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The Journal of Immunology, 2005, 175: 1795-1804.
Copyright © 2005 by The American Association of Immunologists

Polarized Uterine Epithelial Cells Preferentially Present Antigen at the Basolateral Surface: Role of Stromal Cells in Regulating Class II-Mediated Epithelial Cell Antigen Presentation1

Charles R. Wira2,*, Richard M. Rossoll* and Roger C. Young{dagger}

* Department of Physiology and {dagger} Department of Obstetrics and Gynecology, Dartmouth Medical School, Lebanon, NH 03756


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
To study Ag presentation in the female reproductive tract, DO11.10 TCR transgenic mice specific for the class II MHC-restricted OVA323–339 peptide and nontransgenic BALB/c mice were used. We report here that freshly isolated uterine epithelial cells, uterine stromal, and vaginal APCs present OVA and OVA323–339peptide to naive- and memory T cells, which is reduced when cells are incubated with Abs to CD80 and 86. To determine whether polarized primary epithelial cells present Ags, uterine epithelial cells were cultured on cell inserts in either the upright or inverted position. After reaching confluence, as indicated by high transepithelial resistance (>2000 ohms/well), Ag presentation by epithelial cells incubated with memory T cells and OVA323–339 peptide placed on the basolateral surface (inverted) was 2- to 3-fold greater than that seen with epithelial cells in contact with T cells and peptide on the apical surface (upright). In contrast, whereas freshly isolated epithelial cells process OVA, polarized epithelial cells did not. When epithelial cells grown upright on inserts were incubated with T cells and OVA323–339 peptide, coculture with either hepatocyte growth factor or conditioned stromal medium increased epithelial cell Ag presentation (~90% higher than controls). These studies indicate that uterine stromal cells produce a soluble factor(s) in addition to a hepatocyte growth factor, which regulates epithelial cell Ag presentation. Overall, these results demonstrate that polarized epithelial cells are able to present Ags and suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to regulate Ag presentation in the uterus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Epithelial cells at mucosal surfaces throughout the body are the first line of protection against potential bacterial and viral pathogens (1, 2). Acting as sentinels of immune protection, epithelial cells have evolved to provide a physical barrier (3), recognize and respond as a part of the innate immune system by secreting chemokines and cytokines that recruit and activate immune cells (4, 5), secrete microbicidal and virucidal agents that inactivate and/or kill pathogens (6, 7, 8), as well as process and present Ags as part of the adaptive immune system (9, 10). Unique to epithelial cells in the female reproductive tract is the additional demand of responding to potential pathogens, while at the same time, allowing sperm and a fetal placental unit, both of which are allogeneic, to survive (11, 12, 13).

Ag presentation by epithelial cells has been demonstrated at several mucosal surfaces. For example, numerous reports have described the expression of HLA class II Ags on epithelial cells of the intestine and reproductive tract (9, 14, 15, 16, 17). Using intestinal epithelial cell (IEC)3 lines to measure Ag processing and presentation, IECs were shown to internalize Ags and present processed peptides in the context of class II to CD4+ T cells (9). Kaiserlian et al. (18) demonstrated in a murine model that IECs could present Ags to a CD4+ T cell hybridoma and that an anti-class II mAb blocked IL-2 production by T cells. Subsequent studies with other Ags confirmed these results, although most Ags were inefficiently presented (19). Hershberg et al. (20, 21) used a polarized IEC line transfected to express HLA-DR to demonstrate class II-tetanus toxoid Ag presentation to restricted CD4+ T cells. These studies demonstrated that epithelial cells preferentially presented Ag at the basolateral surface (21). We more recently demonstrated that uterine cell suspensions consisting of human epithelium and stromal cells were capable of presenting Ags to autologous T cells (17). These studies further demonstrated that isolated, purified human uterine epithelial cells can process and present Ags to autologous T cells (17).

Using the rat as a model system, we demonstrated that uterine epithelial and stromal cells process and present OVA Ags and that the Ag presentation to CD4+ T cells is mediated through class II (22, 23). In other studies, we found that Ag presentation in the uterus and vagina is under the control of sex hormones and varies with the stage of the reproductive cycle. When given to ovariectomized rats, estradiol enhanced Ag presentation in the uterus (23) and inhibited Ag presentation in the vagina (24, 25). In addition to sex hormones, Ag presentation in the uterus increases when ovariectomized rats are treated with IFN-{gamma} or IL-6 (22). More recently we found that, in response to estradiol, uterine and vaginal epithelial cells inhibit Ag presentation in the uterine stroma by secreting TGF-{beta} (26, 27). These findings indicated that epithelial cells affect stromal cell function through the release of soluble factors such as TGF-{beta}.

Less recognized is the important role of soluble factors, which are produced by underlying stromal cells that regulate uterine epithelial cell function in the female reproductive tract. In the uterus, for example, stroma and epithelium function as a unit with each cell type producing factors that regulate the growth, differentiation, and function of the other (28, 29). The stroma, in particular, heavily influences the epithelium during organ development and in the maintenance of adult homeostasis (30, 31, 32). Previously, we found that underlying stromal cells from the uterine endometrium decreased the barrier function of human polarized epithelial cells (33). More recently, we focused on the role of uterine stromal cells in regulating mouse epithelial cell monolayer electrical integrity and cytokine release (34). These studies indicated that uterine stromal cells produce a soluble factor(s) that increases epithelial cell TER and decreases TNF-{alpha} release into both the apical and basolateral chambers without effecting TGF-{beta} release (34). More recently, we found that hepatocyte growth factor (HGF), a known mesenchymal growth factor that mediates mammary gland epithelial cell proliferation (35), increased transepithelial resistance (TER), while at the same time, decreased apical TNF-{alpha} release (36).

The overall goal of the present study was to more fully define the regulatory control of Ag presentation in the female reproductive tract. Our objectives were to: 1) determine whether freshly prepared epithelial and stromal cells in the mouse uterus and vagina are functionally able to process and present recall Ags to OVA-specific memory T cells; 2) evaluate whether epithelial cells and uterine stromal APCs are able to present Ag to naive T cells; 3) determine whether polarized epithelial cells preferentially process and present Ags at the basolateral surface; and 4) establish whether underlying uterine stromal cells influence epithelial cell Ag presentation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
General procedures

Sexually mature, BALB/cAnNCr, female mice weighing between 15 and 20 g were obtained from the National Cancer Institute colony at Charles River Laboratories. Transgenic BALB/c-TgN(DO11.10)10Loh mice at 10–20 g were purchased from The Jackson Laboratory. Transgenic mice express the mouse {alpha}- and {beta}-chain TCR that pairs with the CD4 coreceptor and is specific for chicken OVA323–339 (OVA323–339 peptide) in the context of I-Ad (class II restricted). Animals were maintained in a constant-temperature room with fixed light/dark intervals (12 h) and allowed food and water ad libitum. Animals were sacrificed by CO2, and uteri and/or vaginae were pooled from 8 to 16 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.

Preparation of CD4+ memory T cells

CD4+ T cells from TCR transgenes homozygous mice were prepared from spleens and peripheral lymph nodes of 6- to 10-wk-old mice as described previously (37, 38). Splenic cells were dispersed by gentle grinding between two sterilized glass slides in HBSS (Invitrogen Life Technologies). Following centrifugation for 8 min at 500 x g, erythrocytes were lysed by NH4Cl treatment. The CD4+ subpopulation of T cells was purified by negative selection using a Mouse T Cell CD4 Subset Column kit (R&D Systems). To prepare memory cells, T cells were stimulated with 0.3 µM OVA peptide and incubated with irradiated BALB/c splenocytes added at 20:1 as APCs in IMDM (Sigma-Aldrich) containing 10% heat-inactivated (56°C for 30 min) FBS (HyClone) supplemented with 100 µM nonessential amino acids, 1 mM sodium pyruvate (both from Invitrogen Life Technologies), 50 µM 2-ME (Sigma-Aldrich), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Mediatech). Two-milliliter cultures (5 x 106 cells/well) were expanded at 72 h by 3-fold dilution into fresh medium. Between days 7 and 10, when T cells were considered to be resting as determined by minimal [3H]thymidine uptake, they were harvested, washed, and frozen at –80°C in IMDM containing 50% FBS and 10% DMSO (Sigma-Aldrich) until used.

Preparation of CD4+ naive T cells

The naive population of T cells was further isolated after the CD4 column by triple-stain sorting with a FACStar-plus Flow Cytometer (BD Biosciences). The cells (30–35 x 106) in 1 ml of IMDM with 10% FBS (heat inactivated, defined) were incubated with Abs to CD4 (final concentration 5 µg/ml, FITC-labeled), CD62L (2 µg/ml, PE), and CD44 (2 µg/ml, Cy5; all directly labeled monoclonals from BD Biosciences/Pharmingen) in IMDM for 30 min on ice in the dark, washed, then resuspended in fresh medium for flow cytometric analysis. Naive T cells were characterized as CD4+,CD62Lhigh,CD44low as described previously (39).

Preparation of uterine and vaginal cells

Uterine epithelial cells. To prepare isolated epithelial cells, uteri were removed, cut open lengthwise, and incubated with 46,500 U/ml of trypsin (Sigma-Aldrich) in 2.5% pancreatin (Invitrogen Life Technologies), at 20 ml/g tissue, for 60 min at 4°C and 60 min at 22°C as described previously (34). Following transfer to ice-cold (3°C) HBSS, uteri were vortexed to release sheets of epithelial cells. Uterine tissues were rinsed and vortexed an additional two times, and resulting cell suspensions were combined. Pooled epithelial sheets were recovered by passing the cell suspension through a 20-µm mesh nylon screen (Small Parts), collected, and centrifuged (500 x g) for 5 min. Epithelial sheets were resuspended in culture medium consisting of DMEM:Ham’s F-12 nutrient mixed 1:1 (without phenol red; Invitrogen Life Technologies) containing 10% defined FBS and supplemented with 20 mM HEPES (Invitrogen Life Technologies), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Sheets were aspirated through 20-g needles to prepare isolated cells for experimentation.

To culture epithelial sheets, ~2 x 105 cells (without needle disruption) were seeded in the inside compartment of 6.4 mm in diameter Falcon cell culture inserts with 0.4-µm pore membranes (Fisher Scientific) coated with diluted Matrigel (growth factor reduced, without phenol red; Collaborative Biomedical Products). Cells were seeded in a 300-µl volume of DMEM:Ham’s F-12 with 10% charcoal-dextran-stripped FBS (HyClone) at a ratio of three to four culture inserts per uterus and placed in 24-well tissue culture companion plates (Fisher Scientific) containing 850 µl of the appropriate culture medium in the outside compartment and incubated at 37°C with 5% CO2. Alternatively, Nunc inserts (10 mm, 0.4-µm pore; Nalge Nunc International) were coated and seeded at the same cell ratio with 300 µl of medium (either DMEM:Ham’s F-12 with 10% stripped FBS or Cellgro complete medium (Mediatech) containing L-glutamine, penicillin, and streptomycin) inside and 500 µl outside in 24-well Nunclon plates (Nalge Nunc International) to adjust for the different volume capacities of the inserts. The purity of epithelial cell cultures prepared as described above was evaluated by three criteria: 1) epithelial cells used in these studies were examined by microscopy and found to consist as 50–500 cell epithelial sheets that were free of isolated cell contaminants; 2) mouse uterine epithelial cells achieve high TER (>2000 ohms/well; background resistance 150 ohms/well), which would not be possible if there was stromal cell contamination; and 3) staining of cell inserts with anti-CD45 Ab indicated that epithelial cells account for >99.5% cells present on each insert.

For some experiments, epithelial cultures were grown on the bottom of coated Nunc inserts. Cell sheets were added in 200 µl of medium to chambers on inverted inserts made by attaching sterile Tygon tubing "collars" around the bottom of the insert. The inside compartments of the inserts had been filled with medium and immersed upside down to just below the collar lip in medium in 6-well Costar culture plates (Corning Glass). Cultures were grown for 2–3 days before collars were removed, and inserts were turned upright in fresh medium in 24-well plates. In all cases, epithelial cell monolayers were monitored daily by TER as an indication of tight junction formation using an Evom epithelial voltohmmeter and electrode (World Precision Instruments) and fed fresh culture medium in both compartments every other day.

Uterine stromal cells. To isolate stromal cells, pooled uteri, following the removal of epithelial cells, were incubated for 30 min at 37°C in 0.05% trypsin and 530 µM EDTA in HBSS (Mediatech) with 400 U/ml deoxyribonuclease I (Worthington Biochemical), using 2 ml/tissue as described previously (34). Tissues were dispersed by gentle rubbing in culture medium on a 40-µm mesh nylon screen (Small Parts), and the resulting cell suspension was centrifuged (500 x g) for 10 min. Stromal cells were resuspended in DMEM:Ham’s F-12 with 10% defined FBS and plated at 5 x 105 cells in 850 µl/well of 24-well plates (Fisher Scientific). To prepare conditioned stromal medium (CSM), culture medium from stromal cells was collected and replaced at 48-h intervals, centrifuged (10,000 x g) for 5 min, stored at –80°C, and diluted 1:1 with fresh DMEM:Ham’s F-12 with 10% stripped FBS when used.

Vaginal cells. Vaginal cells from BALB/c mice were prepared by incubation of tissues, which were hemi-sectioned lengthwise, with 46,500 U of trypsin/ml, 2.5% pancreatin, using 2 ml/vagina, for 60 min at 4°C and 60 min at 22°C with constant rotation (100 rpm) before separation by gentle rubbing in DMEM:Ham’s F-12 culture medium on a 40-µm mesh nylon screen. Cells were aspirated through 18- and 20-gauge needles to prepare isolated cells for experimentation.

Ag presentation assays

To measure Ag presentation by freshly isolated cells, OVA peptide-specific T cells (1 x 105 cells/100 µl) in DMEM:Ham’s F-12 culture medium were seeded in triplicate wells in 96-well flat-bottom microtiter plates (Nunc) with irradiated uterine epithelial or stromal cells as well as vaginal APCs (1 x 105 cells/100 µl) in the presence of OVA (APC+T+OVA) or OVA323–339 peptide (APC+T+peptide). APCs were irradiated before the start of Ag presentation with 5000 rad to prevent their proliferation. Controls included in all experiments were APCs incubated with T cells in the absence of Ag (APC+T), APCs incubated with Ag (APC+OVA/peptide), and T cells incubated with Ag (T+OVA/peptide). Following 48 h of incubation at 37°C, T lymphocyte proliferation was measured by [3H]thymidine uptake as described previously (27). Each well received 1 µCi (50 µl of medium) 20–24 h before the termination of each experiment. Cells in wells were transferred individually onto glass fiber filtermats with a cell harvester (Skatron). Radioactivity incorporated into cells was measured in a liquid scintillation counter (Packard Instrument).

Ag presentation assays in culture inserts

Once epithelial cells achieved high TER (>2000 ohms/well) after ~7 days in culture, inserts of polarized upright and inverted epithelial monolayers were used in Ag presentation assays. T cells were added to the inside chamber (3 x 105 cells/300 µl of final volume of appropriate medium) in the presence of 1200 µg/ml OVA or 5 µg/ml peptide in the inside and/or outside chamber as indicated in the figure legends and incubated as above. Incorporation of [3H]thymidine was measured by pipette resuspension of T cells in individual inserts without disrupting epithelial monolayers, removing the cell suspension to centrifuge away bubbles, then transferring it to 96-well plates to be harvested as above.

Abs and cytokines

Abs and isotype control Abs to CD4, CD62L, and CD44, all directly labeled monoclonals, were purchased from BD Biosciences/Pharmingen. Recombinant human HGF and goat anti-human HGF Ab and isotype control were purchased from R&D Systems. Purified rat anti-mouse B7-1 (CD80) and B7-2 (CD86) mAbs (BD Biosciences/Pharmingen) were added alone or in combination, each at a final concentration of 2.5 µg/ml, to inhibit Ag presentation/proliferation. Rat IgG2a,K isotype was used at the same concentration as a control.

Statistics

Data were compared by one-way ANOVA, followed by a Tukey multiple comparison post test. Differences of p ≤ 0.05 were considered significant. All values are expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Presentation of OVA323–339 peptide by uterine epithelial cells to memory T cells

Previous studies from our laboratory have shown that rat uterine epithelial cells, as well as APCs in the uterine stroma and in the vagina, are able to present Ags to T cells (23, 27). To more fully characterize Ag presentation in the female reproductive tract, studies were undertaken to extend our Ag presentation system to the mouse. To study Ag presentation in the uterus and vagina, DO11.10 TCR transgenic mice were used as a source of T cells that are specific for the class II MHC-restricted OVA323–339 peptide (323–339), as reported previously (38). Uterine epithelial cells were prepared from nontransgenic BALB/c mice as described previously (34, 40). As shown in Fig. 1, when memory T cells, prepared by culture in vitro with OVA peptide (see Materials and Methods for details), are incubated with uterine epithelial cells, increasing concentrations of OVA323–339 peptide resulted in increased Ag presentation, measured as the proliferation of T cells by [3H]thymidine incorporation into OVA-specific memory T cells incubated with irradiated uterine epithelial cells. In the absence of OVA323–339 peptide, very little [3H]thymidine was incorporated when epithelial cells were incubated with T cells or with OVA323–339 peptide or when T cells were incubated with OVA323–339 peptide. Ag presentation by uterine cells was significantly greater with 2.5 and 5 µg/ml than with 0.5 and 1.0 µg/ml Ag. These results indicate that this model system can be used to measure Ag presentation in the female reproductive tract.



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  		FIGURE 1. Effect of increasing doses of OVA323–339 peptide on Ag presentation by uterine epithelial cells to CD4+ DO11.10 memory T cells. Animals (12 BALB/c mice) were sacrificed, and uterine tissues were pooled and cells were prepared by enzymatic digestion. Memory T cells were prepared by incubating spleen cells from DO11.10 mice with OVA323–339 peptide for 7–10 days in culture as described in Materials and Methods. Epithelial cells (APCs; 1 x 105 cells/100 µl) were incubated with memory T cells (T; 1 x 105 cells/100 µl) and increasing concentrations of OVA323–339 peptide (0.5–5 µg/ml) for 3 days. [3H]Thymidine was added for the last 24 h of incubation. Values shown are [3H]thymidine incorporation for APC+T+Ag323–339 peptide as well as control groups APC+T, APC+Ag323–339 peptide, and T+Ag323–339 peptide incubations as mean ± SE (three wells/group). *, Significantly (p < 0.05) greater than controls. Representative of two experiments.

 
Ag presentation by uterine and vaginal cells to memory T cells

To establish that epithelial cells as well as APCs in the uterine stroma and vagina are able to process as well as present Ags, isolated cells from intact animals were prepared and incubated with DO11.10 memory T cells in the presence of OVA or OVA323–339 peptide. As shown in Fig. 2, when OVA323–339 peptide (upper panel) or OVA (lower panel) was added to the incubation medium, Ag presentation by uterine epithelial and stromal cells as well as vaginal cells was observed. The incorporation of [3H]thymidine seen with OVA and OVA323–339 peptide was significantly greater than that seen in the absence of OVA (APC+T). These studies indicate that epithelial cells as well as APCs in the uterus and vagina are able to both process and present recall Ags to memory T cells.



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  		FIGURE 2. Ag processing and presentation by freshly isolated uterine epithelial and stromal cells as well as vaginal cells to CD4+ DO11.10 memory T cells (three wells/group). APCs (1 x 105 cells/100 µl) were incubated with CD4+ memory T cells (1 x 105 cells/100 µl) along with either OVA323–339 peptide (upper panel; 5 µg/ml) or OVA (lower panel; 1200 µg/ml) for 3 days. Values shown are the mean ± SE (three wells/group). [3H]Thymidine was added for the last 24 h of incubation. *, APC+T+Ag was significantly (p < 0.05) greater than controls (APC+T, APC+Ag, and T+Ag). Representative of three experiments.

 
To determine whether costimulatory molecules are involved in Ag presentation in the mouse female reproductive tract, epithelial cells and/or stromal cells were incubated with DO11.10 memory T cells in the presence of OVA and Abs to CD80 (B7.1) and CD86 (B7.2), which participate in T cell costimulation via interactions with CD28 and CD152 (CTLA-4) (41, 42, 43). As seen in Table I, when uterine epithelial cells, as well as uterine stromal and vaginal APCs, were incubated with T cells and OVA in the presence of Abs that interfere with the actions of accessory molecules, Ag presentation is inhibited relative to that seen with isotype controls. Ag presentation of OVA to memory T cells was reduced by 30–40% when anti-B7-1 and B7-2 Abs were added to the assay wells.


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  		Table I. Influence of accessory molecules on Ag presentation by uterine (UT) epithelial cells and APCs in the uterine stroma and vagina

 
Ag presentation to naive T cells

To determine whether uterine and vaginal cells are able to present Ags to naive T cells, epithelial cells as well as uterine stromal and vaginal APCs were incubated with naive T cells recovered from DO11.10 mice. As seen in Fig. 3, reproductive tract APCs were able to process and present Ag to naive T cells. As shown in the upper panel (OVA323–339 peptide) and lower panel (OVA), epithelial cells and stromal cells from the uteri as well as vaginal cells from intact mice processed and presented Ags to naive T lymphocytes. To confirm these findings, our second approach was to prepare naive T cells by flow cytometry because naive DO11.10 mice can have low numbers of memory OVA-reactive T cells (J. Gorham, unpublished observation). To eliminate the possibility that Ags were being presented to these cells, flow cytometry purification and isolation of naive T cells was undertaken. In the current study, naive, Ag-specific CD62LhighCD4+CD44low DO11.10 T cells were isolated from the spleens of transgenic mice. These cells were used to study Ag processing and presentation by uterine epithelial and stromal cells using our T cell proliferation assay. As shown in Fig. 4, when epithelial cells and stromal cells containing APCs were incubated with OVA, both processed and presented OVA as well as presented the OVA323–339 peptide to naive T cells.



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  		FIGURE 3. Stimulation of naive CD4+ T cell proliferation by uterine epithelial cells and APCs in the uterine stroma and vagina in the presence of OVA and OVA323–339 peptide. Reproductive tract APCs were prepared from BALB/c mice (nine animals) as described in Materials and Methods. Naive CD4+ T cells were purified from spleen cells (two to four animals) of DO11.10 mice by negative selection using a Mouse T Cell CD4 Subset Column kit (Materials and Methods). Epithelial cells and stromal cells from uteri as well as vaginal cells (1 x 105 cells/well) were cocultured with naive CD4+ DO11.10 T cells (1 x 105 cells/well) and either OVA323–339 peptide (upper graph; 5 µg/ml) or OVA (lower graph; 1200 µg/ml). After 2 days, T cell proliferation was assessed by the addition of [3H]thymidine to wells for an additional 24 h of incubation. Each bar represents the mean counts per minute ± SE of triplicate wells. *, Significantly (p < 0.05) greater than controls within each cell type.

 


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  		FIGURE 4. Presentation of Ags by uterine epithelial and stromal cells to purified naive DO11.10 T cells. Naive T cells (CD4+,CD62Lhigh,CD44low) from spleen cells (two to four animals) of adult DO11.10 mice were recovered by flow cytometer-positive selection as described in Materials and Methods. Naive T cells were incubated (1 x 105 cells/well) with epithelial (upper graph) or stromal (lower graph) cells (1 x 105 cells/well) prepared from a pool of 12 BALB/c mice along with OVA323–339 peptide (5 µg/ml) or OVA (1200 µg/ml). Following 2 days of incubation, [3H]thymidine was added for an additional 24 h (three wells/group). **, Significantly (p < 0.01) greater than controls.

 
Ag presentation by polarized uterine epithelial cells grown on culture inserts

To determine whether polarized primary epithelial cells present Ags at the apical and basolateral surfaces, uterine epithelial cells were isolated and grown to confluence on cell inserts in either the upright or inverted position. We found that under the culture conditions used in these studies, TER (mean ± SE) values of both insert sets were the same irrespective of whether epithelial cells were grown in the upright or inverted position (2690 ± 581 vs 2227 ± 701 ohms/well). After forming tight junctions, epithelial cells were incubated with DO11.10 TCR memory T cells and OVA323–339 peptide placed either on the apical surface (upright) or basolateral surface (inverted) for 48 h before the addition of [3H]thymidine to the upper chamber for an additional 24 h. As seen in Fig. 5, Ag presentation by epithelial cells in contact with T cells at the basolateral surface (inverted) was 2- to 3-fold greater with OVA323–339 peptide than that seen with epithelial cells in contact with T cells and OVA323–339 peptide on the apical surface (upright). The differences seen between apical and basolateral Ag presentation, as shown in Fig. 5, are representative of our findings in three of three experiments. In contrast, as seen in Fig. 6, whereas freshly isolated epithelial cells presented intact OVA to T cells (Fig. 2), polarized epithelial cells, irrespective of whether T cells were in contact with basolateral or apical (data not shown) surfaces, failed to process and present OVA.



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  		FIGURE 5. Apical and basolateral Ag presentation by polarized uterine epithelial cells to CD4+ DO11.10 memory T cells. Isolated uterine epithelial cells from 12 intact animals were cultured for 8 days on cell inserts (three to four inserts/uterus). Epithelial cells were plated on inserts either above or below the insert membrane as described in Materials and Methods. Following the attainment of high TER values, memory T cells (3.5 x 105 cells/well) along with OVA323–339 peptide (5 µg/ml) were placed inside each cell insert as indicated in the diagrams of cell orientation. [3H]Thymidine was added for the last 24 h of a 3-day incubation. At the end of each experiment, T cells were gently aspirated from inserts for measurement of [3H]thymidine incorporation. Values shown are [3H]thymidine incorporation for APC+T+Ag323–339 peptide as well as control groups APC+T and T+Ag323–339 peptide incubations as mean ± SE (three wells/group). **, Ag presentation at the basolateral surface was significantly (p < 0.01) greater than that seen at the apical surface. Representative of three experiments.

 


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  		FIGURE 6. Presentation of OVA323–339 peptide and OVA Ags to memory T cells by inverted polarized uterine epithelial cells. Isolated uterine epithelial cells from 12 intact animals were cultured for 8 days on cell inserts (three to four inserts/group). Following the attainment of high TER values (2020 ± 248 ohms/well), epithelial cells were incubated with memory T cells (3.5 x 105 cells/well) along with OVA323–339 peptide (5 µg/ml) or OVA (1200 µg/ml) placed inside each cell insert. [3H]Thymidine was added for the last 24 h of a 3-day incubation. Values shown are [3H]thymidine incorporation for APC+T+Ag as well as control groups, including APC+T and T+Ag incubations as mean ± SE. **, Ag presentation at the basolateral surface was significantly (p < 0.01) greater than that seen in controls. Representative of five experiments.

 
In a series of experiments (data not shown), we found that variations in the pore size of inserts (0.4–3.0 µm), coating inserts with different dilutions of Matrigel (1:1–1:4) as well as varying the concentration of OVA used (300–1200 µg/ml), had no effect on the ability of polarized cells to present Ags at either surface. In other studies (data not shown), we found by flow cytometry that whereas fresh epithelial cells and splenocytes are positive for B7.1 and B7.2, 4–13 and 4–9%, respectively, we were unable to detect any evidence of staining by confocal microscopy of polarized epithelial cells grown to confluence on cell inserts. Cells were B7.1 and B7.2 negative irrespective of whether they were incubated with Abs at the apical or basolateral surface as either live or fixed cells. These findings suggest that in the absence of underlying stromal cells and/or estradiol and progesterone, epithelial cell Ag processing is compromised, possibly due to deficiencies in costimulatory molecule expression.

Influence of CSM on Ag presentation by polarized uterine epithelial cells

In previous studies with polarized uterine epithelial cells, we found that the presence of mouse uterine stromal cells or CSM had pronounced effects on epithelial cell TER and TNF-{alpha} secretion in culture (34). When stromal cells and/or CSM were placed in the basolateral chamber below epithelial cells for 24–48 h, epithelial cell TER increased (70% higher than control) and TNF-{alpha} release decreased into both the apical and basolateral chambers (30–50%). These studies indicated that uterine stromal cells produce a soluble factor(s) that regulates epithelial cell TER and release of TNF-{alpha} without effecting TGF-{beta} release. To determine whether uterine stromal cells communicate with epithelial cells via a soluble factor(s) to affect epithelial Ag presentation, epithelial cells were grown upright on cell culture inserts before being transferred to plates containing CSM in the basolateral compartment. As seen in Fig. 7, culture for 24 h before addition of memory T cells and OVA323–339 peptide increased epithelial cell Ag presentation ({approx}90% higher than control). These studies indicate that uterine stromal cells produce a soluble factor(s), which enhances epithelial cell Ag presentation. In other experiments (data not shown), CSM, when added to epithelial cells along with OVA, failed to reverse the inability of polarized cells to present Ags. Overall, these results suggest that uterine stromal cells communicate with epithelial cells via a soluble factor(s) to regulate Ag presentation at apical surfaces in a way similar to that seen with its effect on TER (34, 36).



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  		FIGURE 7. Effect of epithelial cell culture with CSM on Ag presentation at the apical surface. Epithelial cells from 12 intact animals were grown on cell inserts (three to four inserts/treatment group) to confluence and transferred to wells containing CSM (500 µl; diluted 1:1 with fresh medium) in the basolateral compartment, as described in Materials and Methods. Coincident with insert transfer, memory T cells (3.5 x 105 cells/well) along with OVA323–339 peptide (5 µg/ml) were placed inside each cell insert (300 µl). Following 2 days of incubation, [3H]thymidine was added, and cells were incubated for an additional 24 h. At the end of each experiment, T cells were gently aspirated from inserts for measurement of [3H]thymidine incorporation. Results are shown as mean ± SE. **, Ag presentation by epithelial cells incubated with CSM in the basolateral compartment was significantly (p < 0.01) greater than that seen with control groups. Representative of six experiments.

 
Effect of HGF on uterine epithelial cell Ag presentation in culture

HGF, a mesenchymal growth factor made by stromal fibroblasts (44), is known to stimulate mammary epithelial cell proliferation (35). More recently, we found that HGF, when added to the basolateral chamber of polarized uterine epithelial cells, increased TER, while at the same time, decreased apical TNF-{alpha} release (36). When epithelial cells were incubated with anti-HGF or anti-HGFR Ab before HGF or CSM, HGF effects on TER, but not TNF-{alpha} release, were blocked. These findings prompted us to ask whether HGF might be the factor in CSM that affects the ability of epithelial cells to present OVA323–339 peptide to OVA-specific memory T cells. As seen in Fig. 8, when HGF was added to the basolateral surface of epithelial cells grown to high TER on inserts, we found that Ag presentation of OVA323–339 peptide to memory T cells increased significantly beyond that seen with control cells (no HGF). The stimulatory effect of HGF was observed when T cells were placed on the apical as well as the basolateral surfaces of epithelial cells.



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  		FIGURE 8. Effect of HGF on apical and basolateral Ag presentation by epithelial cells. Epithelial cells from 16 intact mice were grown to confluence on apical or basolateral cell insert surfaces. On day 7 of incubation, inserts were transferred to plates containing 500 µl of HGF (100 ng/ml) in the basolateral compartment (three to four inserts/group). At the time of insert transfer, memory T cells (3 x 105 cells/well) along with OVA323–339 peptide (5 µg/ml) were placed inside each cell insert, as indicated in the accompanying figures. [3H]Thymidine was added for the last 24 h of a 3-day incubation, after which T cells were recovered for measurement of [3H]thymidine incorporation. *, Ag presentation (APC+T+peptide+HGF) by epithelial cells incubated in the presence of HGF was significantly (p < 0.05) greater than epithelial cells (APC+T+peptide). Representative of three experiments.

 
To determine whether HGF has a stromal cell-like effect on epithelial cell Ag presentation, epithelial cells were grown to confluence before being incubated in the presence of CSM or HGF. Fig. 9 demonstrates that basolateral addition of HGF (100 ng/ml) for 48 h significantly increases Ag presentation by epithelial cells at the apical surface, as does CSM. As seen in Fig. 9, preincubation of CSM with anti-HGF Ab (5 µg/ml) for 30 min before placement in the basolateral compartment of polarized epithelial cells failed to block the stimulatory effect of CSM on Ag presentation. Addition of anti-HGF Ab to HGF (50 ng/ml) and CSM completely blocked the stimulatory effect of each on TER (data not shown). Overall, these studies indicate that, whereas HGF is the growth factor important for regulating epithelial cell integrity as measured as TER, stimulation of Ag presentation by polarized epithelial cells appears to involve a stromal cell signal(s) other than HGF.



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  		FIGURE 9. Lack of an effect of anti-HGF Ab on the stimulatory effect of CSM on Ag presentation at the apical surface of polarized uterine epithelial cells. Uterine epithelial cells were isolated from 16 mice and cultured for 5 days. After polarized epithelial cells reached high TER inserts, cells were incubated in control medium or transferred into wells containing HGF (100 ng/ml), CSM (diluted 1:1 with fresh medium), or CSM + goat anti-HGF (5 µg/ml; preincubated for 30 min before insert transfer). Epithelial cells were cocultured with memory T cells (3 x 105 cells/well) along with OVA323–339 peptide (5 µg/ml) placed inside each cell insert for 3 days with [3H]thymidine added for the last 24 h. T cells were recovered for measurement of [3H]thymidine incorporation. The results are shown as the mean ± SE. **, Ag presentation by epithelial cells incubated with HGF, CSM, and CSM+Ab were significantly (p < 0.01) higher than control cells. Representative of two experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The research presented demonstrates that uterine epithelial cells, as well as APCs in the uterine stroma and vagina, process and present recall Ags to memory T cells. Furthermore, using naive T cells from DO11.10 mice, these studies indicate that epithelial cells and APCs in the uterine stroma are able to process and present Ags to naive CD4+ T cells. These findings also indicate that polarized epithelial cells present Ags and that Ags are preferentially presented at the basolateral surface relative to that seen when memory T cells are in contact with the apical surface. Lastly, we found that CSM and HGF stimulate epithelial cell Ag presentation but that factor(s) in CSM, in addition to HGF, play a regulatory role in epithelial cell Ag presentation. These findings suggest a key role for stromal cells in regulating Ag presentation by uterine epithelial cells.

In the current study, we asked whether epithelial cells in the uterus are able to present Ags to naive T cells. We found that freshly isolated epithelial cells, as well as APCs in the uterine stroma, are able to process and present Ags as determined by coincubation of APCs with naive T cells from non-OVA-primed mice as well as T cells selected by flow cytometry. Others have shown that lung dendritic cells stimulate naive Ag-specific T cells (39). What was unexpected was their finding that lung B cells were unable to present Ags. These findings indicate that APCs at mucosal surfaces must be analyzed on a case-by-case basis to evaluate Ag presentation function.

Our findings that uterine epithelial cells process and present Ags to naive and memory T cells take on a new importance with the recognition that the uterine lumen is not a sterile environment but rather a site at which bacteria and viruses move continuously from vagina to uterus to the Fallopian tubes (45). This conclusion is based in part on migration studies showing that sperm deposited in the vagina reach the Fallopian tubes within minutes (46). More recently, Parsons et al. (45) demonstrated that radio-opaque dye is seen in the uterine lumen within 2 h of being placed in the vagina. In this study, dye traveled from vagina to uterus irrespective of stage of the menstrual cycle, the use of oral contraceptives, or whether women were pre- or postmenopausal (45). Our studies suggest, that in addition to functioning as a part of the innate immune system, epithelial cells have the capacity to initiate an adaptive immune response under conditions in which pathogenic challenge exceeds the innate immune protection afforded by epithelial cells. For review, see Ref. 47 .

One of the principal aims of this study was to examine the ability of polarized uterine epithelial cells to present Ag to CD4+ memory cells. To test the capacity of epithelial cells to present Ags, epithelial cells were grown to confluence on either the upper or lower surface of cell inserts. Following formation of tight junctions, T cells and OVA323–339 peptide were added to one surface or the other. We initially used OVA323–339 peptide to bypass processing to directly measure Ag presentation to memory T cells. We found that presentation of Ags was significantly greater at the basolateral surface than at the apical surface. These findings extend the important observations of Hershberg et al. (21) who demonstrated preferential basolateral presentation with a human IEC line that expressed HLA-DR. To the best of our knowledge, our findings represent the first demonstration of Ag processing by primary epithelial cells.

What was unexpected in our study was the failure of polarized epithelial cells to process Ags. In contrast, Hershberg et al. (21) reported that polarized IEC process as well as present Ags to HLA-DR-restricted T cells. One explanation for this difference may be that because epithelial cells from the reproductive tract are under hormonal control, the presence of estradiol and/or progesterone may be essential for processing to occur. Previously, we reported that Ag presentation by epithelial cells from estradiol-treated rats is 2- to 3-fold greater than that seen with epithelial cells from ovariectomized (hormone depleted) controls (26, 27). Because polarized cells growth to confluence takes 7–10 days and occurred in our studies in the absence of CSM or physiological concentrations of hormone, the presence of these hormones or CSM factors may be critical for processing to occur.

Our findings in the present study that Ag presentation by polarized epithelial cells increases when CSM is added to the basolateral chamber, to the best of our knowledge, is the first demonstration that stromal cells play are important in regulating Ag presentation by epithelial cells in the uterine lumen. Recognition of epithelial-stromal communication grew out of the pioneering studies of Cunha and Korach (30, 48). Using estradiol receptor knockout mice in uterine cell reconstitution experiments, they demonstrated that estradiol-induced uterine epithelial cell proliferation is mediated through estradiol receptors in stromal fibroblasts rather than through receptors in epithelial cells (48). More recently, we found that epithelial cell integrity, measured as high TER, and cytokine secretion by epithelial cells are influenced by underlying stromal cells and by CSM (34, 49, 50). Just how Ag presentation is affected remains to be determined and is under investigation in our laboratory.

In addition to CSM stimulating epithelial cell Ag presentation, we found that HGF added to the basolateral chamber had a pronounced stimulatory effect on Ag presentation. Molecules such as HGF are produced by the uterine stroma and act via epithelial receptors to affect changes in epithelial cells (48, 51, 52). Studies by Zhang et al. (35) identified HGF as the stromal mediator of estrogen-induced epithelial proliferation in the mouse mammary gland. More recently, we found that HGF increases TER, while at the same time, decreases apical TNF-{alpha} release (36). When epithelial cells and/or stromal cells were incubated with anti-HGF or anti-HGFR Ab before HGF, HGF effects were blocked, indicating that epithelial cells express the receptor for HGF and that HGFR mediates the effects of HGF on TER and TNF-{alpha}. Based on the neutralization of CSM with HGF- and HGFR Abs, we concluded that stromal cell regulation of epithelial cell TER, but not TNF-{alpha} secretion, is mediated through the secretion of stromal HGF. In the present study, we tested HGF in our polarized Ag presentation assay and found that it stimulates Ag presentation. Because anti-HGF Ab added to CSM had no effect, these findings indicate that factor(s), in addition to HGF, affect epithelial cell Ag presentation and as well as cytokine secretion by epithelial cells. Others have shown that stromal cells produce a number of growth factors, including keratinocyte growth factor, epidermal growth factor, and insulin-like growth factor 1, which regulate epithelial cell proliferation (53, 54, 55, 56). These studies, as well as our own findings, suggest that epithelial cells are responsive to multiple stromal signals that act either alone or in combination to affect cell proliferation, barrier function, cell secretion, and Ag presentation.

The present studies demonstrate that uterine epithelial cells present Ag. What remains to be determined is to whom Ag is presented in situ. That epithelial cells make contact with T cells as well as other immune cells in the stroma was demonstrated by Komuro (57, 58) and Hashimoto (58), who identified fenestrations (pores) in the basement membrane immediately below the epithelia in the intestine. Scanning electron microscopy indicated that these fenestrations, which are circular to oval in shape and are 0.5–5 µm in diameter, were passages for migrating cells of the immune system such as lymphocytes, eosinophils, and macrophages. Of equal importance was the identification of protrusions from the basal parts of epithelial cells that passed through these fenestrations into the lamina propria to make physical contact with underlying immune cells. In other studies, they demonstrated the penetration of the epithelial processes and close contact between lymphocytes and so forth and the epithelial cells (59). Our finding that polarized epithelial cells present Ags to T cells placed in contact with the basolateral surface suggest that the anatomical associations seen by Komuro represent a intercellular communication between these different cell types.

In conclusion, our studies indicate that underlying stromal cells markedly influence Ag presentation by epithelial cells in the uterus. These findings further demonstrate that epithelial cells are able to present Ags to naive as well as memory CD4+ T cells. Given the ability of uterine epithelial cells to recruit and activate immune cells through the secretion of cytokines and chemokines in response to potential pathogens, our findings suggest that epithelial cells are in a unique position to recruit cells as part of the innate immune system as well as to present Ags as part of the adaptive immune system.


   Acknowledgments
 
We gratefully thank Dr. James Gorham (Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH) for his assistance in setting the Ag presentation model used in this study. We also express our appreciation to Dr. John Fahey for a critical review of this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI-13541 and AI-34478 and in part by Norris Cotton Cancer Center Support Grant CA-23108. Back

2 Address correspondence and reprint requests to Dr. Charles R. Wira, Department of Physiology, Dartmouth Medical School, Borwell Building, 1 Medical Center Drive, Lebanon, NH 03756-0001. E-mail address: Charles.R.Wira{at}Dartmouth.edu Back

3 Abbreviations used in this paper: IEC, intestinal epithelial cell; HGF, hepatocyte growth factor; TER, transepithelial resistance; CSM, conditioned stromal medium. Back

Received for publication November 16, 2004. Accepted for publication May 16, 2005.


   References
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 Materials and Methods
 Results
 Discussion
 Disclosures
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