B) degrade type IV collagen (Woessner, 1991). In human chorionic cells, tumor necrotic factor alpha (TNF ) has been shown to enhance the production of MMPs and prostaglandin E2 (PGE2) and to suppress tissue inhibitors of metalloproteinase (TIMP); thus, TNF leads to weakening and rupture of the membrane through degradation of collagen in the extracellular matrix (So, 1993).
Neutrophil elastase, a serine protease, is released from activated neutrophils in the presence of inflammation and degrades the components of the extracellular matrix. This enzyme has been isolated in human cervical mucous and is believed to play a role in the pathogenesis of PROM (Helmig et al., 1995). Alpha1 antitrypsin (AAT) inhibits neutrophil elastase, as well as cathepsin G, limiting tissue degradation when these enzymes are released from inflammatory cells. AAT is one of the most abundant proteinase inhibitors in human plasma and is produced mainly in the liver, peripheral blood monocytes and alveolar macrophages (Perlmutter et al., 1985). We previously reported that human amniotic epithelial (hAE) cells also expressed AAT and the expression was increased during the induction of hAE cells toward hepatocyte characteristics with oncostatin M (OSM) and dexamethasone (Takashima et al., 2004). However, the biological function of AAT in the amnion has not been determined. OSM is a member of the interleukin-6 (IL-6) superfamily, and IL-6 levels are elevated in the amniotic fluid of cases with intra-amniotic infection (Romero et al., 1990; Saito et al., 1993). Therefore, like OSM and IL-6, AAT is also thought to be related to an inflammatory reaction occurring in the amnion. Indeed, cases of PROM have a decreased concentration of AAT in their amniotic fluid, which results in an increase in trypsin activity (Kanayama et al., 1986).
In this study, we assessed the localization of AAT in human amnion, its expression and its biological activity in cases with or without PROM delivered preterm and term.
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Preparation of amnion
Amniotic membranes were obtained after deliveries from 23 to 41 week' gestation, with informed consent. The study and the use of the human amnion were approved by the Ethics Committee of the University of Toyama. The diagnosis of membrane rupture was based on pooling of vaginal fluid and a positive nitrazine test. PROM was defined as the spontaneous rupture of the chorioamniotic membranes accompanied by the loss of amniotic fluid at least 1 h before onset of labor. For cell culture and cytokine stimulation experiments, the amnions were obtained from term labor (
37 weeks of gestation) without PROM (TL) cases who underwent Cesarean sections without any other complications, but with a history of repeated Cesarean sections or breech position. For western blotting, immunohistochemical analysis (oxidized AAT) and enzymatic assays, the study groups were defined as follows: group 1: term labor (37 weeks of gestation) without PROM (TL); group 2: term labor with PROM (TPROM); group 3: preterm labor without PROM (PTL) and group 4: PPROM. In this study, only singletons were used. The amnion and chorion were manually separated. In the cell culture experiments, whole membranes were used. In other experiments, each membrane was cut into pieces (3 x 3 cm2) and several of them were used in the experiments.
Cell preparation and cultivation
Human AE cells and mesenchymal (hAM) cells were isolated using the method described in our previous report (Wei et al., 2003) with some modifications. Briefly, the amnion was washed in PBS and then cut into pieces in PBS containing 0.03% hyaluronidase (Sigma-Aldrich Co., St Louis, MO, USA) and 0.025% deoxyribonuclease I (Sigma-Aldrich Co.). The minced amnion was digested with 0.2% trypsin (Sigma-Aldrich Co.) in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich Co.) and incubated for 20 min at 37°C. Trypsinization of the amnion was repeated several times until hAE cells were no longer obtained. Mesenchymal cells were isolated after removal of hAE cells. The tissue pieces were placed in DMEM with 0.075% collagenase and 0.0075% deoxyribonuclease I (Sigma-Aldrich Co.) and were incubated at 37°C with stirring at 600 rpm for 60 min. For cultivation, hAE and hAM cells were resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) (Moregate, Bulimba, QLD, Australia) and Antibiotic-Antimycotic mixture (Invitrogen, Grand Island, NY, USA), and seeded onto culture dishes (Greiner Bio-One GmbH, Frickenhausen, Germany) at a concentration of 5.0 x 104 cells/cm2. For stimulation studies, IL-6 (Sigma-Aldrich Co.), TNF (Dainippon Pharmaceutical Co.) or OSM (Sigma-Aldrich Co.) was added to the culture medium. Stimulation procedure was as follows: primary hAE cells were cultivated for 3–5 days in DMEM supplemented with 10% FBS, until they reached 80% confluency, and then stimulated once with 250 ng/ml IL-6, 1, 10, 102 ng/ml OSM or 1, 10, 102, 103 units/ml TNF for 3 days in the presence of 10% FBS. Then, cells were harvested and RNA was extracted immediately. Each experiment was repeated four times.
RT–PCR analysis
Total RNA was extracted from hAE cells using ISOGEN kits (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Aliquots of 1 µg of total RNA were treated with 0.1 U/µl deoxyribonuclease I (DNase I, GIBCO BRL, Grand Island, NY, USA) at room temperature for 15 min and then used in a first-strand reaction consisting of incubation with oligo (dT)15 primer (Promega, Madison, WI, USA) and Omniscript reverse transcriptase (OmniscriptTM RT Kit, QIAGEN GmbH, Hilden, Germany) at 37°C for 1 h. The resulting cDNA was subjected to PCR using a Taq PCR Core Kit (QIAGEN GmbH). PCR was performed on cDNA under the following conditions. AAT (215 bp): forward 5'-CCACGATATCATCACCAAGTTCC-3', reverse 5'-TGGTCAGCAGCCTTATGC-3', 94°C for 1 min, 58°C for 1 min, 72°C for 1 min, for 35 cycles. MMP-9 (208 bp): forward 5'-GGAAGATGCTGCTGTTCAGC-3', reverse 5'-ACCTGGTTCAACTCACTCACTCCG-3', 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, for 20 or 25 cycles. GAPDH (411 bp): forward 5'-CAAGAAGGTGGTGAAGCAGG-3', reverse 5'-ATGGTACATGACAAGGTGCG-3', 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, for 35 cycles. PCR products were separated in 2% agarose gels followed by ethidium bromide staining, and visualization on a UV illuminator. Visualized bands were scanned intoMulti Gauge Ver. 3.0 (Fujifilm, Tokyo, Japan), and the pixel intensities of the bands were quantified by densitometry according to manufacturer's instructions.
Immunohistochemistry
In order to examine the localization of AAT in the human amnion, a horseradish peroxidase (HRP)-conjugated goat anti-human AAT polyclonal antibody (1:10 000, BETHYL Laboratories. Inc., Montgomery, TX, USA) was used as the primary antibody. Oxidized type AAT was detected using a mouse monoclonal anti-human oxidized AAT antibody (1:50, Ikagaku Co. Ltd, Kyoto, Japan).
Frozen sections (
4 µm-thick) mounted on glass slides were fixed with cold acetone for 20 min. To detect all types of AAT, after washing with PBS, 0.3% hydrogen peroxide in methanol was applied to block endogeneous peroxide activity. After washing with PBS three times for 5 min each time, non-specific binding was blocked with 10% non-immune rabbit serum (Nichirei, Tokyo, Japan). Then, the sections were incubated with the primary antibody overnight at 4°C. After washing with PBS, they were incubated in a solution of 0.03% 3,3'-diaminobenzidine (DAB) in TBS with 0.03% hydrogen peroxide for 5 min. Then, the sections were counterstained with hematoxylin.
For detecting oxidized AAT, after fixation and washing, non-specific antibody binding sites were blocked using 10% non-immune rabbit serum (Nichirei) for 1 h at room temperature and the sections were incubated with the primary antibody overnight at 4°C. After washing, they were incubated with a secondary antibody: biotin-conjugated anti mouse IgG (Nichirei). After washing with PBS, they were incubated with fluorescein isothiocyanate-conjugated streptavidin (1:100, Dako Cytomation, Denmark) in PBS for 30 min at room temperature. The sections were counterstained with propidium iodide (10 µg/ml, Wako Pure Chemical Industries, Ltd, Osaka, Japan), and examined by fluorescence microscopy (Leika DMRBE).
Western blotting analysis for AAT
The freshly isolated hAE cells or pieces of human amnion tissues were homogenized in a lysis buffer (50 mM Tris–HCl, pH 8.0, containing 250 mM NaCl, 0.5% NP-40), including protease inhibitors. These samples were then subjected to SDS-polyacrylamide gel electrophoresis (10% polyacrylamide for resolving) under reducing conditions. Proteins were then transferred onto membranes (ImmunobilonTM-P, MILLIPORE Co., Bedford, MA, USA). The blotted membranes were incubated with blocking solution [2% milk in TBS containing Tween-20 (Wako), (TBST)] for at least 1 h at room temperature. Then, the membranes were incubated with an HRP-conjugated goat anti-human AAT polyclonal antibody (1:10 000 BETHYL Laboratories) in blocking solution at 4°C for 16 h. After washing with TBST, bands were visualized with an ECL plus Western Blotting Detection Kit (Amersham Bioscience Co., Piscataway, NJ, USA) and a LAS-1000 plus Lumino Imaging Analyzer (Fujifilm). After washing with TBST, the membranes were incubated with a mouse anti-β actin monoclonal antibody (1:5000, Sigma-Aldrich Co.) in blocking solution at 4°C for 16 h. Then, the membranes were incubated with an ECL HRP-conjugated anti-mouse IgG antibody (1:4000, Amersham Bioscience Co.) in a blocking solution for 1 h at room temperature. After washing with TBST, bands were visualized as above. Immunoblots were scanned into Multi Gauge Ver. 3.0 (Fujifilm), and the pixel intensities of the immunoreactive bands were quantified by densitometry according to manufacturer's instructions. All experiments were repeated three times.
Gelatin zymography
Gelatinolytic activity in hAE cells with cytokine stimulation was analyzed by electrophoresis using Gelatin zymo electrophoresis kit (Cosmobio, Tokyo, Japan). Total protein was extracted from hAE cells described above without protease inhibitors. 0.1 µg (TNF) or 0.5 µg (OSM and IL-6) protein subjected to zymography gelatin electrophoresis. Zymography was performed following the manufacturer's instructions. White zones on the gels indicate the gelatinolytic activity of proteinases.
Enzymatic assay of AAT activity
AAT activity in the human amniotic membrane was analyzed using an enzymatic assay for trypsin inhibitory capacity (Sigma-Aldrich Co.). The principle is: trypsin hydrolyses a trypsin-substrate, N-benzoyl-L-arginine ethyl ester (BAEE), to N-benzoyl-L-arginine and ethanol, and AAT inhibits this reaction.
Total protein was extracted from the amnion as described earlier without using proteinase inhibitors, and 1 mg of each was subjected to the assay immediately after extraction. Trypsin activity was measured by monitoring the hydrolysis of BAEE (
A253 nm), using a UV spectrophotometer (Yu et al., 2003). Just after adding several concentrations of standard AAT (0.004–0.02 mg at the final assay concentration, Sigma-Aldrich Co.) and trypsin (0.005 mg in final concentration) to BAEE (0.46 mM) solution in HCl (0.06 mM) and sodium phosphate (63 mM), UV absorbance was recorded at 1-min intervals; the first 5 min of the reaction were used to make the standard slope. The A253 nm/min was obtained using the maximum linear rate for each concentration of AAT. Next, instead of standard AAT, 1 mg of sample protein was applied and the activity of AAT in the samples was calculated as the trypsin inhibition rate. The inhibition rate (%) = (A/min AAT=0–A/min sample)/A/min AAT=0x100; A/min AAT=0 was obtained from the standard slope.
Statistical analysis
Bonferroni/Dunn test was used to test for difference in maternal age between the groups (Table I). Mann–whitney U-test was used to evaluate the difference of immunoreactive band (Fig. 2C) and to evaluate the difference of gestational age between the groups (Table I). The rate of cesarean section and CAM were compared between the groups, using Pearson's chi-squared test as appropriate. The AAT activity was analyzed using Scheffe method, and differences were considered significant when P-values were <0.05.
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Production of AAT by human AE cells
In order to investigate whether human amnion produces endogeneous AAT was studied by western blotting in freshly isolated hAE obtained from cases of term delivery without any complications. In order to detect endogeneous AAT, protein was extracted from freshly isolated hAE cells, and not from the whole membrane. In all cases studied, a 52-kDa immunoreactive band was clearly detected, suggesting that hAE cells were producing AAT (Fig. 1A). Beta-actin was used as an internal control.
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Next, we performed immunohistochemical analysis to determine the localization of AAT in the human amnion tissue from cases of term delivery without any complications. Several hAE cells showed a positive cytoplasmic reaction to the AAT antibody. By immunohistochemical analysis for the extension section of human amnion, AAT immunoreactivity is clearly expressed in the cytoplasm of epithelial cells (Fig. 1B). There was no difference in the expression pattern among the membrane sites. In this analysis, an HRP-conjugated goat anti-human AAT polyclonal antibody, which could detect all types of AAT, including the oxidized and cleaved types, was used. These results showed that both hAE and hAM cells produce endogeneous AAT.
MMP-9 expression in human AE cells
As PPROM is closely related to inflammation occurring in the amnion, we studied the relationship between the expression of AAT, MMP-9 and inflammatory cytokines. As MMP-9 degrades type IV collagen, which is a major component of the basement membrane of the amniotic membrane, we confirmed whether hAE cells expressed endogeneous MMP-9 by RT–PCR. Human AE cells cultured for 5 days showed a band for MMP-9. We did not measure active MMP-9, so this data described only change in MMP-9 expression. Three cases that delivered at term without any complications were examined and all specimens showed MMP-9 expression. Although at 20 cycles of PCR reaction the band was weak, at 25 cycles of reaction, it appeared very clearly (Fig. 2B). This was the same for the freshly isolated hAE cells (data not shown). A clear band for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed good mRNA integrity (Fig. 2A and B).
Stimulation of hAE cells with inflammatory cytokines
Next, we studied whether the expression of AAT could be increased by cytokines reported to be related to PPROM. After 3 days of primary cultured, hAE cells were stimulated with OSM, TNF or IL-6 for another 3 days. All these factors increased expression of AAT (Fig. 2B and C). As shown in Fig. 2C, expression level of AAT/GAPDH was significantly increased by all these factors. These results show AAT in human amnion is controlled after these inflammatory cytokines. By stimulation with TNF , expression of MMP-9 was also significantly increased in hAE cells. However, OSM stimulation significantly decreased MMP-9 expression (Fig. 2B and C). We observed only a small increase of MMP-9/GAPDH level by IL-6 stimulation. Thus, the expression of AAT and MMP-9 were both increased by TNF , but AAT differs from MMP-9 after OSM stimulation. GAPDH was used as an internal control and confirmed to have equivalent loading and integrity of cDNA.
MMP-9 activity in hAE cells with cytokine stimulation
Considering that MMP-9 in hAE cells has been related to inflammatory cytokines, we analyzed whether these cytokines stimulated hAE cells constitutively synthesize MMPs by gelatin zymography assays. As shown in Fig. 2D, pro-MMP-9 (92 K) and active MMP-9 (82 K, 76 K) were increased by TNF stimulation. However, OSM and IL-6 stimulation resulted in lower MMP-9 activity. Previous results (Fig. 2B and C) were supported by gelatin zymography.
Analysis of cleaved AAT in human amnion tissue with PPROM
Assuming that in the case of PPROM, AAT expression in the amnion tissue might change, we studied differences in AAT among cases with or without PROM. We divided tissues into four groups: TL, PTL, TPROM and PPROM.
At first, using a goat anti-human AAT polyclonal antibody, we studied the expression-quantity of 52 kDa AAT in each case; however, we observed no significant differences of expression level among the four groups (Fig. 3). Then, we focused on the quality of AAT because several inactive types of AAT have been reported. A previous report demonstrated that at sites of inflammation, by releasing metalloproteinase, neutrophils could proteolytically inactivate AAT, decreasing 4 kDa in molecular size, and that the short type was designated as cleaved-type AAT (Vissers et al., 1988). As shown in Fig. 3, cleaved-type AAT, with a molecular mass of 48 kDa, was seen not only in the amnion with PPROM, but also in the amnion without PROM. By densitometry analysis, there were no statistically significant differences in the amount of AAT protein expression (cleaved AAT/β-actin values), including both the normal type and cleaved type, among specimens with PROM and those without PROM for both gestational groups.
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Presence of oxidized AAT in the amnion with PPROM
We further analyzed the quality of AAT protein, expressed in these cases. Oxidized AAT is another modified form of AAT, which is found in inflammatory exudates at levels of
5–10% of total AAT (Zhang et al., 1993; Sepper et al., 1995). Oxidized AAT promotes tissue destruction not only by the loss of proteinase inhibitory activity, but also by recruiting and activating monocytes in lesions (Vogt, 1995; Moraga et al., 2000). Therefore, we performed immunohistochemical analysis to determine whether or not amnion tissue with PPROM expresses oxidized AAT. Positive staining of oxidized AAT was observed in the amniotic epithelial cells with PROM (Fig. 4A and B), especially in preterm gestation (Fig. 4A). Oxidized AAT was negative in the amnion without PROM (term, n = 5, preterm, n = 2). These findings suggest that AAT in the amnion with PROM was oxidized and lost activity, occurring more markedly in the PPROM cases (n = 5) compared with TPROM (n = 2). The staining pattern was notably different when compared with polyclonal AAT antibody (Fig. 1B), suggesting that the oxidized and active forms were immunologically distinct.
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Measurement of AAT activity using an enzymatic assay
We further analyzed the activity of AAT in the amnion tissue using an enzymatic assay (Fig. 5). The clinical characteristics of the patients are shown in Table I. As for the inhibition rate of trypsin, the level of AAT activity with PPROM [0.79 ± 1.70% (n = 10)] and TPROM [(4.26 ± 5.22% (n = 9)] was significantly lower than that of TL [(30.47 ± 15.67% (n = 13)] (P-value for both comparisons <0.001). AAT activity in PPROM was significantly lower than that of PTL [(19.22 ± 15.8% (n = 16)] (P < 0.01). Four cases in PTL showed low level of AAT activity, all of them had pathological CAM (open square, Fig. 5). There were no differences between term and preterm gestation in AAT activity in the amnion. These results did not depend on the onset or absence of labor (data not shown).
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We demonstrated that AAT was expressed in all human amnion specimens studied here and in the specimens obtained from TL AAT-positive hAE cells were immunostained focally. These results were consistent with a previous report that the amnion secreted AAT into the culture supernatants (Takashima et al., 2004). As both mRNA and protein were detected, as well as localization of AAT protein, contamination from other origins was ruled out and endogeneous AAT expression was shown in the amnion. This raised questions regarding the role of AAT in the amnion, and how its expression is regulated.
AAT has been shown to reduce neutrophil adhesion to matrix protein, inhibit neutrophil chemotaxis and limit tissue degradation when tissue proteinases are released from inflammatory cells. Therefore, it was expected that in the amnionitis leading to PROM, AAT would play an important role and we examined whether cytokines regulated the expression of AAT.
In this study, we showed that TNF , IL-6 and OSM induced AAT. It was reported that significant levels of TNF and IL-6 were found in human amniotic fluids from patients with preterm labor and intra-amniotic infection (Romero et al., 1989, 1990). TNF , which induced AAT in hAE cells, also stimulates human chorionic cells and accelerates the biosynthesis of MMP-1, MMP-3 and PGE2, while MMP-3 has been shown to cleave and inactivate AAT in vitro (Winyard et al., 1991). Perlmutter et al. reported that in a study of hepatoma cells, IL-6 increased AAT mRNA, but not TNF (Perlmutter et al., 1989), suggesting that the expression mechanism for AAT might be different in each tissue.
In our study, TNF induced MMP-9 expression in hAE cells, while OSM reduced it, partially consistent with a previous report that in vitro, IL-1β and TNF induced secretion of MMP-9 in human amnion and trophoblasts (Fortuno et al., 1999; Vadillo-Ortega and Estrada-Gutierrez, 2005). Gelatin zymography showed that active MMP-9 in hAE cells is increased by TNF , but decreased by OSM. MMP-9 is important not only as a proteinase, but also as an inhibitor of AAT, because AAT may be cleaved by MMP-9 and inactivated (Mast et al., 1991; Ghavami et al., 2007). Therefore, among the cytokines we studied here, OSM has the potential to prevent PROM by reducing MMP9 production, as well as inducing AAT.
Although it was shown that AAT expression in hAE cells was regulated by cytokines, there was no difference in the quantitative analysis of AAT expression between the amnion with and without PROM, the major cause of which has been reported to be amnionitis. Therefore, we analyzed AAT protein qualitatively. The aniti-proteinase activity of AAT is inhibited by chemical modification, including inter- or intra-molecular polymerization, oxidation, complex formation with target proteinases (e.g. neutrophil elastase) and/or cleavage by elastase or other enzymes (Carp and Janoff, 1980; Peteropoulou et al., 2003). In vitro, human neutrophil elastase is known to inactivate AAT by cleaving it near the inhibitory site, down-regulating its inhibitory capacity at the site of inflammation, and thus allowing for greater neutrophil elastase activity, such as degradation of connective tissue components, cleavage of serum proteins and alteration cellular functions (Havemann and Gramse, 1984). However, in this study, immunoreactive bands for cleaved-type AAT were shown without any statistical difference in the amnion both with and without PROM.
AAT is oxidized by free radicals released from activated inflammatory cells, including neutrophils, and is thus also inactivated. Oxidization of the methionine residue to methionine sulfoxide, located at its active center, results in AAT inactivation and degradation (Matheson and Travis, 1985; Desrochers et al., 1992; Vogt, 1995). Reactive oxygen species are instable molecules, generating tissue damage that is responsible for PPROM (Woods, 2001). In this study, we demonstrated that the amnion with PPROM and TPROM expressed oxidized AAT more strongly compared with the specimens PTL and TL. In addition, in the PPROM and TPROM cases, its expression was significantly higher in the preterm-delivered amnion compared with term gestation-amnion. Considering that oxidized AAT not only loses its inhibitory functions to proteinase, but also influences leukocyte recruitment through stimulating human neutrophil migration and degranulation (Parmar et al., 2002), its relation to tissue damage in the amniotic membrane in PROM may not be negligible. In this study, we did not investigate the other inactivated forms of AAT, such as inter- or intra-molecular polymerization and complex formation with target proteinases (e.g. neutrophil elastase). It is possible that several large or small bands detected in the western blotting for AAT in Fig. 3 might be other forms of inactivated AAT. Figure 3 showed the most representative data. We repeated experiments using three more samples in each groups, but the results were similar. Several inactivated forms of AAT could appear in the same specimen; however, further study will be necessary to clarify this point.
With the presence of oxidized AAT in the amnion with PROM, it was suggested that AAT activity might be decreased in those cases. Therefore, we studied the AAT activity by calculating the trypsin inhibitory capacity in the extracted protein from amnion. The activity of AAT was significantly decreased in both PPROM and TPROM. To our knowledge, this is the first report measuring the activity of AAT in amnion tissue. Our results suggested inactivated AAT, maybe by oxidization, is one of the causes of PROM. However, there was no significant difference in AAT activity between TPROM and PPROM. Therefore, AAT activity as proteinase inhibitory capacity might be important in the premature rupture of the membrane itself, but it is not specific to preterm events. In contrast, oxidized AAT was more markedly detected in the case with PPROM than TPROM case. Therefore, in addition to a local reduced inhibitory capacity of AAT stimulative influences of oxidized AAT to inflammatory cells may facilitate or prolong amniotic inflammation in PPROM.
In summary, we showed that the amnion expresses endogeneous AAT and its expression was regulated by cytokines, such as TNF , IL-6, OSM, in hAE cells. In the cases with PROM, the oxidized inactive form of AAT was detected and total AAT activity was decreased. Thus, it is thought that AAT in the amnion is a kind of protective shield at inflammatory sites; however, in PROM cases, AAT lost its inhibitory activity, perhaps due to oxidization, so an imbalance of AAT and proteolytic enzymes led to PROM.
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This work was supported in part by Grants-in-Aid 16390473, 16650102 and 20390430 for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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We wish to thank Osamu Higuchi, Makiko Nogami, Teng Zang, and Etsuko Furuichi for technical assistance.
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Submitted on October 24, 2007; resubmitted on November 14, 2008; accepted on November 18, 2008.
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