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Endocrinology Vol. 143, No. 8 2872-2879
Copyright © 2002 by The Endocrine Society

ARTICLE

Antigen Presentation by Vaginal Cells: Role of TGFß as a Mediator of Estradiol Inhibition of Antigen Presentation

Charles R. Wira, Marcie A. Roche and Richard M. Rossoll

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

Address all correspondence and requests for reprints to: Dr. C. R. Wira, Department of Physiology, Dartmouth Medical School, Borwell Building, 1 Medical Center Drive, Lebanon, New Hampshire 03756-0001. E-mail: . Charles.R.Wira{at}Dartmouth.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Estradiol is known to inhibit antigen presentation in the vagina. We report here that TGFß mediates the action of estradiol on vaginal antigen presenting cells (APC). When vaginal APC from ovariectomized rats were incubated with increasing concentrations of TGFß1 and TGFß2 in the presence of ovalbumin-specific T cells and ovalbumin, both TGFß1 and TGFß2 inhibited vaginal cell antigen presentation, whereas IL-6, IL-10, and TNF{alpha} had no consistent effect. In other experiments, estradiol-induced inhibition of antigen presentation by vaginal cells was partially reversed when vaginal APC were incubated with anti-TGFß antibody. In contrast, anti-TNF{alpha}, anti-IL-6, and anti-IL-10 had no effect on antigen presentation. The effect of anti-TGFß was seen with vaginal APC from ovariectomized rats treated with estradiol for 1 d as well as 3 d. Finally, analysis of TGFß in the culture media of vaginal cells from saline- and estradiol-treated rats indicated that the TGFß produced is biologically active. In response to estradiol, vaginal cell production of TGFß was significantly greater than that seen with control cells. These studies suggest that estradiol regulation of antigen presentation by vaginal cells is mediated through the local production of TGFß by vaginal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SUCCESSFUL PROTECTION IN the female reproductive tract and at other mucosal surfaces against pathogenic organisms uses the innate and the adaptive immune systems (1, 2). The innate immune system differs from the adaptive immune system in the immediacy of response, the cell types involved (neutrophils, macrophages), and the nature of the response to antigenic challenge. In contrast, the adaptive immune system consists of both humoral and cell-mediated immunity and involves the production of antibodies by B cells as well as the killing of infected cells by T cell and NK cell contact (3). As a part of the adaptive response, potential pathogens (antigens) are internalized by antigen presenting cells (APC), processed, and presented to T cells to initiate humoral and cell-mediated immune protection (4, 5). In the female reproductive tract, the unique requirements for regulation of immune responses have evolved to distinguish allogeneic spermatozoa and/or an immunologically distinct fetus from potential bacterial and viral pathogens (6, 7). To accomplish this, the female reproductive tract has evolved to be responsive both to the constraints of procreation and to the protection of the mother.

The immune system in the female reproductive tract is precisely synchronized throughout the reproductive cycle to optimize conditions for successful reproduction (7). Following release from the ovary, estradiol affects numerous cell-mediated as well as humoral immunological parameters including the movement of IgA and IgG from blood to tissue to uterine secretions and the function of immune cells in the reproductive tract (8, 9, 10, 11, 12). Transport of IgA in the uterus is mediated through the stimulatory effect of estradiol on polymeric Ig receptor (pIgR) mRNA and pIgR, the receptor responsible for transporting IgA from tissue to lumen (13, 14, 15). In the vagina, movement of IgA and IgG as well as expression of pIgR and its mRNA are also under hormonal control (16). In contrast to the uterus, estradiol inhibits the movement of IgA and IgG into vaginal secretions, possibly to prepare for mating when sperm are deposited into the vaginal lumen of the rat (11).

The afferent (antigen recognition) arm of the immune system, specifically the APC in the uterus and vagina, are also under hormonal control (17, 18, 19). In response to estradiol, antigen presentation by uterine epithelial cells is stimulated at a time when antigen presentation by APC in the uterine stroma and vagina is inhibited (19). Responses at both sites are mediated by estradiol and occur during the normal estrous cycle in a way that reflects the changing pattern of endogenous hormones (18, 19). More recently, we have found that inhibition of antigen presentation is complete within 24 h after a single injection of estradiol (20). Because the number of APC in the vagina and the expression of major histocompatibility complex (MHC) class II on these cells remain unchanged, these findings suggest that factors other than cell migration and/or down-regulation of MHC class II are responsible for the inhibition of antigen presentation by estradiol. Moreover, with repeated injections of estradiol (3 d), APC numbers increase in the vagina at a time when inhibition of antigen presentation persists (21).

Several studies indicate that cytokines, which influence antigen presentation, are produced by immune cells within the female reproductive tract. Included in this category are IL-1{alpha}, IL-1ß, TNF{alpha}, IL-6, TGFß, and granulocyte-macrophage colony stimulating factor (GM-CSF) as well as IL-4, IL-5, and IL-10 (22, 23, 24, 25, 26, 27). Some of these cytokines are differentially expressed during the reproductive cycle and following exogenous hormone treatment, indicating that these factors are under hormonal control. For example, in the rat, TGFß expression in the uterus increases in response to steroid treatment and during the periimplantation period of pregnancy (26, 28). Vaginal epithelium has been identified as the major source of TGFß in the mouse (29). In response to diethylstilbesterol (DES), a synthetic estrogen, TGFß1, 2, and 3 mRNAs and products increase sequentially in vaginal tissues between 2 and 24 h post hormone treatment (29). Similarly, TNF{alpha} and IL-6 are produced in the murine and human uterus (24, 30, 31, 32). In contrast, other cytokines such as IL-1 and GM-CSF promote the maturation of Langerhans cells in skin into potent immunostimulatory dendritic cells, whereas cytokines such as TGFß either maintain Langerhans cells in an immature state to limit their ability to activate T cells or stimulate epithelial-associated dendritic cell development and function (33, 34). Other cytokines produced by female reproductive tract cells that may be relevant to antigen presentation include TNF{alpha} and IL-6 (17). In situ hybridization demonstrated that TNF{alpha} mRNA increases in the basalis glandular epithelium in early proliferative and mid- to latesecretory phase, whereas stromal cell mRNA increases in the progesterone dominant phase of the menstrual cycle. IL-6 is expressed in human proliferative endometrium, with progressive increase in the secretory/menstrual phase. Expression is most pronounced in the glandular and surface epithelial cells. The production of IL-6 by isolated endometrial epithelial and stromal cells is inhibited by physiologic concentrations of estradiol (35, 36).

The overall goal of the present study was to examine the mechanism(s) whereby estradiol regulates antigen presentation in the vagina of the rat. Our objectives were to: 1) determine whether TGFß, TNF{alpha}, IL-6, and IL-10 inhibit vaginal antigen presentation; 2) measure the effects of antibody neutralization of cytokines (TGFß, TNF{alpha}, IL-6, and IL-10) on estradiol inhibition of antigen presentation by vaginal cells; and 3) evaluate whether, under the conditions of hormone treatment, TGFß is made by vaginal cells in culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General procedures
Female adult Lewis rats weighing 150–200 g were purchased from Charles River Breeding Laboratories (Kingston, NY). Animals were maintained in a constant-temperature room with fixed light/dark intervals of 12 h each and allowed food and water ad libitum. Rats were ovariectomized 7 d before each experiment. Animals were killed by decapitation, and vaginal tissues were recovered for isolation of vaginal cells. All procedures involving animals were conducted after approval of the Dartmouth College Institutional Animal Care and Use Committee.

Preparation of vaginal cells
Vaginal cells from ovariectomized and hormone-treated rats were prepared by incubation of vaginal tissues (2 ml/vagina) with 50,000 U trypsin (Sigma, St. Louis, MO) per ml pancreatin (25 mg/ml; Life Technologies, Inc., Grand Island, NY) for 60 min at 4 C and for 60 min at 20 C with constant rotation (120 rpm) before separation by mesh sieve (250-µm pore size) as previously described (20). Cells were aspirated through 18- and 20-gauge needles to prepare isolated cells before resuspension in complete Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Inc.) containing 25 mM HEPES supplemented with 10% fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT), 5% NCTC-109 (BioWhittaker, Inc., Walkersville, MD), 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin.

Antigen presentation assays
To measure antigen presentation, ovalbumin (OVA)-specific T cells (T) (1 x 105 cells/100 µl) in complete RPMI 1640 medium were cultured in triplicate wells in 96-well flat-bottom microtiter plates with irradiated vaginal APC (1 x 105 cells/100 µl) in the presence of OVA (50 µl, 300 µg/ml final concentration) (APC + T + OVA) (19). OVA-specific T cell lines for in vitro studies were prepared from the lymph nodes of Lewis rats injected with OVA-complete Freund’s adjuvant emulsion into the hind footpads (100 µg OVA in 100 µl PBS plus 100 µl complete Freund’s adjuvant per rat) as previously described (19). Vaginal cells were irradiated before the start of antigen presentation with 4000 rads to prevent their proliferation. Controls included in all experiments were APC incubated with T cells in the absence of OVA (APC + T), APC incubated with OVA (APC + OVA) and T cells incubated with OVA (T + OVA). Following 48 h of incubation at 37 C, T lymphocyte proliferation was measured by 3H-thymidine uptake. Each well received 1 µCi of 3H-thymidine (in 50 µl medium) 20–24 h before the termination of each experiment. Cells in individual wells were transferred onto glass fiber filtermats with a cell harvester (Skatron, Sterling, VA). Radioactivity incorporated into cells was measured in a liquid scintillation counter (Packard, Meriden, CT).

Hormone treatment, antibodies and cytokines
Estradiol-17ß from Calbiochem (La Jolla, CA) was initially dissolved in 100% ethanol, evaporated to dryness, and then resuspended in 0.9% saline as previously described (20). Control animals received only saline. To correct for the alcohol present in the estradiol preparation, an equivalent amount of ethanol was evaporated in flasks used to prepare saline. As determined by previous studies, use of 1–2 µg/rat was determined in dose-response studies showing the effect of estradiol on vaginal immune responses are close to saturation (11, 20). Antibodies were purchased from R&D Systems (Minneapolis, MN). Antibodies (mouse antihuman TGFß1, 2, and 3; mouse antirat IL-6 and anti-TNF{alpha}; and goat antirat IL-10) were used at the concentrations indicated in the figure legends. MOPC-21 (Sigma) and P3 myeloma supernatant (a generous gift from Dr. Michael Fanger, Department of Microbiology and Immunology), both mouse IgG1 isotype, and goat IgG (R&D Systems) were used as isotype controls at the appropriate concentrations. Recombinant human TGFß1 and TGFß2, which are cross-reactive with rat TGFß receptors, as well as rat IL-6, IL-10, and TNF{alpha} were purchased from R&D Systems and used at the concentrations indicated in the figure legends.

Measurement of TGFß
Active (mature) TGFß was measured in a bioassay that uses mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 (PAI-1) promoter fused to the luciferase reporter gene (37). This PAI/L cell line, which measures biologically active TGFß, is based on the ability of TGFß to induce PAI-1 expression, resulting in a dose-dependent increase in luciferase activity. The induction of luciferase activity in this cell line is specific and sensitive to picogram quantities of TGFß (37). Previously frozen transfected mink lung epithelial cells were thawed and washed in cold complete RPMI 1640 medium. Cells were seeded at 1 x 105/100 µl per well in a 96-well flat-bottom white opaque plate (USA Scientific, Inc., Ocala, FL), centrifuged at 800 x g (GC-6R centrifuge with a swinging bucket rotor, Beckman Instruments, Inc., Fullerton, CA) for 15 sec and incubated for 3 h at 37 C in 5% CO2 to allow cells to adhere. Following incubation, cells were centrifuged and medium was replaced with 50 µl fresh medium plus 50 µl of cell culture supernatant or serially diluted TGFß (recombinant human TFGß) for a standard curve. Cells were then incubated for an additional 17–20 h before being washed 2 times in Hanks’ balanced salt solution (100 µl; Life Technologies, Inc.). Cell lysates were prepared by treating cells with 50 µl cell culture lysis reagent (Promega Corp., Madison, WI) for 15 min at room temperature. Luciferase activity of cell lysates was measured using luciferase assay reagent (100 µl; Promega Corp.) added to each well. Illumination was recorded for 10 sec following a 2-sec delay in a Microplate Luminometer model LB96V (EG&G Berthold, Gaithersburg, MD).

Statistics
Data were compared by one-way ANOVA, followed by a Tukey multiple comparison posttest. Differences of P <= 0.05 were considered significant. All values are expressed as mean ±SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of TGFß on antigen presentation by vaginal cells
Previous studies by us have shown that estradiol has an inhibitory effect on antigen presentation by APC in the vagina of ovariectomized rats (18, 20). To explore the possibility that this effect might be mediated through TGFß, ovariectomized animals were treated with either saline or estradiol (2.0 µg/d) for 3 d before being killed 24 h after the third injection. As shown in Fig. 1Go, antigen presentation by vaginal cells is inhibited when animals are treated with estradiol (hatched bars). Thymidine incorporation in the presence of OVA when APC from estradiol-treated rats were incubated with T cells and OVA was significantly lower than that seen with APC from saline-treated animals. To determine the effect of TGFß on antigen presentation, cells from saline- and estradiol-treated animals were assayed for their ability to present antigen in the presence of TGFß (APC + T + O + TGFß). Irrespective of whether vaginal cells were from saline- or estradiol-treated rats, antigen presentation was inhibited in three out of three experiments when TGFß was added to the incubation media.



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  		Figure 1. Effect of TGFß1 on antigen presentation by vaginal cells. Isolated vaginal cells (APC) from ovariectomized rats treated with saline (0.1 ml/d) or estradiol (2 µg/d) for 3 d were prepared as described in Materials and Methods. APC (1 x 105 cells/100 µl) were incubated with OVA-sensitized T cells (T; 1 x 105 cells/100 µl) and OVA (300 µg/ml) for 3 d. TGFß1 (10 ng/ml, final concentration) was added to each well at the start of culture. 3H-thymidine was added for last 24 h of incubation. Values shown are 3H-thymidine incorporation for APC + T + OVA and APC + T incubations as mean ± SE of APC from 7–9 animals/per group (n = 3). *, Significantly (P < 0.05) different from APC + T + OVA, **, Significantly (P < 0.01) different from APC + T + OVA.

 
Response of antigen presentation by vaginal cells to TGFß1 and TGFß2
To examine the effects of different isoforms of TGFß on antigen presentation, isolated vaginal cells were prepared from ovariectomized rats before incubation with T cells and OVA. As shown in Fig. 2Go, when TGFß1 (A) or TGFß2 (B) are added to the incubation media, antigen presentation by vaginal cells is inhibited. This inhibitory effect was observed over a dose range from 0.01–10 ng/ml of TGFß1 and TGFß2. The effects of TGFß on 3H-thymidine incorporation in the absence of OVA (APC + T) were minimal compared with that seen when antigen was present (APC + T + OVA).



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  		Figure 2. Influence of TGFß1 and TGFß2 dose on antigen presentation by vaginal cells from ovariectomized rats. Vaginae were pooled (7–8 rats/group) and cells prepared for incubation with OVA-sensitized T cells and OVA for 3 d along with increasing doses of TGFß1 (A) or TGFß2 (B). 3H-thymidine was added for last 24 h of incubation. Each bar represents the mean cpm ± SE of triplicate wells (n = 2). **, Significantly (P < 0.01) less than control.

 
To determine whether TGFß suppresses antigen presentation by vaginal cells from rats treated acutely with estradiol, vaginal cells were prepared from ovariectomized rats treated with a single injection of estradiol. As seen in Fig. 3AGo, TGFß inhibits antigen presentation by vaginal APC from both saline- and estradiol-treated rats. As was observed with animals treated with estradiol for 3 d (Fig. 1Go), TGFß inhibits antigen presentation beyond that seen with estradiol alone when animals are treated with estradiol for 1 d.



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  		Figure 3. Effect of TGFß1, IL-10, TNF{alpha}, and IL-6 on antigen presentation by vaginal cells from saline- and estradiol-treated rats. Ovariectomized rats received a single injection (100 µl) of estradiol (1.0 µg/rat). Control animals were given saline (100 µl). Twenty-four hours after the injection, animals were killed, vaginal tissues were pooled, and cells were prepared as described in Materials and Methods. TGFß1 (A, 10 ng/ml), IL-10 (B, 100 ng/ml), TNF{alpha} (C, 0.1 ng/ml), and IL-6 (D, 1 ng/ml) were added to cell cultures of APC (1 x 105 cells/100 µl), OVA-sensitized T cells (T; 1 x 105 cells/100 µl) and OVA (300 µg/ml) before incubation for 3 d. 3H-thymidine was added for last 24 h of incubation. Values shown are 3H-thymidine incorporation for APC + T + OVA and APC + T incubations as mean ± SE of APC from 3 wells/per group (n = 1–3). **, Significantly (P < 0.01) different from control (APC + T + O) lacking TGFß.

 
To examine the specificity of this response, antigen presentation by vaginal APC was measured in the presence of several selected cytokines. As seen in Fig. 3Go, when IL-10 (B), TNF{alpha} (C), and IL-6 (D) were added to our antigen presentation assay, no pronounced effects were observed relative to that seen in the absence of these cytokines. Of the cytokines examined, only TGFß (A) consistently inhibited antigen presentation by vaginal cells. In other studies (not shown), each of these cytokines was analyzed over a dose range which is known to be immunologically active.

Effect of endogenous TGFß on antigen presentation vaginal cells
The inhibitory effect of TGFß on antigen presentation by vaginal APC prompted us to examine the possibility that endogenous TGFß production by vaginal cells might be influencing antigen presentation by vaginal cells. To examine this possibility, vaginal cells from ovariectomized rats treated with either saline (0.1 ml) or estradiol (2 µg/d for 3 d) were prepared and incubated with T cells and OVA in the presence or absence of anti-TGFß antibody. Controls used in these studies were matched in terms of isotype and amount of antibody added to the incubation media. As seen in Fig. 4Go, the addition of anti-TGFß antibody enhances antigen presentation by vaginal cells beyond that seen with the isotype control. Enhancement of antigen presentation was observed with APC from saline as well as estradiol-treated animals. Anti-TGFß antibody partially reversed the inhibition of antigen presentation by estradiol in four of four experiments. In contrast, anti-TGFß enhancement of antigen presentation by APC from saline-treated animals was statistically significant in only one of four experiments. This finding further supports our hypothesis that TGFß is being made by vaginal cells. The addition of an isotype control to the antigen presentation assay had no effect on antigen presentation (not shown).



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  		Figure 4. Influence of anti-TGFß1, 2, and 3 antibody on antigen presentation by vaginal cells from saline- and estradiol-treated rats. Ovariectomized rats received three injections daily (100 µl) of estradiol (2.0 µg/rat) or saline (100 µl) before being killed 24 h after the third injection. APC and T cells were incubated with OVA and anti-TGFß antibody or isotype control (1.0 µg/ml) for 3 d, 3H-thymidine was added for last 24 h of incubation. Values shown are mean ± SE of APC from three wells per group (n = 4). **, Significantly (P < 0.01) different from isotype control. ++, Significantly (P < 0.01) lower than saline control.

 
To examine the potential effects of other cytokines on vaginal antigen presentation, vaginal cells from saline- and estradiol-treated animals were analyzed for their ability to present antigen in the presence of antibodies to selected cytokines. As shown in Fig. 5Go, the addition of neutralizing antibodies to IL-6 (5A), TNF{alpha} (5B), and/or IL-10 (5C) had no effect on antigen presentation by vaginal cells irrespective of prior hormone treatment. These experiments, which are representative of two to three experiments each in which a range of antibody concentrations were used (not shown), indicate the specificity of the anti-TGFß neutralizing response seen in these studies. In other experiments to determine whether there might be a synergistic effect when these neutralizing antibodies are added together to the antigen presentation assay, no significant difference was observed when pooled antibodies to TGFß, IL-6, TNF{alpha}, and IL-10 were compared with anti-TGFß antibody alone (not shown).



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  		Figure 5. Lack of influence of anti-IL-6, anti-TNF{alpha}, and anti-IL-10 on antigen presentation by vaginal cells from saline- and estradiol-treated rats. Vaginal cells from saline- (0.1 ml/d) and estradiol-treated rats (2 µg/d for 3 d) were incubated with OVA-specific T cells and OVA, along with anti-IL-6 (A, 1.0 µg/ml), anti-TNF{alpha} (B, 30 µg/ml) and anti-IL-10 antibodies (C, 2.5 µg/ml) or an isotype control (A, B: mouse IgG1, 30 µg/ml and C: goat IgG, 2.5 µg/ml), for 3 d with the addition of 3H-thymidine for the last 24 h. Antigen presentation in the presence of antibody was not significantly different from isotype control (n = 2–3).

 
TGFß production by vaginal cells in culture
To determine whether vaginal cells produce TGFß in culture, cells were prepared from ovariectomized rats that were treated with 1, 2, or 3 daily injections of saline or estradiol (2 µg/d) before being killed 24 h after the last injection. As seen in Fig. 6Go, estradiol treatment in vivo significantly increases the production of TGFß by isolated vaginal cells in culture media beyond that seen with vaginal cells from saline controls measured on d 3 of culture. This time interval was selected to correspond exactly to the optimized time interval used in our antigen presentation experiments (18, 19). Further, as determined by the assay protocol used in these studies (37), we conclude that the TGFß produced by vaginal cells from saline- and estradiol-treated animals is biologically active. These findings provide direct evidence in support of our antibody neutralization experiments, which suggested that TGFß is produced by vaginal cells.



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  		Figure 6. Effect of estradiol on TGFß production by vaginal cells in culture. Isolated vaginal cells (5 x 105 cells/0.9 ml) from ovariectomized rats treated with estradiol (2 µg/d) for 1, 2, and 3 d were prepared and incubated in RPMI 1640 media with 10% fetal bovine serum for 3 d. Following a change of media at 1 and 2 d of culture, media was collected 24 h later (d 3), centrifuged at 10,000 x g for 5 min and stored at -20 C until assayed for TGFß as described in Materials and Methods (n = 4). **, Significantly (P < 0.01) greater than TGFß levels in media from saline controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The research presented demonstrates that estradiol regulation of antigen presentation by vaginal cells is mediated through the actions of TGFß. We show that TGFß addition to vaginal APC inhibits antigen presentation and that its neutralization by antibodies in vitro partially reverses the inhibitory effects of estradiol on antigen presentation. That TGFß is a key regulator is further supported by our finding that estradiol stimulates the production of biologically active TGFß by vaginal cells in culture following hormone treatment of ovariectomized rats. These findings suggest a key role for TGFß in mediating the inhibitory effects of estradiol on the afferent arm of the immune system in the rat vagina.

Previously, we observed that the administration of estradiol to ovariectomized rats inhibits the ability of vaginal cells to present antigen to T cells (18, 20). This inhibition occurred at a time when the number of APC in vaginal tissues either remained constant (1 d) or increased with estradiol treatment (3 d) (20, 21, 38). This apparent paradox prompted us to ask if factors produced locally in the lower reproductive tract, in response to estradiol, might selectively down-regulate antigen presentation as APC migrate into the vagina. Our results indicate that TGFß plays a central role in regulating antigen presentation by vaginal cells. Others have shown that TGFßs are a family of peptides that are involved in wound healing, embryogenesis, and carcinogenesis (39, 40, 41, 42, 43) as well as in a number of immunological parameters including alterations in the function of APC (44, 45, 46). For example, when TGF-ß1 is added to the culture media, dendritic Langerhans cells exhibit a more immature phenotype, lose TNFRIp55 expression and are inhibited from maturing in response to nonspecific signals such as LPS, TNF{alpha}, and IL-1 (47). In other studies, TGFß appears to have a dual role in macrophage function in that it acts initially as a proinflammatory agent to recruit and activate resting monocytes (48). Following differentiation, however, the immunosuppressive actions of TGFß predominate, to lead to the resolution of inflammatory responses. Our studies add a new dimension to the actions of TGFß in that under noninflammatory conditions and in response to hormonal stimulation, antigen presentation by vaginal cells is rapidly inhibited by estradiol.

Antigen presentation by vaginal cells is down-regulated within 24 h of a single injection of estradiol (20). This inhibition parallels our in vivo studies showing that at proestrus, when ovulation is imminent and serum estradiol levels are known to be elevated, vaginal antigen presentation is transiently suppressed for 24–48 h only to rise again at diestrous, when serum progesterone levels are predominant (18). While the physiological significance of these findings remains to be established, our antigen presentation results parallel exactly the time (proestrus/estrus) when sperm would be deposited in the vagina. If APC were functionally capable of processing and presenting antigen at this time, one could postulate that an immune response against sperm antigens might occur with infertility the end result.

Our studies with TGFß and antibodies to TGFß demonstrate that TGFß affects both resident vaginal APC as well as those that traffic into the vagina in response to estradiol (20, 21, 38). Consistent with this hypothesis is our finding that, following estradiol priming in vivo, isolated vaginal cells secrete significantly more TGFß than do vaginal cells from saline-treated controls. What remains to be determined is whether TGFß acts as a secreted cytokine or mediates its effect through cell-to-cell contact. For example, in studies to examine the role of cytokines in tolerance, Strober and colleagues (49) have shown that membrane bound TGFß1 mediates the suppression of T and B cells in the intestinal tract. When stimulated, CD4+CD25+T regulatory cells produce TGFß1 and IL-10 with high levels of TGFß expressed on the cell surface (49). These findings indicate that T cells mediate the suppression of both T and B cell function through cell-to-cell contact, most likely through TGFß receptors on target cells. In contrast, TGFß1 is known to down-regulate the induced expression of both class II and B7-1 on epithelial cells (50). In other studies, APC, following in vitro pulsing with antigen in the presence of TGFß, acquire anterior chamber-associated immune deviation properties (51, 52). That is, APC drive T cells to proliferate and secrete IL-4 but not interferon-{gamma}. These changes, when considered along with the ability of TGFß to block APC-induced apoptotic cell death, suggest that TGFß treatment of APC alters their functional program of costimulation (52). Whether TGFß in the vagina acts by altering APC function or through cell-to-cell contact remains to be determined.

Cytokines other than TGFß are well recognized to regulate antigen presentation. For example, IL-10 is known to be immunosuppressive in some systems but not others (53, 54, 55, 56). Whereas treatment with IL-10 inhibits the ability of Langerhans cells to activate T cells (57), our findings, which complement that reported in the gastrointestinal tract (49), demonstrate that the addition of IL-10 or its removal through antibody neutralization has little or no effect on antigen presentation by vaginal APC. Our studies indicate further that the effects of estradiol on antigen presentation are not mediated through IL-6 or TNF{alpha}, which are produced in the vagina and under hormonal control. We cannot exclude the possibility that other cytokines are involved or that estradiol acts directly on APC in the vagina. That the action of estradiol is not a general systemic effect is suggested by findings that, at a time when vaginal and uterine stromal APC are inhibited, antigen presentation by uterine epithelial cells as well as APC from the spleen [and thymus (our unpublished observation)] is stimulated (17, 19, 58).

The source of TGFß produced by vaginal cells in our studies is most likely the squamous epithelial cells that line the vaginal lumen. Using a combination of techniques including immunohistochemistry, in situ hybridization and Northern RNA analysis, Takahashi et al. (29) demonstrated that TGFß1, 2, and 3 are made by vaginal epithelial cells from immature mice in response to the administration of DES, a potent synthetic estrogen. The expression of the mRNAs for these isoforms was distinct in their time-dependent appearance following hormone treatment. Expression in response to DES was observable within 30 min for TGFß3 mRNA and by 6 h for TGFß1 and TGFß2 mRNAs. In contrast, immunohistochemical analysis of vaginal squamous cells indicated that protein expression of the three isoforms was more prolonged. In support of our hypothesis that estradiol mediates antigen presentation by regulating TGFß production is our finding that secretion of TGFß by isolated cells from the vagina is dependent on estradiol. Of particular interest is our finding that estradiol given in vivo increases TGFß production in vitro at a time that parallels the optimal conditions of our assay for antigen presentation (19, 20). Additional experiments, beyond the scope of this study, are needed to identify the TGFß isoforms produced by rat vaginal cells as well as the optimal times after estradiol stimulation that each is produced. Nevertheless, these findings provide a solid foundation to demonstrate that vaginal cells from adult rats produce TGFß in response to estradiol to influence the local stromal environment of the vagina in which APC are known to be present (20, 38). In preliminary studies, we have found that estradiol inhibits antigen presentation in the uterine stroma by stimulating uterine epithelial cell production of TGFß (Wira, C. R., and R. M. Rossoll, unpublished observation). Whether this is a regulatory pathway for immunomodulation at other mucosal surfaces remains to be determined.

In conclusion, we report that the afferent arm of the mucosal immune system in the rat vagina is under hormonal control. These studies indicate that control of antigen presentation by estradiol in the vagina is most likely mediated through cytokine production rather than direct hormone action on APC. Further, these findings suggest that endocrine balance, which varies with the stage of the menstrual cycle, menopause, the use of oral contraceptives, and hormone replacement therapy, may be important determinants in the recognition and response of the mucosal immune system to potential pathogens.


   Acknowledgments
 
The authors gratefully thank Dr. James Gorham, Department of Pathology, Dartmouth Hitchcock Medical Center for his assistance in setting up the TGFß assay used in this study.


   Footnotes
 
This work was supported by Research Grants AI-13541 and AI-34478 from the NIH and in part by the Norris Cotton Cancer Center Support Grant CA-23108.

Abbreviations: APC, Antigen presenting cells; DES, diethylstilbestrol; GM-CSF, granulocyte-macrophage colony stimulating factor; MHC, major histocompatibility complex; OVA, ovalbumin; PAI-1, plasminogen activator inhibitor-1; pIgR, polymeric Ig receptor; RPMI, Roswell Park Memorial Institute; T, T cells.

Received January 17, 2002.

Accepted for publication April 12, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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