Objective
An inflammatory challenge disrupts the cervical epithelial barrier and promotes cervical remodeling.
Study Design
Immortalized ectocervical and endocervical cells were treated with lipopolysaccharide (LPS), and interleukin (IL)-6, IL-8, and soluble E-cadherin (SECAD) were assessed. Cells were then pretreated with dexamethasone prior to LPS exposure, and IL-6, IL-8, and SECAD levels were again assessed. The integrity of the epithelial cell barrier was determined using a permeability assay.
Results
LPS significantly increased IL-6 and IL-8 levels, and SECAD was significantly increased at 24 hours. LPS induced inflammation increased permeability for both cell lines. Dexamethasone pretreatment prior to LPS exposure significantly decreased IL-6 and IL-8 levels in both cell lines. There was no reduction in SECAD levels with dexamethasone pretreatment. Permeability decreased in the presence of dexamethasone for ectocervical cells only.
Conclusion
These studies demonstrate an inflammatory challenge to cervical epithelial cells promotes a cytokine release and functionally alters the cervical epithelial barrier.
Preterm birth remains the leading cause of neonatal morbidity and mortality in the United States. Maternal infections are proposed to account for 25-40% of preterm births. Whereas preterm delivery is believed to be the final common pathway of a multitude of instigating factors, it is clear that infection, particularly with concomitant inflammation, plays a significant role in prematurity. Several proinflammatory cytokines, including interleukin (IL)-6 and IL-8, have been implicated in the onset of spontaneous preterm labor.
Although the precise mechanisms of preterm parturition remain elusive, there are clinical studies suggesting that premature cervical shortening and/or cervical ripening contributes to preterm birth. A short cervix, as detected by transvaginal ultrasound, is a strong predictor of preterm birth, even in the absence of clinical symptoms. Accumulating clinical data suggest that cervical ripening, as indicated by a short cervical length, occurs weeks if not months prior to preterm parturition, suggesting that cervical remodeling is the initial and primary molecular event in preterm birth.
A short cervix is believed to be a physical manifestation of premature cervical remodeling. Cervical remodeling and eventual cervical ripening is a natural process that involves the gradual change of the connective tissue in the cervix throughout pregnancy. The pathways that govern cervical remodeling have been somewhat revealed using animal models and some limited human studies. Based on that body of work, cervical remodeling can be considered as a continuous process that can be divided into 4 unique but overlapping phases: (1) softening, (2) ripening, (3) dilation and labor, and (4) postpartum repair. Maintenance of cervical integrity and appropriate timing of cervical remodeling is critical to maintain pregnancy.
Although this concept of cervical remodeling is believed to occur in normal pregnancy, the precise molecular pathways involved in cervical remodeling, in both term and preterm parturition, are not well understood. There is some evidence that the biological processes governing the epithelial cell barrier may be involved in cervical remodeling for both preterm and term parturition. In other biological systems, such as the gastrointestinal system, there is a greater understanding of the role of the epithelial cell in creating a barrier that supports gastrointestinal health.
This body of literature also reveals specific pathways by which the immune system and the epithelial cell interact and lead to pathophysiological states. Specifically these studies demonstrate that the epithelial barrier is the target for bacterial pathogens and/or their byproducts and that chronic or acute compromise of the epithelial barrier results in disruption of the underlying stroma and can promote inflammation.
Understanding lessons learned in other biological systems and prior work from our laboratory, we hypothesize that interactions between pathogens and cervical epithelial cells results in an inflammatory response and initiates premature cervical remodeling. These studies were performed to assess whether pathogen byproducts can activate an inflammatory cascade in cervical epithelial cells and subsequently lead to compromise of the cervical epithelial cell barrier. We also sought to assess whether preventing the inflammatory response would prevent the disruption of the cervical epithelial cell barrier.
Materials and Methods
Cell culture and preparation
Immortalized HPV 16/E6E7 ectocervical (ATCC CRL-2614) and endocervical (ATCC CRL-2615) cells (American Type Culture Collection, Manassas, VA) were maintained in keratinocyte serum-free medium (GIBCO BRL Life Technologies, Gaithersburg, MD) supplemented with 50 μg/mL bovine pituitary extract (BPE), 0.1 ng/mL epidermal growth factor (EGF), 100 U/mL penicillin, and 100 μg/mL of streptomycin at 37°C in a 5% CO 2 humidified incubator.
Detection of soluble immunobiological mediators
Ectocervical and endocervical cells were plated at a concentration of 9.6 × 10 4 cells/well on 6-well plates for 24 hours in keratinocyte serum-free medium containing BPE, EGF, penicillin, and streptomycin. Cells were then treated with keratinocyte serum-free media containing only penicillin and streptomycin for 24 hours. After 24 hours, cells were then treated with 25 μg/mL of lipopolysaccharide strain Escherichia coli 055:B5 (LPS) (Sigma Chemical Co, St Louis, MO) (n = 6 per treatment group in both ectocervical and endocervical cell lines). LPS is a component of the cell wall of Gram-negative bacteria and is a noted byproduct of these pathogens, which is capable of stimulating an inflammatory response. Media were collected at 6 and 24 hours; the cytokines IL-6 and IL-8 and soluble E-cadherin (SECAD) were assessed at both time points using enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). IL-6 and IL-8 were assessed as markers of inflammation, as it has been shown that cervical epithelial cells can make and generate these cytokines in response to inflammatory stimuli.
In addition to cytokines, we chose to investigate SECAD since E-cadherin plays a critical role in epithelial cell-cell adhesion, morphogenesis, and the maintenance of tissue architecture. E-cadherin is cleaved into SECAD in endothelial cells in the event of injury or inflammation. Consequently, SECAD can be assessed and used as an indicator of the integrity of the epithelial barrier.
To test whether dexamethasone prevented the inflammatory response mediated by LPS, cells were treated with either 100 nM or 1 μM of dexamethasone (Sigma) or ethanol vehicle for 2 hours prior to treatment with LPS (n = 3 per treatment group in both ectocervical and endocervical cell lines). Media were again collected at 6 and 24 hours after LPS exposure; the cytokines IL-6 and IL-8 and SECAD were assessed at both time points.
Analysis of epithelial permeability
The permeability of both ectocervical and endocervical cells was determined by measuring the transepithelial passage of the water-soluble fluorescent probe fluorescein isothiocyanate -dextran from the apical to the basolateral side of the membrane, as has been shown with intestinal epithelial cells. Ectocervical cells were plated at a concentration of 1 × 10 6 cells/well, and endocervical cells were plated at 5 × 10 5 cells/well across a semipermeable membrane (24-well in vitro vascular permeability assay; Millipore, Billerica, MA) for 18 hours in keratinocyte serum-free medium containing BPE, EGF, penicillin, and streptomycin. The cell were then treated with keratinocyte serum-free medium containing only penicillin and streptomycin for 6 hours and then treated with 25 μg/mL, 10 μg/mL, or 2.5 μg/mL of LPS or vehicle for 24 hours. After 24 hours of LPS exposure or control, fluorescein isothiocyanate-dextran in phenol red free media (ScienCell Research Laboratories, Carlsbad, CA) was added to the apical side of the membrane, and after 2 hours media were collected from the basolateral side (n = 3 per treatment group in both ectocervical and endocervical cell lines).
To assess whether dexamethasone mitigated the increase in permeability from LPS, cells were treated with 100 nM of dexamethasone, 1 μM of dexamethasone, or ethanol vehicle for 2 hours prior to treatment with 25 μg/mL of LPS (n = 3 per treatment group for each cell line). The amount of diffused dextran was measured in a fluorometer (excitation wavelength = 485 nM and emission wavelength = 535 nM). The extent of permeability was determined by measuring the fluorescence of the bottom chamber.
Lactate dehydrogenase cytotoxicity assay
The viability of ectocervical and endocervical cells after treatment with 25 μg/mL of LPS was determined by measuring lactate dehydrogenase (LDH) leaking into the medium using the CytoTox 96 nonradioactive cytoxicity assay (Promega Corp, Madison, WI) (n = 3 per treatment group in both cell lines). After collecting cell culture medium, the cells were lysed with radioimmunoprecipitation assay buffer containing protease (Complete protease inhibitor cocktail tablets; Roche Diagnostics, Mannheim, Germany) and phosphatase (Phosphatase Inhibitor Cocktail 2 and 3; Sigma) inhibitors. Released LDH (an indirect measure of loss of cell membrane integrity) was measured with a coupled enzymatic assay that results in the conversion of tetrazolium salt into a red formazan product. The amount of color formed is proportional to the number of lysed cells. The enzymatic reaction was measured spectrophotometrically by absorbance readings at 490 nm. The percentage of cytoxicity was calculated as experimental LDH (cell culture media)/maximum release of LDH (cell lysates).
Statistical analysis
Statistical analyses were performed comparing means or medians depending on whether the data were parametric (1-way analysis of variance [ANOVA]) or nonparametric (ANOVA on ranks). If significance was met, a pair-wise comparison was then performed using a Student-Newman-Keuls (SNK). For results requiring a comparison of multiple groups (ie, LPS and dexamethasone), 2-way, repeated-measures ANOVA was used, followed by Bonferrroni posttests (BPT) for multiple comparisons. Analysis was performed by running GraphPad Prism software (GraphPad, San Diego, CA).
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