Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print March 1, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.050690
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/1/131    most recent
biolreprod.106.050690v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.
Agricola
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.
BIOLOGY OF REPRODUCTION 75, 131–139 (2006)
DOI: 10.1095/biolreprod.106.050690
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Expression of Toll-Like Receptors (TLR) and Responsiveness to TLR Agonists by Polarized Mouse Uterine Epithelial Cells in Culture1

Gisela Soboll 3 ,4, Li Shen 5, and Charles R. Wira 2 4

Departments of Physiology4 and of Microbiology and Immunology,5 Dartmouth Medical School, Lebanon, New Hampshire 03756

ABSTRACT

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1–6 by mouse uterine epithelial cells was demonstrated, with TLRs 7–9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam3Cys-Ser-(Lys)4 and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.

female reproductive tract, uterus, vagina

INTRODUCTION

The mucosal immune system in the female reproductive tract has the unique requirements of protecting the host from invasion by bacterial, fungal, and viral pathogens while also allowing successful procreation of a fetal placental unit that is immunologically distinct from the mother. Central to this protection are the uterine epithelial cells, which provide a physical barrier and produce a spectrum of antimicrobials, including complement, lysozyme, lactoferrin, defensins, and secretory leukocyte protease inhibitor [1, 2]. Studies have demonstrated that epithelial cells from the mouse and rat uterus could be grown on cell inserts; following growth to confluence, these cells form tight junctions and preferentially secrete cytokines in a polarized fashion [3–6]. Epithelial cells also secrete an array of chemokines and cytokines, which are crucial for the recruitment and activation of innate immune cells [7, 8]. For example, epithelial cells of the endometrium produce numerous cytokines, including granulocyte-macrophage colony-stimulating factor 2 (CSF2), macrophage colony-stimulating factor (CSF1), tumor necrosis factor A (TNFA), interleukin (IL) 1, IL6, IL8, leukemia-inhibitory factor, and transforming growth factor B [9]. Chemokines produced by epithelial cells that are responsible for recruiting immune cells into the reproductive tract include cysteine-cysteine ligand (CCL) 20/macrophage inflammatory protein (MIP) 3A, CCL5/RANTES (regulated on activation, normal T-cell expressed and secreted), and IL8 [7, 10, 11]. Should pathogenic challenge exceed the capacity of epithelial cells to contain and control pathogens, then epithelial cells as well as macrophages and dendritic cells signal the adaptive immune system.

To identify pathogens, the innate immune system uses germ line-encoded, Toll-like receptors (TLRs), which recognize microbial antigens commonly referred to as pathogen-associated molecular patterns (PAMPs) [12–14]. Toll-like receptors are expressed on immune cells (lymphocytes, macrophages, and dendritic cells) as well as on epithelial cells [15]. In some cases, recognition of a specific microbe, such as Chlamydia sp., occurs through multiple receptors and adaptors [16], allowing unique downstream events and induction of genes that function in host defense [17]. These include genes for antimicrobials, such as defensins and secretory leukocyte protease inhibitor. In some cases, upregulation of genes encoding major histocompatibility complex and costimulatory molecules, as well as secretion of cytokines and chemokines, can occur [18]. To date, 11 TLRs have been identified in mammals [19, 20]. The TLR2 has the ability to build heterodimers with TLR1 and TLR6 and is able to recognize a broad range of microbial products and components, including gram-positive bacteria, mycobacteria, peptidoglycan, zymosan, or Pam3Cys-Ser-(Lys)4 (Pam3Cys) [21]. The TLR3 recognizes double-stranded RNA [22], and TLR4, in association with CD14 and LY96/MD-2, recognizes gram-negative bacteria [23]. The TLR5 recognizes bacterial flagellin [24], and TLR7 and TLR8 recognize single-stranded RNA and some synthetic components [25, 26]. The TLR9 recognizes CpG oligonucleotide type A (CpG) [27]. Recently, TLR11 was shown to recognize parasite-associated, profilin-like molecules [28, 29], whereas TLR10 remains an orphan receptor [17].

Much attention has been paid to TLR expression and function in airway epithelial cells [30] and intestinal epithelial cells [31, 32], but few studies have investigated TLR expression and function in the female reproductive tract. Human vaginal and cervical epithelial cells have been reported to express TLRs 1–3, 5, and 6 [33]. Pioli et al. [34] reported the constitutive expression of TLRs 1–6 as well as MYD88 and CD14 in human fallopian tube, uterine endometrium, cervix, and ectocervix. Intriguingly, differential expression of TLRs 2 and 4 was observed in distinct compartments of the female reproductive tract, with expression being lowest in the fallopian tubes and highest in the cervix and vagina [34]. Expression of TLRs 1–9 was demonstrated in primary human uterine epithelial cells and in a human uterine epithelial cell line [35]. Others have demonstrated that TLR expression does not necessarily translate to responsiveness to PAMPs [36]. Whereas human primary uterine epithelial cells express TLRs 1–9, only treatment with the TLR3-agonist poly (I:C) elicited the secretion of proinflammatory cytokines and upregulation of human ß-defensins 1 and 2 as well as interferon B and 2',5'-oligoadenylate synthetase mRNA expression [36]. In contrast, ECC-1 cells, which are derived from uterine epithelial cells, express TLRs 1–9 and respond to the TLR-agonists zymosan and flagellin (TLR2 and TLR5, respectively) by secreting increased levels of IL8, CCL2, and IL6. In the rat, treatment of uterine epithelial cells with Escherichia coli, but not with Lactobacillus rhamnosus, increases the secretion of TNFA and CCL20 by epithelial cells [37]. In other studies, the TLR-agonists lipopolysaccharide (LPS) and Pam3Cys increased the secretion of both cytokines by rat uterine epithelial cells in culture [11].

Recognizing that uterine epithelial cells are the first to encounter potential pathogens, the present study was undertaken to define more fully the extent to which TLRs are expressed on polarized mouse uterine epithelial cells and to determine the extent to which these TLRs are responsive to PAMPs. The objectives of the present work were as follows: 1) to determine if mouse uterine epithelial cells express TLR, 2) to establish whether stimulation of TLR by specific PAMPs induces the secretion of cytokines and/or chemokines, and 3) to define the pattern of cytokines and/or chemokines secretion following PAMP exposure that contributes to the initiation of both an inflammatory response and the recruitment of immune cells to the site of infection.

MATERIALS AND METHODS

Animals

Sexually mature BALB/c mice were purchased from the National Cancer Institute colony at Charles River Laboratories. Animals were housed and fed according to the guidelines of the Dartmouth College Institutional Animal Care and Use Committee; all procedures were approved before start of the experiments. Animals were housed in a constant-temperature room with a 12L:12D photoperiod and allowed food and water ad libitum. For each experiment, animals were killed by CO2 asphyxiation, and uterine tissues from 10 to 15 adult animals, representing all stages of the estrous cycle, were isolated and pooled for each experiment.

Isolation and Preparation of Epithelial Cells

Uterine epithelial cell suspensions were prepared by enzymatic digestion and screen disruption as described previously [38]. Briefly, after removal, uteri were slit lengthwise and digested with 46 500 U/ml of trypsin (Sigma) in 2.5% (wt/vol) pancreatin solution (Invitrogen Corp.) for 1 h at 4°C, followed by a 60-min digestion at 22°C. Digested tissues were then vortexed three times in Hanks balanced salt solution (Invitrogen) to release the epithelial sheets before passing the cell suspension through a 20-µm nylon mesh (Small Parts, Inc.) for collection by centrifugation. Uterine epithelial cells were resuspended in Dulbecco modified Eagle medium/Ham F-12 nutrient (mixed 1:1; Invitrogen) plus 10% (vol/vol) charcoal dextran-stripped fetal bovine serum (FBS; Hyclone) and 20 mM Hepes, 100 µg/ml of streptomycin, 100 U/ml of penicillin, and 2 mM L-glutamine (all from Invitrogen) and then seeded onto Nunc cell culture inserts (diameter, 10 mm; pore size, 0.4 µm; Nalgene Nunc International) coated with diluted Matrigel (Collaborative Biomedical Products) at 300 µl/apical chamber of the cell culture insert. Inserts were placed into 24-well tissue culture plates (Nalgene Nunc International) containing 500 µl of culture media per well. When epithelial cells reached confluence, as indicated by transepithelial resistance (>1000 ohms/well), the culture media was switched to Cellgro Complete (Mediatech) plus 100 µg/ml of streptomycin, 100 U/ml of penicillin, and 2 mM L-glutamine before stimulation with TLR agonists. The purity of epithelial cell cultures was evaluated by three criteria and reported previously [39]. Briefly, we found the following: 1) When examined by microscopy, epithelial cells were found to consist of sheets (50–500 cells) that were free of isolated cell contaminants; 2) mouse uterine epithelial cells achieved high transepithelial resistance (>1000 ohms/well; background resistance, 150 ohms/well), which would not be possible with stromal cell contamination; and 3) staining of polarized cell inserts with anti-CD45 antibody indicated that epithelial cells account for more than 99.5% of the cells present on each insert.

RT-PCR Analysis

To investigate the extent to which Tlr1 through Tlr9 mRNAs are expressed in uterine epithelial cells of adult mice, uteri were grown to confluence on culture inserts as described above before recovery of RNA. At the time of recovery, each insert received TRIzol Reagent (250 µl/well; Invitrogen) according to the manufacturer's instructions to extract total RNA before pooling extracts from four replicate inserts. The RNA was purified using an RNeasy Mini Kit (Qiagen Sciences) and treated with DNase (Ambion). After testing the RNA for integrity and possible contaminants, the RNA and no-RT controls were reverse transcribed using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) following the manufacturer's protocol. Murine TLR cDNA was amplified by PCR using the Platinum PCR Supermix (Invitrogen) and Tlr1 through Tlr9 mRNA-specific primers (Applied BioSystems) (Table 1). Primers were designed to be specific for each Tlr mRNA and to ensure that PCR products would have a size of approximately 300 bp. Thirty-five cycles of PCR amplification were performed on the PTC-100 Thermal Cycler (MJ Research, Inc.) as follows: denaturation, 94°C for 30 sec; annealing, 55°C for 30 sec; and extension, 72°C for 1 min. Products were visualized on a 1.5% (wt/vol) agarose gel.


View this table:
[in this window]
[in a new window]
  		TABLE 1. PCR primer pairs used for amplification of mouse Tlr mRNA.

Stimulation with TLR Agonists

The TLR agonists used in these studies included the following: for TLR1/TLR2, Pam3Cys (EMC Microcollections), 10 µg/ml; for TLR2/TLR6, peptidoglycan from Staphylococcus aureus (Invivogen), 10 µg/ml; for TLR3, poly (I:C) (Invivogen), 25 µg/ml; for TLR4, ultrapure LPS from Salmonella minnesota (List Biological Laboratories), 1 µg/ml; for TLR7, loxoribine (Invivogen), 100 µM; and for TLR9, CpG oligonucleotide (Invivogen), 1 µM. Wells containing Cellgro media only were included in all experiments as controls. Following 48 h of incubation, apical and basolateral supernatants were collected and centrifuged at 10 000 x g for 5 min to remove cell debris before storage at –80°C until further analysis. In time-course studies, apical and basolateral media were collected at 4, 8, 12, 24, 48, and 72 h and immediately replaced with media containing fresh TLR agonists. The TLR agonists were tested for LPS contamination using the Limulus assay (BioWhittaker, Inc.) according to manufacturer's instructions before use in cell culture assays (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2. LPS contamination of TLR agonists.

Cytokine/Chemokine Analysis

The cytokines and chemokines measured in the present study included IL1A, IL6, CCL2, TNFA, IL8, CSF2, CSF3, IL1G, CCL3/MIP1A, IL12p40/p70, and interferon (IFN) G. Analysis was performed by Bio-Plex assay as previously described [35]. Briefly, the Bio-Plex suspension array system (Bio-Rad) uses fluorescently dyed Luminex beads (Bio-Rad) to measure simultaneously multiple cytokines/chemokines from one sample. The lower limits of detection for measuring each of the cytokines/chemokines in this assay were as follows: IL1A, 15 pg/ml; IL6, 10 pg/ml; CCL2, 5 pg/ml; TNFA, 5 pg/ml; IL8 (keratinocyte-derived chomokine [KC]; 15 pg/ml); CSF2, 10 pg/ml; CSF3, 15 pg/ml; IL1B, 10 pg/ml; CCL3, 15 pg/ml;, IL12p40/p70, 15 pg/ml; and IFNG (1 pg/ml). Standards were prepared in Cellgro. Cytokine/chemokine-specific antibodies, attached to the Luminex beads (5000 beads per cytokine/chemokine), were incubated with samples, blanks, standards, or internal controls for 30 min. Following a wash step, biotinylated secondary antibodies were added for a further 30 min before washing and detection with a Streptavidin PE-labeled antibody (R & D Systems, Inc.) . Results were read with the Bio-Plex array reader and analyzed with Bio-Plex Manager software (Bio-Rad). A minimum of four cell culture inserts were analyzed per treatment group.

Statistical Analysis

Standard errors of the mean and mean values were calculated for each cytokine/chemokine by averaging the results of between four and six culture inserts per treatment group. For statistical analysis, t-tests comparing apical and basolateral amounts of cytokines released, as well as TLR-agonist induction of cytokines/chemokines versus media control, were performed. For the time-course experiment, ANOVA plus Tukey-Kramer multiple-comparisons test compared levels of cytokines/chemokines collected at different time points. A P value of less than 0.05 was considered to be statistically significant.

RESULTS

Preferential Release of Cytokines/Chemokines

To determine the extent to which polarized epithelial cells grown on inserts constitutively secrete cytokines and chemokines, apical and basolateral media were collected following 48 h of incubation. The formation of tight junctions, as indicated by transepithelial resistance of 1000 ohms/well or greater (control wells had transepithelial resistance of 130–150 ohms/well), was used as an indication of epithelial cell integrity and verification that a polarized monolayer had been formed (data not shown). As seen in Figure 1A, IL1A, CCL2, IL6, CSF2, and TNFA were secreted at moderate levels into the apical chamber and at low levels into the basolateral chamber. In addition, high levels of CSF3 and IL8 were secreted into the apical chamber, and moderate levels of CSF3 and IL8 were found in the basolateral chamber (Fig. 1B). In all cases, apical secretion of IL1A, CCL2, IL6, CSF2, TNFA, CSF3, and IL8 was significantly greater than that measured in the basolateral chamber. As a part of these studies, we found that IL1B, CCL3, IL12p40/p70, and IFNG secretion could not be detected in either the apical chamber or the basolateral chamber (data not shown).


Figure 1
View larger version (15K):
[in this window]
[in a new window]
  		FIG. 1. Preferential cytokine release by polarized mouse uterine epithelial cells. Mouse uterine epithelial cells from 10 to 15 animals were isolated and grown to high transepithelial resistance on cell inserts. The 48-h accumulation of cytokines in the apical chambers (hatched bars) and basolateral chambers (white bars) were analyzed by Bio-Plex assay. Values (mean ± SEM) was calculated from a minimum of four cell inserts. The cytokines analyzed were IL1A, CCL2, IL6, CSF2, and TNFA (A) as well as CSF3 and IL8 (B). Significant differences between apical and basolateral release are indicated (*P < 0.05, **P < 0.01). Representative of three separate experiments.

Expression of TLRs in Mouse Uterine Epithelial Cells

The expression of Tlr1 through Tlr9 mRNAs by uterine epithelial cells was examined using RT-PCR. Purified epithelial cells were isolated from pooled uteri of intact animals as described in Materials and Methods before total RNA isolation and mRNA analysis. Murine uterine epithelial cells were evaluated for the expression of mouse TLR using the TLR-specific primers shown in Table 1. As seen in Figure 2, uterine epithelial cells express mRNAs for Tlr1 through Tlr6. Expression was consistently found in primary mouse uterine epithelial cells from three separate experiments. In these experiments, Tlr7 and Tlr8 mRNAs were expressed in one of three experiments, and Tlr9 mRNA was expressed in two of three experiments (data not shown). In other experiments (not shown), we have compared fresh and polarized uterine epithelial cells and found that expression of Tlr2 through Tlr6 is unaffected by culture [40].


Figure 2
View larger version (26K):
[in this window]
[in a new window]
  		FIG. 2. Expression of Tlr mRNA in primary mouse uterine epithelial cells. Lanes 1–6 correspond to Tlr1 through Tlr6 mRNA and marker, respectively. Total RNA was isolated from mouse uterine epithelial cells, and RT-PCR was used to examine Tlr mRNA expression.

Induction of Cytokine Responses by TLR Agonists

Having demonstrated that epithelial cells express mRNAs for Tlr1 through Tlr6, our aim was to determine if these TLRs were functional and responsive to PAMPs. As shown in Figure 3, treatment of polarized epithelial cells with the TLR1/TLR2-agonist Pam3Cys placed in the apical chamber for 48 h before media collection resulted in a significant increase in both apical and basolateral (bidirectional) secretion of TNFA, CSF3, CSF2, and IL8. As a part of these studies, we found that Pam3Cys significantly increased the apical secretion of IL1A, CCL2, and IL6 relative to controls without affecting basolateral cytokine/chemokine release. To define more fully the response pattern of secretion, cells were treated with the TLR2/TLR6-agonist peptidoglycan. As seen in Table 3, peptidoglycan significantly increased the bidirectional secretion of IL1A, TNFA, CSF3, CSF2 and IL8 and also significantly increased apical secretion of CCL2 and IL6.


Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Cytokine release by polarized mouse uterine epithelial cells following stimulation with 10 µg/ml of the TLR2 (1/6) agonist Pam3Cys-Ser-(Lys)4 (Pam3Cys). The 48-h accumulation of cytokines in the apical and basolateral chambers was determined following stimulation with control media (white bars) or Pam3Cys (hatched bars). The cytokines analyzed were IL1A, CCL2, IL6, and TNFA (A) as well as CSF3, CSF2, and IL8 (B). Values (mean ± SEM) was calculated from a minimum of four cell inserts. Significant differences between Pam3Cys treatment and controls are indicated (*P < 0.05, **P < 0.01). Representative of three separate experiments.


View this table:
[in this window]
[in a new window]
  		TABLE 3. Cytokine release by polarized mouse uterine epithelial cells following stimulation with the TLR2 (1/6) agonist peptidoglycan.a

To determine whether uterine epithelial cells are responsive to a TLR3 agonist, cells were incubated in the presence or absence of the known TLR3-agonist poly (I:C). As seen in Figure 4, poly (I:C) induced the bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and also increased the basolateral secretion of CSF3 (Fig. 4B) relative to control cells. However, in contrast to Pam3Cys or peptidoglycan, poly (I:C) had no effect on the secretion of IL1A and IL8 or on the apical release of CSF3 by epithelial cells.


Figure 4
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 4. Cytokine release by polarized mouse uterine epithelial cells following stimulation with 25 µg/ml of the TLR3-agonist poly (I:C). The 48-h accumulation of cytokines in the apical and basolateral chambers was determined following stimulation with control media (white bars) or poly (I:C) (hatched bars). The cytokines analyzed were IL1A, CCL2, IL6, and TNFA (A) as well as CSF3, CSF2, and IL8 (B). Values (mean ± SEM) was calculated from a minimum of four cell inserts. Significant differences between poly (I:C) treatment and controls are indicated (*P < 0.05, **P < 0.01). Representative of three separate experiments.

The effects of the TLR4-agonist LPS are shown in Figure 5. Treatment with LPS, placed in the apical chamber of polarized epithelial cells, induced the bidirectional release of CCL2, TNFA, CSF3, CSF2, and IL8 and the apical release of IL6, but such treatment had no effect on IL1A secretion. In contrast, cells incubated with the TLR7-agonist loxoribine and the TLR9-agonist CpG were unresponsive in that neither agonist had any effect on the cytokines/chemokines measured in the present study (data not shown). The responsiveness of TLR8 was not analyzed, because in the mouse, no agonist is currently available. In other studies, we found that constitutively, secretion by polarized epithelial cells of IL1B, MIP1A, IL12p40/p70, and IFNG was below the limits of detection for our assays and not affected by the presence of the TLR agonists used in the present study (data not shown).


Figure 5
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 5. Cytokine release by polarized mouse uterine epithelial cells following stimulation with 1 µg/ml of the TLR4-agonist LPS. The 48-h accumulation of cytokines in apical and basolateral chambers was determined following stimulation with control media (white bars) or LPS (hatched bars). The cytokines analyzed were IL1A, CCL2, IL6, and TNFA (A) as well as CSF3, CSF2, and IL8 (B). Statistical significance of LPS induction for each cytokine is indicated (*P < 0.05, **P < 0.01). Graphs shown are representative of three separate experiments.

LPS Contamination of TLR Agonists

Recognizing that contamination of PAMPs with LPS is a potential problem, we tested the PAMPs used in these studies to ensure that the responses observed when uterine epithelial cells were incubated in the presence of TLR agonists were not the result of contamination with LPS. The results shown in Table 2 for PAMPs analyzed by the Limulus assay indicate that only trace amounts (0.05 ng/ml) were found for Pam3Cys, peptidoglycan, poly (I:C), loxoribine, or CpG DNA as well as in control media. We conclude that LPS at 0.05 ng/ml was below the level of detection, because treatment of epithelial cells with CpG and/or loxoribine, which contained trace amounts of LPS, had no effect on cytokine/chemokine secretion. As a TLR4 agonist, LPS was used at a concentration of 1 mg/ml, which is 20 000-fold the concentration found in the Pam3Cys, peptidoglycan, poly (I:C), loxoribine, or CpG reagents, and this dose elicited a unique cytokine/chemokine release pattern. In contrast, we found that the TLR5-agonist flagellin was highly contaminated, and for this reason, it was not included in our study.

Time Course of Cytokine Responses

To define more fully the pattern of epithelial cell responsiveness to TLR agonists, time-course studies were undertaken in which TLR agonists were added to the apical chambers of polarized cells. Apical media containing a given PAMP were replaced at 4-h intervals along with basolateral media. At 4, 8, and 12 h, apical and basolateral media was collected and analyzed for cytokine/chemokine secretion. As seen in Figure 6, peptidoglycan treatment transiently increased the secretion of IL6, which peaked within the first 4 h of treatment and then returned to baseline levels by 8 and 12 h despite the continued presence of peptidoglycan. In contrast (Fig. 6B), secretion of CSF2 increased within 4 h and remained elevated at 8 and 12 h. In other studies (data not shown), we found that secretion of CSF2 in response to peptidoglycan persisted at 24, 48, and 72 h in culture. Given two distinct release profiles (early vs. continuous), we evaluated the pattern of secretion, in response to peptidoglycan, of a number of cytokines/chemokines and found that IL1A displayed an early response pattern, similar to that seen with IL6, when uterine epithelial cells were exposed to peptidoglycan (Table 4). In contrast, we found that CCL2, TNFA, and IL8 secretion was continuous and similar to that of CSF2 following exposure to peptidoglycan (Table 4). Interestingly, TNFA and CCL2 secretion measured at 8 and 12 h was slightly lower that that seen at 4 h. In other studies (Table 4), we found that in response to LPS, IL6 release was early (4 h), but CCL2, TNFA, CSF2, and IL8 secretion was continuous (8 and 12 h). In contrast to peptidoglycan, LPS had no effect on IL1A secretion (data not shown). These studies also demonstrated that poly (I:C) treatment elicited both an early (IL6) as well as a continuous (CCL2) release pattern. As seen in Table 4, IL6 peaked at 4 h and then declined, whereas CCL2 secretion rose and persisted over the course of the experiment.


Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		FIG. 6. Time course of apical cytokine release by polarized mouse uterine epithelial cells. Supernatants were collected at 4, 8, and 12 h during stimulation with peptidoglycan. Controls are indicated as white bars, and hatched bars indicate stimulation with peptidoglycan (PGN). A) Secretion of IL6 is shown as a representative for an early response pattern. B) Secretion of CSF2 is shown as a representative for a continuous release pattern. Further examples of these release patterns following stimulation with PGN, LPS, or poly (I:C) are indicated in Table 4. Values (mean ± SEM) was derived from a minimum of four cell inserts. Significant differences between controls and PGN treatment are indicated (*P < 0.05, **P < 0.01).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Time course of apical cytokine release by polarized mouse uterine epithelial cells.a

DISCUSSION

The results of the present study demonstrate that mouse uterine epithelial cells express Tlr1 through Tlr6 mRNAs and, occasionally, Tlr7 through Tlr9 mRNAs. Under physiological conditions, polarized mouse uterine epithelial cells constitutively secrete IL1A, IL6, TNFA, CCL2, CSF2, CSF3, and IL8. These findings indicated that secretion of cytokines/chemokines was preferential into the apical compartment. Treatment of epithelial cells at the apical surface with PAMPs known to bind to TLRs 1–4 and 6 stimulated selective cytokine/chemokine secretion. Each PAMP elicited a distinct pattern of secretion for each of the agonists used (Pam3Cys, peptidoglycan, poly (I:C), and LPS). In contrast, loxoribine and CpG (TLR7 and TLR9 agonists, respectively) had no effect. Induction of cytokine secretion by PAMPs fell into two distinct release profiles (early vs. continuous): those that were rapidly released within 4 h (IL6 and IL1A) and those that were continuously secreted for 12 h (IL8, CSF2, CCL2, and TNFA).

It has been shown previously that human uterine epithelial cells express Tlr1 through Tlr9 [35, 36] and that uterine epithelial cell lines express Tlr1 through Tlr6 plus Tlr9 [41]. However, whereas epithelial cells express Tlr1 through Tlr9, only the TLR3-agonist poly (I:C) was able to induce cytokine/chemokine responses in primary human uterine epithelial cells [36]. In our study, mouse uterine epithelial cells secreted a variety of cytokines and chemokines in response to PAMPs specific for TLRs 1–4 and 6. This heightened responsiveness may be caused by differences between the mouse and human reproductive tract. In the human, sperm are placed in the vagina, but in the mouse, sperm are deposited directly into the uterus. Under these conditions, the upper reproductive tract of the mouse is exposed to a greater antigenic load (sperm and bacteria) compared with the human uterus [42] and, therefore, may be more responsive to a wider range of potential pathogens. Our findings in the mouse, when compared to those in the human, suggest that responsiveness to PAMPs or pathogens in the reproductive tract may be dependent on the microenvironment and antigenic load that periodically occurs in the reproductive tract.

Others have demonstrated that TLR2, in cooperation with TLR1 and TLR6, is activated by a variety of PAMPs, including products of gram-positive and gram-negative bacteria as well as yeast products [12]. Our studies indicate that mouse uterine epithelial cells are highly responsive to stimulation with peptidoglycan and Pam3Cys, which signal through TLR2 in combination with TLR1 or TLR6. Both PAMPs increased secretion of IL1A, IL6, TNFA, CCL2, IL8, CSF2, and CSF3. The importance of signaling through TLR2 in the female reproductive tract recently has been shown in the pathogenesis of Chlamydia sp. and Neisseria gonorrhoeae [16, 43], which are responsible for the majority of pelvic inflammatory disease cases [2]. Macrophages lacking TLR2 produced significantly less TNFA and IL6 in response to active infection with Chlamydia sp., and in Tlr2 knockout mice, significantly lower levels of TNFA and macrophage inflammatory protein-2 were detected in genital tract secretions during the first week of infection [16]. This, in turn, led to a significant reduction in oviduct and mesosalpinx pathology at late time points [16]. The purified preparation of Lip from N. gonorrhoeae has been discovered to be a potent inflammatory mediator in immortalized human endocervical epithelial cells as well as stimulating the production of IL8 and the activation of the transcription factor NF-{kappa}B by human embryonic kidney 293 cells transfected with Tlr2 [43]. Infection of reproductive epithelial cells with these pathogens is known to induce proinflammatory cytokines, including IL1A, IL8, IL6, CCL2, and TNFA [16, 33, 43–45]. Our findings that TLR2-agonists Pam3Cys and peptidoglycan increase epithelial cell secretion of these cytokines suggest that some of the responses seen with Chlamydia sp. and N. gonorrhoeae may be mediated through TLR2. Because IL1A, TNFA, IL6, and IL8 secretion results in the recruitment and activation of macrophages, dendritic cells, and neutrophils as well as stimulation of T-helper cells and B cells, our findings suggest that uterine epithelial cells are sentinels that respond rapidly to sexually transmitted diseases.

Several studies have demonstrated that TLR4 also plays an important role in the pathogenesis of sexually transmitted diseases. Ligation of TLR4 by whole chlamydial organisms downmodulates signaling by other TLRs and reduces the amount of oviduct and mesosalpinx pathology associated with the inflammatory response induced by TLR2 ligation [16]. The TLR4 has been shown to play a role in Trichomonas vaginalis infection [46], and it contributes to the pathogenesis of inflammation-induced preterm labor [47]. Our findings indicate that stimulation of TLR4 with LPS results in the apical and basolateral secretion of CCL2, TNFA, CSF3, CSF2, and IL8 and apical release of IL6 by polarized uterine epithelial cells. To the best of our knowledge, this is the first time that stimulation through TLR4 has been shown to induce a response by mouse uterine epithelial cells. Our finding of differences in cytokine profiles following stimulation of different TLRs suggests that the innate immune system in the female reproductive tract has evolved to respond to specific pathogens in ways that are unique, distinct, and antigen-specific [48].

The TLR3, along with TLRs 7–9, can be found in intracellular compartments and have been identified as crucial for the detection of invading viruses [49]. Others have shown that a number of viruses, including herpes simplex virus 2, human immunodeficiency virus, and human papilloma virus, affect the female reproductive tract and that activation of TLR3 as well as TLR9 in the vagina of adult animals induces antiviral immunity to herpes simplex virus 2 [50–52]. The data presented here show that TLR3 is expressed in uterine epithelial cells. We found that stimulation of TLR3 resulted in an increased secretion of cytokines/chemokines, whereas stimulation of TLR7 or TLR9, which are inconsistently found, had no effect on the 12 cytokines/chemokines examined. The lack of functional activity likely resulted from the lack of expression, but it also is possible that stimulation of TLR7 or TLR9 resulted in the secretion of cytokines/chemokines not examined in the present study. Another possibility is that secondary molecules and/or signals may be needed for TLR stimulation. For example, in other studies, we have found that inflammatory mediators, such as IFNG or TNFA, enhance the stimulation signal of selected PAMPs [35]. Just why epithelial cells were unresponsive to PAMPs specific for TLR7 or TLR9 remains to be determined.

That uterine epithelial cells were responsive to poly (I:C) suggests that these cells recognize double-stranded as well as cellular RNA, potentially from cells killed by an invading pathogen [22, 53]. In addition to stimulating the secretion of proinflammatory cytokines and chemokines, poly (I:C) is known to induce the expression of the antimicrobial peptides ß-defensins 1 and 2 and antiviral molecules IFNB, myxovirus resistance gene 1, and 2',5'-oligoadenylate synthetase [36]. Clearly, by recognizing the ligand of TLR3, epithelial cells are playing an active role in the maintenance of immune protection in the female reproductive tract.

The epithelium of the uterus, which is similar to that at other mucosal sites, is supported by an underlying population of immune cells. These immune cells are both dispersed and present as lymphoid aggregates made up of a B-cell core surrounded by T cells with an outer halo of macrophages [54, 55]. Our findings in the present study suggest that mouse uterine epithelial cells contribute to immune readiness and protection by constitutively secreting IL1A, IL6, TNFA, CCL2, CSF2, CSF3, and IL8 at the basolateral surface. Each of these cytokines/chemokines is known to play a role in the recruitment of neutrophils, macrophages, monocytes, dendritic cells, and other immune cells and to aid in the differentiation and function of these cells at mucosal surfaces [56–62]. Interestingly, we found that a number of cytokines/chemokines are released preferentially into the apical compartment. This may be important for maintaining the presence of immune cells within the subepithelial layers of the endometrium as well as within the uterine lumen. Previously, we found that polarized uterine epithelial cells in culture preferentially release TNFA into the apical compartment and CCL20 into the basolateral compartment [11]. Our findings in the present study extend these observations by showing that a number of cytokines/chemokines are preferentially released and that under conditions of PAMP treatment, directional release is not altered but, rather, is enhanced with PAMP stimulation.

The present study indicates that epithelial cells respond to some PAMPs with the rapid release of cytokines (4 h) whereas others are released more gradually (8–12 h). The proinflammatory cytokines IL1A and IL6 were secreted and peaked within 4 h of stimulation and returned to baseline levels by 8 h, but IL8 and CSF2, which are crucial for the recruitment, differentiation, and function of neutrophils [63, 64], were continuously secreted. Also continuously released, though slightly reduced, were CCL2 and TNFA, which play a role in the acute response to infection [65, 66]. That this varied pattern of release in response to PAMPs is shared between species is suggested from findings in the rat, in which epithelial cells, in response to LPS and Pam3Cys, rapidly released CCL20 and gradually released TNFA [11]. Whereas cytokine functions can appear to be redundant, a precise orchestration of cytokine/chemokine secretion and action likely is important for eliciting both an innate and an adaptive immune response. The precise patterns of cytokine release seen in the present study following PAMP exposure likely are essential for maintaining the balance needed to protect against invading pathogens while also preparing for fertilization and implantation. Further studies are needed to define more fully the regulatory role of cytokine/chemokine and the impact that alterations in secretion will have on reproductive potential.

In summary, the present study shows that mouse uterine epithelial cells produce a spectrum of cytokines/chemokines in response to stimulation by known agonists of TLR. These results suggest that uterine epithelial cells are able to recognize a variety of pathogens, including bacteria and viruses, and respond by secreting cytokines and chemokines that alert cells of the innate and adaptive immune system to prevent and/or control infection. The rapid response to these antigens suggests that the uterine epithelium plays a crucial role in maintaining both a level of surveillance and protection against potential pathogens to optimize conditions for successful mammalian reproduction.

ACKNOWLEDGMENTS

The authors gratefully thank Richard Rossoll for his assistance in these studies and in reviewing this manuscript.

FOOTNOTES

1 Supported by research grants AI-13541 and AI-33478 from the NIH. Back

2 Correspondence: Charles R. Wira, Department of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756. FAX: 603 650 6130; charles.r.wira{at}darmouth.edu Back

3 Current address: Department of Clinical Sciences, James L. Voss Veterinary Teaching Hospital, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523-1620. Back

Received: 3 January 2006.

First decision: 31 January 2006.

Accepted: 27 February 2006.

REFERENCES

  1. King AE, Critchley HO, Kelly RW, Innate immune defenses in the human endometrium. Reprod Biol Endocrinol 2003 1:116-123[CrossRef][Medline]
  2. Quayle AJ, The innate and early immune response to pathogen challenge in the female genital tract and the pivotal role of epithelial cells. J Reprod Immunol 2002 57:61-79[CrossRef][Medline]
  3. Julian J, Carson DD, Glasser SR, Polarized rat uterine epithelium in vitro: constitutive expression of estrogen-induced proteins. Endocrinology 1992 130:79-87[Abstract/Free Full Text]
  4. Julian J, Carson DD, Glasser SR, Polarized rat uterine epithelium in vitro: responses to estrogen in defined medium. Endocrinology 1992 130:68-78[Abstract/Free Full Text]
  5. Jacobs AL, Carson DD, Uterine epithelial cell secretion of interleukin-1{alpha} induces prostaglandin E2 (PGE2) and PGF2{alpha} secretion by uterine stromal cells in vitro. Endocrinology 1993 132:300-308[Abstract/Free Full Text]
  6. Jacobs AL, Sehgal PB, Julian J, Carson DD, Secretion and hormonal regulation of interleukin-6 production by mouse uterine stromal and polarized epithelial cells cultured in vitro. Endocrinology 1992 131:1037-1046[Abstract/Free Full Text]
  7. Kayisli UA, Mahutte NG, Arici A, Uterine chemokines in reproductive physiology and pathology. Am J Reprod Immunol 2002 47:213-221[Medline]
  8. Robertson SA, Brannstrom M, Seamark RF, Cytokines in rodent reproduction and the cytokine-endocrine interaction. Curr Opin Immunol 1992 4:585-590[CrossRef][Medline]
  9. Robertson SA, Mayrhofer G, Seamark RF, Uterine epithelial cells synthesize granulocyte-macrophage colony-stimulating factor and interleukin-6 in pregnant and nonpregnant mice. Biol Reprod 1993 46:1069-1079
  10. Altman JD, Moss PAH, Goulder PJR, Barouch DH, McHeyzer-Williams MG, Bell JI, McMichael AJ, Davis MM, Phenotypic analysis of antigen-specific T lymphocytes. Science 1996 274:94-96[Abstract/Free Full Text]
  11. Crane-Godreau MA, Wira CR, CCL20/macrophage inflammatory protein 3{alpha} and tumor necrosis factor {alpha} production by primary uterine epithelial cells in response to treatment with lipopolysaccharide or Pam3Cys. Infect Immun 2005 73:476-484[Abstract/Free Full Text]
  12. Medzhitov R, Toll-like receptors and innate immunity. Nat Rev Immunol 2001 1:135-145[CrossRef][Medline]
  13. Medzhitov RM, Janeway CJ, Innate immunity. N Engl J Med 2000 343:338-344[Free Full Text]
  14. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A, The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001 410:1099-1103[CrossRef][Medline]
  15. Takeda K, Kaisho T, Akira S, Toll-like receptors. Annu Rev Immunol 2003 21:335-376[CrossRef][Medline]
  16. Darville T, O'Neill JM, Andrews CW, Jr, Nagarajan UM, Stahl L, Ojcius DM, Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J Immunol 2003 171:6187-6197[Abstract/Free Full Text]
  17. Akira S, Takeda K, Toll-like receptor signaling. Nat Rev Immunol 2004 4:499-511[CrossRef][Medline]
  18. Underhill DM, Toll-like receptors: networking for success. Eur J Immunol 2003 33:1767-1775[CrossRef][Medline]
  19. Zhang D, Zhang G, Hayden MS, Greenblatt MB, Bussey C, Flavell RA, Ghosh S, A Toll-like receptor that prevents infection by uropathogenic bacteria. Science 2004 303:1522-1526[Abstract/Free Full Text]
  20. Barton GM, Medzhitov R, Toll-like receptor signaling pathways. Science 2003 300:1524-1525[Abstract/Free Full Text]
  21. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S, Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 1999 11:443-451[CrossRef][Medline]
  22. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA, Recognition of double-stranded RNA and activation of NF-{kappa}B by Toll-like receptor 3. Nature 2001 413:732-738[CrossRef][Medline]
  23. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, Takeda K, Akira S, Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999 162:3749-3752[Abstract/Free Full Text]
  24. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK, Akira S, Underhill DM, Aderem A, The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 1099 410:1099-1103
  25. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S, Small antiviral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002 3:196-200[CrossRef][Medline]
  26. Lee J, Chuang TH, Redecke V, She L, Pitha PM, Carson DA, Raz E, Cottam HB, Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc Natl Acad Sci U S A 2003 100:6646-6651[Abstract/Free Full Text]
  27. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S, A Toll-like receptor recognizes bacterial DNA. Nature 2000 408:740-745[CrossRef][Medline]
  28. Yarovinsky F, Zhang D, Andersen JF, Bannenberg GL, Serhan CN, Hayden MS, Hieny S, Sutterwala FS, Flavell RA, Ghosh S, Sher A, TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 2005 308:1626-1629[Abstract/Free Full Text]
  29. Lauw FN, Caffrey DR, Golenbock DT, Of mice and man: TLR11 (finally) finds profilin. Trends Immunol 2005 26:509-511[CrossRef][Medline]
  30. Muir A, Soong G, Sokol S, Reddy B, Gomez MI, Van Heeckeren A, Prince A, Toll-like receptors in normal and cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 2004 30:777-783[Abstract/Free Full Text]
  31. Akhtar M, Watson JL, Nazli A, McKay DM, Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-{kappa}B-independent pathway. FASEB J 2003 17:1319-1321[Abstract/Free Full Text]
  32. Cario E, Podolsky DK, Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease. Infect Immun 2000 68:7010-7017[Abstract/Free Full Text]
  33. Fichorova RN, Cronin AO, Lien E, Anderson DJ, Ingalls RR, Response to Neisseria gonorrhoeae by cervicovaginal epithelial cells occurs in the absence of Toll-like receptor 4-mediated signaling. J Immunol 2002 168:2424-2432[Abstract/Free Full Text]
  34. Pioli PA, Amiel E, Schaefer TM, Connolly JE, Wira CR, Guyre PM, Differential expression of Toll-like receptors 2 and 4 in tissues of the human female reproductive tract. Infect Immun 2004 72:5799-5806[Abstract/Free Full Text]
  35. Schaefer TM, Desouza K, Fahey JV, Beagley KW, Wira CR, Toll-like receptor (TLR) expression and TLR-mediated cytokine/chemokine production by human uterine epithelial cells. Immunology 2004 112:428-436[CrossRef][Medline]
  36. Schaefer TM, Fahey JV, Wright JA, Wira CR, Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C). J Immunol 2005 174:992-1002[Abstract/Free Full Text]
  37. Crane-Godreau MA, Wira CR, Effect of Escherichia coli and Lactobacillus rhamnosus on macrophage inflammatory protein 3{alpha}, tumor necrosis factor {alpha}, and transforming growth factor ß release by polarized rat uterine epithelial cells in culture. Infect Immun 2004 72:1866-1873[Abstract/Free Full Text]
  38. Grant KS, Wira CR, Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNF{alpha} and TGFß release in culture. Biol Reprod 2003 69:1091-1098[Abstract/Free Full Text]
  39. Wira CR, Rossoll RM, Young RC, Polarized uterine epithelial cells preferentially present antigen at the basolateral surface: role of stromal cells in regulating class II-mediated epithelial cell antigen presentation. J Immunol 2005 175:1795-1804[Abstract/Free Full Text]
  40. Soboll G, Schaefer TM, Wira CR, Effect of Toll-like receptor (TLR) agonists on TLR and microbicide mRNA expression in uterine and vaginal tissues of the mouse. Am J Reprod Immunol 2006 55:434-446
  41. Young SL, Lyddon TD, Jorgenson RL, Misfeldt ML, Expression of Toll-like receptors in human endometrial epithelial cells and cell lines. Am J Reprod Immunol 2004 52:67-73
  42. Parr EL, Parr MB, Deposition of C3 on bacteria in the mouse uterus after mating. J Reprod Immunol 1988 12:315-319[CrossRef][Medline]
  43. Fisette PL, Ram S, Andersen JM, Guo W, Ingalls RR, The Lip lipoprotein from Neisseria gonorrhoeae stimulates cytokine release and NF-{kappa}B activation in epithelial cells in a Toll-like receptor 2-dependent manner. J Biol Chem 2003 278:46252-46260[Abstract/Free Full Text]
  44. Kaushic C, Grant K, Crane M, Wira CR, Infection of polarized primary epithelial cells from rat uterus with Chlamydia trachomatis: cell-cell interaction and cytokine secretion. Am J Reprod Immunol 2000 44:73-79
  45. Perfettini JL, Darville T, Gachelin G, Souque P, Huerre M, Dautry-Varsat A, Ojcius DM, Effect of Chlamydia trachomatis infection and subsequent tumor necrosis factor {alpha} secretion on apoptosis in the murine genital tract. Infect Immun 2000 68:2237-2244[Abstract/Free Full Text]
  46. Zariffard MR, Harwani S, Novak RM, Graham PJ, Ji X, Spear GT, Trichomonas vaginalis infection activates cells through Toll-like receptor 4. Clin Immunol 2004 111:103-107[CrossRef][Medline]
  47. Elovitz MA, Wang Z, Chien EK, Rychlik DF, Phillippe M, A new model for inflammation-induced preterm birth: the role of platelet-activating factor and Toll-like receptor-4. Am J Pathol 2003 163:2103-2111[Abstract/Free Full Text]
  48. Re F, Strominger JL, Toll-like receptor 2 (TLR2) and TLR4 differentially activate human dendritic cells. J Biol Chem 2001 276:37692-37699[Abstract/Free Full Text]
  49. Boehme KW, Compton T, Innate sensing of viruses by Toll-like receptors. J Virol 2004 78:7867-7873[Free Full Text]
  50. Harandi AM, The potential of immunostimulatory CpG DNA for inducing immunity against genital herpes: opportunities and challenges. J Clin Virol 2004 30:207-210[CrossRef][Medline]
  51. Ashkar AA, Bauer S, Mitchell WJ, Vieira J, Rosenthal KL, Local delivery of CpG oligodeoxynucleotides induces rapid changes in the genital mucosa and inhibits replication, but not entry, of herpes simplex virus type 2. J Virol 2003 77:8948-8956[Abstract/Free Full Text]
  52. Ashkar AA, Yao XD, Gill N, Sajic D, Patrick AJ, Rosenthal KL, Toll-like receptor (TLR)-3, but not TLR4, agonist protects against genital herpes infection in the absence of inflammation seen with CpG DNA. J Infect Dis 2004 190:1841-1849[CrossRef][Medline]
  53. Kariko K, Ni H, Capodici J, Lamphier M, Weissman D, mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem 2004 279:12542-12550[Abstract/Free Full Text]
  54. Morris H, Edwards J, Tiltman A, Emms M, Endometrial lymphoid tissue: an immunohistological study. J Clin Pathol 1985 38:644-652[Abstract/Free Full Text]
  55. Yeaman GR, Guyre PM, Fanger MW, Collins JE, White HD, Rathbun W, Orndorff KA, Gonzalez JL, Stern JE, Wira CR, Unique CD8+ T cell-rich lymphoid aggregates in human uterine endometrium. J Leukoc Biol 1997 61:427-435[Abstract]
  56. Arici A, Head JR, MacDonald PC, Casey ML, Regulation of interleukin-8 gene expression in human endometrial cells in culture. Mol Cell Endocrinol 1993 94:195-204[CrossRef][Medline]
  57. Arici A, MacDonald PC, Casey ML, Regulation of monocyte chemotactic protein-1 gene expression in human endometrial cells in cultures. Mol Cell Endocrinol 1995 107:189-197[CrossRef][Medline]
  58. Ramsay AJ, Husband AJ, Ramshaw IA, Bao S, Matthaei KI, Koehler G, Kopf M, The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science 1994 264:561-563[Abstract/Free Full Text]
  59. Kishimoto T, Taga T, Yamasaki K, Matsuda T, Tang B, Muraguchi A, Horii Y, Suematsu S, Hirata Y, Yawata H, Normal and abnormal regulation of human B cell differentiation by a new cytokine, BSF2/IL-6. Adv Exp Med Biol 1989 254:135-143[Medline]
  60. Joseph SB, Miner KT, Croft M, Augmentation of naive, Th1 and Th2 effector CD4 responses by IL-6, IL-1, and TNF. Eur J Immunol 1998 28:277-289[CrossRef][Medline]
  61. Lorre K, Van Damme J, Verwilghen J, Baroja ML, Ceuppens JL, IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation. J Immunol 1990 144:4681-4687[Abstract]
  62. Rollins BJ, Walz A, Baggiolini M, Recombinant human MCP-1/JE induces chemotaxis, calcium flux, and the respiratory burst in human monocytes. Blood 1991 78:1112-1116[Abstract/Free Full Text]
  63. Shen L, Fahey JV, Hussey SB, Asin SN, Wira CR, Fanger MW, Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis. Cell Immunol 2004 230:23-32[CrossRef][Medline]
  64. Baggiolini M, Loetscher P, Chemokines in inflammation and immunity. Immunol Today 2000 21:418-420[CrossRef][Medline]
  65. Meter RA, Fahey JV, Wira CW, Secretion of monocyte chemotactic protein-1 by human uterine epithelium directs monocyte migration in culture. Fertil Steril 2005 84:191-201[CrossRef][Medline]
  66. Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewska E, Kagnoff MF, A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clin Invest 1995 95:55-65[Medline]



This article has been cited by other articles:


Home page
 Infect. Immun.Home page
R. N. Fichorova, R. T. Trifonova, R. O. Gilbert, C. E. Costello, G. R. Hayes, J. J. Lucas, and B. N. Singh
Trichomonas vaginalis Lipophosphoglycan Triggers a Selective Upregulation of Cytokines by Human Female Reproductive Tract Epithelial Cells.
Infect. Immun., October 1, 2006; 74(10): 5773 - 5779.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
75/1/131    most recent
biolreprod.106.050690v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.
Agricola
Right arrow Articles by Soboll, G.
Right arrow Articles by Wira, C. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS