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BOR - Papers in Press, published online ahead of print December 1, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.035618
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BIOLOGY OF REPRODUCTION 72, 814–821 (2005)
DOI: 10.1095/biolreprod.104.035618
© 2005 by the Society for the Study of Reproduction, Inc.

Hepatocyte Growth Factor Regulation of Uterine Epithelial Cell Transepithelial Resistance and Tumor Necrosis Factor {alpha} Release in Culture1

Katherine S. Grant-Tschudy, and Charles R. Wira2,

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Underlying stromal cells are essential for the normal development of epithelial cells (ECs) at mucosal surfaces. Recent studies from our laboratory have shown that uterine stromal cells regulate EC integrity, measured as transepithelial resistance (TER) as well as tumor necrosis factor (TNF) {alpha} {alpha} secretion by ECs in culture. Using stromal cells in coculture with polarized ECs grown on inserts, we found that stromal cells produce soluble mediators that increase TER and decrease TNF{alpha} secretion. The purpose of the present study was to identify the mechanisms whereby stromal cells exert their effects on uterine epithelium. We report that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates EC proliferation, increases TER but, at the same time, decreases apical TNF{alpha} release. When ECs and/or stromal cells were incubated with anti-HGF or anti-HGF receptor (HGFR) antibody before HGF, the effects of HGF were blocked. These findings indicate that ECs express the HGFR at their basolateral surfaces and that HGFR mediates the effects of HGF on TER and TNF{alpha}. Neutralization of stromal cell secretions with antibodies for HGF and HGFR demonstrate that stromal-derived HGF is the mediator of EC TER. In contrast, neither anti-HGF antibody nor HGFR antibody had any effect on stromal cell-induced decreases in TNF{alpha} secretion. From these results, we conclude that stromal cell regulation of EC TER is mediated through the secretion of stromal HGF. Furthermore, because neutralization of stromal media failed to affect TNF{alpha} secretion, these findings suggest that other growth factors, in addition to HGF, affect EC cytokine production.

estradiol, female reproductive tract, growth factors, immunology, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epithelial cells form a highly ordered monolayer that serves as a primary barrier, which separates the body proper from the external environment, to protect against potential bacterial and viral pathogens [1]. This barrier is critical to reproductive health, because movement of antigens beyond the lumen of the uterus into the submucosa can lead to systemic infection and a disruptive inflammatory cascade [2]. Recognized as the first line of defense, epithelial cells act as sentinels of the innate immune system in that they express Toll-like receptors that recognize conserved pathogen-associated molecular patterns synthesized by microorganisms, bacteria, and viruses [3–5]. Toll-receptor binding, in turn, leads to the rapid secretion of chemokines and cytokines, such as tumor necrosis factor (TNF) {alpha}, that signal the recruitment and activation of immune cells to the underlying mucosa [6]. As a part of this initial response, epithelial cells secrete a family of bactericidal and virucidal agents, including secretory leukocyte protease inhibitor (SLPI), defensins, and so on, that provide initial protection [7–10]. Beyond innate immunity, epithelial cells stimulate adaptive immune responses that are gradual and lead to memory responses [11]. Acting through class II molecules, epithelial cells induce humoral immune protection, which can lead to protection by antibody neutralization of antigens and antibody-dependent cell cytotoxicity [12].

Epithelial cell interactions with underlying stromal cells are essential for normal development and reproductive function of the female reproductive tract [13]. During embryonic development, the mullerian duct epithelium in the mouse gives rise to the epithelial lining of the oviducts, uterus, cervix, and upper vagina [14]. Under the influence of stromal cells, epithelial cells differentiate in ways unique to each region of the tract [14, 15]. Stromal cell influences on the epithelium begin during embryonic differentiation and continue through uterine development into adulthood [16]. In addition to providing a physical support, stromal cells affect epithelial cell proliferative and secretory activities through the release of paracrine factors, cell-extracellular matrix contact, and direct cell-cell contact [13, 17, 18].

Within the uterus, epithelial and stromal cells produce factors that regulate the growth, differentiation, and function of each other [16, 19, 20]. Molecules such as insulin-like growth factor (IGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and keratinocyte growth factor (KGF) are produced by the uterine stroma and act via epithelial receptors at distances of 50–350 nm to affect changes in epithelial cells [13, 17, 21, 22]. Stromal signals modulate proliferation and secretory activities of epithelium as well as mediate the effects of estradiol on epithelial cells in the uterus [18, 23]. Recent studies by Zhang et al. [24] identified HGF as the stromal mediator of estrogen-induced epithelial proliferation in the mouse mammary gland. This conclusion was based on the findings that proliferative activity in estradiol-17ß-stimulated mammary fibroblast-conditioned medium was inhibited completely by neutralizing antibody to HGF.

The HGF is a pleiotropic, mesenchymal-derived growth factor that has epithelial cell-specific mitogenic and morphogenic properties [25–29]. Expressed by uterine stromal cells of the human, mouse, cow, and sheep uterus [28, 30– 32], HGF effects are mediated by its high-affinity receptor (HGFR) or c-met, a transmembrane type I tyrosine kinase receptor expressed in epithelial tissues [27]. Low-affinity, high-capacity binding sites also have been reported in primary epithelial cells and epithelial cells lines, some of which were unresponsive to HGF [33]. The synthesis of HGF by stromal cells and its reported effects on epithelia suggest a paracrine mode of action and role for HGF as a mediator of epithelial-stromal interactions [34].

In previous studies, we found that mouse uterine epithelial cells grown in coculture with stromal cells secrete less TNF{alpha} than epithelial cells grown alone [35]. As a part of these studies, stromal cells were shown to increase uterine epithelial cell barrier function measured as transepithelial resistance (TER) [35]. Moreover, when conditioned stromal medium (CSM), prepared by incubating stromal cell fibroblasts in media, was substituted for stromal cells, it had the same effect on polarized epithelial cells, as did stromal cells. These findings led to the conclusion that soluble factors secreted by stromal cells suppress epithelial secretion of TNF{alpha} but increase TER.

To date, and to the best of our knowledge, stromal-derived growth factors have not been examined in terms of their ability to affect epithelial cell immune function. Recognizing that TNF{alpha} is an important cytokine produced by uterine epithelial cells that acts as an immune inflammatory mediator [36], our goal in the present study was to identify the stromal cell factors involved in modulating TNF{alpha} release and barrier function. Specifically, our objectives were to examine the effects of HGF on epithelial cell TER and TNF{alpha} release, to establish whether the effects of HGF on epithelial cell TER and TNF{alpha} secretion are mediated through HGFRs on epithelial cells, and to determine whether HGF is the paracrine factor produced by uterine stromal cells that decreases epithelial cell TNF{alpha} release and increases TER.


   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 a 12L:12D photoperiod and allowed food and water ad libitum. For each experiment following death by CO2, uteri were pooled from 8 to 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, slit 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 as previously described [35]. Briefly, following transfer to ice-cold (3°C) Hanks balanced salt solution (Gibco-BRL/Invitrogen), digested uteri were vortexed to release sheets of epithelial cells. Epithelial sheets were recovered by passing the cell suspension through a 20-µm 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)/Ham F-12 nutrient mixed 1:1 (Gibco-BRL/Invitrogen) plus 10% defined fetal bovine serum (FBS; Hyclone, Logan, UT) supplemented with 20 mM Hepes, 100 µg/ml of streptomycin, 100 U/ml of penicillin, and 2 mM L-glutamine (all from Gibco-BRL/Invitrogen). Cell sheets were seeded in the apical compartment of Falcon (diameter, 6.4 mm; power size, 0.4 µm) or Nunc (diameter, 10 mm; pore size, 0.4 µm) 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-culture inserts per uterus and incubated at 37°C with 5% CO2. Inserts were placed in 24-well tissue-culture plates (Falcon/Nunc; Fisher Scientific) containing 850 or 500 µl of medium in the basolateral compartment and incubated at 37°C with 5% CO2.

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 (Gibco-BRL/Invitrogen) plus 400 U/ml of DNase Worthington, Lakewood, NJ). Tissues were dispersed by gentle rubbing on 40-µm nylon mesh (Small Parts), and the resulting cell suspension was centrifuged (500 x g) for 10 min. Stromal cells were resuspended in 850 µl of complete DMEM/Ham F12 plus 10% defined FBS and plated at 5 x 105 cells per 850 µl per well in 24-well plates. Medium was replaced at 48-h intervals over 4 days to remove nonadherent cells. Purity of the adherent stromal cell preparation was established by immunohistochemistry by staining stromal cells for CD45 (Pharmingen, San Diego, CA). As reported previously, fresh stromal preparations contain 5–20% leukocytes, but stromal cultures at 4 days are devoid of CD45-positive cells [37]. Based on these findings and those of morphological analysis, we concluded that the stromal cells in culture were 99% fibroblasts at the time of epithelial coculture.

Coculture of Epithelial Cells with Stromal Cells and CSM

In experiments involving the coculture of epithelial and stromal cells, epithelial and stromal cells were grown separately to confluence on cells inserts and/or in 24-well plates as described above. Once epithelial cells achieved high TER (>800–1000 {Omega}/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. 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.

To prepare CSM, stromal cells were isolated and cultured as described above. Stromal cells were grown in complete medium (i.e., CSM) and replaced at 48-h intervals. The CSM used in these experiments was the medium collected from cells between Days 4 and 6 of culture. Medium was centrifuged (10 000 x g), stored at –80°C, diluted 1:1 with fresh DMEM/Ham F-12 plus 10% FBS, and placed in the basolateral compartment of confluent uterine epithelial cells.

TER Measurements

The TER was monitored as an indication of tight junction formation and epithelial monolayer integrity using a set of Ag:AgCl electrodes and an EVOM epithelial Voltohmmeter (World Precision Instruments, Inc., New Haven, CT). Electrical resistance measurements were taken daily after alcohol sterilization of the electrode probe. Background TER of Matrigel-coated cell-culture insert was approximately 180 {Omega}/well. Epithelial cells were considered to be confluent and polarized when TER reached greater than 800 {Omega}/well.

Growth Factor and Cytokine Analysis

Analysis of TNF{alpha} was carried out on supernatants collected from epithelial cells as previously described [38]. Briefly, supernatants (100 µl) collected from epithelial cells grown alone or in coculture with stromal cells and/or CSM were assayed by TNF{alpha} ELISA (R&D Systems, Minneapolis, MN). The ELISAs were performed according to the commercial kit protocol. Recombinant human HGF (NSO-derived; R&D Systems) was added to the basolateral compartment of confluent, polarized, uterine epithelial cells with high TER. The HGF was used at 50 ng/ml as well as at 5, 50 100, 200, and 400 ng/ml in dose-response experiments. The TER measurements were taken at 24 and 48 h postaddition. In neutralization studies, HGF (100 ng/ml) was incubated with 5 µg/ml of affinity-purified goat anti-human HGF (R&D Systems) or 5 µg/ml of appropriate isotype control (goat immunoglobulin G; Caltag Laboratories, San Francisco, CA) for 1 h at 37°C before addition to the basolateral compartment. To block the HGFR, epithelial cells were preincubated with goat anti-mouse HGFR antibody (15 µg/ml; R&D Systems) or isotype control antibody (15 µg/ ml) in the basolateral compartment for 2 h before addition of HGF (100 ng/ml) to the basolateral compartment (100 ng/ml). The TER readings were taken following 24-h incubation. Apical supernatants were collected at the end of 48-h coculture for TNF{alpha} ELISA.

Epithelial-Stromal Cell Coculture: Blocking Epithelial Cell HGFR and Neutralizing Stromal HGF

Epithelial and stromal cells were isolated and cultured separately to confluence as described above. Epithelial and stromal cells were cocultured when epithelial cells reached high TER. At the onset of coculture, stromal cells were fed with control medium, medium containing goat anti-human HGF antibody (5 µg/ml), or medium containing an isotype control (5 µg/ml). Fresh medium was added to the apical compartment. The TER was measured at 24 h of coculture. Epithelial cells also were preincubated with goat anti-mouse HGFR antibody (15 µg/ml) or isotype control antibody (15 µg/ml) in the basolateral compartment for 2 h before stromal cell addition. Apical supernatants were collected from all epithelial cells after 48 h of coculture and analyzed by TNF{alpha} ELISA.

Statistics

The data were calculated as the mean ± SEM. INSTAT for Macintosh (GraphPad Software, San Diego, CA) was used to perform a one-way, repeated-measures ANOVA as well as Dunnett multiple-comparison tests and unpaired Student t-tests. SYSTAT 9 for Windows (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 considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of HGF on Uterine Epithelial Cell Function

Estrogen stimulates mammary epithelial cell proliferation indirectly via regulation of HGF produced by mammary stromal cells [24]. The HGF acts on epithelial cells to promote cell growth, morphogenesis, migration, and motility [29]. As a stromal-derived paracrine growth factor, HGF is involved in epithelial-stromal interactions of numerous tissues, including the intestine, mammary gland, and endometrium [17, 27, 28, 34, 39]. To test the hypothesis that HGF affects epithelial cell TER, uterine epithelial cells were grown on inserts to form high TER before incubation with recombinant human HGF (NSO-derived; 50 ng/ml). As seen in Figure 1, when added for 24 h to the basolateral chamber of polarized epithelial cells, epithelial cells incubated with HGF had TERs significantly higher than those of cells incubated with medium alone. This increase in TER was maintained at 48 h of exposure. To examine more fully the effect of HGF on TER, a dose-response experiment was carried out. As shown in Figure 2, HGF added to the basolateral compartment increases epithelial cell TER in a dose-dependent manner, with 100 and 200 ng/ml treatments being significantly higher than control epithelial cells. The TER response was maximal at 200 ng/ ml, in that 400 ng/ml of HGF did not further increase TER.



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  		FIG. 1. Effect of HGF on uterine epithelial cell TER. Following TER measurements of polarized epithelial cells at baseline (0 h), cells were incubated with control medium or recombinant human HGF (50 ng/ml) in the basolateral chamber (n = 4–6 inserts/group). The TER measurements were taken 24 h following treatment. The results are shown as the mean ± SEM and are representative of five experiments. **TER significantly (P < 0.05) higher than for control epithelial cells



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  		FIG. 2. Influence of HGF dose on uterine epithelial cell TER. Epithelial cells were isolated from mouse uteri and polarized on cell-culture inserts. Following baseline TER measurements, epithelial cells were incubated with control medium or medium containing increasing doses of HGF (5, 50, 100, or 200 ng/ml) in the basolateral chamber (n = 4–6 inserts/group). The TER measurements were taken 24 h following treatment. The results are shown as the mean ± SEM and are representative of three experiments. **TER significantly (P < 0.05) higher than for control epithelial cells

To establish that the effect of HGF on uterine epithelial cell TER was mediated by HGF and not by a contaminant of the preparation, epithelial cells were incubated with HGF (100 ng/ml) alone, HGF that had been preincubated with affinity-purified goat anti-mouse HGF antibody (5 µg/ml), or HGF preincubated with isotype control antibody (5 µg/ ml) for 1 h at 37°C before addition to the basolateral chamber of polarized epithelial cells. As seen in Figure 3, HGF alone and HGF plus isotype control significantly increased TER, but coculture with HGF preincubated with goat anti-human HGF antibody had no effect on TER relative to that seen with control cells.



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  		FIG. 3. Effect of antibody neutralization of HGF on the ability of recombinant HGF to enhance epithelial cell TER. Epithelial cells were isolated from mouse uteri and grown to confluence. On Day 6, after epithelial cells had reached high TER, epithelial cells were incubated with control medium, HGF (100 ng/ml), HGF plus isotype control (5 µg/ml), or HGF plus affinity-purified goat anti-HGF antibody (5 µg/ml) in the basolateral compartment (n = 4–6 inserts/group). The HGF plus isotype control and the HGF plus anti-HGF antibody were mixed and incubated at 37°C for 1 h before addition to basolateral compartment of epithelial cells. The TER measurements were taken 24 h later. The results are shown as the mean + SEM and representative for three experiments. Both HGF alone and HGF with or without isotype control significantly (P ≤ 0.05) increased TER relative to control. **TER significantly (P < 0.05) lower than for HGF plus isotype control cells. NS, No significant difference from control epithelial cells. Representative of 3 experiments

The HGFR (or c-met) has been demonstrated on the basolateral membranes of polarized T84 intestinal epithelial cells in culture [40]. To determine if mouse uterine epithelial cells express HGFR on the basolateral surface, polarized epithelial cells were preincubated for 2 h at 37°C with affinity-purified goat anti-mouse HGFR antibody or isotype control added to the basolateral compartment before the addition of HGF. As shown in Figure 4, epithelial cells incubated with HGF (100 ng/ml) with or without isotype control (15 µg/ml) for 24 h in the basolateral compartment had significantly higher TERs than epithelial cells in control medium. In contrast, when epithelial cells were preincubated with goat anti-mouse HGFR antibody (15 µg/ml) before HGF addition, TERs remained unchanged in the presence of HGF, indicating that antibody to HGFR interfered with the action of HGF.



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  		FIG. 4. Effect of anti-HGFR antibody on HGF induced increase in TER. Epithelial cells were isolated, cultured, and grown to high TER. Epithelial cells were incubated with control medium, HGF (100 ng/ml), HGF plus isotype control antibody (15 µg/ml), or HGF plus goat anti-mouse HGFR antibody (15 µg/ml) in the basolateral compartment (n = 4–6 inserts/ group). Epithelial cells were preincubated at 37°C for 2 h with isotype control or anti-HGFR antibody in the basolateral compartment before addition of HGF to the same compartment. The TER measurements were taken 24 h later. The results are shown as the mean + SEM and representative of three experiments. **TER significantly (P < 0.05) lower than for HGF plus isotype control-treated cells. NS: not significantly different than control epithelial cells

Previous studies from our laboratory have shown that stromal cells and CSM, when placed in the basolateral chamber of epithelial cells, inhibited both the apical and basolateral secretion of TNF{alpha} by polarized epithelial cells [35]. To determine if HGF has a stromal cell-like effect on epithelial cell release of TNF{alpha}, epithelial cells were grown in the presence of stromal cells, CSM, and HGF (50 ng/ ml). Figure 5 demonstrates that basolateral addition of HGF (50 ng/ml) for 48 h significantly decreases TNF{alpha} release by epithelial cells, as does CSM. As seen in Figure 6, preincubation of HGF (100 ng/ml) with anti-HGF antibody (5 µg/ml) for 1 h before placement in the basolateral compartment of polarized epithelial cells blocked the inhibitory effect of HGF on TNF{alpha} release. To test the hypothesis that the action of HGF is mediated through the HGFR, uterine epithelial cells were preincubated with goat anti-mouse HGFR blocking antibody (15 µg/ml) for 2 h before HGF addition to the basolateral compartment. As shown in Figure 6, HGFR blocking antibody reversed the inhibitory effect of HGF on TNF{alpha} release relative to that seen with HGF in the presence of isotype controls (5 and 15 µg/ml).



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  		FIG. 5. Effect of HGF on epithelial cell release of TNF{alpha}. Epithelial and stromal cells were isolated from mouse uteri and cultured. The CSM was prepared on Day 4 of culture from confluent stromal cells. Polarized epithelial cells on cell-culture inserts were transferred to plates containing stromal cells or CSM in the basolateral chamber (n = 4–6 inserts/group). Epithelial cells also were incubated with control medium or HGF (50 ng/ ml) in the basolateral chamber. Following 48 h of incubation, apical supernatants were collected from all treatment groups and analyzed for TNF{alpha}. The results are shown as the mean + SEM and are representative of three experiments. **TER significantly (P < 0.05) lower than for control cells



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  		FIG. 6. Effect of anti-HGF antibody and anti-HGFR antibody on HGF-induced decrease in TNF{alpha} release by epithelial cells. Epithelial cells were isolated, cultured, and grown to high TER on cell-culture inserts. Cells were incubated with control medium, HGF (100 ng/ml), HGF plus isotype control antibody (5 and 15 µg/ml), HGF plus anti-HGF antibody (5 µg/ ml), or HGF plus anti-HGFR antibody (15 µg/ml) in the basolateral compartment. Both HGF plus isotype control and HGF plus anti-HGF antibody were mixed and incubated at 37°C for 1 h before addition to basolateral compartment of epithelial cells (n = 4–6 inserts/group). Epithelial cells were preincubated at 37°C for 2 h with isotype control (15 µg/ ml) or anti-HGFR antibody in the basolateral compartment. Following preincubation, HGF (100 ng/ml) was added to the same compartment. Following 48 h of treatment, apical supernatants from all treatment groups were collected and analyzed for TNF{alpha}. The results are shown as the mean + SEM and are representative of three experiments. **TNF{alpha} significantly (P < 0.05) lower than for control cells

Effect of Neutralizing Stromal HGF on Epithelial Cell Function

Previous studies from our laboratory indicate that epithelial cell coculture with stromal cells leads to an increase in TER and a reduction in epithelial TNF{alpha} release [35]. To test the hypothesis that uterine stromal cells produce HGF, which, in turn, regulates epithelial TER, as seen with recombinant HGF, goat anti-human HGF antibody was added to stromal cell cultures to neutralize HGF. Following the formation of high TER, epithelial cell inserts were transferred to plates containing stromal cells, stromal cells plus anti-HGF antibody (5 µg/ml), and stromal cells plus isotype control (5 µg/ml). Other cells inserts were incubated with HGF as a positive control. As seen in Figure 7, the addition of goat anti-human HGF antibody to stromal cells at the start of coculture with epithelial cells blocks the ability of stromal cells to increase epithelial cell TER at 24 h. In contrast, HGF (100 ng/ml) in the basolateral compartment as well as stromal cells cultured alone or with isotype control immunoglobulin (5 µg/ml) significantly (P ≤ 0.05) increased TER relative to that seen in control wells.



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  		FIG. 7. Effect of antibody neutralization of HGF on stromal cell-induced increase in TER. Epithelial and stromal cells were isolated from mouse uteri and cultured for 4 days until reaching confluence as previously described. Polarized epithelial cells were incubated with medium alone or HGF (200 ng/ml) in the basolateral compartment (n = 4–6 inserts/group). Cell-culture inserts of polarized epithelial cells were also transferred to 24-well plates containing stromal cells alone, stromal cells plus anti-HGF antibody (5 µg/ml), or stromal cells plus isotype control antibody (5 µg/ ml) in the basolateral compartment. The TER measurements were taken 24 h following treatment. The results are shown as the mean + SEM and representative of two experiments. **TER significantly (P < 0.05) higher than control cells. NS, Not significantly different than control cells

To determine whether stromal-derived HGF, in addition to affecting TER, is responsible for reducing TNF{alpha} release by epithelial cells, polarized epithelial cells were cocultured with stromal cells, stromal cells plus goat anti-human HGF antibody (5 µg/ml), and stromal cells plus isotype control antibody (5 µg/ml) for 48 h. Unexpectedly, we found that neutralization of stromal cell HGF had no effect on epithelial release of TNF{alpha}. As seen in Figure 8, when anti-HGF antibody was added to the basolateral compartment of epithelial cells cocultured with stromal cells, neutralization had no effect on TNF{alpha} release, despite the fact that the dose used was sufficient to completely block stromal cell-induced TER effects. As a part of these studies, the goat anti-mouse HGFR antibody, which blocks HGF action, was added to stromal cell cultures at the time of epithelial cell insert transfer. Similar to that seen with goat anti-human HGF antibody, the presence of goat anti-mouse HGFR antibody had no effect on stromal inhibition of epithelial cell TNF{alpha} release. Overall, these studies indicate that whereas HGF is the growth factor important for regulating epithelial cell integrity, measured as TER, reduced secretion of TNF{alpha} appears to involve a stromal cell signal (or signals) other than HGF.



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  		FIG. 8. Lack of effect of anti-HGF antibody and anti-HGFR antibody on the inhibitory effect of stromal cells on TNF{alpha} release by epithelial cells. Mouse uterine epithelial and stromal cells were isolated and cultured for 4 days. After polarized epithelial cells reached high TER, cell-culture inserts of epithelial cells were cultured in control medium or transferred to plates containing stromal cells alone, stromal plus anti-HGF antibody (5 µg/ml), stromal plus anti-HGFR antibody (15 µg/ml), or stromal plus isotype control (5 and 15 µg/ml; n = 4–6 inserts/group). Epithelial cells were cocultured with or without treatment for 48 h. Apical supernatants were collected from all treatment groups and analyzed for TNF{alpha} by ELISA. The results are shown as the mean + SEM and are representative of three experiments. **TNF{alpha} significantly (P < 0.001) lower than control cells


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented demonstrate that HGF, produced by uterine stromal cells, affects normal uterine epithelial cell integrity, measured as TER as well as the secretion of TNF{alpha}, a key cytokine involved with immune protection at mucosal surfaces of the female reproductive tract. Based on antibody neutralization data, the stromal-derived factor that regulates epithelial cell TER is shown to be HGF. Our studies indicate that HGF acts via receptors located on the basolateral surfaces of polarized uterine epithelial cells in culture. Whereas HGF decreased epithelial cell TNF{alpha} release but, at the same time, increased TER, antibody neutralization of stromal media failed to reverse the inhibitory effect of stromal cells on TNF{alpha} secretion by epithelial cells. These findings suggest that whereas HGF most likely is the stromal growth factor that regulates epithelial cell integrity (i.e., TER), regulation of TNF{alpha} secretion appears to involve a signal (or signals) other than HGF. Recognizing that epithelial cell integrity and secretion of the proinflammatory cytokine TNF{alpha} are central to barrier protection and recruitment of immune cells of the innate and adaptive immune systems at mucosal surfaces, our findings suggest that protection against potential viral, bacterial, and fungal pathogens in the female reproductive tract may, in part, be mediated indirectly by soluble factors produced by underlying stromal cells.

The synthesis of HGF by stromal cells [28, 30–32] together with its reported mitogenic, motogenic, and morphogenic effects on epithelia [25–29] led to the suggestion that HGF may be a key mediator of epithelial-stromal interactions [34]. For example, treatment of confluent T84 and MDCK cells with HGF decreased TER [40]. In other studies involving subconfluent MDCK cells, HGF inhibited the assembly of epithelial cell-cell junctions [41]. In contrast, when treated basolaterally with HGF (50–500 ng/ml), polarized MDCK cells increased TER within 24 h [25]. Our studies extend these findings by demonstrating that normal primary epithelial cells respond to HGF with dose- and time-dependent increases in TER. Pollack et al. [25] determined that HGF-induced increases in TER by MDCK cells was the result of individual cells of the monolayer crawling over each other to form a pseudostratified layer, during which time barrier function and tight junctions remained functionally intact. Our results of increased epithelial cell TER in response to HGF treatment most likely represent a direct effect on tight junctions or conductance, because histological examination of epithelial inserts indicated that cells retain their monolayer conformation during the course of incubation (data not shown). Studies are presently under way to identify the mechanisms whereby HGF increases epithelial TER. Whether the TER increases observed in our study involve the regulation of tight junction proteins, including ZO-1, occludin, and several claudin family members, which are essential components of tight junctions, remains to be determined [42].

Results from treatment of uterine epithelial cells with HGF indicate that HGF inhibits TNF{alpha} secretion. To the best of our knowledge, the present study is the first report of in vitro HGF regulation of TNF{alpha} secretion by polarized uterine epithelial cells. In a murine model of graft-versus-host disease, HGF was shown to suppress interferon-{gamma} and TNF{alpha} expression in the intestine and liver as well as to ameliorate disease [43]. As an essential cytokine of the innate and adaptive immune systems, TNF{alpha} acts as an inflammatory mediator and regulator of both physiologic and pathophysiologic processes [44, 45]. For example, TNF{alpha} enhances phagocytosis and cytotoxicity in neutrophilic granulocytes as well as induces synthesis of a number of chemoattractant cytokines, including interferon-{alpha}-inducible protein-10, macrophage chemotactic factor-1, and the chemokine KC [37, 46]. The extent to which this growth factor affects epithelial cell secretory function remains to be determined. Clearly, however, uterine epithelial cells, which are responsive to a number of growth factors and sex hormones, have evolved both to protect against potential pathogens as well as to accept a blastocyst that is allogeneically distinct. Our findings suggest that regulatory control of reproductive and immunological processes involves the communication of epithelial cells with underlying stromal cells.

Recognizing that TNF{alpha} has been reported to impair barrier function and to decrease TER of epithelial cells in other systems [14–17], studies were carried out in which epithelial cells were incubated with TNF{alpha} (unpublished results). We found that addition of TNF{alpha} to polarized uterine epithelial cells in culture increased TER in a dose-dependent manner. Moreover, we unexpectedly found that antibody neutralization to eliminate epithelial cell TNF{alpha} had no effect on TER. These results indicate that the effects of stromal cells and HGF on TNF{alpha} and TER are both separate and distinct.

Our results indicate that the effects of HGF on uterine epithelial cell TER and TNF{alpha} secretion are mediated through basolaterally expressed HGFR, because preincubation of cell monolayers with a blocking antibody to HGFR in the basolateral compartment before HGF addition inhibited the ability of HGF to affect TER and TNF{alpha} release. These findings suggest that HGF, by binding to its receptor, regulates both epithelial cell integrity and cytokine secretion by polarized uterine epithelial cells. Other investigators have shown that HGF effects are mediated by its high-affinity receptor c-met, a transmembrane type I tyrosine kinase receptor expressed in epithelial tissues [27]. The concentrations of HGF required to increase uterine epithelial cell TER and TNF{alpha} secretion in our study were sufficient to saturate c-met, but other investigators have reported that hepatocytes, keratinocytes, and melanocytes contain low-affinity, high-capacity HGF-binding sites [33]. More recent studies suggest that the high concentrations of HGF used to stimulate MDCK TER are mediated through HGF interactions with low-affinity sites [25]. Interestingly, we find that preincubation with goat anti-mouse HGFR antibody, which recognizes high-affinity sites (c-met), blocks the effect of HGF on epithelial cell TER. If low-affinity sites do exist on mouse uterine epithelial cells, then our studies suggest that the antibody blocking HGFR /c-met recognizes both types of receptors (i.e., low and high affinity).

Our findings that HGF regulate uterine epithelial cell TER and TNF{alpha} in the same way as uterine stromal cells and CSM do led to the hypothesis that HGF is the stromal cell factor affecting epithelial cell function. Unexpectedly, we found that antibody neutralization of HGF in stromal media had no effect on stromal cell-induced decreases in TNF{alpha} release. Consistent with this observation was our finding that antibody neutralization of the HGFR failed to reverse the inhibitory effect of stromal cells on epithelial TNF{alpha} release. In contrast, HGF as well as HGFR antibodies reversed stromal cell-induced increases in epithelial cell TER. These results indicate that HGF is, presumably, the key regulator of epithelial cell integrity (i.e., TER). The HGF has been proposed to be a mediator of stromal-induced epithelial cell proliferation [34]. More recent findings with epithelial cells from the mammary gland support this hypothesis [24]. Our findings of increased TER in response to HGF and CSM extend these observations by demonstrating that in addition to cell proliferation, cell tight junction formation is enhanced. That TNF{alpha} secretion was not affected by anti-HGF antibody neutralization of CSM indicates that other growth factors and/or cytokines produced by stromal cells contribute to the regulation of TNF{alpha} secretion by epithelial cells. Consistent with these observations are studies that showed stromal cells produce numerous growth factors, including KGF, EGF, and IGF-I, all of which regulate epithelial cell proliferation [47–50]. What is suggested from these as well as our studies is that epithelial cells have evolved to be responsive to discrete stromal signals that, on the one hand, alter cell proliferation, integrity, and barrier function and, on the other, regulate epithelial cell secretory function.

In conclusion, these studies demonstrate that HGF, an important growth factor, regulates both epithelial cell integrity and cytokine secretion by polarized uterine epithelial cells in culture. Produced by uterine stromal cells, HGF appears to be the growth factor responsible for regulating epithelial cell integrity, measured by TER. In contrast, TNF{alpha} secretion, which also is under stromal cell control, appears to be regulated by one or more stromal cell signals other than HGF. Our findings that stromal cells suppress epithelial TNF{alpha} at a time when epithelial cell tight junction resistance increases suggests that in the absence of stromal influences, epithelial cells may overproduce this proinflammatory cytokine, which, in turn, could lead to increased risk of infection, implantation failure, and immunological pregnancy loss. At the same time, lowering TER could, potentially, increase the risk of bacterial and/or viral invasion. These studies suggest that by controlling these two physiological properties, stromal cell regulation of epithelial cell function has evolved both to protect against potential pathogens and to maintain proper cytokine balance and, thereby, prevent reproductive failure. These studies demonstrate a new level of complexity in our understanding of epithelial-stromal interactions in the uterine endometrium as they affect immune functions in the reproductive tract. Understanding the ways in which epithelial cells are responsive to stromal cells in the reproductive tract is crucial to our understanding the role of the immune system in mammalian reproduction as well as in protection against potential pathogens, including human immunodeficiency virus, which threaten reproductive health and survival.


   ACKNOWLEDGMENTS
 
The authors gratefully express their appreciation to Richard Rossoll for his assistance in these studies. They also thank Dr. James C. Leiter for his assistance with the statistical analysis of the data.


   FOOTNOTES
 
1 Supported by research grant AI-13541 from the NIH. Back

2 Correspondence: Charles R. Wira, Department of Physiology, Dartmouth Medical School, Borwell Building, 1 Medical Center Drive, Lebanon, NH 03756-0001. FAX: 603 650 6130; charles.r.wira{at}dartmouth.edu Back

Received: 23 August 2004.

First decision: 20 September 2004.

Accepted: 19 October 2004.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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C. R. Wira, R. M. Rossoll, and R. C. Young
Polarized Uterine Epithelial Cells Preferentially Present Antigen at the Basolateral Surface: Role of Stromal Cells in Regulating Class II-Mediated Epithelial Cell Antigen Presentation
J. Immunol., August 1, 2005; 175(3): 1795 - 1804.
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