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BOR - Papers in Press, published online ahead of print May 28, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.015495
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BIOLOGY OF REPRODUCTION 69, 1091–1098 (2003)
DOI: 10.1095/biolreprod.103.015495
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Effect of Mouse Uterine Stromal Cells on Epithelial Cell Transepithelial Resistance (TER) and TNF{alpha} and TGFß Release in Culture1

Katherine S. Grant2, and Charles R. Wira

Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recognizing that uterine stromal cells regulate several uterine epithelial cell function(s), the current study was undertaken to more fully define cell-cell communication in the uterus and to examine the role of uterine stromal cells in regulating epithelial cell monolayer integrity and cytokine release. Uterine epithelial and stromal cells from adult intact mice were isolated and cultured separately on cell culture inserts and/or in culture plates. Epithelial cells, which reach confluence as indicated by high transepithelial resistance (TER > 1000 ohms/well), preferentially release transforming growth factor-beta (TGFß) into the basolateral chamber ({approx}70% > apical) and tumor necrosis factor-alpha (TNF{alpha}) into the apical compartment ({approx}30% > basolateral). When epithelial cells on cell culture inserts were transferred to plates containing stromal cells, coculture for 24–48 h increased epithelial cell TER ({approx}70% higher than control) and decreased TNF{alpha} release into both the apical and basolateral chambers ({approx}30%–50%). In contrast, TGFß release was not affected by the presence of stromal cells. In other studies, the effects of stromal cells on epithelial cell TER and TNF{alpha} release persisted for 5–7 days following the removal of stromal cells and were also seen in coculture studies in which conditioned stromal media (CSM) was placed in the basolateral chamber. These studies indicate that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNF{alpha} without effecting TGFß release. These results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to maintain uterine integrity and epithelial secretory function.

cytokines, female reproductive tract, immunology, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The endometrium of the uterus is lined by a columnar epithelium that is supported by a stromal cell foundation [1]. Uterine epithelial cells are connected by tight junctions and form a highly organized monolayer responsible for separating the host from potentially harmful bacterial and viral pathogens [2–4]. In addition to serving a crucial barrier role, uterine epithelial cells actively take part in mucosal immune responses, including antigen presentation, the transport of IgA, and the production of a variety of growth factors, antibacterial factors, chemokines, and cytokines [5–12].

The relationship between epithelial cells and stromal cells and their precursor mesenchymal cells is tissue specific and established during a critical period of development. In the female reproductive tract, stromal cells affect epithelial development and function throughout the course of organ development and homeostasis. Stromal cells have been shown to direct the phenotypic expression of epithelium in the uterus and vagina [13–16]. For example, stroma from the neonatal mouse uterus can induce the differentiation of vaginal squamous epithelium into uterine columnar epithelium, whereas vaginal stroma can induce uterine columnar epithelium to become stratified squamous [15–17]. Previous studies from our laboratory have shown that adult rat uterine stromal cells regulate epithelial cell expression of the polymeric immunoglobulin receptor (pIgR), the receptor responsible for transporting polymeric IgA and IgM from the tissue to the lumen [18]. In response to the presence of stromal cells, uterine epithelial expression of pIgR was markedly reduced within 24–48 h of coculture. The way in which epithelial-stromal cell interactions are transmitted is not well understood but is thought to include direct cell-cell contact, signals via the extracellular matrix (ECM), and soluble signals, such as growth factors and cytokines [9].

Rather than acting directly on epithelial cells, some effects of estradiol are mediated through uterine stromal cells. Cunha and Young [19] demonstrated that despite the absence of estradiol receptors in normal neonatal mouse uterine epithelial cells, in vivo estradiol (E2) treatment stimulated uterine epithelial cell proliferation. These studies led to the hypothesis that estradiol might stimulate uterine epithelial mitogenesis indirectly, possibly through the estrogen receptor-alpha (ER{alpha}) positive stroma [19]. More recently, Cooke et al. [20] confirmed this hypothesis using ER-knockout mice. In recombination studies using epithelial and stromal cells from wild-type and ER{alpha} knockout mice, uterine epithelial mitogenesis was shown to be an indirect effect of estradiol that was mediated through ER{alpha} positive stromal cells [20]. In contrast, Buchanan et al. [21] demonstrated that both epithelial and stromal ER{alpha} were essential in the estradiol regulation of uterine epithelial cell lactoferrin synthesis.

Cytokines and their receptors are critical in regulating the reproductive and immunological environment of the uterus. Cytokines expressed by cells in the mouse uterus include GM-CSF, IL-6, IL-1{alpha}, IL-1ß, TNF{alpha}, and TGFß [7, 22–24]. These polypeptides most commonly act in an autocrine or paracrine fashion and are necessary for intercellular signaling to control local immune events. Two cytokines with potential to be central regulators of reproductive and immune events in the uterus are TGFß and TNF{alpha}. The TGFß family is ubiquitously expressed and involved in numerous processes, including embryogenesis, wound healing, and modifying the function of antigen presenting cells [25, 26]. Three mammalian isoforms TGFß, TGFß 1, 2, and 3, are present in the mouse reproductive tract and under estradiol control [23]. Takahashi et al. [23] demonstrated that estradiol stimulates mRNA and protein expression of all three isoforms by epithelial cells. TNF{alpha} is produced by a variety of cell types and acts as an inflammatory mediator and is a regulator of both physiologic and pathophysiologic processes [27, 28]. In the reproductive tract, TNF{alpha} mRNA and protein are produced by both epithelial and stromal cells [27, 29–31]. Moreover, as with TGFß, TNF{alpha} message and protein in mouse uterine tissues fluctuate with the estrous cycle and are dependent on estradiol and progesterone [27, 32].

With the recognition that underlying stromal cells have the potential to regulate epithelial cell function, the present study was undertaken to more fully define epithelial-stromal cell communication in the uterus and the role these interactions have in regulating the mucosal immune system of the female reproductive tract. The objectives of this work were to 1) determine if mouse uterine epithelial cells grown on cell inserts maintain high transepithelial resistance (TER) and release TGFß and TNF{alpha}, 2) define whether stromal cells affect uterine epithelial monolayer integrity and secretory function, and 3) determine whether cell-cell contact is essential for the communication between epithelial and stromal cells.


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

Sexually mature, Balb/c, female mice were obtained from the National Cancer Institute colony at Charles River Laboratories (Kingston, NY). Animals were housed in a constant-temperature room with 12L:12D intervals and allowed food and water ad libitum. For each experiment, animals were killed by CO2, and uteri were pooled from 8–12 animals at all stages of the estrous cycle. All procedures involving animals were conducted after approval of the Dartmouth College Institutional Animal Care and Use Committee.

Epithelial and Stromal Cell Preparation

To prepare epithelial cells, uteri were removed, cut open lengthwise, pooled, and incubated with 0.25% trypsin (Sigma, St. Louis, MO)/2.5% pancreatin (Gibco-BRL/Invitrogen, Grand Island, NY) for 60 min at 4°C and 60 min at 22°C. Following transfer to ice-cold (3°C) Hanks Balanced Salt Solution (HBSS; Gibco-BRL/Invitrogen), digested uteri were vortexed to release sheets of epithelial cells. Uterine tissues were rinsed and vortexed an additional three times and resulting cell suspensions pooled. Epithelial sheets were recovered by passing the cell suspension through a 20-micron nylon mesh (Small Parts Inc., Miami Lakes, FL), collected, and centrifuged (500 x g). Epithelial sheets were resuspended in complete medium consisting of Dulbecco Modified Eagle Medium (DMEM) (without phenol red)/Hams F-12 nutrient mixed 1:1 (Gibco/Invitrogen, Grand Island, NY) +10% fetal bovine serum (FBS; Hyclone, Logan, UT) supplemented with 20 mM Hepes, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine (all from Gibco/Invitrogen). Cell sheets were seeded in the apical compartment of 0.4-micron-pore-size/6.4-cm-diameter Falcon cell culture inserts (Fisher Scientific, Pittsburgh, PA) coated with Matrigel (without phenol red; Collaborative Biomedical Products, Bedford, MA). Cells were seeded in a volume of 300 µl at a ratio of approximately three to four cell inserts per uterus and incubated at 37°C with 5% CO2. Inserts were placed in 24-well tissue culture plates (Fisher Scientific) containing 850 µl of medium in the basolateral compartment and incubated at 37°C with 5% CO2. Throughout each experiment, medium was collected from the basolateral and apical chambers and replaced at 48-h intervals, centrifuged (10 000 x g), and stored at -80°C until assayed. A minimum of four to six inserts per treatment group was used in each experiment.

To isolate stromal cells, pooled uteri, following the removal of epithelial cells, were incubated for 30 min at 37°C in 0.05% trypsin+0.02% EDTA+400 U/ml DNase (Gibco-BRL/Invitrogen; Worthington, Lakewood, NJ). Tissues were dispersed by gentle rubbing in medium on a 40-micron nylon mesh (Small Parts Inc, Miami Lakes, FL); the resulting cell suspension was centrifuged (500 x g) for 10 min. Stromal cells were resuspended in (850 µl) complete DMEM/Hams F12+10% FBS and plated at 5 x 105/850 µl per well in 24-well plates. Medium was collected from each well, replaced at 48-h intervals, centrifuged (10 000 x g), and stored at -80°C until assayed. To prepare conditioned stromal medium (CSM), stromal cells were grown in complete medium and replaced at 48-h intervals. CSM used in these experiments was the medium collected from cells between Days 2 and 4 of culture. Medium was centrifuged (10 000 x g), stored at -80°C, and used in cocultures diluted 1:1 with fresh DMEM/Hams F12+10% FBS. Purity of the stromal cell preparation was established by immunohistochemistry. Following 4 days in culture, with media changes at 48-h intervals to remove nonadherent cells, stromal cells were stained for CD45 (Pharmingen, San Diego, CA). Whereas fresh stromal preparations contain 5%–20% leukocytes, stromal cultures at 4 days were devoid of CD45 positive cells. Based on these findings, we conclude that the stromal cells in culture were 99% fibroblast at the time of epithelial coculture.

Coculture of Epithelial and Stromal Cells

In experiments involving the coculture of epithelial and stromal cells, epithelial and stromal cells were grown separately to confluence on cell inserts and/or in 24-well plates, as described previously. Once epithelial cells achieved high TER (>800–1000 ohms/well), inserts of polarized epithelial cells were transferred to 24-well plates containing stromal cells. Epithelial and stromal cells were not in direct contact in any coculture experiments. Cell inserts were removed from plates containing stromal cells and transferred to plates containing fresh media to determine the long-term effect of stromal cell exposure on both TER and cytokine release at both the apical and basolateral surfaces. Throughout each experiment, medium was collected from the apical and basolateral compartments and replaced at 48-h intervals, centrifuged (10 000 x g), and stored at -80°C until assayed.

Transepithelial Resistance Measurements

Transepithelial resistance (TER) was monitored daily as an indication of tight junction formation in the epithelial monolayer using an EVOM epithelial voltohmmeter and electrode (World Precision Instruments Inc., New Haven, CT).

Cytokine Analysis

Aliquots (50 µl) of supernatants collected from epithelial cells growing alone or in coculture with stromal cells or CSM were assayed by TGFß bioassay as previously described [33]. Briefly, active (mature) TGFß was measured using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. These cells were generously provided by Dr. James Gorham (Dartmouth Medical School, Lebenon, NH). This quantitative bioassay is based on the ability of TGFß to induce plasminogen activator inhibitor-1, resulting in a dose-dependent increase in luciferase activity. Luciferase activity induction in mink lung epithelial cells is specific and sensitive to picogram quantities of TGFß [34]. Cells were thawed, washed, and plated at 1 x 105/100 µl in opaque, flat-bottom 96-well plates (USA Scientific, Inc., Ocala, FL), centrifuged (800 x g) for 15 sec, and allowed to adhere for 3 h at 37°C. Following incubation, cells were centrifuged, and the medium was replaced with 50 µl fresh medium and 50 µl of serially diluted standards (recombinant human TGFß) or cell supernatants containing TGFß. Cells were incubated overnight (17–20 h), washed two times in HBSS (100µl), and lysed with 50 µl lysis reagent (Promega Corp., Madison, WI) for 15 min at room temperature. Luciferase activity of lysates was measured by adding luciferase reagent (100 µl; Promega Corp.) to each well and recording illumination for 10 sec following a 2-sec delay in a Microplate Luminometer model LB96V (EG&G Berthold, Gaithersburg, MD). Supernatants (100 µl) collected from epithelial cells growing alone or in coculture with stromal cells or CSM were also assayed by TNF{alpha} ELISA (R&D Systems, Minneapolis, MN). ELISAs were performed according to the commercial kit protocol.

Statistics

The data were calculated as the mean ± standard error of the mean. GraphPad Software (San Diego, CA) was used to perform a one-way repeated-measures analysis of variance (ANOVA), and SYSTAT 9 (SPSS Science, Chicago, IL) was used to perform a two-way ANOVA or a two-way repeated-measures ANOVA. When an ANOVA indicated that significant differences existed among means, preplanned paired comparisons were made using the Bonferonni method to adjust P-values. A P-value of less than 0.05 was taken as indicative of statistical significance. The details of each particular analysis are described at the appropriate point in the next section.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Epithelial Cell Cytokine Release

To study the release of cytokines by polarized epithelial cells, isolated mouse uterine epithelial cells were grown to confluence on cell inserts (four to six inserts/treatment group) prior to the analysis of media in the apical and basolateral chambers. Within 3–4 days of culture, cells grow to confluence and form tight junctions as indicated by transepithelial resistance measurements (TER) that approach 1000 ohms/well (background resistance 160–180 ohms/well) (Fig. 1A). One-way ANOVA indicated that significant differences existed among means (P < 0.0001). Specific pairwise comparisons were made between TER measurements on the different days of incubation (P < 0.001). Moreover, there were linear increases in TER over the course of the experiment.



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  		FIG. 1. Preferential release of TNF{alpha} and TGFß by mouse uterine epithelial cells in the presence of high TER. Isolated uterine epithelial cells from 8–12 intact animals were cultured for 10 days on cell inserts (four to six inserts/treatment group) and TER measurements were taken daily (A), as described in Materials and Methods. Culture media in apical (300 µl) and basolateral (850 µl) compartments was replaced at 48-h intervals prior to collection on Days 4, 6, 8, and 10. Following centrifugation at 10 000 x g for 5 min and storage at -80°C, samples were assayed for TNF{alpha} by ELISA (B) and TGFß by bioassay (C), as described in Materials and Methods. The results are shown as the mean ± SEM. *, Significant increases in TER over time (*P < 0.001). **, Apical TNF{alpha} release significantly (*P < 0.05) greater than Basolateral release. *, Basolateral TGFß release significantly (*P < 0.05) higher than apical release (representative of five experiments: n = 5)

The production and distribution of TNF{alpha} and TGFß in media collected from the apical and basolateral chambers of cell culture inserts at 48-h intervals between Days 4 and 10 of culture are shown in Figure 1, B and C. The two-way ANOVA, in which time was a repeated measure and chamber (apical/basolateral) was a between-subject factor, indicates that TNF{alpha} concentrations varied over time. However, regardless of the time interval analyzed, polarized epithelial cells released significantly more TNF{alpha} into the apical chamber than into the basolateral chamber (P < 0.003). In contrast, under identical culture conditions, TGFß concentrations were stable, and epithelial cells preferentially released more TGFß into the basolateral chamber compared to the apical compartment (P < 0.0002).

Effect of Stromal Cells on Epithelial Cell Function

To examine the effect of stromal cells on TER, epithelial cells were grown alone or in the presence of stromal cells and resistance measurements taken daily (six to eight inserts/treatment group) (Fig. 2). As determined by TER, cells reached confluence by Day 4 (TER > 800–1000 ohms/well), after which TER gradually increased from Days 4–13 to levels of 4000–5000 ohms/well. On Day 4, inserts containing epithelial cells were transferred to plates containing stromal cells. The two-way ANOVA, in which time was a repeated measure and each treatment group was a between-subject factor, indicated that there was a significant time effect with and without stromal cells (P < 0.0001), as TER increased with time in both conditions. TER was not different in the two groups on control Days 3 and 4. However, after epithelial inserts were transferred to plates containing stromal cells, the TER, with rare exceptions, was significantly greater (P < 0.05) in the cells cocultured with stromal cells compared to control epithelial cells cultured alone. As a part of these studies, it was found that removal of the stromal cells on Day 8 did not reverse the increase in TER seen when epithelial cells were cocultured with stromal cells. This increase in epithelial cell TER following coculture with stromal cells persisted for the duration of the experiment.



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  		FIG. 2. Effect of uterine stromal cells on transepithelial resistance (TER). Uterine epithelial cells from 8–12 intact mice were grown to confluence on cell inserts (six to eight inserts/treatment group). Epithelial cell formation of tight junctions was determined by measuring TER. Following TER readings on Day 4, when cells had reached high TER (>800–1000 ohms/well), inserts were transferred to plates containing stromal cells. Stromal cells were removed on Day 8. TER of epithelial cells was monitored daily for 13 days. The results are shown as the mean ± SEM. **, TER significantly (*P < 0.05) higher than epithelial cells growing alone (n = 5).

To determine the effect of stromal cells on epithelial TNF{alpha} and TGFß release, studies involving the coculture of epithelial and stromal cells were carried out (Fig. 3). Apical supernatants were collected on Days 4, 6, 8, and 10 from epithelial cells grown alone and those grown in the presence of stromal cells and analyzed for TNF{alpha} release (four to six inserts/treatment group). The two-way ANOVA, in which time was a repeated measure and treatment group was a between-subject factor, indicated that the TNF{alpha} levels were not different in the two groups at baseline (Day 4). While TNF{alpha} release did vary over time, apical supernatants collected on Days 6 and 8 from epithelial cells cocultured with stromal cells contained significantly less TNF{alpha} (P < 0.001) compared to epithelial cells grown alone (Fig. 3A). To determine whether stromal cell effects on TNF{alpha} release would persist, previously cocultured cell culture inserts were transferred to fresh medium. As shown in Figure 3A, at 48 h post-coculture (Day 10), TNF{alpha} levels of those cells that had been grown in the presence of stromal cells were significantly reduced (P < 0.001) compared to epithelial cells grown alone. In contrast to TNF{alpha}, when apical media were analyzed for TGFß, the presence of stromal cells, as well as their removal, had no effect on TGFß release, as shown in Figure 3B. Concentrations of TGFß measured in apical supernatants of epithelial cells cocultured with stromal cells were not different from those seen with epithelial cells alone.



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  		FIG. 3. Effect of uterine stromal cell coculture on apical TNF{alpha} and TGFß release by epithelial cells. Epithelial and stromal cells were isolated from 8–12 intact mice and grown to confluence separately. Culture medium was replaced at 48-h intervals. Supernatants from the apical compartments (four to six inserts/treatment group) were collected on Day 4 prior to transfer of inserts to stromal cells. Stromal cells were removed following collection on Day 8 (A) or Day 6 (B), and apical supernatants were collected 48 h following that removal. Samples were assayed for TNF{alpha} ELISA (A) and TGFß by bioassay (B). The results are shown as the mean ± SEM. *, Apical TNF{alpha} release by epithelial cells grown in the presence of stromal cells is significantly (**P < 0.001) less than those grown alone (n = 4)

Effect of Conditioned Stromal Media on Epithelial Cell Function

To determine whether the effects of stromal cells are mediated through the release of soluble factors, conditioned stromal media (CSM) was prepared, as described previously. Figure 4 shows the TER of epithelial cells cocultured with stromal cells and epithelial cells grown in the presence of CSM in the basolateral chamber (four to six inserts/treatment group). The two-way ANOVA, in which time was a repeated measure and treatment group was a between-subjects factor, indicates that there is a significant time effect, with and without treatment, on TER (P < 0.0001). The TER measurements were not different among the three groups at baseline (Day 6). When stromal cells were cocultured with epithelial cells, TER increased significantly (P < 0.001) within 24 h relative to that seen with epithelial cells grown alone. While the addition of CSM did increase TER compared to control epithelial cells, this difference was not significant (P = 0.056) at 24 h of coculture. The stimulatory effect of stromal cell coculture on TER persisted following the removal of stromal cells. The TER of cells previously cocultured with CSM was significantly higher (P < 0.001) than epithelial cells grown alone by 24-h post-coculture; this increase was not significantly different than the increase seen with stromal cell coculture.



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  		FIG. 4. Effect of epithelial cell coculture with stromal cells and conditioned stromal media on transepithelial resistance (TER). Epithelial cells from 8–12 intact animals were grown on cell inserts (four to six inserts/treatment group) to confluence and transferred to plates containing stromal cells or conditioned stromal media (850 µl) in the basolateral compartment, as described in Materials and Methods. TER measurements of epithelial cells were taken at baseline, 24 h of coculture, and 24 h post-coculture. The results are shown as the mean ± SEM. *, TER significantly (**P < 0.001) higher than epithelial cells growing alone (n = 3); {dagger}, P = 0.056

The effect of stromal cells and CSM on apical TNF{alpha} and TGFß release by uterine epithelial cells is shown in Figure 5 (four to six inserts/treatment group). The two-way ANOVA, in which time was a repeated measure and treatment group was a between-subjects factor, indicated that epithelial cells grown in the presence of CSM for 48 h released significantly less (P < 0.05) apical TNF{alpha} compared to control cells. This effect is comparable to the significant decrease in TNF{alpha} release when epithelial cells were cocultured with stromal cells (P < 0.05) (Fig. 5A). In contrast, neither stromal cells nor CSM had any effect on apical TGFß release following 48 h of coculture (Fig. 5B).



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  		FIG. 5. Effect of uterine stromal cells and stromal conditioned stromal media on apical release of TNF{alpha} and TGFß by epithelial cells. Epithelial cells from 8–12 intact mice were grown and cocultured with stromal cells or conditioned stromal media in the basolateral compartment on Day 4 (four to six inserts/group). Apical supernatants were collected at baseline (Day 4) and following 48 h of coculture on Day 6 and analyzed for TNF{alpha} (A) and TGFß (B). The results are shown as the mean ± SEM. **, Apical release of TNF{alpha} by epithelial cells grown in the presence of stromal cells and CSM is significantly (**P < 0.05) less than those grown alone (n = 3–4).

Effect of Stromal Cell Coculture on Epithelial Cell Function: Apical and Basolateral Release

Recognizing that the effect of stromal cells on the apical release of TNF{alpha} persists following stromal cell removal (Fig. 3), a study was undertaken to determine whether stromal cells regulate the release of TNF{alpha} and TGFß by epithelial cells at the basolateral surface (four to six inserts/treatment group). A two-way ANOVA, comparing apical and basolateral release by epithelial cells grown alone and those previously grown in the presence of stromal cells, indicated that prior stromal cell exposure resulted in a significant decrease in both apical and basolateral TNF{alpha} release (P < 0.0001). This indicates that stromal cell coculture affects release of TNF{alpha} at both the apical and the basolateral surface in the same way. Thus, total TNF{alpha} release (combined total of apical and basolateral release) by uterine epithelial cells cocultured with stromal cells was decreased following exposure to stromal cells, compared to epithelial cells grown alone. The pattern of preferential release of TNF{alpha} into the apical compartment (apical >= basolateral) was not affected by the presence of stromal cells. In contrast to stromal cell effects on TNF{alpha} release, stromal cells had no effect on either apical or basolateral TGFß release by epithelial cells (Fig. 6B).



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  		FIG. 6. Effect of coculture with stromal cells on apical and basolateral release of TNF{alpha} and TGFß. Epithelial and stromal cells from 8–12 intact mice were grown to confluence prior to transfer to 24-well plates containing stromal cells (four to six inserts/group). Following 96 h of coculture, inserts were transferred to wells without stromal cells and with fresh medium. Apical and basolateral supernatants were collected at 48 h post-stromal cell exposure and analyzed for TNF{alpha} (A) and TGFß (B). Mean ± SEM. *, Apical and basolateral TNF{alpha} release by epithelial cells grown in the presence of stromal cells and is significantly (**P < 0.001) less than TNF{alpha} release by epithelial cells grown alone (n = 4)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented demonstrate that primary mouse uterine epithelial cells grown in culture form polarized monolayers that maintain high transepithelial resistance (TER) as well as release TNF{alpha} and TGFß. These findings indicate that uterine epithelial cells preferentially release TNF{alpha} to the apical media. In contrast, TGFß is preferentially released from the basolateral surface relative to that measured in the apical compartment. Coculture of epithelial cells with stromal cells increases epithelial cell integrity, as measured by TER. In contrast, coculture with stromal cells significantly decreased apical and basolateral release of TNF{alpha} and had no effect on either apical or basolateral release of TGFß. Further, when added to the basolateral compartment, conditioned medium from stromal cells affected TNF{alpha} and TER, but not TGFß, in a similar manner to coculture with stromal cells. These studies indicate that uterine stromal cells act via a soluble factor(s) to regulate uterine epithelial cell integrity and secretory function.

It has been previously shown that polarized cultures of epithelial cells preferentially release secretory products. Others have demonstrated that polarized epithelial cells grown in culture preferentially release interleukin-1 alpha, interleukin-6, as well as pIgR to the apical compartment, while prostaglandins have been shown to be released preferentially at the basolateral surface [18, 35–38]. Previous studies from our laboratory have shown that rat epithelial cell preferential apical secretion of pIgR, the receptor essential for IgA transport, is inhibited when cells are cocultured with uterine stromal cells [18]. Stromal cell inhibition of apical secretion paralleled a decrease in the intracellular concentration of pIgR, as determined by confocal microscopy, and occurred under conditions in which cell integrity and nutritional requirements were not compromised. To more fully define the interactions between epithelial and stromal cells of the uterus, this approach was adapted to a mouse model. The work presented here extends these findings by demonstrating the effect of stromal cells on the regulation of two biologically important cytokines, TNF{alpha} and TGFß, as well as on the integrity and barrier function of uterine epithelial cells.

The finding that polarized uterine epithelial cells release TNF{alpha} both apically and basolaterally raises an important question about the physiologic role of this cytokine in the uterus. TNF{alpha} is part of a critically important cytokine communication network necessary for coordinating proper uterine function [37, 39]. Basolateral release of this cytokine may activate underlying stromal cells and facilitate leukocyte recruitment, including antigen presenting cells [27]. The finding that TNF{alpha} is preferentially released at the apical surface extends the immunohistochemical findings of Roby and Hunt [27], who reported TNF{alpha} is localized to the apical surface of the uterine epithelial cells. One possible consequence of apical release is that TNF{alpha} functions in an autocrine and/or paracrine manner on neighboring epithelial cells to support blastocyst attachment and implantation as well as regulate tissue degradation, reorganization, and repair during the estrous cycle [27, 40]. Alternatively, since TNF{alpha} is known to induce the synthesis of a number of chemoattractant cytokines, including interferon-alpha inducible protein-10, macrophage chemotactic factor-1, and the chemokine KC, TNF{alpha} released into the uterine lumen may, along with uterine secretions, flow into the cervix and vagina. The release of this cytokine may affect, either directly or indirectly, the chemotaxis of leukocytes in the lower reproductive tract [41, 42]. Consistent with the observation that uterine epithelial cells in culture secrete TNF{alpha} apically is our recent finding that uterine fluid collected from intact mice at proestrous contains significant amounts of TNF{alpha} (unpublished results).

The preferential release of biologically active TGFß at the basolateral surface of epithelial cells raises the possibility that through this cytokine, epithelial cells regulate underlying stromal cell function. It has been demonstrated that intrauterine injection of TGFß induces GM-CSF release and the initiation of an influx of leukocytes [43]. Others have shown that TGFß treatment of mouse uterine stromal cells increases PGE2 and PGE2{alpha} (PGs) secretion, molecules important for successful implantation and initiation of the stromal decidual reaction [36]. The demonstration that removal of the uterine epithelium interferes with decidualization implies that epithelial cells are responsible for transmitting decidual signals to the stroma [44]. Basolateral release of TGFß may be necessary for transmitting such a signal. Takahashi [23] has reported that epithelial cells of the mouse female reproductive tract produce TGFß, possibly to regulate epithelial homeostasis. Others have shown that it plays a key role in angiogenesis and cyclic tissue growth in the uterus [24]. More recently, we have found that TGFß produced by uterine epithelial cells is a key regulator of antigen presentation by APC in the underlying uterine stroma [45]. Estradiol increases epithelial cell TGFß release that in turn inhibits antigen presentation by APC in the stroma. This effect is specific for TGFß and parallels the finding that antigen presentation by vaginal cells following in vivo treatment with estradiol is mediated through the action of TGFß, most likely produced by squamous epithelial cells [33].

Epithelial cell integrity and maintenance of tight junctions are essential for protection against potential bacterial and viral pathogens [46]. The data presented here indicate that underlying stromal cells in the uterine endometrium modulate barrier function of polarized epithelial cells. To the best of our knowledge, this study is the first demonstration that uterine stromal cells, acting through a soluble factor(s), enhance epithelial barrier function, as measured by TER. Our laboratory has previously shown that when human uterine epithelial cells are cocultured with stromal cells, TER is reduced. This effect was found to be reversible in that when human uterine epithelial cells that had been cocultured with stromal cells are transferred to fresh medium, TER returned to control values [6]. In contrast, coculture of intestinal lamina propria cells with polarized epithelial cells has no effect on TER [47]. These studies further reported that when bacterial LPS was added to the culture medium, epithelial cell TER was reduced in the presence of stromal cells. The data presented in this paper demonstrating increased TER in the presence of stromal cells indicate that cell-cell interactions are species and tissue specific. Our findings of increased epithelial TER in the presence of stromal cells most likely involves the regulation of tight junction proteins, including ZO-1, occludin, and several claudin family members [48]. Endogenous cytokines and growth factors have been shown to regulate tight junction function, but the mechanism by which such agents affect TER remains to be determined [49]. What is clear is that by enhancing TER, stromal cells are clearly playing an active role in the maintenance of cell integrity and barrier protection at mucosal surfaces [50, 51].

To the best of our knowledge, this study is the first demonstration that TNF{alpha} release by uterine epithelial cells is regulated by stromal cells. It was found that stromal cell coculture significantly decreased both apical and basolateral TNF{alpha} release. While TNF{alpha} in the reproductive tract appears to be necessary for normal physiologic function, dysregulation and overproduction of this cytokine has been attributed to increased risk of infection, implantation failure, spontaneous abortion, and endometriosis [52]. Underlying uterine stromal cells may provide a necessary regulatory signal to epithelial cells to decrease TNF{alpha} release to prevent such abnormalities.

The results presented demonstrate that cell-cell contact is not required for epithelial cells to respond to underlying stromal cells. Using cell culture inserts for separation and conditioned media from stromal cells in culture, these results indicate that a soluble factor(s) is released by stromal cells that acts at the basolateral surface of the epithelial cells to increase epithelial cell TER and decrease TNF{alpha} release. Jacobs et al. [37] demonstrated that coculture with uterine stromal cell conditioned media significantly decreased interleukin-6 release by polarized mouse uterine epithelial cells; however, unlike the findings presented here, the authors paradoxically found that coculture with uterine stromal cells had no effect on interleukin-6 release. Stromal cells are known to produce numerous growth factors and cytokines that affect epithelial function. For example, epidermal growth factor, insulin-like growth factor-1, and hepatocyte growth factor are all produced in the uterine stroma and have been found to stimulate epithelial mitogenesis and development [16, 20, 53, 54]. More recently, Zhang et al. [55] demonstrated that hepatocyte growth factor, produced by stromal fibroblasts, is responsible for mediating estrogen-induced epithelial proliferation in the mammary gland. Stromal cell regulation of TER and TNF{alpha} without affecting TGFß suggests that multiple stromal cell factors may be acting in concert to elicit the observed effects. Studies are currently under way to identify the factor(s) responsible.

In conclusion, these studies indicate that uterine stromal cells exert a differential effect on epithelial cells and play a precise role in regulating both uterine integrity and epithelial function. These results provide evidence that cell-cell contact is not required and that these effects are mediated by a soluble factor(s) that influences epithelial cell function and secretory activity. A better understanding of epithelial-stromal interactions in the uterus is necessary to obtain a clearer understanding of the mucosal immune system in the female reproductive tract.


   ACKNOWLEDGMENTS
 
The authors gratefully thank Dr. James Gorham, Department of Pathology, Dartmouth Hitchcock Medical Center, and Mr. Richard Rossoll for their assistance in setting up the TGFß assay used in these studies. We would also like to thank Dr. James C. Leiter for his assistance with the statistical analysis of our data.


   FOOTNOTES
 
1 This work was supported by National Institutes of Health research grant AI-13541. Back

2 Correspondence: Katherine S. Grant, Department of Physiology, Dartmouth Medical School, Borwell Building, 1 Medical Center Drive, Lebanon, NH 03756-0001. FAX: 603 650 6130; katherine.grant{at}dartmouth.edu Back

Received: 16 January 2003.

First decision: 14 February 2003.

Accepted: 13 May 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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