| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Obstetrics and Gynecology (T.H., Y.O., Y.H., K.K., O.Y., M.H., C.M., T.Y., O.N., O.T., Y.T.), Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan; and Core Research for Evolutional Science and Technology (O.T.), Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan
Address all correspondence and requests for reprints to: Yutaka Osuga, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail: yutakaos-tky{at}umin.ac.jp.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B in ESCs, which was also inhibited by these antibodies. On the other hand, LPS did not stimulate IL-8 secretion in cultured EECs. However, LPS stimulated IL-8 secretion from EECs in the presence of soluble CD14. Flow cytometric analysis revealed that CD14 was expressed on the cell surface of ESCs but not EECs. In addition, immunohistochemical analysis showed that CD14 was stained in ESCs but not EECs. Pretreatment of interferon-
(IFN-) enhanced LPS-induced IL-8 secretion from ESCs. IFN- increased the expression of TLR4 mRNA. It also increased the amounts of mRNA for CD14, MD2, and MyD88, which are needed for LPS recognition in concert with TLR4. In summary, we demonstrated that both ESCs and EECs express TLR4 and respond to LPS through TLR4. We further showed that EECs, not ESCs, required soluble CD14 for TLR4 activation. Interestingly, IFN-, an antiinfectious cytokine, was found to activate the TLR4 system in ESCs. Altogether, the results imply that the TLR4 system might represent local immunity in the human endometrium with differential modes of TLR4 actions between ESCs and EECs. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In recent years increasing attention has been directed to innate immunity, the primary defense system against pathogens. In particular, toll-like receptors (TLRs), which comprise a family of membrane proteins consisting of at least 10 members, are suggested to play significant roles in innate immunity (2). TLRs respond to invading microorganisms by recognizing pathogen-associated molecular patterns of their products. Among the TLR members, TLR4 is well known for its capability of recognizing lipopolysaccharide (LPS), an integral component of the outer membrane of Gram-negative bacteria (3).
TLR4 recognizes LPS through a complex mechanism entailing several accessory molecules. For instance, CD14, a high-affinity LPS receptor, plays a central role in this mechanism. CD14 is secreted into serum as a soluble CD14 (sCD14). It is also expressed as a glycophosphoinisitol-linked protein on the cell surface. Furthermore, MD2 is required for LPS recognition. MD2 is expressed on the cell surface, forming the complex with the ectodomain of TLR4. MyD88, a common adaptor protein for TLRs, mediates intracellular signal transduction after TLR4 activation. The intracellular signals, then, culminate in the activation of nuclear factor-B (NF-B) as well as MAPK, leading to subsequent induction of various genes that function in host defense.
Alternate systems without CD14 and/or TLR4 have been suggested to exist for transducing LPS-induced stimulation. For example, heterogeneous receptor complex comprised of heat shock proteins 70 and 90, chemokines receptor 4, and growth differentiation factor 5 can recognize LPS. It is speculated that different cell types use different ways of recognizing LPS, depending on the activation state of the cells (4).
In view of the findings that LPS stimulates the production of multiple bioactive molecules by endometrial cells (5, 6, 7, 8, 9), we speculated that TLR4 plays a role in the endometrium. However, the presence and function of TLR4, as well as other known TLRs, in the endometrium has not been studied. Thus, we tested whether the TLR4 system could be at work in the human endometrium. To address this, we sought to examine the presence and possible function of TLR4 in human endometrial cells. In the present study, we characterized the TLR4 system in human endometrial cells. In addition, the effect of interferon (IFN)-, an immunoinflammatory modulator, on the TLR4 system was investigated, aiming to get an insight into the regulation of TLR4 system in the endometrium.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS from Escherichia coli serotype O127:B8, type I collagenase, and antibiotics (mixture of penicillin, streptomycin, and amphotericin B) were purchased from Sigma (St. Louis, MO). DMEM/Ham’s F12 (F-12) medium was from Gibco (Grand Island, NY). Human recombinant CD14 and IFN- were obtained from R&D systems (Minneapolis, MN). Sheep antihuman CD14 (azide free) was purchased from R&D Systems (Minneapolis, MN). Sheep IgG was obtained from ICN Biomedicals (Aurora, OH). Mouse antihuman CD14 (MY4) and isotype control mouse IgG2b were obtained from Beckman-Coulter (Fullerton, CA). Mouse antihuman CD14 (TUK4) was purchased from Dako Cytomation (Glostrup, Denmark). Mouse antihuman TLR4 (HTA125, azide free) was purchased from Cosmo Bio (Tokyo, Japan). Isotype control mouse IgG2a (azide free) was purchased from R&D. Rabbit polyclonal antihuman TLR4 antibody (H-80) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Charcoal-stripped fetal bovine serum (FBS) was from Hyclone (Logan, UT). Deoxyribonuclease I was from Takara (Tokyo, Japan).
Patients and samples
Endometrial tissues were obtained from patients undergoing hysterectomy for benign gynecological conditions. All patients had regular menstrual cycles, and none had received hormonal treatment for at least 6 months before surgery. The tissues collected under sterile conditions were processed for primary cell cultures. The stages of the menstrual cycles were determined according to the patients’ menstrual history and standard histological criteria (10). The experimental procedures were approved by the institutional review board of the University of Tokyo and signed informed consent for use of the endometrium was obtained from each woman.
Isolation and culture of human endometrial stromal and epithelial cells
Isolation and culture of human endometrial stromal cells (ESCs) and epithelial cells (EECs) were processed as described previously (11, 12). Fresh endometrial biopsy specimens collected in sterile medium were rinsed to remove blood cells. The tissues were minced into small pieces and incubated in DMEM/F-12 containing type I collagenase (0.25%) and deoxynuclease I (15 U/ml) for 120 min at 37 C. The resultant dispersed endometrial cells were separated by filtration through a 40-µm nylon cell strainer (Becton Dickinson and Co, Franklin Lakes, NJ). Endometrial epithelial glands that remained intact were retained by the strainer, whereas dispersed ESCs passed through the strainer into the filtrate.
ESCs in the filtrate were collected by centrifugation and resuspended in phenol-red free DMEM/F-12 containing 10% charcoal-stripped FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. ESCs were seeded in a 100-mm culture plate and kept at 37 C in a humidified 5%CO2-95% air atmosphere. At the first passage, the cells were plated into 6- or 48-well culture plates (Becton Dickinson) at a density of 2 x 105 cells/ml. The cells reached confluence in 2 or 3 d and then were used for the experiments.
EECs were collected by backwashing the strainer with DMEM/F-12 containing 10% charcoal-stripped FBS, seeded in a 100-mm plate, and incubated at 37 C for 60 min to allow contaminated ESCs to attach to the plate wall. The nonattached EECs were recovered and cultured in the culture medium at a density of 2 x 105 cells/ml into 6- or 48-well culture plates. The cells, which reached confluence in 3 or 4 d, were used for experiments. The purity of both the stromal and epithelial cell preparations was more than 95%, as judged by positive cellular staining for vimentin or cytokeratin, respectively.
Treatment of the cells
First, to examine effects of LPS and sCD14 on IL-8 production, the cells were incubated in serum-free DMEM/F12 medium with various doses of LPS and sCD14 for 24 h. Second, to examine effects of anti-CD14 neutralizing antibody (R&D) and anti-TLR4 neutralizing antibody (HTA-125), ESCs were preincubated in 1% FBS (used as carrier proteins for antibodies) DMEM/F12 with the antibodies for 30 min and then stimulated with 100 ng/ml LPS for 45 min to observe NF-B nuclear translocation and for 24 h to evaluate IL-8 secretion. Next, to examine priming effects of IFN- on LPS-induced IL-8 production by ESCs, the cells were incubated with or without IFN- (100 ng/ml) for 12, 24, 48, or 72 h in DMEM/F-12 supplemented with 5% FBS and then stimulated with LPS (100 ng/ml) in serum-free medium for 24 h. Finally, to examine effects of IFN- on TLR4, CD14, MD2, and MyD88 mRNA expression, the cells were incubated with or without IFN- (100 ng/ml) for 0, 2, 6, 12, 24, or 48 h in medium supplemented with 5% FBS.
RNA extraction, reverse transcription (RT), and real-time quantitative PCR of TLR4, CD14, MD2, and MyD88
Using an RNeasy minikit (QIAGEN, Hilden, Germany), we extracted total RNA from ESCs and EECs, both of which had been cultured in 6-well plates. One microgram of total RNA was reverse transcribed in a 20-µl volume using an RT-PCR kit (TOYOBO, Osaka, Japan). Standard PCR was performed using Rever Tra Dash (TOYOBO) according to the manufacturer’s instruction. Human glyceraldehyde dehydrogenase (GAPDH) primers (TOYOBO) were used to ensure RNA quality and amounts. For negative controls, RNA without RT was used.
Primer pairs of TLR4, CD14, MD2, and MyD88 used in PCR are shown in Table 1
. PCR conditions for amplifications of TLR4, CD14, MD2, MyD88, and GAPDH were 28 cycles (for TLR4, CD14, and GAPDH) or 30 cycles (for MD2 and MyD88) at 98 C for 10 sec, 60 C for 2 sec, and 74 C for 15 sec.
|
Each PCR product was purified with a QIAEX II gel extraction kit (QIAGEN), and their identities were confirmed using an ABI PRISMTM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Western blotting
Cultured cells in 6-well plates were homogenized in the lysis buffer containing 50 mM Tris HCl (pH 6.8), 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue and were diluted to 1 mg total protein per milliliter. Samples and lysate of THP-1, a human monocytic leukemia cell line (Santa Cruz Biotechnology) were resolved in 8% SDS-PAGE. Proteins were blotted onto a nitrocellulose membrane and incubated with a rabbit antibody to TLR4 (1:300) as a primary antibody and an antirabbit antibody (1:1000; Amersham Pharmacia Biotech, Little Chalfont, UK) as a secondary antibody. Immune complexes were visualized by use of an enhanced chemiluminescence Western blotting system (Amersham Pharmacia Biotech).
In situ hybridization
For preparation of the digoxigenin (DIG)-labeled RNA probe for TLR4, the 317-bp fragment of the human TLR4 complementary DNA, obtained by RT-PCR with the primers described in Table 1
, was subcloned into the appropriate restriction site of the PCR II-TOPO vector (Invitrogen, Carlsbad, CA). After linearization of the plasmid with an appropriate restriction enzyme, the linearized vectors were used as templates for the synthesis of DIG-labeled RNA probes using SP6 or T7 RNA polymerase. In situ hybridization was performed using an ISHR starting kit (Nippon Gene, Toyama, Japan) as described previously (16). Briefly, tissue collected for in situ hybridization (proliferative phase endometrium, n = 5; secretory phase endometrium, n = 4) was fixed by immersion in 10% neutral buffered saline overnight at 4 C before routine paraffin embedding. The paraffin-embedded specimens were sliced at a 5-µm thickness. These sections were mounted on poly-L-lysine-treated slides, deparaffinized, and rehydrated. They were further digested with 5 mg/ml proteinase K for 10 min at room temperature, treated with acetic anhydride, and then subjected to treatment with prehybridization solution for 30 min at 42 C. Hybridization was carried out by applying the diluted probe to each slide section. Each section was incubated in a humidified chamber overnight at 42 C. Slides were washed and then treated with RNase for 30 min at 37 C. After being blocked with blocking solution, the sections were incubated with an anti-DIG, alkaline phosphatase-conjugated antibody (1:500, Roche) for 1 h at room temperature. Color development was carried out by overlaying them with nitroblue tetrazolum/5-bromo-4-ccholoro-3-indolyl phosphate (Roche), and they were incubated in a humidified container in the dark for 12 h. Sense probe hybridization was used as a control for background level.
Measurement of IL-8
Concentrations of IL-8 in conditioned media were measured using a specific ELISA kit (Genzyme/Techne, Minneapolis, MN). The sensitivity of the assay was 15.6 pg/ml. The intraassay and interassay coefficients of variation were less than 5%.
Flow cytometric analysis
Cell suspensions were prepared from confluent cells grown as a monolayer in 6-well plates and detached by using 0.25% trypsin-0.02% EDTA. The cells (2 x 105 cells/sample) were washed twice with PBS containing 2% FBS and stained with the mouse antihuman CD14 monoclonal antibody MY4 or isotype control mouse IgG2b for 30 min on ice. Then the cells were washed twice and incubated with fluorescein isothiocyanate-conjugated antimouse IgG (H+L) antibody (Beckman-Coulter) for 30 min on ice. Being washed twice, the cells were analyzed using EPICS XL flow cytometer (Beckman-Coulter) and EXPO 32 software (Beckman-Coulter).
Immunohistochemistry
Endometrial tissue samples (n = 9) were washed in PBS, embedded in OCT compound (Sakura, Tokyo, Japan), and snap frozen in liquid nitrogen. Cryosections were cut at an 8-µm thickness and mounted on poly-L-lysine-treated slides. Sections were fixed in acetone for 30 min on ice and washed in PBS for 5 min twice. Sections were treated with 3% H2O2 for 15 min to eliminate endogenous peroxidase. After blocking with nonspecific staining blocking reagent (Dako), the sections were incubated with 1 µg/ml anti-CD14 antibody (TUK4) or 1 µg/ml mouse IgG2a isotype control for 60 min at room temperature and incubated with peroxidase-conjugated secondary antibody (goat antimouse Envision plus, Dako) for 30 min. Staining was detected with the diamino-benzidine chromogen after 5 min. All sections were counterstained with hematoxylin and evaluated under a light microscope.
Immunofluorescence detection of NF-B translocation
ESCs were cultured in 16-well chamber slides (Nunc, Naperville, IL) in a humidified 5% CO2-95% air environment and allowed to grow to about 50% confluence. In specific experiments, the cells were pretreated for 30 min with 20 µg/ml anti-TLR4 antibody (HTA125), 20 µg/ml mouse IgG2a, 10 µg/ml anti-CD14 antibody, or 10 µg/ml sheep IgG before LPS treatment. After 45 min of LPS (100 ng/ml) treatment, the cells were fixed with cold methanol/acetone (1:1) at –20 C for 20 min, washed twice with PBS, blocked for 20 min with 5% bovine serum in PBS, and incubated with an anti-NF-B (p65) antibody (2 µg/ml in 1.5% bovine serum in PBS) for 60 min. After two washes with PBS, the slides were incubated in fluorescein isothiocyanate-conjugated secondary antibody (1:200 in 1.5% bovine serum) for 45 min. The slides were then washed two more times and mounted with coverslips using VectorShield (Vector Laboratories, Burlingame, CA). Analysis of the specimens was performed using a fluorescence microscope (BX50; Olympus, Tokyo, Japan).
Statistical analysis
Data were evaluated using ANOVA with Scheffé’s post hoc analysis for multiple comparisons. P < 0.05 was accepted as statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
RT-PCR analysis demonstrated the expression of TLR4, CD14, MD2, and MyD88 mRNA in both EECs and ESCs in culture (Fig. 1). No signal was detected in negative controls using RNA without RT. The expression levels of TLR4, CD14, and MD2 appeared to be higher in ESCs, compared with the levels of corresponding molecules in EECs, whereas the expression levels of MyD88 were virtually the same between ESCs and EECs.
|
Using Western blotting, we demonstrated the expression of TLR4 protein as a band at about 100 kDa in both EECs and ESCs. A band of the same size was detected in THP-1, which is known to express TLR4 (17) (Fig. 2).
|
TLR4 mRNA was detected in glandular and luminal epithelial cells and stromal cells using in situ hybridization (Fig. 3). The intensity of staining in these three cell types was essentially equivalent in the same section throughout the menstrual cycle. No specific hybridization products were observed when using the sense riboprobes.
|
LPS between 10 and 1000 ng/ml did not change significantly the amount of IL-8 secretion from EECs. On the other hand, as shown in Fig. 4, LPS at 1 ng/ml and higher doses noticeably increased the secretion of IL-8 from ESCs. The maximal effect was observed with LPS at 100 ng/ml. The magnitude of increase with 100 ng/ml LPS was variable, depending on the cells of different patients, showing between 1.4- and 10.0-fold, with the median 2.7-fold (n = 10). Preincubation of ESCs with polymyxin B (Sigma), a known inhibitor of LPS by binding it, for 30 min before LPS treatment completely abolished LPS-induced IL-8 secretion.
|
). On the other hand, LPS-stimulated IL-8 secretion from ESCs was not augmented by the addition of sCD14 at 0.01 and 0.1 µg/ml (Fig. 5B). The augmentative effect was observed with sCD14 at as high as 1 µg/ml in ESCs. sCD14 (1 µg/ml) alone did not have any effect on IL-8 secretion from both ESCs and EECs.
|
As demonstrated in Fig. 6, the fluorescence intensity of ESCs stained with anti-CD14 antibody was markedly higher than that of control, whereas the intensity was equivalent in EECs, irrespective of anti-CD14 staining. The data show that CD14 is expressed on the cell surface of ESCs but not on EECs.
|
As shown in Fig. 7, the presence of immunoreactive CD14 in the human endometrium was demonstrated. Diffuse and intense CD14 immunoreactivity was localized in stromal cells. In contrast, no CD14 immunoreactivity was visualized in both glandular and luminal epithelial cells. No staining was seen when mouse IgG2a was used as a primary antibody.
|
Treatment with the neutralizing antibodies for CD14 or TLR4 significantly diminished LPS-induced increase in IL-8 secretion, whereas the control IgGs (mouse IgG2a or sheep IgG) had no effect (Fig. 8). Cotreatment with both anti-CD14 and anti-TLR4 antibodies did not show the additive effect.
|
B nuclear translocation in ESCs
NF-B, when activated, translocates from the cytoplasm to the nucleus. Untreated ESCs showed cytoplasmic staining of NF-B (p65) (Fig. 9A), whereas LPS-treated ESCs exhibited nuclear staining of NF-B (Fig. 9B). Pretreatment with the anti-TLR4 antibody (Fig. 9C) or the anti-CD14 antibody (Fig. 9E) inhibited the LPS-induced nuclear translocation of NF-B. Pretreatment with the control IgGs [mouse IgG2a (Fig. 9D) or sheep IgG (Fig. 9F)] did not effect the LPS-induced nuclear translocation. Pretreatment with polymyxin B completely blocked the LPS-induced nuclear translocation (Fig. 9G).
|
pretreatment on the production of IL-8 by ESCs
ESCs were preincubated with or without IFN- (100 ng/ml) for 12–72 h, and then the cells were further incubated for 24 h with or without LPS (100 ng/ml) (Fig. 10). Preincubation with IFN- for 12 h enhanced LPS-induced IL-8 secretion. LPS stimulated IL-8 secretion from the IFN--pretreated cells with the length of preincubation time, the increase being 7.9-fold at 12 h and 23.2-fold at 72 h, compared with the controls.
|
up-regulates TLR4, CD14, MD2, and MyD88 mRNA expression in ESCs
Real-time quantitative PCR analysis demonstrated that IFN- up-regulated TLR4, CD14, MD2, and MyD88 mRNA expression (Table 2). When looking at the amounts of mRNA 2 h after the addition of IFN-, the increase in TLR4 mRNA was 3.9-fold, whereas mRNA of CD14, MD2, and MyD88 increased about 2-fold. The amount of TLR4 mRNA was elevated with time up to 48 h. On the other hand, maximal increases in CD14, MD2, and MyD88 mRNA were observed within 6–12 h in culture, followed by a decrease with time up to 48 h.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS alone is capable of inducing the secretion of IL-8 in ESCs, whereas LPS requires sCD14 to stimulate the secretion in EECs. Consistent with this, CD14 was found to be expressed on the cell surface of ESCs but not EECs. In addition, the immunohistochemical analysis revealed the presence of CD14 protein in ESCs but not in EECs. The findings that antibodies for CD14 and TLR4 suppressed LPS-induced IL-8 secretion and nuclear translocation of NF-B in ESCs implicate that the effects of LPS are actually mediated by TLR4 as well as CD14 in ESCs. These results further reinforce that CD14, either membranous or soluble, play an essential role in LPS-induced TLR4 activation in both ESCs and EECs.
The disparity of sCD14 requirement for TLR4 activation between ESCs and EECs seems to have physiological relevance. TLR4 activation with the aid of sCD14 is also known in intestinal epithelial cells and bladder epithelial cells, in which the tolerant status for LPS in a basal condition is supposed to suppress unnecessary inflammation induced by commensal bacteria (18, 19). Although the contact of epithelial cells with bacteria in the endometrium seems to be less frequents compared with those of the intestine and the bladder, the absence of membranous CD14 in EECs might similarly prevent harmful hyperresponsiveness in response to microorganisms. However, EECs constitutively expressing TLR4 suggest that the cells are poised to ward off bacteria once sCD14 is provided. In this context, sCD14 present in seminal plasma (20) may contribute to TLR4-mediated immunity against possible contaminated microorganisms in semen.
Then comes a question as to why ESCs but not EECs possess cell surface CD14. In answer to this, we speculate the following scenario. In case the epithelial barrier is destroyed, the resultant exposure of ESCs to bacteria induces TLR4-mediated inflammatory responses, thus representing dual defensive systems against pathogens. Concurrently with inflammation in the endometrial stromal layer, sCD14 derived from migrating leukocytes and exudates (21, 22, 23) may evoke TLR4-mediated reactions in EECs on one hand and further inflammatory reactions in ESCs on the other hand, thus effectively eliminating pathogens. Likewise, different responses to LPS between stromal cells and epithelial cells has been observed in the oral mucosa in which the responses have been suggested to be at work in immune defense (24, 25).
The notion that the TLR4 system might be inactive in EECs relative to ESCs in basal status also appears to be consistent with the finding presented here that expression levels of TLR4, CD14, and MD2 mRNA were lower in EECs, compared with ESCs. No difference of MyD88 mRNA expression levels between EECs and ESCs might be due to the unique attribute of MyD88 that it is not an intermediary specific to TLR4.
The present study showed that IFN- increases LPS-induced secretion of IL-8 in ESCs. The production of IFN- in response to infection is a hallmark of innate and adaptive immunity, which serves to activate macrophages and induce Th1-type responses. In view of the antiinfectious properties of IFN-, it may be reasonable that IFN- up-regulates TLR4-mediated responses so as to enhance host defense mechanisms against microorganisms. IFN- is expressed in the leukocytes of the endometrium (26, 27), and the secretion of IFN- from endometrial leukocytes is augmented by IL-18 (15). Furthermore, IFN- expression in the endometrium increases in the first-trimester decidua, especially in the decidua basalis (28, 29). Therefore, it is tempting to speculate that antiinfectious mechanisms involving TLR4 and IFN- may contribute to the protection of the conceptus during pregnancy.
IFN- up-regulating LPS-stimulated IL-8 secretion has been reported in gingival fibroblasts and oral epithelial cells (24, 30). IFN- markedly up-regulates CD14 and MyD88 expression, but not TLR4 and MD2 expression, in the gingival fibroblasts (24), whereas increased expression of CD14, MyD88, TLR4, and MD2 by IFN- was observed in the oral epithelial cells (30). These findings suggest that the effect of IFN- on the TLR4 system may be exerted through different mechanisms, depending on the cell type.
In the present study, the amounts of TLR4, MD2, CD14, and MyD88 mRNA in ESCs were all increased by IFN- treatment for 2 h. However, only the expression levels of TLR4 further increased stepwise up to 48 h, whereas the levels of MD2, CD14, and MyD88 decreased after reaching the maximal level within 6–12 h. These findings may suggest that the regulatory mechanism of mRNA expression by IFN- is distinct between TLR4 vs. MD2, CD14, and MyD88.
TLR4 might have roles other than the receptor for LPS in the endometrium. The finding that chlamydial heat shock protein 60 activates NF-B through TLR4 (31) may imply a possible role of TLR4 in the endometrium in host defense against chlamydia trachomatis, a typical pathogen in pelvic inflammatory disease. Moreover, recent studies have shown that TLR4 can be activated by several endogenous ligands, such as fibronectin domains (32), fibrinogen (33), and hyaluronan oligosaccharides (34). These findings lead us to suggest as-yet-undescribed roles of TLR4 in the endometrium.
In the present study, treatment with antibodies for CD14 or TLR4 apparently completely inhibited the LPS-induced translocation of NF-B in ESCs. On the other hand, treatment with these antibodies, even in combination, showed partial inhibition on IL-8 secretion by ESCs. In view of the current notion that alternative pathways other than TLR4 system exist for LPS recognition, our findings imply the presence of such alternative pathways in ESCs.
The expression levels of TLR4 mRNA were seemingly less in EECs than ESCs in culture, whereas relatively high levels of protein were present in EECs as well as ESCs. The expression levels of TLR4 mRNA appeared to be equivalent in EECs and ESCs in the in situ hybridization experiment. The difference of experimental status, e.g. in vivo or in culture, might cause these apparent discrepancies.
In summary, the present study demonstrated the presence of TLR4 in human endometrial cells, pointing to its possible roles in innate immunity in the human endometrium. In addition, given the pronounced expression of IFN- in the first-trimester decidua, an observed modulatory effect of IFN- on TLR4-mediated responses might be relevant to preventive mechanisms against infection during pregnancy.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Abbreviations: DIG, Digoxigenin; EEC, endometrial epithelial cell; ESC, endometrial stromal cell; F-12, Ham’s F12; FBS, fetal bovine serum; GAPDH, glyceraldehyde dehydrogenase; IFN, interferon; LPS, lipopolysaccharide; NF-B, nuclear factor-B; RT, reverse transcription; sCD14, soluble CD14; TLR, Toll-like receptor.
Received February 10, 2004.
Accepted October 18, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-inducible protein-10 in human endometrial stromal cells. Mol Hum Reprod 8:176–180B through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes. J Biol Chem 274:7611–7614 interferon increase production of interleukin-8 in response to lipopolysaccharide through up-regulation of membrane CD14 and MyD88 mRNA expression. Infect Immun 70:1272–1278 interferon-immunoreactive cells in normal endometrium and myometrium. Virchows Arch A Pathol Anat Histopathol 420:419–424[CrossRef][Medline]
is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J Immunol 160:5145–5153 to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J Med Microbiol 51:626–634This article has been cited by other articles:
 |
|
 |
|
  K. N. Khan, M. Kitajima, T. Imamura, K. Hiraki, A. Fujishita, I. Sekine, T. Ishimaru, and H. Masuzaki Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70 Hum. Reprod., July 1, 2008; (2008) den195v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. W Horne, S. J Stock, and A. E King Innate immunity and disorders of the female reproductive tract Reproduction, June 1, 2008; 135(6): 739 - 749. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Koga and G. Mor Review Article: Expression and Function of Toll-Like Receptors at the Maternal--Fetal Interface Reproductive Sciences, March 1, 2008; 15(3): 231 - 242. [Abstract] [PDF]
|
|
|
|
 |
|
 T. Hirata, Y. Osuga, K. Hamasaki, O. Yoshino, M. Ito, A. Hasegawa, Y. Takemura, Y. Hirota, E. Nose, C. Morimoto, et al. Interleukin (IL)-17A Stimulates IL-8 Secretion, Cyclooxygensase-2 Expression, and Cell Proliferation of Endometriotic Stromal Cells Endocrinology, March 1, 2008; 149(3): 1260 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Herath, E. J Williams, S. T Lilly, R. O Gilbert, H. Dobson, C. E Bryant, and I M. Sheldon Ovarian follicular cells have innate immune capabilities that modulate their endocrine function Reproduction, November 1, 2007; 134(5): 683 - 693. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Yang, M.-Y. Wu, C.-D. Chen, M.-J. Chen, Y.-S. Yang, and H.-N. Ho Altered apoptosis and proliferation in endometrial stromal cells of women with adenomyosis Hum. Reprod., April 1, 2007; 22(4): 945 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hirota, Y. Osuga, K. Koga, O. Yoshino, T. Hirata, C. Morimoto, M. Harada, Y. Takemura, E. Nose, T. Yano, et al. The Expression and Possible Roles of Chemokine CXCL11 and Its Receptor CXCR3 in the Human Endometrium J. Immunol., December 15, 2006; 177(12): 8813 - 8821. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. V. Fahey, T. M. Schaefer, and C. R. Wira Sex hormone modulation of human uterine epithelial cell immune responses Integr. Comp. Biol., December 1, 2006; 46(6): 1082 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. N. Evans, L. Nguyen, J. Chan, B. A. Innes, J. N. Bulmer, M. D. Kilby, and M. Hewison Effects of 25-Hydroxyvitamin D3 and 1,25-Dihydroxyvitamin D3 on Cytokine Production by Human Decidual Cells Biol Reprod, December 1, 2006; 75(6): 816 - 822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Takemura, Y. Osuga, T. Yamauchi, M. Kobayashi, M. Harada, T. Hirata, C. Morimoto, Y. Hirota, O. Yoshino, K. Koga, et al. Expression of Adiponectin Receptors and Its Possible Implication in the Human Endometrium Endocrinology, July 1, 2006; 147(7): 3203 - 3210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Herath, D. P. Fischer, D. Werling, E. J. Williams, S. T. Lilly, H. Dobson, C. E. Bryant, and I. M. Sheldon Expression and Function of Toll-Like Receptor 4 in the Endometrial Cells of the Uterus Endocrinology, January 1, 2006; 147(1): 562 - 570. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||
