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BOR - Papers in Press, published online ahead of print September 17, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016204
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BIOLOGY OF REPRODUCTION 70, 253–259 (2004)
DOI: 10.1095/biolreprod.103.016204
© 2004 by the Society for the Study of Reproduction, Inc.

Pregnancy

Epithelial Cell-Derived Neutrophil-Activating Peptide-78 Is Present in Fetal Membranes and Amniotic Fluid at Increased Concentrations with Intra-amniotic Infection and Preterm Delivery1

J.A. Keelan2, J. Yang, R.J. Romero, T. Chaiworapongsa, K.W. Marvin, T.A. Sato, and M.D. Mitchell

Liggins Institute and Department of Pharmacology and Clinical Pharmacology3 Department of Obstetrics and Gynaecology,4 University of Auckland Faculty of Medical and Health Sciences, Auckland, New Zealand NICHD Perinatal Research Branch,5 Hutzel Hospital, Detroit, Michigan 48201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased (~4-fold) with term labor in amnion (n = 15), and with preterm labor (~30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1ß and tumor necrosis factor {alpha}. Production of ENA-78 by choriodecidual explants was increased modestly after 2–4 h of exposure to lipopolysaccharide (5 µg/ml). An immunoreactive doublet (~8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.

cytokines, decidua, parturition, placenta, pregnancy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although preterm labor and birth can be associated with multiple etiologies, intrauterine infection and/or inflammation is believed to be the most common cause of the more clinically relevant preterm deliveries (<=32 wk of gestation) [1]. A large number of studies have been conducted regarding various fluids and tissues from preterm deliveries, and our understanding at the cellular level of the processes associated with intrauterine infection-driven preterm labor has advanced considerably over the past two decades or so. Leukocytosis of the gestational membranes—amnion, chorion, and decidua—is the histological hallmark of an ascending intrauterine infection and a strong correlate with microbial invasion of the amniotic cavity (MIAC), elevated amniotic fluid cytokine and prostaglandin levels, evidence of matrix protein remodeling associated with premature rupture of membranes (PROM), and shortened pregnancy length [2–6].

The factors responsible for recruiting leukocytes, predominantly neutrophils and macrophages, to the gestational membranes have not been confirmed, although a number of likely candidates have been identified and studied. These include interleukin (IL)-8; macrophage chemotactic protein-1; growth-related oncogene (GRO)-{alpha}, -ß, and -{gamma}; and chemoattractant lipids, such as leukotriene B4 and prostaglandins [7–11]. In a cDNA array study of membranes from infected and noninfected placentas, we recently reported that a large number of chemokine genes were upregulated in association with preterm delivery, particularly when chorioamnionitis was present [12]. One of these genes encoded epithelial cell-derived neutrophil-activating peptide (ENA)-78 (also named CXCL5), a relatively recently discovered member of the CXC chemokine family that contains a Glu-Leu-Arg (ELR) motif essential for neutrophil-stimulating activity [13, 14]. As its name suggests, ENA-78 is a product of epithelial cells, and it has been implicated in the pathogenesis of a number of diseases [15–17]. Its expression is not limited to epithelia, however, having been identified in hepatocytes, endothelial cells, endometrial stromal cells, and a variety of marrow-derived cells [18–22]. Within the uterus, ENA-78 expression and production have been identified in the endometrial stroma [23]. The bioactivity of the full-length, 78-amino-acid form of the molecule is increased by N-terminal proteolysis, yielding stable isoforms (ENA-5-78 and -8,9-78) that are 3- to 9-fold more potent chemoattractants than the full-length polypeptide [24, 25]. ENA-78 and its derived peptides are ligands for CXCR2 (IL-8RB), a chemokine receptor that is also activated by IL-8 and the GRO proteins [26]. The CXCR2 is expressed predominantly by neutrophils but also by some nonimmune tissues; in the uterus, CXCR2 has been localized to amnion epithelial cells, placenta, and uterine epithelium [27, 28].

The fetal membranes are composed primarily of epithelial tissue: amniotic epithelial cells and chorionic trophoblasts. Hence, we hypothesized that ENA-78 derived from these tissues might be an important factor in the recruitment and activation of neutrophils to the fetal membranes following exposure to microbial products and the resultant release of proinflammatory mediators. The aim of the present study, therefore, was to assess the ability of the gestational membranes to produce ENA-78, both basally and in response to proinflammatory cytokines, and to determine whether ENA-78 levels within the uterus (membranes and amniotic fluid) are elevated in pregnancies complicated by preterm labor both with and without chorioamnionitis and intrauterine infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Culture medium (Ham F12/Dulbecco Modified Eagle) was obtained from Irvine Scientific (Santa Ana, CA). Fetal calf serum was purchased from Life Technologies Ltd. (Auckland, New Zealand). Bovine gamma-globulin and bacterial lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St Louis, MO). Matched-pair antisera to ENA-78 (monoclonal and polyclonal) were obtained from R&D Systems (Minneapolis, MN), as was recombinant human ENA-5-78, which was used as a calibrator. Donkey anti-goat immunoglobulin (Ig) G horseradish peroxidase conjugate and biotinylated anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Streptavidin biotin-alkaline phosphatase was purchased from DAKO A/S (Glostrup, Denmark). Maxisorb ELISA plates and disposable plastic tissue culture consumables were supplied by Nalge Nunc International (Roskilde, Denmark). The protein-G Sepharose 4B was from Amersham Biosciences (NZ) Ltd. (Auckland, New Zealand).

Immunohistochemistry

Full-thickness gestational membranes from term placentae delivered by cesarean section before the onset of labor were fixed in 4% paraformaldehyde and embedded in paraffin. After clearing and dewaxing in xylene/graded alcohols, sections (thickness, 7 µm) were subjected to antigen retrieval (boiling for 20 min in 0.5 M Tris-HCl [pH 10]), then blocked for 20 min with 1% H2O2 in 50% methanol. Antisera (goat anti-ENA) was applied to the sections in PBS/0.5% Tween-20/5% normal horse serum and incubated overnight at 4°C. Following washing in PBS/0.5% Tween-20, immunoperoxidase detection was accomplished using tyramide amplification with a TSA kit (NEN, PerkinElmer, Wellesley, MA) according to the manufacturer's instructions and developed with diaminobenzidine. Digital photomicrographs were taken using a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan) fitted with a JVC TK-C1381 (JVC, Yokohama, Japan) color video camera. Negative controls, consisting of either no primary antisera or nonreactive goat antisera, were run in parallel.

ENA-78 ELISA

Concentrations of ENA-78 were measured by two-site sandwich fluoroimmunoassay using matched-pair antisera from R&D Systems. Recombinant human ENA-5-78 standard was used to calibrate the assay. After incubation with biotinylated anti-goat IgG second antibody followed by streptavidin-alkaline phosphatase complex, the generation of fluorescent signal from 4-methylumbelliferyl phosphate substrate was determined fluorometrically (355/460 nm) in a Victor2 multiplate reader (Wallac Oy, Turku, Finland). Curve fitting and data extrapolation were performed using on-board software (Workout; Wallac Oy). The limit of detection of the assay was ~20 pg/ml. Intra- and interassay precision (coefficient of variation) was 5.6% and 11.4%, respectively. Amniotic fluid samples were assayed in duplicate; culture media from replicate culture wells were assayed in singletons. The level of IL-8 was similarly measured using ELISA as detailed previously[29].

Immunoprecipitation and Immunoblotting

Samples (1 ml) of conditioned media from choriodecidual explants were incubated with monoclonal anti-ENA-78 antibody (30 µg) for 1 h at room temperature, then immunoprecipitated by incubation with protein-G Sepharose (75 µl) overnight at 4°C. The affinity gel was washed four times in PBS by resuspension/centrifugation (5000 x g, 5 min), then heated to 95°C for 5 min in SDS-PAGE loading buffer (40 µl) containing ß-mercaptoethanol. Aliquots (35 µl) were electrophoresed on an 18% SDS-PAGE gel and transferred to nitrocellulose membrane using a semidry transfer unit (Bio-Rad Laboratories Pty Ltd., Auckland, New Zealand). After blocking overnight in 2.5% nonfat milk powder, membranes were incubated with goat anti-ENA-78 antibody (0.2 µg/ml) overnight at 4°C, followed by horseradish peroxidase-conjugated anti-goat IgG (1:6000) for 1 h. Images were developed by incubation with Super-Signal West Dura enhanced chemiluminescence reagent (Pierce, Rockford, IL) and exposure to radiographic film (Hyperfilm, Amersham Biosciences [NZ] Ltd.). Recombinant human ENA-5-78 was run on the gel as a positive control (1–5 ng). Molecular weights were estimated by comparison with Rainbow prestained size markers (Amersham Biosciences [NZ] Ltd.).

Amniotic Fluid Collection

Amniotic fluid samples were collected by transabdominal amniocentesis. All women provided informed consent before the collection of amniotic fluid. The collection and use of amniotic fluid was approved by the human investigation committees of the participating institutions (i.e., Wayne State University, Hutzel Hospital, MI; Sotero del Rio Hospital, Puente Alto, Chile) and approved for research purposes by the Institutional Review Board of the National Institutes of Child Health and Human Development. Several studies using these fluids have been published recently that include full details of their collection and clinical categorization [30, 31]. In brief, samples were divided into four main groups: Group 1 consisted of women in the midtrimester (15–17 wk) of pregnancy who underwent amniocentesis for genetic indications and delivered an appropriate-for-gestational-age infant at term (n = 23). Group 2 consisted of women with preterm labor and intact membranes; these patients were subdivided into the following categories: group 2a, those with preterm labor without MIAC who delivered at term (n = 29); group 2b, those with preterm labor without MIAC who delivered preterm (<37 wk; n = 30); and group 2c, those with preterm delivery with MIAC (n = 19). Group 3 consisted of women with preterm PROM, either with (n = 30) and without (n = 43) MIAC. Group 4 consisted of women with term gestations (>37 wk) without MIAC; these patients were subdivided into the following categories: group 4a, those with intact membranes not in labor (n = 26), and group 4b, those with intact membranes in labor (n = 25). Because of sample volume limitations, amniotic fluids were diluted 1:5 in assay diluent before ELISA.

Tissue Extraction and Analysis

Placentas were collected at National Women's Hospital (Auckland, New Zealand) with informed consent under the approval of the Auckland Ethics Committee. Amnion as well as choriodecidual and placental tissues from pregnancies delivered by cesarean section at term before the onset of labor, after spontaneous labor at term, or following spontaneous preterm labor, both with and without intrauterine infection, were collected and frozen in liquid nitrogen for subsequent processing as detailed previously [12, 32]. For protein extraction, tissue aliquots were sonicated and homogenized in an aqueous hypotonic lysis buffer supplemented with protease inhibitors as previously described [32]. Aliquots of homogenate extracts, stored at -80°C, were thawed and diluted in assay diluent before immunoassay analysis. Leukocyte infiltration was assessed histologically using CD45 immunoperoxidase staining and graded (0–4) as described previously [12].

Explant Culture

Comparative rates of production of ENA-78 and IL-8 by gestational membranes in vitro were studied using an explant model [33, 34]. All placentas used in these experiments were obtained after cesarean section at term before the onset of labor (indications: previous section or malpresentation) and were devoid of clinical evidence of intrauterine infection. Explant cultures from amnion and choriodecidual tissues were prepared as previously described [33, 34], cultured in 24-well culture dishes in serum-free media (1 ml/well), and treated after a 24-h equilibration period with stimuli for a further 2–24 h. Media were stored at -20°C before assay, then thawed and diluted for assay as appropriate. Explant tissue wet weights (mg/well) were recorded at the end of the experiments for normalization purposes.

Amnion Monolayer Culture

To determine the responsiveness of amnion ENA-78 production to stimulation by proinflammatory cytokines, cultures of amnion epithelial cells from membranes from normal pregnancies delivered by cesarean section at term before onset of labor were prepared as described previously [29]. Experiments were performed on Days 3–4 of culture using four wells per treatment on three individual cultures.

Statistical Analysis and Presentation of Data

Production of ENA-78 in explant culture was derived as picograms per milligram wet weight of tissue per time point and, when appropriate, expressed as a percentage of the control value to allow the results of multiple experiments to be pooled and analyzed collectively. Similarly, production by amnion monolayer cultures was expressed as picograms per microgram of cellular protein per time point. Statistical significance was assessed by ANOVA followed by the Dunnett test or the Bonferroni t-test post hoc. Differences in amniotic fluid concentrations of ENA-78 were assessed by the Kruskal-Wallis test for nonparametric data. Pairwise group comparisons (tissue homogenates) were performed by the Mann-Whitney U-test. A P value of less than 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Data from our previous cDNA expression array studies indicated that ENA-78 expression occurs in gestational membranes and is increased with preterm labor, particularly when associated with chorioamnionitis [12]. To extend these preliminary observations and establish that such changes occur at the protein level, soluble extracts were prepared from amnion and choriodecidual membranes from term deliveries (with and without labor; n = 15 each) and preterm deliveries (n = 31), and ENA-78 concentrations were measured by ELISA. Levels of ENA-78 were similar in amnion and choriodecidual homogenates (Fig. 1). A significant increase was observed in amnion with spontaneous labor at term, with a further increase with preterm labor, whereas in choriodecidua, preterm tissues showed a significant increase in ENA-78 abundance relative to term with no effect of labor (P < 0.05, Mann-Whitney U-test). In amnion (but not choriodecidual) tissue homogenate extracts (n = 27) from preterm deliveries, ENA-78 concentrations were positively correlated with degree of leukocyte infiltration (r2 = 0.481). In these same tissues, ENA-78 abundance was more weakly correlated (r2 = 0.379) with IL-8 (described in [32]).



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  		FIG. 1. ENA-78 abundance in amnion and choriodecidual membranes from term and preterm deliveries. Aliquots of amnion and choriodecidual tissues were lysed and extracted, and levels of ENA-78 in the supernatants were determined by ELISA. Data are displayed as the median ± interquartile range (boxes), with the 10th and 90th percentiles shown as error bars. Differences between groups were determined by the Mann-Whitney U-test (*P < 0.05). TNL, term, no labor (n = 15); TSL, term, spontaneous labor (n = 15); PTL, preterm labor (n = 31)

Paraffin-embedded tissues from placentas delivered at term before the onset of labor were examined by immunohistochemistry using polyclonal anti-ENA-78 antiserum to establish the cellular source of ENA-78 in the membranes. The ENA-78 immunoperoxidase staining was localized predominantly to chorionic trophoblasts, with weaker and less consistent staining in the amniotic epithelium and decidual cells (Fig. 2). No significant staining was observed in the negative controls (antibody omitted or replaced with irrelevant goat antibody). These data are consistent with the decidua and fetal membranes all being a source of ENA-78.



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  		FIG. 2. Top) Immunoperoxidase staining of ENA-78 in full-thickness, term gestational membranes. Antigen was localized to amnion epithelium (A) and chorionic trophoblast (C), with occasional staining in decidua (D). No consistent differences in staining were observed between term or preterm tissues. Bottom) Negative controls (no primary antibody) were devoid of staining. Magnification x200

Monolayer cultures of dispersed amnion cells were prepared to determine concentration-dependent effects of proinflammatory cytokines on amnion ENA-78 production (multiple-dose experiments are easier to perform on monolayer cultures and give more precise results than explants). Production of ENA-78 by amnion monolayer cultures was stimulated in a dose-dependent fashion by both IL-1ß and TNF{alpha}, with maximal production (3- to 10-fold that of control) observed at 5 ng/ml or more of IL-1ß and at 10 ng/ml or more of TNF{alpha} (Fig. 3).



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  		FIG. 3. Dose-dependent stimulation of ENA-78 production by cultured amnion epithelial cells by proinflammatory cytokines. Experiments were performed in quadruplicate wells. Data (production as pg/µg protein over 16 h) are displayed as the mean ± SEM from a representative experiment performed three times with similar results. *P < 0.05 by the Dunnett test after ANOVA

Amnion and choriodecidual membrane explants were used to compare ENA-78 and IL-8 production rates between tissues. Production of ENA-78 by choriodecidual and amnion explants were similar under basal conditions (~0.3 ng/mg tissue over 24 h; n = 4 independent experiments). Production of IL-8 was several-fold greater than that of ENA-78, with choriodecidual ENA-78 production being only 14.4% ± 3.7% (mean ± SEM) of IL-8 production after 24 h of incubation. Time-course studies to investigate the rate and relative responsiveness of ENA-78 and IL-8 production to LPS revealed that ENA-78 production was stimulated by LPS at the initial time points (2–4 h), but by 24 h of incubation, no difference was observed between control and LPS-treated cultures (Fig. 4). In contrast, IL-8 production was significantly elevated in response to LPS after 24 h but not at earlier time points. The degree of response by either ligand to LPS stimulation was modest (<3-fold).



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  		FIG. 4. Effects of LPS on production of ENA-78 and IL-8 over a 24-h time course. Choriodecidual explants (n = 3 replicates) were treated with LPS (5 µg/ml) at the time points indicated, and media were harvested for determination of ENA-78 and IL-8 concentrations by ELISA. Production data, normalized to milligrams wet weight of tissue, are displayed as the mean ± SD. A representative of three experiments is shown. *P < 0.05 by the Dunnett test after ANOVA

Initial attempts using immunoblotting to characterize the molecular weight of the ENA-78 isoform(s) secreted by amnion and choriodecidual tissues were unsuccessful because of the lack of sensitivity of the method. Subsequent analysis by immunoprecipitation/immunoblotting of LPS-stimulated choriodecidual-conditioned media (n = 2) revealed the presence of two immunoreactive bands with very similar molecular weights (~8000 Da) that comigrated closely with the ENA-5-78 standard (Fig. 5). Both ENA species appeared to be present in approximately equal amounts, with one of the bands running slightly slower than the standard, possibly representing the full-length ENA-78 peptide.



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  		FIG. 5. Immunoprecipitated ENA-78 from choriodecidual (CD) explant-conditioned media analyzed by immunoblotting. Samples of LPS-stimulated conditioned media (n = 2 experiments) were immunoprecipitated with monoclonal anti-ENA-78 antibody and immunoblotted after electrophoresis on 18% SDS-PAGE with goat-anti-ENA-78 polyclonal antibody. Blots were developed by enhanced chemiluminescence and exposed to radiographic film for visualization. Recombinant ENA-5-78 (1 and 5 ng) was run as a positive control. The migration point of the 10-kDa molecular weight standard is indicated by the arrow

The majority (89.3% [201/225]) of amniotic fluid samples had ENA-78 concentrations above the detection limit (>100 pg/ml after sample dilution). Labor at term had no effect on ENA-78 levels in amniotic fluid (Table 1). However, the median concentration of ENA-78 in amniotic fluid at term (regardless of labor status) was higher than that at midtrimester, which is indicative of increased levels with advancing gestational age. Indeed, a positive correlation was found between gestational age at amniocentesis and ENA-78 concentration (r5 = 0.7, P < 0.001, Spearman rank correlation) in the following groups: 1) patients presenting with preterm labor and intact membranes who delivered at term (n = 29), 2) patients at midtrimester who had normal pregnancy outcomes (n = 23), and 3) patients at term with intact membranes without labor (n = 26). Of those patients presenting with preterm labor with intact membranes, those who delivered preterm had a higher median ENA-78 concentration in amniotic fluid than those who delivered at term (Fig. 6). In patients with MIAC, amniotic fluid concentrations of ENA-78 were even higher, regardless of other obstetric factors (Fig. 7 and Table 1). Preterm PROM was also associated with increased amniotic fluid ENA-78 concentrations (Table 1), particularly in patients with preterm PROM and MIAC (Fig. 7).


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  		TABLE 1. ENA-78 concentrations in amniotic fluid from women with normal and abnormal pregnancies



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  		FIG. 6. Amniotic fluid ENA-78 concentrations, determined by ELISA, in pregnancies presenting with preterm labor. Significance between groups was assessed by the Kruskal-Wallis test for nonparametric data



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  		FIG. 7. ENA-78 concentrations in amniotic fluid from patients with preterm PROM, both with and without MIAC. Comparisons between groups were performed using the Kruskal-Wallis test


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To our knowledge, this is the first study to describe the presence of ENA-78 in the amniotic cavity and to assess its production by intrauterine tissues in both normal and infected pregnancies. A number of interesting observations have arisen from the present study. First, we have confirmed that ENA-78 immunolocalizes to amnion epithelial cells and chorionic trophoblast; taken together with our earlier cDNA array data, this provides strong evidence that these cells are the source of ENA-78 measured in tissue culture and membrane homogenates. The production rate of ENA-78 by amnion and choriodecidual explants was 3- to 8-fold lower than that of IL-8, which is consistent with the IL-8 abundance data regarding the tissue extracts established during previous studies [32]. IL-8 has been actively studied in the context of leukocytosis and intrauterine inflammation, and it is widely considered to have a major role in recruiting neutrophils into the gestational membranes and cervix in both normal and infection-associated pregnancies. Our findings suggest that ENA-78 is also likely to perform this role, although it may be secondary in importance to IL-8 in light of their relative differences in abundance. The size of the immunoreactive ENA-78 material (two bands of very similar size) secreted by the choriodecidua suggests that the peptide is proteolytically cleaved after secretion and, hence, is likely to be biologically potent. The presence of CXCR2 in amniotic epithelium [35] raises the possibility that amnion-derived ENA-78 may act on the amnion itself in an autocrine fashion, although the nature of its effects on amnion epithelial cells is not yet known.

As anticipated, production of ENA-78 was responsive to cytokine stimulation. Our observations of elevated levels of ENA-78 in extracts of membranes from pregnancies delivered preterm are consistent with the well-accepted model of intrauterine infection/inflammation, with the liberation of cytokines, such as TNF{alpha} and IL-1ß, being critical to the inflammatory response elicited as part of the host response to microbial invasion. Although LPS treatment acts on choriodecidual membranes through the secondary liberation of cytokines [36], we observed only a modest ability of LPS to stimulate either ENA-78 or IL-8 production. We are unaware of any studies that have explored the ability of IL-8 to regulate ENA-78 production. Interestingly, ENA-78 production was more rapid in its response to LPS than was IL-8, suggesting they might act at different stages of the inflammatory response. The significant correlation between leukocyte count (i.e., histological chorioamnionitis grade) and ENA-78 abundance in amnion tissues delivered preterm supports a role for ENA-78 in this process, although it is not possible from these data to rule out the idea that increased ENA-78 levels are a consequence rather than a cause of leukocyte activation.

In amniotic fluid, we found that ENA-78 levels were markedly elevated in women who had evidence of intrauterine infection that had progressed to infection of the amniotic cavity (i.e., MIAC), particularly when their membranes had ruptured prematurely. Studies of a number of other chemokines in amniotic fluid from both normal and pathological pregnancies, such as IL-16, [31], RANTES [30], IL-8 [37–39], GRO-{alpha}, [38, 40], and MIP-1{alpha} [41, 42], have revealed similar findings, although in contrast to the results of some chemokine studies [30, 39, 41], ENA-78 abundance in amniotic fluid did not exhibit a significant increase with onset of labor at term. Nevertheless, elevated chemokine levels in the presence of intra-amniotic infection, preterm PROM, and preterm labor are a consistent observation in the published literature. Even in women without a positive amniotic fluid culture, a significant proportion are likely to have an ascending intrauterine infection contributing to their presentation with preterm labor and/or preterm PROM, representing the normal pathophysiological spectrum of the condition. Overall, our data are consistent with the paradigm of the production of an infection-driven chemokine (i.e., ENA-78) being responsible, at least in part, for membrane leukocytosis, resulting in inflammatory activation, matrix remodeling, membrane rupture, and initiation of uterine contractions.

The ENA-78 was also detectable in amniotic fluid at midpregnancy, and levels increased significantly with gestational maturity, as has been reported for IL-16 [31], GRO-{alpha}, [40], and IL-8[39]. The lack of change in ENA-78 levels in amniotic fluid with term labor is interesting, and this may reflect the state of inflammatory activation of the tissue of origin. Because amnion and chorion probably are the major contributors to amniotic fluid ENA-78, and because leukocyte infiltration of the fetal membranes (as opposed to the decidua) is minimal with term labor [43], a lack of increase in amniotic fluid ENA-78 concentrations with term labor is not unexpected. The corollary of this is that static concentrations of ENA-78 in the amniotic cavity might not be sufficient to recruit neutrophils into the fetal membranes at term, explaining the relative lack of leukocytosis observed. Opposing this hypothesis are several reports presenting evidence of increased abundance of cytokines and chemokines in the fetal membranes or amniotic fluid with term labor, which is consistent with inflammatory activation occurring in the amniotic cavity at term [32, 44–47]. It would appear, therefore, that differing chemokines and cytokines exhibit different levels of involvement in the inflammatory processes associated with term labor, the significance and implications of which remain to be seen.

In conclusion, we have demonstrated that the epithelial cell-derived chemoattractant ENA-78 is produced by the fetal membranes during both normal and pathological pregnancies and accumulates in amniotic fluid at significant concentrations during pregnancy. Intrauterine infection, probably mediated via the release of proinflammatory cytokines as part of host response, is associated with elevated production of ENA-78 by the fetal membranes and increased concentrations of ENA-78 in amniotic fluid. This chemokine likely plays a role in recruiting leukocytes to the amniotic cavity during pregnancies complicated by intrauterine infection. It may also play a role in normal pregnancy, although this remains to be determined.


   ACKNOWLEDGMENTS
 
The assistance of the patients, theater staff, and nursing staff of National Women's Hospital, Auckland, is gratefully acknowledged.


   FOOTNOTES
 
1 Supported by Programme Grant 99/277 from the Health Research Council of New Zealand. Back

2 Correspondence: J.A. Keelan, Liggins Institute & Dept Pharmacology and Clinical Pharmacology, University of Auckland, 2-6 Park Ave., Grafton, Auckland, New Zealand. FAX: 649 373 7497; j.keelan{at}auckland.ac.nz Back

Received: 11 February 2003.

First decision: 14 March 2003.

Accepted: 5 September 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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