The goal of this study was to examine the role of E 2 in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E 2 on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human β-defensin-2 (HBD2), tumor necrosis factor (TNF)-α, IL-8, and nuclear factor (NF)-kB. When incubated with E 2 for 24–48 h, we found that E 2 stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus , E 2 increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E 2 had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1β on mRNA expression of TNF-α, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E 2 inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E 2 . Further, it suggests that with E 2 regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.

PROBLEM:Uterine epithelial cells produce cytokines that stimulate leukocytes in response to a microbial insult. The goals of this study were to determine if uterine epithelial cells produce the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), and to see if toll-like receptor (TLR) agonists stimulate MIF secretion.METHODS OF STUDY:Human uterine epithelial cells were isolated and grown in cell culture inserts. Levels of MIF secretion were examined by ELISA and MIF messenger RNA (mRNA) expression was examined using real time RT-PCR.RESULTS:Uterine epithelial cells constitutively secrete MIF and exposure to the TLR3 agonist poly (I:C) resulted in enhanced apical secretion of MIF. MIF secretion appeared to be from pre-formed intracellular stores, since exposure of epithelial cells to poly (I:C) had little effect on the expression of MIF-mRNA.CONCLUSIONS:These results demonstrate that uterine epithelial cells constitutively produce MIF and stimulation with poly (I:C) results in enhanced MIF production. This suggests that MIF secretion by uterine epithelial cells may play a critical role in innate immune responses against viral pathogens mediated through TLR3.

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist, poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelia cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.

The objective of this study was to examine the effects of sex hormones on IL-1beta-mediated responses by uterine epithelial cells. The mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells was examined following stimulation with IL-1beta in the presence of estradiol or progesterone. Estradiol inhibited the IL-1beta-mediated mRNA expression and secretion of human beta-defensin-2 and CXCL8 by uterine epithelial cells while progesterone had no effect. Inhibition of the IL-1beta-mediated response by estradiol was dose dependent, with maximal inhibition observed using 10(-7) to 10(-10) M, and was shown to be mediated through the estrogen receptor because addition of a pure estrogen receptor antagonist abrogated this effect. The mechanism by which estradiol inhibits IL-1beta-mediated responses by uterine epithelial cells appears to be the down-modulation of the IL-1R type I, thereby reducing the uterine epithelial cell's ability to respond to IL-1beta. These results suggest that the inhibitory effect of estradiol on IL-1beta-mediated inflammatory responses by uterine epithelial cells indicates a link between the endocrine and immune systems and may be crucial for dampening proinflammatory responses during the time of ovulation or pregnancy.

Uterine epithelial cells express toll-like receptors (TLR) that mediate innate immune responses. A critical regulator of innate immune responses is MIF. This study was performed to measure TLR mRNA expression by the uterine epithelial cell lines ECC-1 and RL95-2 and the effect of TLR agonists on MIF secretion by uterine epithelial cells. These studies demonstrate that ECC-1 and RL95-2 uterine epithelial cells express TLR3 and TLR5 mRNA, while only ECC-1 cells express TLR2 mRNA. Furthermore, only the TLR3 agonist poly (I:C) induced the secretion of MIF by ECC-1 and RL95-2 cells. These results indicate that epithelial cells lining the uterine cavity are sensitive to viral dsRNA and may be crucial to defending the female reproductive tract against viral pathogens.