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a Perinatal Research Centre, Department of Perinatal Medicine and Department of Obstetrics and Gynaecology, University of Melbourne, The Royal Women's Hospital, Carlton, Victoria, 3053, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-8 displays both inflammatory and growth-regulating properties (reviewed in [6, 7]) but is notable for its selective chemotaxis, degranulation, and activation of neutrophils [8, 9]. Although the role of IL-8 in human pregnancy and labor has yet to be established, IL-8 may contribute to cervical ripening and/or rupture of fetal membranes because of its activation of collagenase and elastase [10]. This mechanism has been suggested as a possible cause of premature rupture of the membranes [11, 12].
IL-8 is released from human placental explants [13, 14] and cervical explants [15], as well as from primary cultures of choriodecidual cells [16], decidual stromal cells, and cytotrophoblasts [17]. Furthermore, IL-8 has also been measured in supernatants of myometrium, decidua, and amnionchorion homogenates [17]. By in situ hybridization and immunohistochemistry, IL-8 has been localized in cytotrophoblast and Hofbauer cells of the placenta [13, 18] in decidual stromal cells and lymphocytes of the decidua [18], as well as in amnion and chorion cells of the fetal membranes [19].
Although IL-8 has been found in intrauterine tissues, little is known about the effects of gestation and labor on the release of IL-8 from human gestational tissues. Previous studies have demonstrated an increased IL-8 concentration in amniotic fluid in association with term gestation and/or term labor onset [20, 21], and in association with chorioamnionitis [20, 22]. The labor-associated elevation in IL-8 has been observed in myometrium [17] but not in placenta or decidua [13]. Nevertheless, we recently reported a labor-associated increase in the release of IL-1
[23], IL-6 [24], and tumor necrosis factor (TNF) [25] from human gestational tissue explants, and some of these cytokines are known to up-regulate IL-8 [4, 26]. The aim of this study was to test the hypothesis that IL-8 release from human gestational tissue explants is increased in association with gestation, labor onset, and chorioamnionitis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Placentae with attached fetal membranes were collected at the Royal Women's Hospital, Victoria, Australia. Relevant institutional ethics approval was obtained along with informed consent from all patients.
Effect of gestation and labor
To test the hypothesis that IL-8 release from human gestational tissue explants is increased with gestation and labor onset, placentae were collected at term (T; 
37 wk gestation) and preterm (P; 20–36 wk gestation, inclusive), before or after spontaneous labor onset. Placentae were collected from six different groups: preterm not-in-labor (PNIL, n = 6), preterm in-labor (PIL, n = 5), preterm after-labor (PAL, n = 6), term not-in-labor (TNIL, n = 6), term in-labor (TIL, n = 6), and term after labor (TAL, n = 6).
TNIL samples were obtained from women at term before labor onset who were undergoing elective cesarean section because of previous cesarean section, cephalo-pelvic disproportion, or breech presentation. TIL samples were collected from women after spontaneous onset of labor at term who delivered by cesarean section either because an elective cesarean was already planned or because of obstetric complications, including obstructed labor, failure to progress, breech presentation, or cephalo-pelvic disproportion. TAL samples were collected after spontaneous onset of labor at term and normal vaginal delivery. PNIL samples were collected from women undergoing cesarean section preterm before labor onset because of maternal and/or fetal conditions, including fetal growth retardation, pregnancy-induced hypertension, and severe pre-eclampsia. PIL samples were collected after spontaneous onset of preterm labor, at unscheduled cesarean section performed because of obstetric complications as listed above for the TIL group. Finally, PAL samples were collected from women after spontaneous onset of idiopathic preterm labor and vaginal delivery.
Effect of chorioamnionitis To test the hypothesis that IL-8 release from human gestational tissue explants is increased by chorioamnionitis (inflammation of the fetal membranes), placentae (n = 6) were collected from women with a provisional diagnosis of chorioamnionitis. Confirmation of the diagnosis of chorioamnionitis was made by a blinded, retrospective case note review performed by an obstetric member (S.P.B.) of the research team. Diagnostic criteria of chorioamnionitis included histological evidence of acute inflammation of the fetal membranes, isolation of microbiological pathogens from fetal membrane culture, peripartum maternal pyrexia, leukocytosis, and/or elevated serum C-reactive protein, maternal and/or fetal tachycardia, and/or purulent amniotic fluid.
Preparation of explants Amnion, choriodecidual, and placental explant cultures were prepared as previously described [24]. The explants were incubated in RPMI 1640 (100 U/ml penicillin, 100 µg/ml streptomycin) at 37°C in a humidified atmosphere of carbogen (5% CO2, 95% O2). After 18 h of culture, the explant-conditioned media and the tissue were collected and stored separately at ;ms20°C until assayed for IL-8 and DNA content, respectively. All explant incubations were performed in triplicate wells for each tissue type and placenta collected.
Assays
IL-8 ELISA. IL-8 concentration in culture media was quantified using a commercially available ELISA kit, IL-8 SL Screening Line (Medgenix Diagnostics, Brussels, Belgium). The assay protocol followed that recommended by the manufacturer. Recombinant human IL-8 (source Escherichia coli code no 58.130.10) was used as the standard, with a working range between 27 pg/ml and 3.5 ng/ml.
The IL-8 assay sensitivity was 27 pg/ml, and the inter- and intraassay coefficients of variation were 17% and 8%, respectively. According to the manufacturers, the antibodies used in the IL-8 assay demonstrated no cross-reactivity with the following cytokines: IL-1, IL-1ß, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, interferon 
, interferon-inducible protein, granulocyte colony-stimulating factor (CSF), granulocyte/macrophage-CSF, growth-regulated oncogene, leukemia inhibitory factor, oncostatin M, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 and -1ß, platelet factor 4, regulated and normal T-lymphocyte expressed and secreted protein (RANTES), stem cell factor (SCF), ß thromboglobulin, transforming growth factor ß, TNF, and TNFß.
DNA Assay
Tissue DNA content was used to normalize the measured release of IL-8 from human gestational tissues. Tissue DNA was quantified using a modified diphenylamine method [27, 28]. A salmon sperm DNA was used for preparation of the assay standard. The assay sensitivity was 20 µg/ml, and the intra- and interassay coefficients of variation were 2.4% and 8.6%, respectively.
Statistics
Statistical computations were performed using a commercially available statistics package (Statgraphics Plus, STSC Inc., Rockville, MD). Data were expressed as mean ± SEM and were initially assessed for homogeneity of variance (Bartlett's test) and transformed as required. Subsequently, data were analyzed using multiple ANOVA (M-ANOVA). Post hoc analysis was performed using the Student-Neuman-Keuls test (SNK). Correlation analysis was also performed. Statistical significance was indicated by a p value of less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunoreactive IL-8 was detected in all explant samples examined. A comparison of IL-8 release from human amnion, choriodecidua, and placenta before and after spontaneous labor onset either delivered by cesarean section or vaginal delivery at term or preterm are presented in Figure 1. Clinical data comparisons for the different labor and gestation groups are represented in Table 1. IL-8 release did not change significantly with labor onset and mode of delivery from amnion, choriodecidual, or placental explants, prepared from either term or preterm tissues.
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IL-8 release from human amnion, choriodecidua, and placenta was significantly different from each tissue type (M-ANOVA, p < 0.00001, Fig. 2). Post hoc analysis using SNK identified significant differences between all the tissues. The tissue differences were noted both at term and preterm.
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Effect of Gestation on IL-8 Release In Vitro
The effect of gestation was assessed by comparing the release of IL-8 from explants prepared from term and preterm tissues. IL-8 release was significantly different at term compared to preterm (M-ANOVA, p < 0.03; Fig. 2). Further analysis demonstrated that choriodecidual release of IL-8 was significantly (M-ANOVA, p < 0.001) increased at term compared to preterm. Correlation analysis of choriodecidual IL-8 release at preterm with the presence or absence of antenatal corticosteroidal treatment for fetal lung maturation did not demonstrate any significant correlations (r = -0.173, n = 17, p = 0.49). In contrast to IL-8 release from choriodecidua, IL-8 release from either amnion (M-ANOVA, p = 0.432) or placental (M-ANOVA, p = 0.185) explants did not differ significantly.
Effect of Chorioamnionitis on IL-8 Release In Vitro
The effect of chorioamnionitis was investigated in preterm tissues. IL-8 release from amnion, choriodecidual, and placental explants did not differ significantly (M-ANOVA, p = 0.200) between noninfected and chorioamnionitis tissues at preterm. A comparison of IL-8 release from preterm human gestational tissues in association with chorioamnionitis is reported in Table 2. After 18 h of culture, conditioned media from chorioamnionitis choriodecidual explants were cloudy, despite the presence of penicillin-streptomycin in the media.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The data obtained establish that the release of IL-8 from gestational tissues obtained at term is greater than that from tissues obtained at preterm; however, an effect of labor onset or chorioamnionitis on IL-8 release could not be confirmed.
IL-8 was released from all tissue explants examined. These data are consistent with previous findings of IL-8 mRNA expression in amnion and chorion cells [19], as well as IL-8 mRNA and protein expression in decidual stromal cells and lymphocytes, and in cytotrophoblast, syncytiotrophoblast, and Hofbauer cells of placenta [18]. Our findings identified the choriodecidua as the most abundant tissue source of IL-8, in comparison to amnion and placenta. This may reflect the different cell types and the cellular source of IL-8 in the tissues.
IL-8 release from human choriodecidual explants was significantly greater in tissues obtained at term compared to those obtained preterm. This novel finding is consistent with previously reported gestation-associated changes in amniotic fluid IL-8 concentrations, which are also increased at term gestation compared to early pregnancy [21]. These data support the proposal that IL-8 may play a maturational role during pregnancy and/or facilitate the process of labor. An effect of exogenous glucocorticoid on IL-8 release in the preterm group, however, cannot be negated. In this study, however, no correlation between exogenous glucocorticoid treatment and IL-8 release could be identified.
Although the precise role of IL-8 in gestational tissues is still to be defined, IL-8 has been reported to induce collagenase and elastase expression and, therefore, to contribute to the process of fetal membrane rupture [10, 11] and cervical dilatation [29]. Regulation of the release of IL-8 from human gestational tissue may be multi-factorial. Recently, stretching of the fetal membranes was demonstrated to significantly increase IL-8 concentration and collagenase activity [30]. Whether stretch of fetal membranes during late gestation affects IL-8 induction and/or release remains to be established. The synthesis of IL-8 may also be affected by steroid hormones. The IL-8 gene contains a glucocorticoid-progestin responsive element, and progesterone inhibition of IL-8 has been demonstrated in choriodecidual [31] and endometrial [17] cell cultures. Progesterone withdrawal has been proposed as a prelude to normal spontaneous onset of labor at term [32]. In the intrauterine environment, the existence of such progesterone withdrawal at term, and hence IL-8 inhibition, may account for the elevated release of IL-8 at term compared to preterm.
Intrauterine IL-8 concentrations have been reported in amniotic fluid and observed to change with labor [20, 21]. Hence in this study, the changes in gestational tissue release of IL-8 in association with labor were determined. IL-8 release from amnion, choriodecidua, and placenta did not change with labor onset and mode of delivery either at term or preterm. These results confirm and extend previous reports, including a study by Shimoya et al. [33] reporting no effect of labor on IL-8 release from placental explants. Similarly, IL-8 release from preterm gestational tissues did not differ significantly with labor and delivery status.
In a subset of preterm tissues, chorioamnionitis was identified clinically and/or histologically. IL-8 release from these tissues was not significantly different from that from gestational tissue explants compared to the other preterm tissues. This is in contrast to a previous report of increased placental IL-8 release in association with chorioamnionitis [33]. However, these investigators could not demonstrate increased IL-8 mRNA expression in chorioamnionitis placentae. The reason for the difference between these results is unclear, but it may relate to differences in incubation time (18 h vs. 24 h, respectively) and method of standardization (ng/mg DNA vs. ng/ml/10 mg dry weight). The responsiveness of IL-8 release to bacterial endotoxin has been previously established [14]. In this study, the failure to identify an effect of chorioamnionitis on IL-8 release may indicate that the induction of IL-8 is an early event in the onset of chorioamnionitis and/or labor that precedes clinically overt symptoms.
In summary, the aims of this study were to investigate the effects of gestation, spontaneous labor onset, and chorioamnionitis on in vitro IL-8 release from human gestational tissue explants. IL-8 was released from human amnion, choriodecidual, and placental explants, but its release was not affected by labor onset and mode of delivery. Choriodecidua IL-8 appeared to be a good candidate for a maturational role in human pregnancy and labor. In particular, the anatomical proximity of the choriodecidua to the cervix, the abundance of IL-8 release from choriodecidua, and the term-associated increase in choriodecidual IL-8 release implicate choriodecidual IL-8 in the regulation of cervical softening, that is, the attraction of the initial cellular infiltrate and the consequential release of enzymes in the cervix, and eventual cervical ripening.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Gregory E. Rice, Perinatal Research Centre, Department of Perinatal Medicine, The Royal Women's Hospital, 132 Grattan Street, Carlton, 3053, Victoria, Australia. FAX: 61 3 9347 2472; gerice{at}ariel.its.unimelb.edu.au
Accepted: April 19, 1999.
Received: June 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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: a paracrine mediator of human term labour? Placenta 1994; 15:A41. during human pregnancy and labour: maternal plasma and amniotic fluid concentrations and release from intrauterine tissues. Eur J Endocrinol 1994; 131:607–614.[Abstract]This article has been cited by other articles:
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  H. Tatemoto, N. Muto, I. Sunagawa, A. Shinjo, and T. Nakada Protection of Porcine Oocytes Against Cell Damage Caused by Oxidative Stress During In Vitro Maturation: Role of Superoxide Dismutase Activity in Porcine Follicular Fluid Biol Reprod, October 1, 2004; 71(4): 1150 - 1157. [Abstract] [Full Text] [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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 K.W. Marvin, J.A. Keelan, R.L. Eykholt, T.A. Sato, and M.D. Mitchell Use of cDNA arrays to generate differential expression profiles for inflammatory genes in human gestational membranes delivered at term and preterm Mol. Hum. Reprod., April 1, 2002; 8(4): 399 - 408. [Abstract] [Full Text] [PDF]
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A. Young, A. J. Thomson, M. Ledingham, F. Jordan, I. A. Greer, and J. E. Norman Immunolocalization of Proinflammatory Cytokines in Myometrium, Cervix, and Fetal Membranes During Human Parturition at Term Biol Reprod, February 1, 2002; 66(2): 445 - 449. [Abstract] [Full Text] [PDF]
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T.S. Nandhra and R.J. Carson {beta}-endorphin inhibits the production of interleukin-8 by human chorio-decidual cells in culture Mol. Hum. Reprod., June 1, 2000; 6(6): 555 - 560. [Abstract] [Full Text] [PDF]
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