Journal of Reproductive Immunology
Volume 84, Issue 1 , Pages 75-85, January 2010

A role for IL-17 in induction of an inflammation at the fetomaternal interface in preterm labour☆

  • Mika Ito

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Akitoshi Nakashima

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Takao Hidaka

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Motonori Okabe

      Affiliations

    • Department of Regenerative Medicine, University of Toyama, Toyama, Japan
    • ,
  • Nguyen Duy Bac

      Affiliations

    • Department of Anatomy, Vietnam Military Medical University, Hatay, Vietnam
    • Department of Genomics and Cytogenetics, Vietnam Military Medical University, Hatay, Vietnam
    • ,
  • Shihomi Ina

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Satoshi Yoneda

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Arihiro Shiozaki

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • ,
  • Shigeki Sumi

      Affiliations

    • Center for the Advancement of Medical Training, Division of Biostatistics and Clinical Epidemiology, University of Toyama, Toyama, Japan
    • ,
  • Koichi Tsuneyama

      Affiliations

    • Department of Diagnostic Pathology, University of Toyama, Toyama, Japan
    • ,
  • Toshio Nikaido

      Affiliations

    • Department of Regenerative Medicine, University of Toyama, Toyama, Japan
    • ,
  • Shigeru Saito

      Affiliations

    • Department of Obstetrics and Gynecology, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
    • Corresponding Author InformationCorresponding author. Tel.: +81 76 434 7355; fax: +81 76 434 5036.

Received 10 April 2009; received in revised form 17 September 2009; accepted 20 September 2009. published online 19 November 2009.

Article Outline

Abstract 

Chorioamnionitis (CAM) is a major cause of preterm delivery. Inflammatory cytokines and chemokines play important roles in the pathogenesis of preterm delivery. Interleukin (IL)-17 is a key cytokine which induces inflammation and is critical to host defense. In this study, we examined the role of IL-17 in the pathogenesis of preterm delivery. The levels of cytokines including IL-17, IL-8 and tumor necrosis factor (TNF) α were measured by ELISA in amniotic fluid from 154 cases of preterm labor. Flow cytometry and immunohistochemical staining were performed to determine the distribution of IL-17-producing cells. IL-8 secretion was evaluated in primary cultured human amniotic mesenchymal (HAM) cells and human amniotic epithelial (HAE) cells stimulated with IL-17, TNFα or IL-1β. We also studied the signaling pathway of IL-17 and TNFα in HAM cells. Levels of inflammatory cytokines in amniotic fluid were higher in preterm delivery cases than in term delivery cases. Furthermore, IL-8, IL-17 and TNFα levels were significantly higher in the preterm cases with CAM stage II or III than those without CAM. Flow cytometry and immunohistochemical staining revealed that CD3+CD4+ T cells were the main source of IL-17 in the chorioamniotic membrane. Interestingly, TNFα-induced IL-8 secretion was enhanced by IL-17 in a dose-dependent manner in HAM cells. The IKK inhibitor BMS-345541 and mitogen-activated protein kinase (MAPK) inhibitors p38, JNK and p42/44 (ERK1/2 pathway) reduced IL-8 secretion by IL-17-stimulated and TNFα-stimulated HAM cells. These results indicate that IL-17, produced by T cells, promotes inflammation at the fetomaternal interface in preterm delivery.

Keywords: Amniotic fluid, Chorioamnionitis, IKK, MAPK, Preterm delivery, Th17

 

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1. Introduction 

Chorioamnionitis (CAM) is a risk factor for preterm delivery (Goldenberg et al., 2002, Hillier et al., 1988) and is associated with brain damage in preterm infants (Verma et al., 1997, Yoon et al., 1997). Chorioamniotic tissues are characterized by an intense infiltration of maternal neutrophils (Blanc, 1981, Steel et al., 2005). The amniotic fluid of CAM patients contains increased levels of pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), interleukin(IL)-1β (Romero et al., 1989a, Romero et al., 1989b), granulocyte-colony stimulating factor (G-CSF), and IL-8 (Saito et al., 1993). The secretion of IL-8, a chemotactic factor for neutrophils, is enhanced by TNFα or IL-1β in several cell types including leukocytes, endothelial cells, fibroblasts, trophoblasts, and decidual cells (Beck et al., 1999, Macdermott, 1996, Mahalingam and Karupiah, 1999, Saito et al., 1993).

IL-17, a pro-inflammatory cytokine, induces the expression of many mediators of inflammation (Moseley et al., 2003, Witowski et al., 2004). In humans, IL-17 was identified first in CD4+T cells (Shin et al., 1999, Yao et al., 1995), and then in CD8+ T cells (Shin et al., 1999), NKT cells (Michel et al., 2007), γδ T cells (Lockhart et al., 2006), and monocytes (Starnes et al., 2001). This cytokine is reported to be associated with various chronic inflammatory conditions, including rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and cancer (Witowski et al., 2004). In chronic inflammatory lung disorders, IL-17 played a very important role in the recruitment and activation of neutrophils (Linden et al., 2005). IL-17 binds to its receptor, IL-17 receptor A (IL-17 RA) (Yao et al., 1995). Signals are transmitted through both NF-κB and the mitogen-activated protein kinase (MAPK) (Moseley et al., 2003).

We firstly examined the correlation between the level of IL-17 in amniotic fluid and the severity of CAM. We also examined the cellular origin of the IL-17. Finally we studied the effects of IL-17 on the secretion of IL-8 by primary cultured human amniotic mesenchymal (HAM) cells and human amniotic epithelial (HAE) cells. In this study, we examined the role of IL-17 in the pathogenesis of preterm delivery.

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2. Materials and methods 

2.1. Amniotic fluid samples 

Pregnant women at 22–34 weeks of gestation, who were treated at Toyama University Hospital for preterm labor, and/or premature preterm rupture of the membranes, were enrolled in this study. Preterm labor was defined according to the Canadian Preterm Labor Investigators group (Richter, 1977) as the presence of regular uterine contractions (six per 60min documented by external tocography) or any uterine activity associated with a cervix effaced by at least 50% or dilated by 2cm or more. Cases complicated by preeclampsia, placental abruption, gestational diabetes, autoimmune disease and fetal genetic anomaly were excluded. After written informed consent was obtained, ultrasonically guided transperitoneal amniocentesis was performed to estimate the intrauterine inflammation in 154 cases (Fig. 1). Amniocentesis was usually performed within a week of admission (Table 1). The amniotic fluid samples were stored at −80°C until assays of IL-8, IL-17 and TNFα concentrations. Chorioamnionitis was confirmed histologically in 103 placental samples from 107 preterm delivery cases. Chorioamnionitis was subclassified according to Blanc's system as follows (Blanc, 1981)—Stage I: Patchy-diffuse accumulation of neutrophils in the subchorionic plate or decidua. Stage II: More than a few scattered neutrophils in the chorionic plate or membranous chorionic connective tissue. Stage III: Neutrophils reach the subamniotic connective tissue and the amnionic epithelium. All of the sampling and use of the amniotic fluid for this study were approved by the Toyama University Ethics Committee.

  • View full-size image.
  • Fig. 1. 

    Profiles of subject included in the study. Total 154 cases were admitted in our University Hospital and amniocentesis was performed within 2 weeks. We performed tocolysis, and 107 cases resulted in preterm delivery and 47 cases resulted in term delivery.

Table 1. Clinical profile of subjects.
Preterm deliveryTerm delivery
CAM negative (n=41)CAM positive (n=62)p-Valuen=47p-Value
Mother's age (years)30.8±4.830.5±4.7N.S.30.9±4.3N.S.
Multiparity19 (46%)33 (53%)N.S.24 (51%)N.S.
Gestational age at admission (weeks)26.1±4.325.7±4.1N.S.28.1±5.3*p<0.05
Gestational age at sampling (weeks)27.0±2.926.7±3.5N.S.28.7±4.2*p<0.05
Gestational age at delivery (weeks)32.2±3.929.6±4.1p<0.0138.1±1.1*,†p<0.0001

NS: Not significant. Data are presented as the mean±S.D.

*p-Value is for CAM-positive group.

†p-Values are for the CAM-negative group.

2.2. Blood and tissue samples 

Heparinized venous blood and decidual samples were obtained from cases whose abortion was induced in early pregnancy (6–9 gestational weeks). Decidual mononuclear cells (leukocytes) were purified by the Ficoll-Hypaque method after homogenization and filtration through a 32μm nylon mesh as reported (Saito et al., 1992). To prevent the effect of enzymatic treatment on the fluorescence intensity of surface antigens, decidual tissues were not enzymatically digested. Peripheral blood mononuclear cells were also isolated by the standard Ficoll-Hypaque method. Neutrophils were separated from whole blood by the dextran precipitation method. Informed written consent was obtained from all patients. All of the sampling and use of the tissues for this study were approved by the Toyama University Ethics Committee.

2.3. Enzyme-linked immunosorbent assay (ELISA) 

The concentrations of IL-8 in amniotic fluid and conditioned medium were measured by ELISA (Luo et al., 2000). We used mouse monoclonal anti-human IL-8 antibody (R&D Systems, Minneapolis, MN) as 1st antibody and rabbit polyclonal anti-human IL-8 antibody (PEPROTECH INC., Rocky Hill, NJ, USA) as 2nd antibody. The limit of detection was 32pg/ml; on average, intra-assay and interassay coefficients of variation were 4.8% and 7.5%, respectively. The levels of IL-17 in amniotic fluid and conditioned medium were measured using an ELISA kit (eBiosciences, San Diego, CA, USA). The detection limit of the IL-17 assay was 4pg/ml. TNFα levels were also measured using a high sensitivity ELISA kit (R&D Systems). Due to an insufficient amount of sample, TNFα levels could not be measured in 51 of 154 cases. The detection limit of the assay was 0.5pg/ml.

2.4. Flow cytometry 

Peripheral blood mononuclear cells (PBMCs) were stimulated with phorbol myristate acetate (PMA; 10ng/ml, Sigma Chemical Co., Deisenhofen, Germany) and 2μg/ml of ionomycin (Sigma Chemical Co.) in the presence of 10μg/ml of brefeldin A (Sigma Chemical Co.) for 4h at 37°C in an atmosphere containing 5% CO2. The peripheral blood granulocytes were incubated in the presence of brefeldin A (10μg/ml) alone for 4h at 37°C in an atmosphere with 5% CO2. Decidual mononuclear leucocytes were stimulated with PMA (5ng/ml, Sigma Chemical Co.) and ionomycin (1μg/ml, Sigma Chemical Co.) in the presence of brefeldin A (10μg/ml, Sigma Chemical Co.) for 4h at 37°C in 5% CO2. These mononuclear cells were stained for 20min at room temperature with FITC-conjugated mAbs to CD4, CD8, CD56, CD14 and CD15 (BD PharmingenTM, San Diego, CA, USA). The cells were washed and fixed in 4% formaldehyde/PBS for 5min at room temperature, and treated with permeabilizing solution buffer (BD Bioscience, SanJose, CA, USA) for 10min at room temperature. They were then stained with PE-conjugated anti-IL-17 (eBioscience) for 30min on ice. After being washed, the cells were analyzed on a FACS Calibur flow cytometer using CellQuest software (BD Bioscience). We counted 50,000 cells. A gate was set on the PBMCs or decidual mononuclear leukocytes using characteristic forward scatter parameters (FSC) and side scatter parameters (SCC). Intracellular cytokine patterns were analyzed by flow cytometry. An isotype-matched PE-conjugated mouse IgG1 antibody was used as a control (eBioscience).

2.5. Immunohistochemistry 

Five micrometer sections from formalin-fixed, paraffin-embedded human chorionic tissues were deparaffinized in xylene, rehydrated in graded solution of alcohol, subjected to antigen retrieval by immersion in 1% Sodium Citraconic Acid in Aqueous Solution (Nissin EM, Tokyo, Japan) and irradiated with standard microwave equipment (maximum 500W; SHARP Co., Tokyo, Japan) for 15min. After tissue samples had cooled to 37°C at room temperature, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 5min. Next non-specific staining was prevented by soaking in 10% rabbit serum, and sections were incubated with goat polyclonal anti-human IL-17 (1:100; R&D Systems). Subsequently, samples were irradiated intermittently (4s on and 3s off) using specialized microwave equipment (MI33, Azumaya, Tokyo, Japan) (Kumada et al., 2004) for 15min and incubated for 30min at room temperature. The intermittent microwave irradiation enhanced immunostaining. Further processing of the sections was performed using the dextran-polymer method (Dako, Glostrup, Denmark) and diaminobenzidine (DAB; Sigma, St. Louis, MO, USA). After being washed, sections were counterstained with Mayer's hematoxylin, washed in water, and successively immersed in graded ethanol solutions and xylene before coverslipping. In control sections, the primary antibody was replaced by control non-immune goat IgG (Vector Laboratories, Burlingame, CA, USA). All samples were processed under the same conditions.

For two color immunofluorescent staining, the samples were reacted with an anti-human CD3 mouse mAb (1:100; Novocastra, Newcastle, UK) and a biotin-labeled anti-human IL-17 goat polyclonal antibody (R&D Systems). They were secondarily stained with a donkey anti-mouse IgG-specific antibody conjugated to Alexa-488 (1:2000; Molecular Probes Inc., Eugene, DR) and streptavidin conjugated to Alexa Fluor 594 (1:1000; Molecular Probes Inc.). They were mounted with coverslips using mounting medium with 4′,6-diamino-2-phenylindole (DAPI; Vector Laboratories). Confocal images of fluorescent materials were collected using a TCS-SP5 confocal laser scanning microscope (Leica Microsystems, Wetzler, Germany).

2.6. Immunocytochemistry 

The cells were fixed in 4% paraformaldehyde–PBS for 15min and labeled with the primary antibody, an anti-human IL-17 receptor A antibody (R&D Systems) at a dilution of 1:100. Negative controls were performed with mouse IgG instead of the primary antibody. The cells were then stained with the donkey anti-mouse IgG-specific antibody conjugated to Alexa-594 (1:2000; Molecular Probes Inc.). They were mounted with coverslips using mounting medium with DAPI (Vector Laboratories) and observed under a TCS-SP5 (Leica Microsystems).

2.7. Isolation and culture of amniotic cells 

Human amniotic epithelial cells and human amniotic mesenchymal cells were isolated as reported (Toda et al., 2007). Briefly, the amniotic membrane was peeled mechanically from the chorion of a placenta obtained from pregnant females with an uncomplicated cesarean section. The tissue was rinsed in phosphate-buffered saline containing 0.03% hyaluronidase (Sigma, Poole, UK) and 0.025% DNase (Sigma), collected into a 50-ml tube with 500ml of Dulbecco's minimum essential medium (DMEM, Sigma) and 0.2% trypsin 1:250 (Sigma), and stirred at 120rpm for 30min at 37°C. Dispersed HAE cells were centrifuged and then cultured in DMEM with heat-inactivated fetal bovine serum (10%, v/v, Biological Industries, Haemek, Israel) and a 1% antibiotic–antifungal solution (Gibco BRL, Gaithersburg, MD, USA). After the HAE cells were completely removed (>98%), HAM cells were isolated from the amniotic tissue. The tissue pieces were placed in DMEM containing collagenase (0.75mg/ml) and DNase (0.075mg/ml) and incubated at 37°C for 60min with shaking (100rpm). The dispersed HAM cells were collected by filtration of the mixture through gauze and centrifugation. Cell viability was over 95% as determined by trypan blue dye exclusion, and the yield of HAM cells was approximately 1×106/g tissue. The HAE cells and HAM cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antifungal solution (GIBCO BRL, Grand Island, NY, USA) and incubated in an atmosphere containing 5% CO2 at 37°C.

2.8. Treatment of the cells 

HAM cells were cultured in DMEM supplemented with 10% FCS, penicillin (50IU per ml) and streptomycin (50IU per ml). The cells were cultured to an exponential phase of growth and treated with 0.1% trypsin and 0.2% EDTA. Thereafter, they (2×105 cells/well) were incubated in 12-well plates (Falcon, Lincoln Park, NJ, USA). At 50–70% confluence, the medium was changed to phenol red-free medium prior to starting treatments to eliminate the influence of estrogen activity. Recombinant human IL-1β and TNFα were obtained from R&D Systems. The p38 MAPK inhibitor SB202190, the ERK1/2 inhibitor PD98059, and the IKK2 inhibitor BMS-345541were obtained from Calbiochem Co. (San Diego, CA, USA). The JNK1/2 inhibitor SP600125 was purchased from Sigma–Aldrich (Brondby, Denmark). Inhibitors were dissolved in dimethylsulfoxide (DMSO) and added directly to cell cultures at concentrations of 10μM of SB202190, 20μM of SP6000125, 5μM of PD98059 and 5μM of BMS-345541 respectively. As a control, an equal volume of DMSO (0.1% of volume) was added to the conditioned medium.

2.9. Statistical analysis 

The data from clinical profiles of subjects are presented as the mean±standard deviation (SD), and were analyzed with an unpaired t-test. The data on cytokine levels in amniotic fluid are presented as median values with ranges, and were analyzed with the Mann–Whitney's U-test. Pearson's correlation coefficient was used to determine the associations between different cytokines. Cytokine levels in conditioned medium were analyzed with the Mann–Whitney's U-test and ANOVA test.

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3. Results 

3.1. Concentrations of IL-17, TNFα and IL-8 in amniotic fluid 

Clinical profiles of subjects are presented in Table 1. To examine the role of IL-17 in the pathogenesis of CAM, the concentrations of IL-17, IL-8 and TNFα in amniotic fluid obtained from preterm labor cases were measured (Table 2). The median concentration levels of IL-17, IL-8 and TNFα were higher in preterm delivery cases than in term delivery cases, with significant differences in IL-8 and IL-17 levels.

Table 2. Cytokine levels in amniotic fluid in cases whose deliveries were terminated preterm or term.
Term delivery (n=47)Preterm delivery (n=107)p-Value
IL-17 (pg/ml)0.2 (0.0–17.3)1.3 (0.0–307.2)0.0210
IL-8 (ng/ml)3.6 (0.1–148.9)22.3 (0.0–480.0)<0.0001
TNFα (pg/ml)0.0 (0.0–238.3)0.4 (0.0–6807)0.0608

Data presented as median values with ranges in parentheses. The number of cases being measured TNFα in term and preterm delivary cases were 25 and 73, respectively. The Mann–Whitney U-test was used for comparisons between groups.

We then examined the correlation between cytokine levels and the severity of CAM. According to Blanc's classification, we divided the preterm delivery cases into four categories, CAM negative [CAM(−)], CAM stage I (CAM I), CAM stage II (CAM II) and CAM stage III (CAM III). IL-17, IL-8 and TNFα levels in amniotic fluid at each CAM stage are shown in Table 3. IL-17 levels were significantly higher in CAM II and III than CAM(−). Similar results were obtained for IL-8 and TNFα levels. However, there were no significant differences between CAM(−) and CAM I. There was no correlation between amniotic IL-17 levels and gestational age at sampling (r=−0.072; p=0.378). Steroid treatment was performed in 14 cases before the amniocentesis (steroid pretreatment group). Because steroid treatment affects immunological functions, we usually administered glucocorticoid after the amniocentesis. Therefore the sample number in the steroid pretreatment group was small. No significant difference was observed between steroid pretreated and untreated groups in CAM(−), CAM I, CAM II and CAM III cases (see Supplementary Table 2). Similarly, antibiotic treatment or pPROM did not affect IL-17 levels in amniotic fluid in CAM(−), CAM I, CAM II and CAM III cases (see Supplementary Tables 1 and 3). In amniotic fluid culture-positive cases, the IL-17 levels [5.5 (0.0–61.8)pg/ml, n=12] were significantly higher than those in culture-negative cases [1.1 (0.0–307.2)pg/ml, n=70] (see Supplementary Table 4). These results suggested that levels of inflammatory-related cytokines in amniotic fluid were correlated with the severity of CAM, especially in stage II and III. Additionally, the IL-17 level in amniotic fluid might be associated with bacterial infection.

Table 3. Relationship between cytokines levels in amniotic fluid and histologic findings in chorioamniotic membranes.

Data presented as median values with ranges in parentheses. The number of cases being measured TNFα in CAM(−), CAM I, CAM II and CAM III were 35, 14, 19 and 10. respectively. The Mann–Whitney U-test was used for comparisons between groups. Significant differences were as follows: *p<0.05, **p<0.01, ***p<0.001.

3.2. Correlation between IL-17 and IL-8 or TNFα levels in amniotic fluid 

Levels of inflammatory cytokines were significantly higher in CAM II and III, however, the relation among the three cytokines was unclear. Consequently, we plotted graphs and calculated the coefficients of correlations between the cytokines (Fig. 2). A significant positive correlation was observed between IL-17 and IL-8 levels (r=0.374; p<0.0001) and IL-8 and TNFα levels (r=0.466; p<0.0001). There was no significant correlation between IL-17 and TNFα levels. These results suggest that IL-8 levels correlate with both IL-17 and TNFα levels, and that the secretion of IL-17 is regulated separately from that of TNFα.

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  • Fig. 2. 

    Correlation between amniotic fluid cytokine levels (a) IL-17 levels and IL-8 levels, (b) IL-8 levels and TNFα levels and (c) IL-17 levels and TNFα levels. A significant, positive correlation was observed between amniotic fluid IL-17 and IL-8 levels and IL-8 and TNFα levels.

3.3. IL-17-producing cells in peripheral blood and decidua in early pregnancy 

To investigate the identity of the IL-17-producing cells, we evaluated IL-17-positive peripheral blood mononuclear cells and decidual lymphocytes after stimulation with PMA and ionomycin by flow cytometry. In early pregnancy, the major population of IL-17-producing T cells was CD4+ T cells in both peripheral blood and decidual lymphocytes (Fig. 3). Very few IL-17-producing cells were detected among CD8+ T cells, CD56dim NK cells, CD56bright NK cells, CD14+ monocytes or CD15+ neutrophils in peripheral blood and amongst decidual leukocytes. These results showed that CD4+ cells were the main source of IL-17 in early pregnancy.

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  • Fig. 3. 

    Intracellular IL-17 pattern in CD4+ T cells, CD8+ T cells, CD56+ NK cells, CD14+ monocytes and CD15+ granulocytes in peripheral blood (a) and decidual lymphocytes (b) during early pregnancy. The right upper panel means IL-17 positive cells among each marker-positive cells. Numbers represent the percentages of the dots in each gated area.

3.4. Localization of IL-17-producing cells in the chorioamniotic membrane 

To investigate the distribution of IL-17-producing cells in the chorioamniotic membrane, immunostaining for IL-17 was performed. IL-17-positive mononuclear cells were detected in the chorionic plate and just below the amniotic membrane in CAM III cases (Fig. 4A, arrowheads). On the other hand, few IL-17-positive cells were detected in CAM(−) cases (Fig. 4B). To estimate the specificity of the IL-17 antibody, samples of synovial membrane from rheumatoid arthritis (RA) patients were used (Fig. 4F). Many IL-17-positive cells were detected in the synovial membranes (Fig. 4F arrow head). When normal goat serum was used as the primary antibody, no IL-17 staining was detected in CAM III samples (Fig. 4C). The addition of recombinant human IL-17 completely blocked the reaction of the IL-17 antibody in CAM III cases (Fig. 4D and E). To verify which cells possess IL-17 in the chorioamniotic membrane, double immunostaining for IL-17 and CD3 was conducted in CAM III cases. A small proportion of CD3-positive cells (green) had IL-17 staining (red) (Fig. 4G, yellow arrow). We did not observe any IL-17 staining in CD3-negative lymphocytes. Furthermore, an immunohistochemical examination using a number of sections indicated that some T cells were IL-17-producing cells in CAM patients. These results showed the presence of IL-17-positive T cells in the chorioamniotic membrane of CAM patients.

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  • Fig. 4. 

    Immunostaining of IL-17 in chorioamniotic membrane of CAM (A, C–E and G), a case of CAM-negative preterm delivery case (B) and synovial membrane in a case of rheumatoid arthritis (RA; F). Sections of tissue were incubated with anti-IL-17 antibody and were stained using the immunoperoxidase technique. Many positive mononuclear cells were observed in chorioamniotic membrane (A). The arrowhead shows IL-17 positive cells. But these IL-17-possitive cells were very few in chorioamniotic membrane of CAM negative case (B). (C) Goat IgG as a negative control. IL-17 positive mononuclear cells were recognized in CAM case (D), but disappeared when the tissue was pretreated with rIL-17 (E). Synovial membrane of a RA patient as positive control for IL-17 immunostaining (F). Many lymphocytes showed with IL-17 staining in synovial membrane. (G) Double immunohistochemical staining for IL-17 (red) and CD3 (green) under a confocal microscope. Arrowheads (green) indicate CD3+ cells, and the arrow (yellow), a double positive cell. Original magnification ×200 (A–C and F), ×400 (D and E) and ×1890 (G).

3.5. Effect of IL-17 on TNFα-induced IL-8 secretion in HAM cells and HAE cells 

To examine the biological function of IL-17 in the chorioamniotic membrane in vitro, we isolated both HAE and HAM cells from chorioamniotic membranes. The IL-17 receptor A (IL-17RA) was clearly detected on the surface of HAM cells, but not HAE cells (Fig. 5A and B). Next, IL-8 levels were measured in culture media of HAM cells and HAE cells stimulated with IL-17, IL-1β or TNFα. The basal secretion of IL-8 was much lower in HAE cells than HAM cells (Fig. 5C). The concentration of IL-17 and TNFα added to cultures was based on the physiological concentration in the amniotic fluid (maximum concentration: 307pg/ml and 6807pg/ml). The concentration of IL-1β added was considered to be within the physiological range, as reported in the literature (Cox et al., 1997). Neither IL-17 nor IL-1β alone affected the secretion of IL-8 in both HAE cells and HAM cells (Fig. 5C). On the other hand, TNFα increased IL-8 secretion significantly in both cells (Fig. 5C).

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  • Fig. 5. 

    IL-17 effects on HAM cells. Condition medium was collected after 24h of culture and the concentration of IL-8 was determined by ELISA. Results are expressed as the mean±SD for five separate experiments. The statistical significance of the modulatory effect of IL-17 was determined with an ANOVA test. Immunofluorocytochemistory for IL-17 receptor (red) on HAM cells (a) and HAE cells (b). Nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Original magnification: ×400. The concentration of IL-8 in conditioned medium of HAE cells and HAM cells stimulated with IL-17, IL-1β or TNFα (c). Vertical bar shows the fold increase in IL-8 levels of non-stimulated HAM cells. HAM cells were stimulated with various concentrations of IL-17 in combination with 1000pg/ml of TNFα or 1000pg/ml of IL-1β (d). IL-17 augmented the TNFα-induced secretion of IL-8 in a dose-dependent manner. **p<0.0001, compared with controls by two-way ANOVA. Suppresion rate of IKK and MARKs in TNFα- and TNFα+IL-17-induced IL-8 secretion from HAM cells (e and f). Cells were incubated for 1h with the IKK inhibitor BMS-345541 (5μM), the p38 MAPK inhibitor SB202190 (10μM), the ERK1/2 inhibitor PD98059 (5μM) or the JNK1/2 inhibitor SP6000125 (20μM) before being stimulated with TNFα (1000pg/ml) (e) or TNFα (1000pg/ml)+IL-17(1000pg/ml) (f) for 24h. *p<0.01, **p<0.0001, compared with controls by an ANOVA test.

Because the basal secretion of IL-8 was much lower in HAE cells than HAM cells, HAM cells were considered appropriate for further study. To estimate the function of IL-17 as an inflammatory co-stimulatory factor, various concentrations of IL-17 were added to conditioned medium with IL-1β or TNFα. IL-17 significantly augmented the TNFα-induced secretion of IL-8 in a dose-dependent manner (p<0.0001), but did not affect the IL-1β-induced secretion of IL-8 in HAM cells (Fig. 5D). This effect of IL-17 and TNFα was observed in HAM cells, but not in HAE cells (data not shown).

3.6. The effect of MAPK inhibitors on IL-8 secretion stimulated with TNFα and/or IL-17 

To determine which pathway is critical to the secretion of IL-8, HAM cells stimulated with IL-17 (1000pg/ml) and/or TNFα (1000pg/ml) were incubated with, the p38 MAPK inhibitor SB202190 (10μM), the JNK1/2 inhibitor SP6000125 (20μM), the ERK1/2 inhibitor PD98059 (5μM), or the IKK inhibitor BMS-345541 (5μM). We chose inhibitors of the pathways reported in the literatures (Qian et al., 2007, Hunter, 2007). The amount of IL-8 secreted by the cells stimulated with TNFα was significantly reduced by all four inhibitors (Fig. 5E). Similar results were obtained with cells stimulated with IL-17 and TNFα (Fig. 5F). The inhibition by MAPK inhibitors was similar for TNFα alone and TNFα plus IL-17. However, with the IKK-inihibitor (BMS-345541), the inhibition was greater in HAM cells stimulated with TNFα and IL-17 was than with TNFα alone (Fig. 5E and F). These results mean that the IKK-NF-κB pathway is most critical for the signal transduction by both IL-17 and TNFα.

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4. Discussion 

In this study, we first showed that amniotic IL-17 levels were significantly higher in cases of preterm delivery than term delivery, and significantly increased in CAM stage III cases. In CAM stage III cases, inflammatory cells were detected in the amniotic membrane, close to the fetus. Second, we showed the presence of IL-17-positive PBMCs during normal pregnancy in a flow cytometric analysis. Interestingly, IL-17-positive cells were observed in the amniotic membrane in CAM cases, but not in case of normal pregnancy. These findings suggest an association between Th17 cells and preterm delivery due to inflammation-related disease. Third, we demonstrated in vitro that IL-17 induced IL-8 secretion in primary HAM cells, which stably expressed the IL-17 receptor A. TNFα also induced IL-8 secretion in the HAM cells. Furthermore, IL-17 significantly enhanced the TNFα- but not IL-1β-induced secretion of IL-8 in a dose-dependent manner. This effect was prevented by an inhibitor of IKK and p38 MAPK. The results indicate that Th17 cells may play an important role in the induction of CAM.

Chow et al. (2008) reported that median IL-17 concentrations were very low in amniotic fluid and maternal sera in women at 14–16 weeks gestation without evidence of infection. Gargano et al. (2008) demonstrated that plasma IL-17 levels in mid-pregnancy were slightly elevated in cases of spontaneous preterm birth, but this was not associated with histological CAM. Considering that intrauterine infection can cause a local inflammatory process and initiate preterm labor, the level of IL-17 in amniotic fluid may be useful for evaluating the severity of CAM, namely local inflammation in the chorioamniotic membrane. In our study, IL-17 protein was detected in almost none of the amniotic fluid samples in cases of preterm delivery without CAM. Furthermore, concentrations of IL-17 were significantly higher in cases of preterm delivery with CAM stage II and III than in CAM-negative cases. These findings could support the following hypothesis. When IL-17-producing lymphocytes infiltrate the chorionic plate, IL-17 protein is secreted into the surrounding tissue (CAM stage II). In CAM stage III, IL-17-producing lymphocytes extend to the fetal membrane, and the IL-17 level rapidly increases in amniotic fluid through the amniotic membrane.

Which cell type produces IL-17 protein in the chorioamniotic membrane? Pongcharoen et al. (2007) reported that IL-17 was expressed in human term placentas. Additional studies found that lymphocytes, particularly CD4+ T cells, NKT cells and γδ T cells, are the main source of IL-17. In our study, IL-17-positive cells were detected among CD3+ T cells and CD4+ T cells. No IL-17-positive extravillous or villous trophoblasts were detected. Furthermore, neither HAE cells nor HAM cells stimulated with IL-1β or TNFα were found to produce IL-17 by ELISA (data not shown). Consequently, our results indicated that CD4+ T cells are the main source of IL-17 in the chorioamniotic membrane. As mentioned above, the level of IL-17 in amniotic fluid significantly correlated with CAM stage. Taken together, this correlation may be due to the infiltration of T cells, not trophoblasts.

Previous papers have emphasized the importance of IL-17 production by T cells in inducing inflammation (Albanesi et al., 2000, Shin et al., 1999). This study showed that intense infiltration by T cells is one of the causes of chronic inflammation in CAM cases. A previous study found that the IL-8 level in the amniotic cavity was elevated in cases of preterm delivery with a microbial infection (Romero et al., 1991). It was also reported that TNFα produced by monocyte/macrophages or trophoblasts, induced IL-8 production via many types of cell (Beck et al., 1999, Lockwood et al., 2006). Our results showed that in combination with IL-17, TNFα significantly enhances the secretion of IL-8 in HAM cells, whereas IL-1β does not. This effect of IL-17 may be due to IL-17RA expression. In our experiments, the amount of IL-17 added was based on the physiological concentration of IL-17 found in amniotic fluid in CAM. This concentration of IL-17, which was adequate for enhancing of TNFα-induced IL-8 production, is lower than in other reports (∼100ng/ml) (Hata et al., 2002, Hirata et al., 2008). These results suggest that a population of IL-17-positive cells may initiate inflammation in the chorioamniotic membrane. Additionally, amniotic TNFα levels were positively correlated with amniotic IL-8 levels, but not with IL-17 levels. IL-17 and TNFα might be produced by different cells, and potentially play an important role in inducing migration of neutrophils through induction of IL-8.

IL-17 and TNFα could activate NF-κB and MAPK pathways in target cells (Shen and Gaffen, 2008, Wajant et al., 2003). MAPK- (p38, ERK 1/2 and JNK) and NF-κB-mediated signaling pathways are differentially activated by LPS in human chorioamniotic cells (Jung et al., 2005, Shoji et al., 2007). Three subgroups of the MAPK family have been identified, all of them phosphorylated on tyrosine and threonine residues by upstream kinases, the MAPK kinases. The p44 and p42 ERK1/2 mediate responses mainly to mitogenic stimuli, whereas the c-Jun NH2-terminal kinases and p38 mediate responses to cellular stress (Cobb and Goldsmith, 1995). In this study, the IKK-inhibitor and MAPK-inhibitors significantly inhibited IL-17 with TNFα-induced IL-8 secretion in HAM cells. IL-8 levels in HAM cells stimulated with IL-17 and TNFα were reduced to <10% of control values by the IKK-specific inhibitor, BMS-345541. This reduction was marked compared with that in cells stimulated with TNFα alone, and greater than the rate of reduction obtained with the MAPK-specific inhibitors. A recent study demonstrated that IL-17 alone did not induce the expression of IκB-ζ, but co-stimulation with TNFα and IL-17 did (Yamazaki et al., 2005), which supported our findings. Taken together, the IKK-NFκB pathway could be most important for IL-8 expression in HAM cells.

In conclusion, this is the first report that IL-17 levels in amniotic fluid were significantly higher in CAM stage II or III cases than in cases of without CAM, that IL-17-positive lymphocytes exist in the chorioamniotic membrane, and that IL-17 enhances the TNFα-induced secretion of IL-8 in HAM cells in vitro. The importance of pro-inflammatory cytokines in the pathogenesis of CAM is gradually becoming apparent, and we here emphasize the functions of IL-17. Although it is unknown whether infection results in IL-17-positive cells in the chorioamniotic membrane, further investigations using HAM cells are required to clarify the importance of the microenvironment including the role of lymphocytes in placentas with CAM.

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Appendix A. Supplementary data 

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☆ Grant support: This work was supported by Grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan [Grant-in-Aid for Scientific Research (B) -20390431] and Grants from the Ministry of Health Labour and Welfare, Japan. Health Labour Sciences Research Grant -H20-kodomo-ippan-004 and H20-kodomo-ippan-002.

PII: S0165-0378(09)00494-X

doi:10.1016/j.jri.2009.09.005

Journal of Reproductive Immunology
Volume 84, Issue 1 , Pages 75-85, January 2010

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