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*Pregnancy
The Journal of Immunology, 2005, 175: 8096-8104.
Copyright © 2005 by The American Association of Immunologists

A Role for TLRs in the Regulation of Immune Cell Migration by First Trimester Trophoblast Cells1

Vikki M. Abrahams*, Irene Visintin*, Paulomi B. Aldo*, Seth Guller*, Roberto Romero{dagger} and Gil Mor2,*

* Department of Obstetrics, Gynecology & Reproductive Sciences, Yale University School of Medicine, New Haven, CT 06520; and {dagger} Perinatology Research Branch, National Institute of Child Health and Human Development, Detroit, MI 48202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Normal pregnancy is characterized by the presence of innate immune cells at the maternal-fetal interface. Originally, it was postulated that the presence of these leukocytes was due to an immune response toward paternal Ags expressed by the invading trophoblasts. Instead, we and others postulate that these innate immune cells are necessary for successful implantation and pregnancy. However, elevated leukocyte infiltration may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Furthermore, such conditions have been attributed to an intrauterine infection. Therefore, we hypothesize that first trimester trophoblast cells, upon recognition of microbes through TLRs, may coordinate an immune response by recruiting cells of the innate immune system to the maternal-fetal interface. In this study, we have demonstrated that human first trimester trophoblast cells constitutively secrete the chemokines growth-related oncogene, growth-related oncogene {alpha}, IL-8, and MCP-1 and are able to recruit monocytes and NK cells, and to a lesser degree, neutrophils. Following the ligation of TLR-3 by the viral ligand, poly(I:C), or TLR-4 by bacterial LPS, trophoblast secretion of chemokines is significantly increased and this in turn results in elevated monocyte and neutrophil chemotaxis. In addition, TLR-3 stimulation also induces trophoblast cells to secrete RANTES. These results suggest a novel mechanism by which first trimester trophoblast cells may differentially modulate the maternal immune system during normal pregnancy and in the presence of an intrauterine infection. Such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Normalpregnancy is characterized by a strong immunological presence at the maternal-fetal interface which is dominated by cells of the innate immune system (1). During the first trimester of pregnancy, 70% of decidual leukocytes are NK cells, 20–25% are macrophages, and ~1.7% are dendritic cells (2, 3, 4). From the adaptive immune system, B cells are absent, but T lymphocytes constitute ~1–3% of the decidual immune cells (5). Originally, it was postulated that the presence of these immune cells at the implantation site was due to an immune response toward paternal Ags expressed by the invading trophoblast (6). More recent studies, however, have prompted the notion that the presence of immune cells, such as NK cells or macrophages, within the endometrium are necessary for successful implantation (7, 8, 9, 10, 11).

Embryonic implantation consists of three consecutive phases; apposition, adhesion, and invasion, and in each of these steps the trophoblast confronts different immune cell types and microenvironments. Therefore, the appropriate communication between the maternal immune system and the invading trophoblast at the fetal-maternal interface may be crucial for successful pregnancy. However, alterations in such communication, as in the case of an infection, could result in a complicated pregnancy. Indeed, elevated and altered distributions of immune cells at the maternal-fetal interface have been observed in pregnancy complications (12, 13, 14, 15). Furthermore, intrauterine infections have been associated with cases of preterm labor (16, 17, 18), intrauterine growth restriction (IUGR), and preeclampsia (19, 20, 21, 22). Thus, an infection at the maternal-fetal interface represents a significant threat to both fetal well-being and the success of a pregnancy. However, the precise mechanisms of pathogenesis are largely unknown.

Cells of the innate immune system respond to infectious microorganisms through a system of pattern recognition (23). Pattern recognition receptors recognize conserved sequences known as pathogen-associated molecular patterns (PAMPs).3 PAMPs, such as bacterial LPS or viral dsRNA, are unique to and expressed by microbes (24). One of the main families of pattern recognition receptors are the TLRs. To date, 10 human TLRs have been identified and designated, TLR 1–10 (25). Although extracellularly each TLR is distinct in its specificity, all receptors signal to a common intracellular pathway. Following ligation, TLRs signal through the adapter molecule MyD88 to activate the NF-{kappa}B pathway, which results in an immune response characterized by the production of cytokines, antimicrobial products, and the regulation of costimulatory molecules (24, 25).

Recently, TLRs have been identified in both first trimester and term placenta, suggesting that the placenta may recognize pathogens through these receptors and induce an immune response (26, 27, 28). Therefore, we hypothesize that trophoblast cells, upon recognition of PAMPs through TLR, may coordinate an immune response by recruiting cells of the innate immune system to the site of an infection at the maternal-fetal interface. In this study, we report that first trimester trophoblast cells generate differential chemokine profiles in response to bacterial LPS, through TLR-4 and poly(I:C), through TLR-3. Furthermore, we demonstrate that TLR-3 or TLR-4 stimulation in first trimester trophoblast cells enhances their ability to recruit innate immune cells. These results suggest a novel mechanism by which trophoblast cells may modulate the maternal immune system in the presence of an intrauterine infection, and such altered trophoblast cell responses might contribute to the pathogenesis of certain pregnancy complications.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

The TLR-4 agonist LPS isolated from Escherichia coli (0111:B4) was purchased from Sigma-Aldrich. Poly(I:C), the synthetic analog of viral dsRNA and a TLR-3-specific agonist, was purchased from InvivoGen. Recombinant human IL-8 and MCP-1 were obtained from R&D Systems. The green fluorescent linker dye PKH67 was purchased from Sigma-Aldrich. The MCP-1, growth-related oncogene {alpha} (GRO-{alpha}), and RANTES ELISA kits were obtained from R&D Systems, and the IL-8 ELISA kit was purchased from Assay Designs.

Patient samples

First trimester placentas (8–12 wk of gestation) were obtained from elective terminations of normal pregnancies performed at Yale-New Haven Hospital. Maternal serum was obtained by collecting blood samples from normal pregnant women ages between 19 and 28 years in their first trimester of pregnancy (8–12 wk of gestation; n = 15) who visited the clinic as part of their normal medical care and did not develop any signs of complications throughout gestation. Peripheral blood serum was also obtained from healthy non-pregnant aged-matched female donors (n = 8). All patients signed consent forms and the use of patient samples was approved under Yale University’s Human Investigations Committees.

Cell culture

The human first trimester extravillous trophoblast cell line HTR8 (referred to from hereon as H8) (29) was a gift from Dr. C. Graham (Queens University, Kingston, Ontario, Canada). The human first trimester-like villous cytotrophoblast cell line 3A was purchased from American Type Culture Collection. The monocytic cell line THP-1 was a gift from Dr. P. Guyre (Dartmouth Medical School, Lebanon, NH). Cell lines were cultured at 37°C in 5% CO2 in RPMI 1640 (Invitrogen) supplemented with 10% FBS (HyClone), 10 mM HEPES, 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, and 100 µg/ml penicillin/streptomycin (Invitrogen).

Innate immune cell isolation

Neutrophil isolation Neutrophils were isolated from the peripheral blood of normal donors by density gradient as previously described (30). Whole blood was diluted 1/2 with HBSS (Invitrogen) and layered over an equal volume of a double-density gradient consisting of Histopaque density 1.077 and 1.119 g/ml, respectively (Sigma-Aldrich). This was centrifuged at 400 x g for 25 min at room temperature, after which the lower granulocyte layer was collected and washed with HBSS. Contaminating RBC were removed by resuspending the cell pellet with sterile distilled water, followed by cell rescue with 1.8% sodium chloride solution. After two washes with HBSS, neutrophils were resuspended in OptiMEM (Invitrogen) to a concentration of 5 x 105 cells/ml.

NK cell isolation NK cells were isolated from the peripheral blood of normal donors using positive selection and a modification of the method described previously (31). Briefly, whole blood was diluted 2/1 with HBSS. This was then layered over an equal volume of Histopaque (density, 1.077g/ml) and centrifuged at 400 x g for 25 min at room temperature. The mononuclear layer was then collected and washed and then resuspended with a mouse anti-CD56 mAb conjugated to magnetic microbeads (Miltenyi Biotec). Following a 15-min incubation at 4°C, the suspension was passed down a MACS separation column attached to a magnetic field. Once the column had been washed three times, the bound NK cells were eluted, resuspended in RPMI 1640/10% FBS, and cultured overnight. The next day, the isolated NK cells were washed and resuspended in OptiMEM to a concentration of 5 x 105 cells/ml. The purity of the CD56+ cells was >90% as determined by flow cytometry.

RT-PCR

Total RNA was isolated from cells and tissues using a RNeasy kit from Qiagen. Reverse transcription was performed on 5 µg of total RNA using the First-Strand cDNA Synthesis kit from Amersham Biosciences according to the manufacturer’s directions. The primers used for amplification of human TLR-3 have been described previously (32) and have the following sequences; 5'-AGCCGCCAACTTCACAAG-3' and 5'-AGCTCTTGGAGATTTTCCAGC-3'. Thirty cycles of PCR were performed at 95°C for 15 s, 58°C for 20 s, and 72°C for 30 s. The size of the product was 425 bp.

Chemokine studies

The effect of TLR ligation on trophoblast chemokine production was determined using the Human Cytokine Array kit, 3.1 (for cell lysates) and III (for cell culture supernatants) (RayBiotech). Briefly, 100 µg of protein from whole cell lysates or 1 ml of cell-free supernatant was incubated with the array membrane. A single array was used for each individual sample from an experiment and each experiment was performed twice. Following incubation with primary biotin-conjugated Abs and HRP-conjugated streptavidin, detection of signals was performed by ECL. An example of a single array is shown in Fig. 1. The intensity of the signals was quantified by densitometry using a digital imaging analysis system and 1D Image Analysis Software (Eastman Kodak). The software quantifies the signals and expresses the results as OD units. The signal intensities were then adjusted for the internal negative controls (background) and then normalized against the internal positive controls on each array membrane, which were given the arbitrary unit of 1. Any expression levels below 0.2 U were considered below the detection limit of the assay, as determined by the software. The concentrations of the chemokines, GRO-{alpha}, IL-8, MCP-1, and RANTES were evaluated by ELISA according to the manufacturer’s instructions. The detection limits of the IL-8 ELISA ranged between 8 and 1000 pg/ml.



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  		FIGURE 1. Human cytokine protein array. Shown is an example of the cytokine protein array used in these studies. Each array was incubated with a single sample from an experiment. Each array contains three duplicate negative controls which generate background ODs and three duplicate positive controls against which all signals were normalized. Remaining signals are the 42 cytokines/chemokines in duplicate.

 
RNA interference (RNAi)

H8 cells were transiently transfected with a GFP expression plasmid containing small-interfering RNA directed against MyD88 (InvivoGen). Briefly, 5 x 105 cells were seeded into a 60-mm dish and cultured overnight until 40–60% confluent. Cells were then transfected for 18 h with 2 µg of DNA using Fugene 6 (Roche Applied Science) in a 1:2 ratio. Following transfection, cells were allowed to recover in growth medium for 24 h before a treatment experiment was performed. The transfection efficiency of this plasmid was 40%, as determined by GFP expression, and knockdown of MyD88 was monitored by Western blot.

Preparation of first trimester trophoblast cell conditioned medium (CM)

H8 trophoblast cells (1 x 106) were seeded into 35-mm tissue culture dishes and cultured overnight in RPMI 1640/10% FBS until 80% confluent. The medium was then replaced with 1 ml of OptiMEM, with or without stimuli. Following a culture of 48 h, the cell-free CM was collected by centrifugation at 400 x g for 10 min and stored at –80°C.

Immune cell migration assay

Immune cell migration toward trophoblast CM One milliliter of immune cells (5 x 105/ml) in OptiMEM was seeded into cell culture inserts with an 8-µm pore size membrane (BD Biosciences). The lower chamber for this assay consisted of 24-well tissue culture plates (Falcon; BD Biosciences) which contained 1 ml of OptiMEM alone, 1 ml of trophoblast CM, or 1 ml of 20% FBS in OptiMEM as a positive control. The inserts, loaded with immune cells, were then placed into the wells and this coculture was incubated for 24 h. Following this incubation, the inserts were removed and cell migration into the lower chamber was determined using a colorimetric assay (Chemicon). Briefly, migrated cells were stained and then lysed according to the manufacturers instructions. The resulting colored mixture was transferred to a 96-well plate and ODs were read at 560 nm using a Spectramax M2 plate reader (Molecular Devices).

Immune cell migration toward trophoblast cells

H8 trophoblast cells (1 x 105) were seeded into wells of a 24-well tissue culture plate and cultured overnight in RPMI 1640/10% FBS until 80% confluent. The H8 cells were then changed to 1 ml of OptiMEM, with or without stimuli, and cultured for 48 h. At 48 h, immune cells were stained with the green fluorescent linker dye PKH67 (2 x 10–5 M), after which cell concentration was adjusted to 5 x 105 cells/ml in OptiMEM. One milliliter of this cell suspension was placed into each cell culture insert with an 8-µm pore size membrane (BD Biosciences). The inserts, loaded with fluorescently labeled immune cells, were then placed into the wells containing the trophoblast cells and the coculture was incubated for 24 h. Following this incubation, the inserts were removed and cell migration into the lower chamber was determined by fluorescent microscopy. Three fields of each well were photographed and cell numbers were determined using Kodak MI software (Eastman Kodak).

Statistical analysis

Data are expressed as mean ± SD. Statistical significance (p < 0.05) was determined using one-way ANOVA with the Bonferroni correction.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chemokine profile of first trimester trophoblast cells following TLR-4 ligation with bacterial LPS

The first objective of this study was to determine the normal cytokine profile expressed by first trimester trophoblast cells and what effect TLR-4 ligation by bacterial LPS would have on trophoblast cytokine production. Using a 42-cytokine array, we determined both the intracellular cytokine content of trophoblast cells by evaluating whole cell lysates and trophoblast cytokine secretion by evaluating the cell-free culture supernatants. From this protein array, 13 chemokines were identified and their intracellular expression levels were compared with those secreted. As shown in Table I, the baseline intracellular and secreted chemokine profiles of first trimester trophoblast cells were remarkably different. Intracellularly, trophoblast cells constitutively expressed IL-8 and MIP-1{delta}, but constitutively secreted GRO, GRO-{alpha}, IL-8, and MCP-1, although their expression levels varied from one another. Following stimulation of TLR-4 with LPS (10 µg/ml), the intracellular content of IL-8 and MIP-1{delta} were increased, whereas the expression of the following chemokines was induced: epithelial neutrophil-activating protein 78 (ENA78), GRO, GRO-{alpha}, MCP-1, MCP-2, MCP-3, and stromal cell-derived factor 1 (SDF-1) (Table I). However, only the secreted levels of GRO, GRO-{alpha}, IL-8, and MCP-1 were significantly elevated following TLR-4 ligation (Table I). These results were confirmed and quantified by ELISA. As shown in Fig. 1, IL-8 and MCP-1 secretion from trophoblast cells following treatment with LPS occurred in a dose (Fig. 2, A and B)- and time-dependent manner (Fig. 2, C and D). In addition, GRO-{alpha} secretion was significantly increased following the treatment of trophoblast cells with LPS (Fig. 2E) and also occurred in a time-dependent manner (data not shown). Similar results were obtained using primary isolated first trimester trophoblast cells (data not shown).


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  		Table I. Expression and secretion of chemokines by first trimester trophoblast cellsa

 


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  		FIGURE 2. TLR-4 ligation induces chemokine secretion from first trimester trophoblast cells. H8 cells (1 x 106) were treated with LPS at a range of concentrations. Cell-free culture supernatants were collected at various time points and assayed for IL-8, MCP-1, and GRO-{alpha}. Treatment with LPS increased the levels of IL-8 (A) and MCP-1 (B) secretion in a dose-dependent manner. Both the constitutive and LPS-induced secretion of IL-8 (C) and MCP-1 (D) by trophoblast cells occurred in a time-dependent manner. Furthermore, the secretion of GRO-{alpha} (E) was also increased following ligation of TLR-4 with LPS (100 µg/ml). *, p < 0.05; **, p < 0.001.

 
Monocytes migrate toward LPS-stimulated first trimester trophoblast cells

The observation that trophoblast cells secrete high levels of IL-8 and MCP-1, which are both known to be chemotactic for monocytes (33, 34), suggests that trophoblast cells may regulate monocyte migration to the maternal-fetal interface. Using an in vitro migration assay, we first sought to determine whether the levels of IL-8 and MCP-1 that were detected in the trophoblast supernatants were sufficient to induce monocyte chemotaxis and transmigration. Indeed, monocyte transmigration was significantly increased by 5.5-fold toward IL-8 at a concentration of 2000 pg/ml and by 6.1-fold toward MCP-1 at a concentration of 1000 pg/ml; when compared with random monocyte migration toward medium alone. Next, the effect of trophoblast-derived CM on monocyte transmigration was evaluated. As shown in Fig. 3A, there was significant monocyte migration toward the CM from untreated trophoblast cells, compared with the random monocyte transmigration toward medium alone (p < 0.001). Moreover, there was a 2.3-fold increase in monocyte migration toward the CM from LPS-treated trophoblast cells compared with that of untreated trophoblast cells (p < 0.001). LPS alone did not induce monocyte transmigration (data not shown). Next, we evaluated the effect of trophoblast cells on monocyte migration using a two-chamber coculture system. For this, trophoblast cells in the lower chamber and monocytes in the upper chamber were separated by an 8-µm pore membrane. Trophoblast cells were pretreated with or without LPS for 48 h before the inserts, loaded with fluorescently labeled monocytes, placed into the wells, and incubated for another 24 h. As shown in Fig. 3B, there was a 1.4-fold increase in monocyte migration toward untreated trophoblasts (panel ii), when compared with migration toward medium alone (panel i). Moreover, pretreatment of trophoblast cells with LPS further increased monocyte transmigration by 1.6-fold (p < 0.05) (Fig. 3B, panel iii).



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  		FIGURE 3. Monocytes migrate toward LPS-stimulated first trimester trophoblast cells. A, Bar chart shows the percentage of THP-1 cells migrated toward medium, the CM from untreated trophoblast cells (CM H8/medium), or the CM from LPS-treated trophoblast cells (CM H8/LPS). **, p < 0.001. B, H8 cells (1 x 106) were incubated with either no treatment or LPS (10 µg/ml) for 48 h, after which inserts loaded with fluorescently labeled THP-1 cells (5 x 105) were placed into the wells and the coculture was incubated for another 24 h. The images show migration of THP-1 cells toward medium (i), untreated trophoblast cells (ii), and trophoblast cells pretreated with LPS (iii). Representative of at least three independent experiments.

 
Chemokine profile of first trimester trophoblast cells following TLR-3 ligation with poly(I:C)

Previous observations have reported TLR-3 expression by term placenta, suggesting that cells from the placenta may recognize and respond to viral products (35). We evaluated the expression of TLR-3 in first trimester human placenta and determined whether receptor expression was associated with trophoblast cells. As shown in Fig. 4, first trimester placental tissue and first trimester trophoblast cells expressed high levels of TLR-3 mRNA. In contrast, the monocytic THP-1 cell line was negative for this receptor. Primary first trimester trophoblast cells were also found to express TLR-3 mRNA (data not shown). We next evaluated the effect of TLR-3 ligation with poly(I:C), a synthetic analog of viral dsRNA, on the secreted chemokine profile of trophoblast cells. When first trimester trophoblast cells were treated with poly(I:C) (25 µg/ml), the secretion of GRO, GRO-{alpha}, IL-8, and MCP-1, as determined by the cytokine array, was significantly up-regulated. However, treatment of trophoblast cells with poly(I:C) also induced the secretion of an additional chemokine, RANTES (p < 0.001; Table II).The results gained from these arrays were again confirmed by ELISA. GRO-{alpha} secretion was significantly increased following TLR-3 stimulation with poly (I:C) (Fig. 5A), as was MCP-1 secretion, and this occurred in a dose-dependent manner (Fig. 5B). RANTES was undetectable in the cell-free supernatants from untreated or LPS-treated trophoblast cells, but high concentration levels were detectable in the supernatants from poly(I:C)-treated trophoblast cells (p < 0.001), confirming this to be a TLR-3-specific response (Fig. 5C).



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  		FIGURE 4. First trimester trophoblast cells express TLR 3. TLR-3 mRNA expression was evaluated in first trimester trophoblast cells by RT-PCR. Upper panel, TLR-3 expression (425 bp) and lower panel,{beta}-actin expression (650 bp). Lane 1, THP-1 cells; lane 2, 3A cells; lane 3, H8 cells; lane 4, 8 wk placenta; and lane 5, mock RT-PCR.

 

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  		Table II. Secretion of chemokines by first trimester trophoblast cells following TLR-3 stimulationa

 


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  		FIGURE 5. Poly(I:C) induces chemokine secretion from first trimester trophoblast cells. H8 cells (1 x 106) were treated with either no treatment (NT), poly(I:C) (25 µg/ml), or LPS (100 µg/ml). Cell-free CM was collected after a 48-h incubation and assayed for GRO-{alpha} (A), MCP-1 (B), and RANTES (C). Treatment of trophoblast cells with poly(I:C) significantly increased the release of GRO-{alpha} and MCP-1. Although RANTES was undetectable in either the untreated or LPS-treated cultures, poly(I:C) induced trophoblast cells to secrete high levels of RANTES. *, p < 0.05; **, p < 0.001.

 
Monocytes migrate toward poly(I:C)-stimulated first trimester trophoblast cells

Since TLR-3 stimulation induced the secretion of IL-8, MCP-1, and RANTES, that is also known to be chemotactic for monocytes (36), the effect of poly(I:C) stimulation on the ability of trophoblast cells to recruit monocytes was evaluated. As shown in Fig. 6A, monocyte migration toward the CM from poly(I:C)-treated trophoblast cells was 2.3-fold more than the monocyte migration toward the CM from untreated trophoblast cells (p < 0.001). Furthermore, trophoblast cells pretreated with poly(I:C) (Fig. 6B, panel iii) induced a 2-fold increase in monocyte transmigration when compared with monocyte migration toward untreated trophoblast cells (Fig. 6B, panel ii) (p < 0.01).



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  		FIGURE 6. Monocytes migrate toward poly(I:C)-stimulated first trimester trophoblast cells. A, Bar chart shows the percentage of THP-1 cells migrated toward medium, the CM from untreated trophoblast cells (CM H8/medium), or the CM from poly(I:C)-treated trophoblast cells (CM H8/poly(I:C)). **, p < 0.001. B, H8 cells (1 x 106) were incubated with either no treatment or poly(I:C) (25 µg/ml) for 48 h, after which inserts loaded with fluorescently labeled THP-1 cells (5 x 105) were placed into the wells and the coculture was incubated for another 24 h. The images show the migration of THP-1 cells toward medium (i), untreated trophoblast cells (ii), and trophoblast cells pretreated with poly(I:C) (iii). Representative of at least three independent experiments.

 
Neutrophils and NK cells migrate toward factors derived from TLR-3- and TLR-4-stimulated trophoblast cells

Since the chemokines GRO-{alpha}, IL-8, MCP-1, and RANTES are all known to be chemotactic for other innate immune cells (37, 38, 39), the effect of trophoblast-derived factors, following either TLR-3 or TLR-4 stimulation, on neutrophil and NK cell migration was investigated. Neutrophil transmigration was significantly increased toward the CM from untreated trophoblast cells by 1.7-fold (p < 0.05) when compared with random migration. A further increase in neutrophil chemotaxis was observed toward the CM from TLR-4-stimulated trophoblasts (1.3-fold; p < 0.05) and TLR-3-stimulated trophoblasts (1.6-fold; p < 0.001) (Fig. 7A). However, it is important to note that at its maximum, the percentage of neutrophils undergoing transmigration (15.83 ± 0.13%) was markedly less than the amount of monocyte migration observed toward the CM from untreated trophoblast cells (24.07 ± 0.63%). In contrast, the degree of NK cell migration toward unstimulated trophoblasts was comparable to that of monocytes. NK migration, in response to the CM from untreated trophoblast cells (32.2 ± 0.93%), was 2.3-fold higher than random NK cell migration (14.31 ± 1.04%; p < 0.001). However, stimulation of trophoblast cells through either TLR-3 or TLR-4 had no effect on NK cell chemotaxis (Fig. 7B).



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  		FIGURE 7. Neutrophils and NK cells migrate toward trophoblast cell-derived factors. Bar charts show the percentage of neutrophils (A) and NK cells (B) migrated toward medium, the CM from untreated trophoblast cells (CM H8/medium), the CM from LPS-treated trophoblast cells (CM H8/LPS), or the CM from poly(I:C)-treated trophoblast cells (CM H8/Poly(I:C)). *, p < 0.05; **, p < 0.001.

 
Effect of MyD88 knockdown on trophoblast chemokine production

TLRs transduce intracellular signals by recruiting the TLR signaling adapter protein MyD88 (40). To determine whether TLR signaling in first trimester trophoblast cells involves the MyD88 pathway, RNAi was used to evaluate the signaling mechanisms of TLR-3- and TLR-4-mediated chemokine secretion. Knockdown of MyD88 expression in first trimester trophoblast cells significantly decreased LPS-induced MCP-1 secretion by 48% (p < 0.05; Fig. 8A). In contrast, knockdown of MyD88 had no effect on the levels of MCP-1 secreted by trophoblast cells following TLR-3 stimulation by poly(I:C) (Fig. 8B).



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  		FIGURE 8. Effect of MyD88 RNAi on TLR-induced MCP-1 secretion by trophoblast cells. H8 cells were transiently transfected with siRNA-human MyD88 or control, after which cells were treated for 48 h with LPS (100 µg/ml, A) or poly(I:C) (25 µg/ml, B). Following incubation, cell-free supernatants were collected and assayed for MCP-1. Bar charts show MCP-1 secretion adjusted for the no treatment control. Knockdown of MyD88 significantly reduced LPS-induced MCP-1 secretion (*, p < 0.05) (A), while knockdown of MyD88 had no effect on poly(I:C)-induced MCP-1 secretion (B).

 
Expression levels of IL-8 and MCP-1 in serum from pregnant women

We have demonstrated that first trimester trophoblast cells constitutively secrete high levels of chemokines. To determine whether our in vitro observations correlate with normal pregnancy conditions, we compared the expression of IL-8 in serum collected from normal pregnant women during their first trimester of gestation with serum from healthy non-pregnant aged-matched controls. IL-8 was undetectable in the serum from all nonpregnant women but highly detectable in the serum from pregnant women during their first trimester of pregnancy (361 ± 573 pg/ml) (Fig. 9).



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  		FIGURE 9. Serum IL-8 levels are high during the first trimester of pregnancy. Bar chart shows IL-8 concentrations in the serum from normal pregnant women during their first trimester (PS) and in healthy nonpregnant women (NPS). IL-8 was undetectable (UD) in all nonpregnant sera, but detectable in the first trimester sera.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The appropriate influx of innate immune cells into the endometrium is thought to be a prerequisite for successful implantation and pregnancy. However, elevated leukocyte infiltration and inappropriate activation may be an underlying cause of pregnancy complications, such as preterm labor or preeclampsia. Such conditions may also be attributed to an infectious microorganism gaining access to gestational tissues. In this study, we have demonstrated that first trimester trophoblast cells constitutively secrete chemokines and are able to recruit immune cells such as monocytes and NK cells. Furthermore, we have demonstrated that following the ligation of TLRs by bacterial or viral products, trophoblast expression and secretion of chemokines is significantly increased and differentially modulated, and this in turn results in elevated and differential leukocyte chemotaxis.

The migration of immune cells into the maternal-fetal interface during normal pregnancy is thought to occur in response to the invading trophoblast which expresses paternal Ags that may be recognized as foreign by the maternal immune system (6). Contrary to this hypothesis, our results suggest that the presence of innate immune cells at the implantation site may be due to chemokines produced by the trophoblast. Indeed, first trimester trophoblast cells have been demonstrated to express the chemokines SDF-1, macrophage migration inhibitory factor, IL-8, and thymus and activation-related chemokine (TARC) (41, 42, 43, 44). When the cytokine profile of unstimulated first trimester trophoblast cells was evaluated, it was notable to find the expression and secretion of a number of chemokines. Unstimulated first trimester trophoblast cells constitutively expressed IL-8 and MIP-1{delta} and secreted GRO, GRO-{alpha}, IL-8, and MCP-1, suggesting that the trophoblast has the capacity to promote the chemotaxis and recruitment of immune cells. This was confirmed by coculture experiments in which both unstimulated first trimester trophoblast CM as well as unstimulated trophoblast cells had the capacity to incite the chemotaxis of monocytes and NK cells. These results are in keeping with the study by Drake et al. (45), who demonstrated, using trophoblast CM, that MIP-1{alpha} could induce the migration of monocytes and NK cells. Similarly, SDF-1 has been shown to be released from invasive trophoblast cells and to preferentially recruit CD16– NK cells (46).

Interestingly, the secretion of chemokines from unstimulated first trimester trophoblast cells correlated with our in vivo observations. IL-8 serum concentrations in normal pregnant women were high, yet undetectable in the serum from nonpregnant women, suggesting that chemokine production is a hallmark of normal pregnancy. The high levels of IL-8 secretion by trophoblast cells, demonstrated by us and others (43), would suggest that neutrophil recruitment by trophoblast cells should also be high, since IL-8 is a potent neutrophil chemoattractant. Although neutrophil chemotaxis toward the CM from unstimulated trophoblast cells was observed, it was significantly lower than that for monocytes and NK cells. Gestational tissues are not normally infiltrated by granulocytes, suggesting that the high levels of IL-8 generated throughout pregnancy has either reduced activity, or that there are other factors simultaneously released by trophoblast cells counteracting the effect of IL-8 on neutrophil migration. In keeping with this, a recent study reported the inhibition of neutrophil and monocyte chemotaxis by amniotic epithelial cell supernatants (47). We could also speculate that by attracting neutrophils early in pregnancy, the trophoblasts may then inhibit neutrophil function and activation. Indeed, it has been shown that neutrophils from pregnant women cannot undergo activation to the same level as neutrophils from nonpregnant women (48). Furthermore, there is the recently described phenomenon of trophoblast-induced contact inactivation of neutrophils within the intervillous space. For this mechanism to occur, the neutrophil entering such space must be attracted by the villous tree, otherwise contact inactivation will not occur. This trophoblast-induced change in neutrophil function is now considered to be an adaptive mechanism, protecting the villous trophoblast from destruction by the innate immune system (49). However, in the case of an infection, such inactivation mechanisms may be lost.

Our next objective was to evaluate the effect of bacterial and viral products on first trimester trophoblast chemokine production. Throughout pregnancy, trophoblast cells express TLRs that recognize microbial products. Initial studies reported mRNA expression of TLR-1 to TLR-10, as well as protein expression of TLR-2 and TLR-4 in term placenta (27, 28, 35). We have observed that in first trimester placental tissues, TLR-2 and TLR-4 are highly expressed in the villous cytotrophoblast and extravillous trophoblast populations (26). In this current study, we have demonstrated that first trimester trophoblast cells also express TLR-3 which recognizes viral dsRNA. Following TLR-3 ligation by viral poly(I:C) and TLR-4 ligation by bacterial LPS, both the constitutive intracellular expression and secretion of chemokines by first trimester trophoblast cells were up-regulated. Interestingly, the secreted chemokine profiles following bacterial and viral stimulation were distinct. TLR-4 activation by LPS enhanced the secretion of GRO, GRO-{alpha}, IL-8, and MCP-1. This finding correlates with early reports showing that trophoblast cells treated with LPS secrete IL-8 (27, 50). TLR-3 ligation by poly(I:C) had a similar effect on chemokine secretion, but also specifically induced the secretion of RANTES.

A number of studies have demonstrated a critical role for both NK cells and macrophages in the establishment of a healthy pregnancy (8, 9, 10, 11). Nevertheless, in abnormal pregnancies, such as prematurity or preeclampsia, decidual tissues contain elevated levels of macrophages, neutrophils, and NK cells, and leukocyte distributions are altered (12, 13, 14, 15, 51, 52). Such conditions have also been associated with the presence of intrauterine infections (18, 19, 20, 21, 22, 53). Similarly, in animal models of preterm labor and pregnancy failure, where the delivery of microbial products are used to initiate disease, the decidua becomes infiltrated with these same innate immune cells (54, 55, 56). The up-regulation of chemokine secretion observed following trophoblast TLR-3 and TLR-4 activation by microbial products correlated with their influence on immune cell chemotaxis. Monocyte transmigration toward trophoblast cells was significantly elevated following stimulation of the trophoblasts through either TLR-3 or TLR-4. Similarly, neutrophil migration was also elevated. Interestingly, NK cell chemotaxis toward unstimulated trophoblast cells was not further enhanced by the treatment of trophoblasts with either viral or bacterial products. Other cell types that are present at the maternal-fetal interface, such as decidual stromal cells or innate immune cells themselves, may provide the additional stimuli required to elevate NK cell migration.

To determine the signaling mechanism by which TLR-3 and TLR-4 mediate chemokine secretion from first trimester trophoblast cells, the expression of the signaling adapter protein MyD88 was inhibited using RNAi. TLR-3 and TLR-4 are both able to transduce signals through either MyD88-dependent or MyD88-independent mechanisms (40). We found that in first trimester trophoblast cells, MCP-1 secretion induced by TLR-4 ligation was dependent on MyD88, whereas MCP-1 secretion triggered through TLR-3 occurred in a MyD88-indepedent manner. This TLR-3 MyD88-indepedent signaling mechanism is further supported by our observations that first trimester trophoblast cells produce IFN-{beta} in response to poly(I:C), but not LPS (57). Such production of type I IFNs through TLR-3 and TLR-4 is known to occur in a MyD88-independent fashion (40).

Although innate immune cells may be important during normal pregnancy for promoting successful implantation and for resolving infections at the maternal-fetal interface, these same leukocytes may contribute to the pathology of certain pregnancy complications. Our findings that TLR-3 and TLR-4 stimulation in trophoblast cells is characterized by an increase in chemokine production and immune cell migration suggest that an intense inflammatory response may be initiated by the trophoblast. Therefore, although TLRs function as important sensors for the trophoblast, allowing it to coordinate a local immune response and to promote cell invasion and placental formation, TLRs may also provide the bridge for placental recognition of danger signals and a subsequent shift in the type of response generated may have harmful consequences for the pregnancy.


   Acknowledgments
 
We thank Dr. John Pizzonia of Eastman Kodak Company for his technical assistance and Dr. Susan Richmond of Yale University for tissue procurement.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant RO1HD049446-01 (to V.M.A.) and the Intramural Research Program of the National Institute of Child Health and Human Development, National Institutes of Health, Department of Health and Human Services, Bethesda, MD. Back

2 Address correspondence and reprint requests to Dr. Gil Mor, Department of Obstetrics, Gynecology & Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, New Haven, CT 06520. E-mail address: gil.mor{at}yale.edu Back

3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern; GRO-{alpha}, growth-related oncogene {alpha}; RNAi, RNA interference; CM, conditioned medium; ENA78, epithelial neutrophil-activating protein 78; SDF-1, stromal cell-derived factor 1; TARC, thymus and activation-related chemokine. Back

Received for publication July 15, 2005. Accepted for publication October 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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