Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells

Exposure to pathogens induces dendritic cells to release inflammatory cytokines and chemokines. The inflammatory response is controlled by endogenous agents such as anti-inflammatory cytokines, glucocorticoids, anti-inflammatory neuropeptides, and lipid mediators. This study is the first report on the inhibition by prostaglandin E2 (PGE2) of TNF release from bone marrow-derived dendritic cells stimulated with lipopolysaccharide (LPS), a TLR4 ligand, or peptidoglycan, a TLR2 ligand. The inhibition of TNF occurs at both mRNA and protein level. The inhibitory effect of PGE2 is mediated by the EP2 and EP4 receptors, and involves both PKA signaling and mediation by DC-derived IL-10. Intraperitoneal administration of PGE2 together with LPS results in a reduction in serum TNF and intracellular TNF in peritoneal exudate cells, compared to LPS alone. In addition, administration of PGE2 in vivo reduces the numbers of CD11c+ DCc that accumulate in the peritoneal cavity in response to LPS. The various implications of the PGE2-induced reduction in TNF are discussed.     

Human intestinal mast cells are a potent source of multiple chemokines

Mast cells are key effector cells of immediate type allergic reactions. Upon activation they release a broad array of pre-stored and de novo synthesized mediators including immunoregulatory cytokines and chemokines. Here, we analyzed the chemokine profile expressed by mature human mast cells. Human mast cells were isolated from intestinal tissue and cultured with stem cell factor (SCF) in the presence or absence of IL-4 for 10d. Cells were stimulated by cross-linking of the high affinity IgE receptor (FcεRI) and/or by SCF. Chemokine and chemokine receptor mRNA expression was determined by real-time RT-PCR and chemokine release was measured by multiplex bead immunoassay. Out of 43 chemokines and 19 chemokine receptors human intestinal mast cells express 27 chemokines and nine chemokine receptors. Twelve chemokines (CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL18, CCL20, CXCL2, CXCL3, CXCL8, and XCL1) were more than four-fold up-regulated in response to FcεRI cross-linking. Combination of pre-culture with IL-4 and/or stimulation with SCF in addition to FcεRI cross-linking further increased the antigen-dependent expression of mRNA for most chemokines. In contrast, the expression of CCL20, CXCL2, and CXCL3 was strongly inhibited by IL-4 treatment. In conclusion, human intestinal mast cells express a broad spectrum of different chemokines underlining their important role as immunoregulatory cells. Furthermore, combined treatment with IL-4 and SCF increases the antigen-mediated expression and release of multiple chemokines, but IL-4 priming inhibits the expression of CCL20, CXCL2, and CXCL3.     

Chemokines and chemokine receptors expression in the lesions of patients with American cutaneous leishmaniasis

American cutaneous leishmaniasis (ACL) presents distinct active clinical forms with different grades of severity, known as localised (LCL), intermediate (ICL) and diffuse (DCL) cutaneous leishmaniasis. LCL and DCL are associated with a polarised T-helper (Th)1 and Th2 immune response, respectively, whereas ICL, or chronic cutaneous leishmaniasis, is associated with an exacerbated immune response and a mixed cytokine expression profile. Chemokines and chemokine receptors are involved in cellular migration and are critical in the inflammatory response. Therefore, we evaluated the expression of the chemokines CXCL10, CCL4, CCL8, CCL11 and CXCL8 and the chemokine receptors CCR3, CXCR3, CCR5 and CCR7 in the lesions of patients with different clinical forms of ACL using immunohistochemistry. LCL patients exhibited a high density of CXCL10+, CCL4+ and CCL8+ cells, indicating an important role for these chemokines in the local Th1 immune response and the migration of CXCR3+ cells. LCL patients showed a higher density of CCR7+ cells than ICL or DCL patients, suggesting major dendritic cell (DC) migration to lymph nodes. Furthermore, DCL was associated with low expression levels of Th1-associated chemokines and CCL11+ epidermal DCs, which contribute to the recruitment of CCR3+ cells. Our findings also suggest an important role for epidermal cells in the induction of skin immune responses through the production of chemokines, such as CXCL10, by keratinocytes.     

Local production of chemokines and prostaglandin E2 in the acute, chronic and recovery phase of murine experimental colitis

Increased levels of chemokines and prostaglandins have been reported in patients with inflammatory bowel disease, although their changes during disease development are less understood. The aim of this study was to investigate the local production of nine selected chemokines and prostaglandin E(2) (PGE(2)) to elucidate their role in colitis progression in BALB/c and C57BL/6 mice exposed to dextran sulphate sodium. The acute inflammation in both strains was accompanied by a significant up-regulation of CXCL1, CXCL2/3, CXCL10, CCL2, CCL4 and CCL22 and a downregulation of PGE(2). In the recovery phase in BALB/c, one-week post-DSS, PGE(2) levels were significantly increased with a concomitant downregulation of CXCL1, CXCL2/3, CXCL10, CCL2, and CCL4. In contrast, in C57BL/6 mice CXCL1, CXCL2/3, CXCL10, CCL2, CCL3 and CCL4 production remained high during the chronic phase, without any up-regulation of PGE(2). In addition, CCL5 was significantly increased at d26 and 33 compared to d5. Interestingly, the number of macrophages was significantly increased during the acute phase, whereas T cells were significantly increased in both the acute and chronic phase in C57BL/6 mice. Thus, our results show that chemokines are produced in a dynamic manner during colitis progression.     

Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients

Rheumatoid arthritis (RA) is a chronic symmetric polyarticular joint disease that primarily affects the small joints of the hands and feet. The inflammatory process is characterized by infiltration of inflammatory cells into the joints, leading to proliferation of synoviocytes and destruction of cartilage and bone. In RA synovial tissue, the infiltrating cells such as macrophages, T cells, B cells and dendritic cells play important role in the pathogenesis of RA. Migration of leukocytes into the synovium is a regulated multi-step process, involving interactions between leukocytes and endothelial cells, cellular adhesion molecules, as well as chemokines and chemokine receptors. Chemokines are small, chemoattractant cytokines which play key roles in the accumulation of inflammatory cells at the site of inflammation. It is known that synovial tissue and synovial fluid from RA patients contain increased concentrations of several chemokines, such as monocyte chemoattractant protein-4 (MCP-4)/CCL13, pulmonary and activation-regulated chemokine (PARC)/CCL18, monokine induced by interferon-gamma (Mig)/CXCL9, stromal cell-derived factor 1 (SDF-1)/CXCL12, monocyte chemotactic protein 1 (MCP-1)/CCL2, macrophage inflammatory protein 1alpha (MIP-1alpha)/CCL3, and Fractalkine/CXC3CL1. Therefore, chemokines and chemokine-receptors are considered to be important molecules in RA pathology.     

Prostaglandin E2 suppresses lipopolysaccharide-stimulated IFN-beta production

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE(2), which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE(2) on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE(2) strongly suppresses LPS-induced IFN-beta production at the mRNA and protein levels. Poly (I:C)-induced IFN-beta and LPS-induced CCL5 production were also suppressed by PGE(2). The inhibitory effect of PGE(2) on LPS-induced IFN-beta expression is mediated through PGE(2) receptor subtypes EP(2) and EP(4), and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3beta. In contrast, PGE(2) inhibits LPS-induced TNF-alpha production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-beta concentration in mice. Collectively, data demonstrate that PGE(2) is a negative regulator for IFN-beta production in activated macrophages and during endotoxemia.     

Mast Cells Release Chemokine CCL2 in Response to Parkinsonian Toxin 1-Methyl-4-Phenyl-Pyridinium (MPP<Superscript>+</Superscript>)

Microglial activation and release of inflammatory cytokines and chemokines are crucial events in neuroinflammation. Microglial cells interact and respond to other inflammatory cells such as T cells and mast cells as well as inflammatory mediators secreted from these cells. Recent studies have shown that neuroinflammation causes and accelerates neurodegenerative disease such as Parkinson’s disease (PD) pathogenesis. 1-methyl-4-phenyl-pyridinium ion (MPP⁺), the active metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine activates glial cells and mediate neurodegeneration through release of inflammatory mediators. We have shown that glia maturation factor (GMF) activates glia and induces neuroinflammation and neurodegeneration and that MPP⁺ activates mast cells and release proinflammatory cytokines and chemokines. The chemokine (C-C motif) ligand 2 (CCL2) levels have been shown to be elevated and play a role in PD pathogenesis. In the present study, we analyzed if MPP⁺ activates mouse and human mast cells to release chemokine CCL2. Mouse bone marrow-derived mast cells (BMMCs) and human umbilical cord blood-derived cultured mast cells (hCBMCs) were incubated with MPP⁺ (10 µM) for 24 h and CCL2 levels were measured in the supernatant media by ELISA. MPP⁺-significantly induced CCL2 release from BMMCs and hCBMCs. Additionally, GMF overexpression in BMMCs obtained from wild-type mice released significantly more CCL2, while BMMCs obtained from GMF-deficient mice showed less CCL2 release. Further, we show that MPP⁺-induced CCL2 release was greater in BMMCs–astrocyte co-culture conditions. Uncoupling protein 4 (UCP4) which is implicated in neurodegenerative diseases including PD was detected in BMMCs by immunocytochemistry. Our results suggest that mast cells may play role in PD pathogenesis.     

TLR4 signaling in cancer cells promotes chemoattraction of immature dendritic cells via autocrine CCL20

Toll-like receptors (TLRs) are involved in the production of inflammatory mediators upon specific ligands stimuli. Chemokines are important inflammatory mediators capable of chemoattracting diverse immune cells. In addition to normal immune cells, the expression of TLRs and chemokines has been detected in various tumor cells. However, the roles of TLRs and chemokines expressed by tumor cells in the processes of tumor progression and immune escape have not been fully elucidated. Here we report that TLR4 ligation by lipopolysaccharide (LPS) significantly promotes CT-26 colon cancer cells to produce chemokine CCL20 via activation of TLR4 signaling pathways. We find that LPS treatment of CT-26 cells can significantly increase the chemoattraction of immature dendritic cells (DC) by the autocrine CCL20. Our studies suggest that TLR4 expressed by tumor cells may be involved in the induction of chemokines like CCL20, providing a potential linkage between chronic inflammation and tumor immune escape.     

Bryostatin-I: A dendritic cell stimulator for chemokines induction and a promising adjuvant for a peptide based cancer vaccine

Bryostatin-1 (Bryo-1), a PKC modulator, was previously shown to activate monocytes and lymphocytes to produce cytokines. In this report, we investigated the adjuvanticity of Bryo-1 both in vitro and in vivo. First, Bryo-1 was found to induce the release of CCL2 and CCL3 from mouse bone marrow-derived dendritic cells (BMDC) in a dose-dependent manner. As little as 0.1 nM Bryo-I induced release of chemokines from BMDC and the maximal induction could be achieved at 5–10 nM. Both PKC and ERK inhibitors attenuated the release of CCL2 and CCL3. Consistently, Western blot indicated that Bryo-I activated ERK in a dose- and time-dependent manner. Experiments with the NF-κB inhibitor, MG-132, demonstrated that NF-κB was involved in the induction of CCL2 but not CCL3. Because chemokines have been demonstrated to have profound effects on immune reactions by regulating the trafficking of DC and other lymphocytes into lymphoid organs, Bryo-I was tested as an adjuvant in an E7 peptide (MHC class I-restricted peptide epitope derived from human papillomavirus (HPV) 16 E7 protein)-based cancer vaccine. Mice immunized by s.c. injection with Bryo-I/E7 had enlarged draining lymph nodes and showed an antigen specific T-cell response demonstrated by the release of IFN-γ from isolated splenocytes and in vivo CTL activity. Finally, immunization with Bryo-I/E7 totally prevented the E7-expressing TC-1 tumor growth in mice. In conclusion, for the first time, we demonstrated that Bryo-I induced chemokine release from dendritic cell and was an effective adjuvant for peptide cancer vaccine.     

Prostaglandin E2 Represses Interleukin 1 Beta-Induced Inflammatory Mediator Output from Pregnant Human Myometrial Cells Through the EP2 and EP4 Receptors

Inflammatory mediators, including prostaglandins, cytokines, and chemokines, are strongly implicated in the mechanism of human labor, though their precise roles remain unknown. Here we demonstrate that interleukin 1 beta (IL-1beta) significantly increased the expression and release of interleukin-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), and granulocyte macrophage colony-stimulating factor (CSF2) by primary human myometrial cells. However, this effect was repressed by prostaglandin E(2) (PGE(2)). As PGE(2) can activate four distinct PGE(2) receptors (EP(1), EP(2), EP(3), and EP(4)) to elicit various responses, we sought to define the EP receptor(s) responsible for this repression. Using selective EP receptor agonists and a selective EP(4) antagonist, we show that PGE(2) mediates the repression of IL-1beta-induced release of CXCL8, CCL2, and CSF2 via activation of the EP(2) and EP(4) receptors. The use of siRNA gene-specific knockdown further confirmed a role for both receptors. Real-time RT-PCR demonstrated that EP(2) was the most highly expressed of all four EP receptors at the mRNA level in human myometrial cells, and immunocytochemistry showed that EP(2) protein is abundantly present throughout the cells. Interestingly, PGE(2) does not appear to reduce mRNA expression of CXCL8, CCL2, and CSF2. Our results demonstrate that PGE(2) can elicit anti-inflammatory responses via activation of the EP(2) and EP(4) receptors in lower segment term pregnant human myometrial cells. Further elucidation of the EP receptor-mediated signaling pathways in the pregnant human uterus may be beneficial for optimizing the maintenance of pregnancy, induction of labor or indeed treatment of preterm labor.     

Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3^−/− mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.     

Differential recognition and scavenging of native and truncated macrophage-derived chemokine (macrophage-derived chemokine/CC chemokine ligand 22) by the D6 decoy receptor

The promiscuous D6 receptor binds several inflammatory CC chemokines and has been recently proposed to act as a chemokine-scavenging decoy receptor. The present study was designed to better characterize the spectrum of CC chemokines scavenged by D6, focusing in particular on CCR4 ligands and analyzing the influence of NH(2)-terminal processing on recognition by this promiscuous receptor. Using D6 transfectants, it was found that D6 efficiently bound and scavenged most inflammatory CC chemokines (CCR1 through CCR5 agonists). Homeostatic CC chemokines (CCR6 and CCR7 agonists) were not recognized by D6. The CCR4 agonists CC chemokine ligand 17 (CCL17) and CCL22 bound to D6 with high affinity. CCL17 and CCL22 have no agonistic activity for D6 (chemotaxis and calcium fluxes), but were rapidly scavenged, resulting in reduced chemotactic activity on CCR4 transfectants. CD26 mediates NH(2) terminus processing of CCL22, leading to the production of CCL22 (3-69) and CCL22 (5-69) that do not interact with CCR4. These NH(2)-terminal truncated forms of CCL22 were not recognized by D6. The results presented in this study show that D6 recognizes and scavenges a wide spectrum of inflammatory CC chemokines, including the CCR4 agonists CCL22 and CCL17. However, this promiscuous receptor is not engaged by CD26-processed, inactive, CCL22 variants. By recognizing intact CCL22, but not its truncated variants, D6 expressed on lymphatic endothelial cells may regulate the traffic of CCR4-expressing cells, such as dendritic cells.     

Gene expression profiling defines ATP as a key regulator of human dendritic cell functions

Extracellular ATP and PGE2 are two cAMP-elevating agents inducing semimaturation of human monocyte-derived dendritic cells (MoDCs). We have extensively compared the gene expression profiles induced by adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) and PGE2 in human MoDCs using microarray technology. At 6 h of stimulation, ATPgammaS initiated an impressive expression profile compared with that of PGE2 (1125 genes compared with 133 genes, respectively) but after 24 h the number of genes regulated by ATPgammaS or PGE2 was more comparable. Many target genes involved in inflammation have been identified and validated by quantitative RT-PCR experiments. We have then focused on novel ATPgammaS and PGE2 target genes in MoDCs including CSF-1, MCP-4/CCL13 chemokine, vascular endothelial growth factor-A, and neuropilin-1. ATPgammaS strongly down-regulated CSF-1 receptor mRNA and CSF-1 secretion, which are involved in monocyte and dendritic cell (DC) differentiation. Additionally, ATPgammaS down-regulated several chemokines involved in monocyte and DC migration including CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL8/MCP-2, and CCL13/MCP-4. Interestingly, vascular endothelial growth factor A, a major angiogenic factor displaying immunosuppressive properties, was secreted by MoDCs in response to ATPgammaS, ATP, or PGE2, alone or in synergy with LPS. Finally, flow cytometry experiments have demonstrated that ATPgammaS, ATP, and PGE2 down-regulate neuropilin-1, a receptor playing inter alia an important role in the activation of T lymphocytes by DCs. Our data give an extensive overview of the genes regulated by ATPgammaS and PGE2 in MoDCs and an important insight into the therapeutic potential of ATP- and PGE2-treated human DCs.     

The proinflammatory effect of prostaglandin E2 in experimental inflammatory bowel disease is mediated through the IL-23-->IL-17 axis

Although Crohn's disease has been traditionally considered to be Th1-mediated, the newly identified Th17 cells emerged recently as crucial participants. Th1/Th17 differentiation is controlled primarily by the IL-12 family of cytokines secreted by activated dendritic cells (DCs) and macrophages. IL-23 and IL-12/IL-27 have opposite effects, supporting the Th17 and Th1 phenotypes, respectively. We found that PGE(2), a major lipid mediator released in inflammatory conditions, shifts the IL-12/IL-23 balance in DCs in favor of IL-23, and propose that high levels of PGE(2) exacerbate the inflammatory process in inflammatory bowel disease through the IL-23-->IL-17 axis. We assessed the effects of PGE(2) on IL-12, IL-27, and IL-23 and found that PGE(2) promotes IL-23, inhibits IL-12 and IL-27 expression and release from stimulated DCs, and subsequently induces IL-17 production in activated T cells. The effects of PGE(2) are mediated through the EP2/EP4 receptors on DCs. In vivo, we assessed the effects of PGE analogs in an experimental model for inflammatory bowel disease and found that the exacerbation of clinical symptoms and histopathology correlated with an increase in IL-23 and IL-17, a decrease in IL-12p35 expression in colon and mesenteric lymph nodes, and a substantial increase in the number of infiltrating neutrophils and of CD4(+)IL-17(+) T cells in the colonic tissue. These studies suggest that high levels of PGE(2) exacerbate the inflammatory process through the preferential expression and release of DC-derived IL-23 and the subsequent support of the autoreactive/inflammatory Th17 phenotype.     

The direct action of 1alpha,25(OH)2-vitamin D3 on purified mouse Langerhans cells

The action of vitamin D(3) on Langerhans cells (LCs) is not well understood. Using highly purified murine LCs (>95%), we investigated the direct action of 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on their functions. 1,25(OH)(2)D(3) inhibited the expression of cell surface molecules including I-A(d), CD40, CD80, and CD86, leading to impaired ability of LCs to stimulate allogenic T cells in the mixed leukocyte reaction. Furthermore, this reagent inhibited chemotaxis of LCs to CCL21 and their survival. Interestingly, 1,25(OH)(2)D(3) reduced the IL-10 production by LCs, whereas the production of IL-6 and IL-12p40 upon activation by CD40 ligation was enhanced. With regard to inflammatory cytokines and chemokines, 1,25(OH)(2)D(3) upregulated the production of IL-1beta, CCL3, CCL4, and CCL5. The production of Th2-type chemokines, represented by CL17 and CCL22, was inhibited, whereas IFN-gamma-triggered production of Th1-type chemokines, represented by CXCL9, CXCL10, and CXCL11, was augmented. These data indicate that the mode of regulation of cytokine and chemokine production in association with 1,25(OH)(2)D(3) treatment seems to be another characteristic discriminating LCs from classical myeloid dendritic cells.     

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.     

Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C)

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 epithelial 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.