The Stress Hormone Cortisol Enhances Interferon-υ-Mediated Proinflammatory Responses of Human Immune Cells.

BACKGROUND: Cortisol is a prototypical human stress hormone essential for life, yet the precise role of cortisol in the human stress response to injury or infection is still uncertain. Glucocorticoids (GCs) such as cortisol are widely understood to suppress inflammation and immunity. However, recent research shows that GCs also induce delayed immune effects manifesting as immune stimulation. In this study, we show that cortisol enhances the immune-stimulating effects of a prototypical proinflammatory cytokine, interferon-υ (IFN-υ). We tested the hypothesis that cortisol enhances IFN-υ-mediated proinflammatory responses of human mononuclear phagocytes (monocyte/macrophages [MOs]) stimulated by bacterial endotoxin (lipopolysaccharide [LPS]). METHODS: Human MOs were cultured for 18 hours with or without IFN-υ and/or cortisol before LPS stimulation. MO differentiation factors granulocyte-macrophage colony stimulating factor (GM-CSF) or M-CSF were added to separate cultures. We also compared the inflammatory response with an acute, 4-hour MO incubation with IFN-υ plus cortisol and LPS to a delayed 18-hour incubation with cortisol before LPS exposure. MO activation was assessed by interleukin-6 (IL-6) release and by multiplex analysis of pro- and anti-inflammatory soluble mediators. RESULTS: After the 18-hour incubation, we observed that cortisol significantly increased LPS-stimulated IL-6 release from IFN-υ-treated undifferentiated MOs. In GM-CSF-pretreated MOs, cortisol increased IFN-υ-mediated IL-6 release by >4-fold and release of the immune stimulant IFN-α2 (IFN-α2) by >3-fold, while suppressing release of the anti-inflammatory mediator, IL-1 receptor antagonist to 15% of control. These results were reversed by either the GC receptor antagonist RU486 or by an IFN-υ receptor type 1 antibody antagonist. Cortisol alone increased expression of the IFN-υ receptor type 1 on undifferentiated and GM-CSF-treated MOs. In contrast, an acute 4-hour incubation of MOs with IFN-υ and cortisol showed classic suppression of the IL-6 response to LPS. CONCLUSIONS: These results reveal a surprisingly robust proinflammatory interaction between the human stress response hormone cortisol and the immune activating cytokine IFN-υ. The results support an emerging physiological model with an adaptive role for cortisol, wherein acute release of cortisol suppresses early proinflammatory responses but also primes immune cells for an augmented response to a subsequent immune challenge. These findings have broad clinical implications and provide an experimental framework to examine individual differences, mechanisms, and translational implications of cortisol-enhanced immune responses in humans.

Glucocorticoids enhance the in vivo migratory response of human monocytes.

Glucocorticoids (GCs) are best known for their potent anti-inflammatory effects. However, an emerging model for glucocorticoid (GC) regulation of in vivo inflammation also includes a delayed, preparatory effect that manifests as enhanced inflammation following exposure to an inflammatory stimulus. When GCs are transiently elevated in vivo following exposure to a stressful event, this model proposes that a subsequent period of increased inflammatory responsiveness is adaptive because it enhances resistance to a subsequent stressor. In the present study, we examined the migratory response of human monocytes/macrophages following transient in vivo exposure to stress-associated concentrations of cortisol. Participants were administered cortisol for 6h to elevate in vivo cortisol levels to approximate those observed during major systemic stress. Monocytes in peripheral blood and macrophages in sterile inflammatory tissue (skin blisters) were studied before and after exposure to cortisol or placebo. We found that exposure to cortisol induced transient upregulation of monocyte mRNA for CCR2, the receptor for monocyte chemotactic protein-1 (MCP-1/CCL2) as well as for the chemokine receptor CX3CR1. At the same time, mRNA for the transcription factor IκBα was decreased. Monocyte surface expression of CCR2 but not CX3CR1 increased in the first 24h after cortisol exposure. Transient exposure to cortisol also led to an increased number of macrophages and neutrophils in fluid derived from a sterile inflammatory site in vivo. These findings suggest that the delayed, pro-inflammatory effects of cortisol on the human inflammatory responses may include enhanced localization of effector cells at sites of in vivo inflammation.

Estradiol suppresses NF-kappa B activation through coordinated regulation of let-7a and miR-125b in primary human macrophages.

Previous findings suggest that 17beta-estradiol (estradiol) has a suppressive effect on TNF-alpha, but the mechanism by which estradiol regulates TNF-alpha expression in primary human macrophages is unknown. In this article, we demonstrate that pretreatment of human macrophages with estradiol attenuates LPS-induced TNF-alpha expression through the suppression of NF-kappaB activation. Furthermore, we show that activation of macrophages with LPS decreases the expression of kappaB-Ras2, an inhibitor of NF-kappaB signaling. Estradiol pretreatment abrogates this decrease, leading to the enhanced expression of kappaB-Ras2 with LPS stimulation. Additionally, we identified two microRNAs, let-7a and miR-125b, which target the kappaB-Ras2 3' untranslated region (UTR). LPS induces let-7a and inhibits miR-125b expression in human macrophages, and pretreatment with estradiol abrogates these effects. 3'UTR reporter assays demonstrate that let-7a destabilizes the kappaB-Ras2 3'UTR, whereas miR-125b enhances its stability, resulting in decreased kappaB-Ras2 in response to LPS. Our data suggest that pretreatment with estradiol reverses this effect. We propose a novel mechanism for estradiol inhibition of LPS-induced NF-kappaB signaling in which kappaB-Ras2 expression is induced by estradiol via regulation of let-7a and miR-125b. These findings are significant in that they are the first to demonstrate that estradiol represses NF-kappaB activation through the induction of kappaB-Ras2, a key inhibitor of NF-kappaB signaling.

PILAR is a novel modulator of human T-cell expansion.

Robust T-cell responses without autoimmunity are only possible through a fine balance between activating and inhibitory signals. We have identified a novel modulator of T-cell expansion named proliferation-induced lymphocyte-associated receptor (PILAR). Surface PILAR is markedly up-regulated on CD4 and, to a lesser extent, on CD8 T cells on T-cell receptor engagement. In absence of CD28 costimulation, PILAR signaling through CD161 supports CD3 antibody-dependent and antigen-specificT-cell proliferation by increasing the expression of antiapoptotic Bcl-xL and induces secretion of T helper type 1 cytokines. These effects are abrogated by PILAR blockade with specific antibodies, which decrease surface levels of CD28. In contrast, PILAR induces apoptotic death on naive and early activated T cells if CD161 engagement is blocked. PILAR is expressed by approximately 7% to 10% of CD4 T cells in 2 samples of inflammatory synovial fluid, suggesting a potential role in the pathogenesis of joint inflammation. In addition, in the ovarian cancer microenvironment, effector T cells express PILAR, but not CD161, although expression of both can be augmented ex vivo. Our results indicate that PILAR plays a central role in modulating the extent of T-cell expansion. Manipulation of PILAR signaling may be important for treatment of autoimmune diseases and cancer.

Pretreatment with stress cortisol enhances the human systemic inflammatory response to bacterial endotoxin.

OBJECTIVE: There is continuing controversy regarding the effect of glucocorticoids on a systemic inflammatory process. Based ona model of glucocorticoid action that includes both pro- and anti-inflammatory effects, we used the human experimental endotoxemia model to test the hypothesis that a transient elevation of plasma cortisol to stress-associated levels would enhance a subsequent (delayed) systemic inflammatory response to bacterial endotoxin. DESIGN: Prospective, randomized, double-blind, placebo-controlled clinical investigation. SETTING: Academic medical center. SUBJECTS: Thirty-six healthy human volunteers. INTERVENTIONS: Participants were randomized to receive a 6-hr intravenous infusion of saline (control), an intermediate dose of cortisol (Cort80; 6.3 mg/hr/70 kg), or a high dose of cortisol (Cort160; 12.6 mg/hr/70 kg) on day 1. On day 2, participants received an intravenous injection of 2 ng/kg Escherichia coli endotoxin followed by serial measurements of plasma cytokine concentrations. MEASUREMENTS AND MAIN RESULTS: Baseline participant characteristics and cortisol and cytokine concentrations were similar in all three groups. The plasma cortisol response to endotoxemia on day 2 was similar in all three groups. The interleukin-6 response to endotoxemia was significantly increased in the Cort80 Group compared with the control Group (p = .004), whereas the interleukin-10 response was significantly suppressed (p = .034). Corresponding results for the Cort160 Group were not significantly different from control Group values. CONCLUSIONS: In this study, transient elevation of in vivo cortisol concentrations to levels that are observed during major systemic stress enhanced a subsequent, delayed in vivo inflammatory response to endotoxin. This appeared to be a dose-dependent effect that was more prominent at intermediate concentrations of cortisol than at higher concentrations of cortisol.

In vivo exposure to high or low cortisol has biphasic effects on inflammatory response pathways of human monocytes.

BACKGROUND: Recent studies demonstrate that glucocorticoids (GCs) have both supportive (stimulatory) and suppressive effects on immune responses, depending upon the GC concentration. Since some GC effects on inflammation are stimulatory, we hypothesized that acute in vivo GC depletion would decrease inflammatory responses of human monocytes. METHODS: Monocytes were isolated from healthy volunteer participants before and after in vivo treatment with; 1) IV saline, 2) IV high dose hydrocortisone (8 microg x kg(-1) x min(-1)) followed by oral hydrocortisone overnight, and 3) oral RU486 (200 mg at 0400 and 1600 h) to block the intracellular GC receptor and IV etomidate (1.5 mg x kg(-1) x h(-1)) for 12 h to prevent compensatory adrenal cortisol synthesis. Plasma adrenocorticotropic hormone, plasma, and salivary cortisol were measured serially. Monocytes were tested for; 1) cytokine responses, 2) expression of CD163, CD119, and CD54, and 3) mRNA levels of GC-responsive inflammatory mediators. All measurements were made with and without in vitro stimulation of monocytes by lipopolysaccharide. RESULTS: Cortisol and adrenocorticotropic hormone measurements demonstrated effective manipulation of in vivo cortisol. In vivo hypercortisolemia and in vivo GC depletion had reciprocal effects on monocyte mRNA levels of 4 important GC-responsive molecules: 1) GC receptor, CD163, interleukin-10, and suppressor of the cytokine synthesis-3. Monocyte cytokine responses and protein expression were not affected by GC depletion. CD163 expression was increased by hypercortisolemia. CONCLUSIONS: Short-term GC depletion affects mRNA levels of GC-responsive molecules but does not affect monocyte protein expression or cytokine responses.

Up-regulation of human monocyte CD163 upon activation of cell-surface Toll-like receptors.

The hemoglobin (Hb) scavenger receptor, CD163, is a cell-surface glycoprotein that is expressed exclusively on monocytes and macrophages. It binds and internalizes haptoglobin-Hb complexes and has been implicated in the resolution of inflammation. Furthermore, the regulation of CD163 during an innate immune response implies an important role for this molecule in the host defense against infection. LPS, derived from the outer membrane of Gram-negative bacteria, activates TLR4 to cause acute shedding of CD163 from human monocytes, followed by recovery and induction of surface CD163 to higher levels than observed on untreated monocytes. We now report that the TLR2 and TLR5 agonists--Pam3Cys and bacterial flagellin--have similar effects on CD163 surface expression. Up-regulation of CD163 following treatment of human PBMC with TLR2, TLR4, and TLR5 agonists parallels increased production of IL-6 and IL-10, and neutralization of IL-6 and/or IL-10 blocks CD163 up-regulation. Furthermore, simultaneous stimulation of TLR2 or TLR5 in combination with TLR4 activation results in enhanced up-regulation of CD163. It is notable that exogenous recombinant IFN-gamma (rIFN-gamma) suppresses cell-surface, TLR-mediated IL-10 production as well as CD163 up-regulation. Sustained down-regulation of CD163 mediated by rIFN-gamma can be partially rescued with exogenous rIL-10 but not with exogenous rIL-6. This divergent regulation of CD163 by cytokines demonstrates that human monocytes react differently to infectious signals depending on the cytokine milieu they encounter. Thus, surface CD163 expression on mononuclear phagocytes is a carefully regulated component of the innate immune response to infection.

Pivotal advance: activation of cell surface Toll-like receptors causes shedding of the hemoglobin scavenger receptor CD163.

The hemoglobin scavenger receptor (HbSR) CD163 is a monocyte/macrophage-specific glycoprotein that binds and facilitates uptake of haptoglobin-hemoglobin (Hp-Hb) complexes, which are rapidly formed in the circulation upon hemolysis of red blood cells. Hemolysis can be caused by a diverse range of infectious agents and provides pathogens a source of iron to enhance their survival and replication. Previous work demonstrated that lipopolysaccharide (LPS) activates monocytes to cleave cell-bound HbSR into a soluble mediator that retains the capacity to bind Hp-Hb complexes. We report that blocking LPS activation of Toll-like receptor 4 prevents LPS-mediated shedding of CD163. Furthermore, activation of two other cell surface Toll-like receptors (TLR), TLR2 and TLR5, induces shedding of the HbSR from human monocytes. In contrast, treatment of monocytes with intracellular TLR3, TLR7, and TLR9 agonists failed to cause HbSR shedding, suggesting that this shedding event is selective to cell surface TLR activation. These data demonstrate that the soluble HbSR is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections.

TGF-beta regulation of human macrophage scavenger receptor CD163 is Smad3-dependent.

Tight regulation of the inflammatory response is essential for the maintenance of physiologic homeostasis. A potentially important mediator of this process is CD163, a macrophage-specific member of the scavenger receptor cysteine-rich family. CD163 surface expression is up-regulated by glucocorticoids and the anti-inflammatory cytokine interleukin-10, and CD163 is shed acutely from the cell surface in response to lipopolysaccharide. We now demonstrate that transforming growth factor-beta (TGF-beta) markedly reduces expression of CD163. Treatment of primary human monocytes with TGF-beta inhibited basal as well as dexamethasone-induced CD163 mRNA and protein expression. De novo protein synthesis was not required for this inhibition, suggesting that TGF-beta regulates CD163 expression transcriptionally. To delineate this transcriptional regulation, a 2.5-kb fragment of the CD163 promoter was isolated. This promoter was inhibited by TGF-beta, and suppression was dependent on Smad3 expression. These results define a novel function for TGF-beta and implicate an important role for CD163 in the host response to inflammation.

Cross-linking of FcgammaR triggers shedding of the hemoglobin-haptoglobin scavenger receptor CD163.

CD163, the hemoglobin (Hb)-haptoglobin scavenger receptor, is a monocyte/macrophage-restricted member of the scavenger receptor, cysteine-rich family of proteins. In addition to being expressed on the cell surface, a soluble form of CD163 has also been reported. Like tumor necrosis factor alpha (TNF-alpha), surface CD163 is proteolytically cleaved from the plasma membrane in response to lipopolysaccharide (LPS) stimulation. As cross-linking of the Fcgamma receptor (FcgammaR) is similarly known to induce TNF-alpha shedding, the effect of FcgammaR stimulation on CD163 shedding was investigated. We found that FcgammaR stimulation resulted in a rapid release of surface CD163 into the supernatant that was blocked by inhibitors of protein kinase C and tyrosine kinases. Although LPS and FcgammaR stimulation in short-term cultures suppressed CD163 mRNA expression, long-term cultures of monocytes treated with LPS-but not with a FcgammaR cross-linking reagent-resulted in an interleukin-10-dependent recovery of surface CD163 expression. These studies suggest that the presence of immune complexes in infection or autoimmunity may radically alter the nature of CD163-dependent monocyte/macrophage processes. This may be particularly important in disease states in which immune complexes and high levels of free Hb are present, such as in autoimmune hemolytic anemia, transfusion reactions, or infections by hemolytic bacteria.

Endotoxin induces rapid metalloproteinase-mediated shedding followed by up-regulation of the monocyte hemoglobin scavenger receptor CD163.

CD163, a monocyte and macrophage-specific surface glycoprotein, which is increased by interleukin-10 and glucocorticoids, is a scavenger receptor for hemoglobin/haptoglobin complexes. We report a rapid and highly reproducible rise in soluble CD163 in the plasma of human volunteers given intravenous lipopolysaccharide (LPS). We also show that LPS induces shedding of CD163 from the surface of isolated monocytes, identifying shedding from monocytes and macrophages as a likely mechanism for the endotoxemia-associated rise in plasma CD163 in vivo. Studies using the inhibitor TAPI-0 indicate that a metalloproteinase is responsible for LPS-mediated shedding of CD163. Finally, we demonstrate a marked increase in surface CD163 expression on circulating monocytes 24 h following experimental endotoxemia. These findings show that CD163 is rapidly mobilized in response to bacterial endotoxin. As hemoglobin can bind LPS and enhance its toxicity, it will be important to determine how cell surface and soluble CD163 influence inflammatory processes during sepsis.

Increase in plasma and surface CD163 levels in patients undergoing coronary artery bypass graft surgery.

Although haptoglobin polymorphism has been shown to be a genetic risk factor in coronary artery disease, its mechanisms of action are incompletely defined. Recently, a macrophage scavenger receptor for the uptake of haptoglobin-hemoglobin (Hp-Hb) complexes was cloned and designated CD163. Macrophage expression of CD163 is increased by glucocorticoids, IL-10 and IL-6. To better understand the in vivo response of CD163 to an inflammatory stimulus and glucocorticoid treatment, we studied 18 patients who underwent elective coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass (CPB). We report a rapid increase in plasma levels of soluble CD163 by 1 h post-declamping the aorta during CABG surgery with CPB. Furthermore, we demonstrate significant increases in monocyte CD163 on post-operative day 1; 14-fold for patients pre-treated with methylprednisolone and 3-fold for those who did not receive exogenous glucocorticoids. These findings show CD163 to be rapidly mobilized in response to systemic inflammatory stimuli and to be affected significantly by glucocorticoids in vivo. The proposed role of CD163 as a Hp-Hb scavenger and anti-inflammatory molecule, in conjunction with the results of this study, make CD163 an intriguing target for potential manipulation of the acute response to inflammation.

A subset of human uterine endometrial macrophages is alternatively activated.

PROBLEM: Human uterine macrophages must maintain an environment hospitable to implantation and pregnancy and simultaneously provide protection against pathogens. Although macrophages comprise a significant portion of leukocytes within the uterine endometrium, the activation profile and functional response of these cells to endotoxin are unknown. METHOD OF STUDY: Flow cytometric analysis of surface receptors and intracellular markers expressed by macrophages isolated from human endometria was performed. Uterine macrophages were stimulated with LPS. Cytokines, chemokines, and growth factors expressed by these cells were analyzed using Bio-Plex analysis. RESULTS: CD163(high) human endometrial macrophages constitutively secrete both pro- and anti-inflammatory cytokines as well as pro-angiogenic factors and secretion of these factors is LPS-inducible. CONCLUSION: A major population of human uterine macrophages is alternatively activated. These cells secrete factors in response to LPS that are involved in the activation of immune responses and tissue homeostasis.

Cortisol exerts bi-phasic regulation of inflammation in humans.

Natural and synthetic glucocorticoids (GCs) have been used for decades to suppress inflammation. In this paper, we re-examine the role of the endogenous GC, cortisol, as a primary homeostatic regulator of the human inflammatory response to injury. Our data show that cortisol regulation of innate immunity can be both pro-inflammatory and anti-inflammatory. Using a human model of in vivo cortisol depletion, we first show that baseline (diurnal) cortisol concentrations do not exert an anti-inflammatory effect. This is the first clue that cortisol regulation of inflammation is not represented by a linear dose-response relationship. We next show in surgical patients that cortisol does exert an acute anti-inflammatory effect over a carefully regulated range of physiologic cortisol concentrations. Finally, transient pre-treatment of healthy humans with cortisol induces a bi-phasic response during a later, delayed systemic inflammatory response: an intermediate cortisol concentration augments inflammation while a high cortisol concentration is neither pro- nor anti-inflammatory. Based on these findings and the work of others, we propose a new paradigm that identifies cortisol regulation of human inflammation as both dualistic-it is pro- and anti-inflammatory-and dynamic, it evolves over time.

Hydrocortisone at stress-associated concentrations helps maintain human heart rate variability during subsequent endotoxin challenge.

PURPOSE: We evaluated the differential impact of stress-associated vs high pharmacologic concentrations of hydrocortisone pretreatment on heart rate variability (HRV) during a subsequent systemic inflammatory stimulus. MATERIALS AND METHODS: Healthy volunteers were randomized to receive placebo (Control) and hydrocortisone at 1.5 µg/kg per minute (STRESS) or at 3.0 µg/kg per minute (PHARM) as a 6-hour infusion. The STRESS dose was chosen to replicate the condition of physiologic adrenal cortical output during acute systemic stress. The PHARM dose was chosen to induce a supraphysiologic concentration of cortisol. The next day, all subjects received 2 ng/kg Escherichia coli endotoxin (lipopolysaccharide). Heart rate variability was analyzed with the statistic approximate entropy (ApEn). A lower ApEn correlates with decreased HRV. RESULTS: At the 3-hour nadir, the decrease in ApEn in the STRESS group was significantly less compared to placebo (P < .03), whereas ApEn in the PHARM group was not statistically different. We also found that the maximal decrease in ApEn preceded maximal increase in heart rate in all groups. The decrease in R-R interval was maximal at 4 hours, whereas the ApEn nadir was 1 hour earlier at 3 hours. CONCLUSIONS: Pretreatment with a stress dose of hydrocortisone but not a higher pharmacologic dose maintained a significantly higher ApEn after endotoxin exposure when compared to a placebo. In addition, decreases in ApEn preceded increases in heart rate.

Human uterine epithelial cell secretions regulate dendritic cell differentiation and responses to TLR ligands.

The balance between immunity and tolerance in the endometrium is governed by dynamic interactions of UEC and immune cells including DC. In this study, we tested the hypothesis that soluble immune mediators secreted by UEC modulate the differentiation and functions of human DC. We found that DC differentiated with CM from polarized UEC (i.e., CM-DC) expressed significantly lower surface CD86. Upon activation with LPS or PIC, the expression of CD80, CD86, and CD83 was decreased significantly on CM-DC relative to Con-DC. Further, mRNA for TLR3, TLR4, and TLR5 was decreased significantly in CM-DC relative to Con-DC. As a functional read-out of the effect of CM on DC, we determined the following parameters: First, analysis of cytokine production showed that when compared with Con-DC, CM-DC responded to LPS or PIC stimulation with enhanced IL-10 production but undetectable IL-12p70 secretion. Second, RT-PCR analysis showed that CM-DC significantly expressed higher mRNA for IDO, an immune tolerance-promoting enzyme. Lastly, in a MLR assay, CM-DC induced significantly lower allogeneic proliferative responses compared with Con-DC. These findings indicate collectively that epithelial cells confer a tolerogenic phenotype to DC in the endometrium. Our results suggest novel cellular and molecular mechanisms for the regulation of adaptive immunity within the FRT.

Estradiol regulates expression of estrogen receptor ERalpha46 in human macrophages.

BACKGROUND: Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17beta-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we quantify expression of ERalpha and ERbeta in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERalpha variant, ERalpha46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERalpha in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERalpha expression in macrophages but has no effect on ERbeta levels in either cell type. During monocyte-to-macrophage differentiation, ERalpha is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERalpha expression levels parallel the ability of E2 to suppress CXCL8 production. CONCLUSIONS/SIGNIFICANCE: This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERalphap46, which is estradiol-inducible. This is mediated through usage of the ERalpha F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERalpha expression and regulation in the ability of E2 to modulate innate immune responses.

Functional expression of pattern recognition receptors in tissues of the human female reproductive tract.

The human female reproductive tract (FRT) must balance the requirements of procreation with the demands of protection from pathogen invasion. We hypothesize that the FRT expresses functional pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) proteins that may mediate these tasks. Expression of PRRs was evaluated in FRT tissues by RT-PCR. PRR function within FRT tissue cells was determined by CXCL8 (IL-8) production in response to treatment with PRR agonists. We now report that TLRs7-9 are expressed in Fallopian tube, uterine endometrium, cervix and ectocervix, while TLR10 expression is restricted to Fallopian tube. NOD1 and NOD2 and the signal transducer RICK were detected in all FRT tissues. Stimulation of FRT tissue cells with PRR ligands resulted in secretion of CXCL8. Results of these studies indicate that PRRs are functionally expressed in FRT tissues, and suggest that these receptors mediate microbial recognition and immune defense in the reproductive tract.

Estradiol attenuates lipopolysaccharide-induced CXC chemokine ligand 8 production by human peripheral blood monocytes.

Regulation of the inflammatory response is imperative to the maintenance of immune homeostasis. Activated monocytes elaborate a broad variety of proinflammatory cytokines that mediate inflammation, including CXCL8. Release of this chemokine attracts neutrophils to sites of bacterial invasion and inflammation; however, high levels of CXCL8 may result in excessive neutrophil infiltration and subsequent tissue damage. In this study, we demonstrate that 17beta-estradiol (E2) attenuates LPS-induced expression of CXCL8 in human peripheral blood monocytes. Treatment of monocytes with estradiol before administration of LPS reduces CXCL8 message and protein production through an estrogen receptor-dependent mechanism, and luciferase reporter assays demonstrate that this inhibition is mediated transcriptionally. Importantly, the ability of estradiol-pretreated LPS-activated monocytes to mobilize neutrophils is impaired. These results implicate a role for estradiol in the modulation of the immune response, and may lead to an enhanced understanding of gender-based differences in inflammatory control mechanisms.

Antigen presentation by human uterine epithelial cells to autologous T cells.

PROBLEM: Epithelial cells, as sentinels of immune protection in the endometrium, use innate immune mechanisms to protect against infection from pathogenic microbes. Our goal in this study was to assess the ability of human uterine epithelial cells to present antigen to cells of the adaptive immune system. METHOD OF STUDY: Highly purified preparations of uterine epithelial cells from 11 patients were assessed for their ability to present tetanus toxoid (TT) to autologous T cells. Leukocyte contamination in the epithelial cell preparations was numerically and functionally determined. Using confocal microscopy, epithelial cells were tested for the expression of CD40 and CD1d. RESULTS: Purified preparations of endometrial epithelial cells isolated from every patient presented TT recall antigen to autologous T cells. Leukocyte contamination of epithelial cell preparations was insignificant. Uterine epithelial cells express CD40 and CD1d. CONCLUSION: Antigen presentation is an additional aspect of uterine epithelial cell function in maintaining women's health.