Richard M. Rossoll

Influence of the Estrous Cycle on the Presence and Distribution of Immune Cells in the Rat Reproductive Tract

PROBLEM: Previous studies have shown that the uterus and vagina contain cells that can present antigen to ovalbumin‐specific T‐cells. The objective of the present study was to systematically characterize the immune cells [major histocompatibility complex (MHC) class‐II+, macrophages, granulocytes, dendritic cells, and CD8+ cells] present in the uterus and vagina of the rat and to examine their distribution at various stages of the estrous cycle.

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

Estradiol is known to inhibit antigen presentation in the vagina. We report here that TGFβ mediates the action of estradiol on vaginal antigen presenting cells (APC). When vaginal APC from ovariectomized rats were incubated with increasing concentrations of TGFβ1 and TGFβ2 in the presence of ovalbumin-specific T cells and ovalbumin, both TGFβ1 and TGFβ2 inhibited vaginal cell antigen presentation, whereas IL-6, IL-10, and TNFα 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-TGFβ antibody. In contrast, anti-TNFα, anti-IL-6, and anti-IL-10 had no effect on antigen presentation. The effect of anti-TGFβ was seen with vaginal APC from ovariectomized rats treated with estradiol for 1 d as well as 3 d. Finally, analysis of TGFβ in the culture media of vaginal cells from saline- and estradiol-treated rats indicated that the TGFβ produced is biologically active. In response to estr...

Oestradiol regulation of antigen presentation by uterine stromal cells: role of transforming growth factor-β production by epithelial cells in mediating antigen-presenting cell function

We have previously shown that oestradiol treatment of ovariectomized rats for 3 days inhibits antigen presentation by uterine stromal cells at a time when oestradiol increases the numbers of antigen‐presenting cells (APC) in the uterine stroma. In the present study, we found that oestradiol treatment for 1 day is sufficient to inhibit antigen presentation by stromal cells. To define the mechanism(s) of this inhibition, we examined the effect of cytokines and found that exogenous transforming growth factor‐β (TGF‐β) inhibits antigen presentation when stromal cells from saline‐ but not oestradiol‐treated animals are incubated with ovalbumin (OVA)‐specific T cells and OVA. In contrast, antigen presentation by uterine epithelial cells was not affected by TGF‐β. In other studies, the acute inhibitory effect of oestradiol (1 day) on stromal antigen presentation is fully reversed when anti‐TGF‐β antibody is added to the culture media. When given for 3 days, oestradiol inhibition of antigen presentation is partially reversed by anti‐TGF‐β antibody at a time when antibodies to tumour necrosis factor‐α and interleukin‐10 have no effect. To determine whether uterine epithelial cells produce TGF‐β, epithelial cells were grown to confluence on transwell inserts. Our findings indicate that uterine epithelial cells produce biologically active TGF‐β which is preferentially released basolaterally in the direction of underlying stromal cells. When oestradiol is given to ovariectomized rats 1 day before sacrifice, TGF‐β production by epithelial cells increases within 24 hr in culture, relative to saline controls. Taken together, these results suggest that oestradiol inhibition of stromal cell antigen presentation is mediated through the stimulatory effect of oestradiol on TGF‐β production by epithelial cells.

Innate Immunity in the Vagina (Part I): Estradiol Inhibits HBD2 and Elafin Secretion by Human Vaginal Epithelial Cells

Vaginal epithelial cells (VEC) are the first line of defense against incoming pathogens in the female reproductive tract. Their ability to produce the anti‐HIV molecules elafin and HBD2 under hormonal stimulation is unknown.

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

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.

Influence of stage of the reproductive cycle and estradiol on thymus cell antigen presentation.

The objective of this study was to determine whether thymus cells present antigen and if endocrine balance influences antigen presentation. We report here that antigen presenting cells (APC) from the thymus glands of male and female rats, when incubated with ovalbumin (OVA)-specific T cells and OVA, are functionally able to present antigen via MHC class II. To determine whether antigen presentation in the thymus is under hormonal control, tissues from female rats at different stages of the estrous cycle were analyzed. Antigen presentation was higher at estrus and proestrus than that seen at diestrus when estradiol levels are low. Estradiol given to ovariectomized animals for 3 days stimulated antigen presentation by adherent thymus cells compared to saline controls. Flow cytometry studies indicated that the adherent thymus cell preparations consisted of DC, T cells, B cells and cells of the myeloid lineage all of which expressed MHC class II, as did a small population of non-leukocytes. Antibody neutralization studies indicated that thymus cell antigen presentation involves the expression of transmembrane proteins B7.1 and B7.2. These studies demonstrate that sex hormones play a central role in regulating antigen presentation in the thymus.

Modulation of hepatocyte growth factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol.

Citation Coleman KD, Ghosh M, Crist SG, Wright JA, Rossoll RM, Wira CR, Fahey JV. Modulation of Hepatocyte Growth Factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol. Am J Reprod Immunol 2012; 67: 44–53

Estradiol regulation of nucleotidases in female reproductive tract epithelial cells and fibroblasts.

The use of topical and oral adenosine derivatives in HIV prevention that need to be maintained in tissues and cells at effective levels to prevent transmission prompted us to ask whether estradiol could influence the regulation of catabolic nucleotidase enzymes in epithelial cells and fibroblasts from the upper and lower female reproductive tract (FRT) as these might affect cellular TFV-DP levels. Epithelial cells and fibroblasts were isolated from endometrium (EM), endocervix (CX) and ectocervix (ECX) tissues from hysterectomy patients, grown to confluence and treated with or without estradiol prior to RNA isolation. The expression of nucleotidase (NT) genes was measurable by RT-PCR in epithelial cells and fibroblasts from all FRT tissues. To determine if sex hormones have the potential to regulate NT, we evaluated NT gene expression and NT biological activity in FRT cells following hormone treatment. Estradiol increased expression of Cytosolic 5′-nucleotidase after 2 or 4 h in endometrial epithelial cells but not epithelial cells or fibroblasts from other sites. In studies using a modified 5′-Nucleotidase biological assay for nucleotidases, estradiol increased NT activity in epithelial cells and fibroblasts from the EM, CX and ECX at 24 and 48 h. In related studies, HUVEC primary cells and a HUVEC cell line were unresponsive to estradiol in terms of nucleotidase expression or biological activity. Our findings of an increase in nucleotidase expression and biological activity induced by estradiol do not directly assess changes in microbicide metabolism. However, they do suggest that when estradiol levels are elevated during the menstrual cycle, FRT epithelial cells and fibroblasts from the EM, CX and ECX have the potential to influence microbicide levels that could enhance protection of HIV-target cells (CD4+T cells, macrophages and dendritic cells) throughout the FRT.

Innate immunity in the vagina (Part II): Anti-HIV activity and antiviral content of human vaginal secretions.

Whether the concentrations of antiviral proteins, and anti‐HIV activity, within human vaginal secretions change across the menstrual cycle is unknown.

Sex hormone regulation of anti-bacterial activity in rat uterine secretions and apical release of anti-bacterial factor(s) by uterine epithelial cells in culture

In mature female rats, sex hormones regulate the reproductive (estrous) cycle to optimize mating and fertility. During the part of the estrous cycle when mating occurs, and when estrogen is the dominant sex hormone, the uterus is susceptible to infection with bacteria that can be deleterious for survival and fertility. The present study investigated whether sex hormones regulate innate immunity in the female reproductive tract by affecting the secretion of an anti-bacterial factor(s) in the rat uterus. Uterine fluids from intact rats at the proestrous stage of the estrous cycle significantly inhibited Staphylococcus aureus growth. When ovariectomized rats were treated with estradiol, anti-bacterial activity against both S. aureus and Escherichia coli increased in uterine secretions with hormone treatment. In contrast, rats injected with either progesterone and estradiol or progesterone alone displayed no bactericidal activity indicating that progesterone reversed the stimulatory effect of estradiol on anti-bacterial activity. In other studies, isolated uterine epithelial cells from intact animals were grown to confluence and high transepithelial resistance on cell inserts. Analysis of apical secretions indicated that a soluble factor(s) is released by polarized epithelial cells which inhibits bacterial growth. These results demonstrate that sex hormones influence the presence of a broad-spectrum bactericidal factor(s) in luminal secretions of the rat uterus. Further these studies suggest that epithelial cells which line the uterine lumen are a primary source of anti-bacterial activity.