BIOLOGY OF REPRODUCTION 62, 1075–1083 (2000)
Human Villous Macrophage-Conditioned Media Enhance Human Trophoblast Growth and Differentiation In Vitro Salma Khan,1,2 Hidetaka Katabuchi,1,2 Masako Araki,2 Ryuichiro Nishimura,3 and Hitoshi Okamura2 Department of Obstetrics and Gynecology,2 Kumamoto University School of Medicine, Kumamoto-City 860-8556, Japan Hyogo Institute of Clinical Research,3 Akashi-City 673-0021, Japan ABSTRACT In human chorionic villi, numerous macrophages, so-called Hofbauer cells, are located adjacent to trophoblasts. To determine the role of the macrophages in the proliferation and differentiation of trophoblasts, cytotrophoblast cells were cultured in serum-free culture-conditioned media of villous macrophages (VMCM), peritoneal macrophages (PMCM), and villous fibroblasts (VFCM). In VMCM, proliferation of cytotrophoblast cells was detected at 24 h by immunocytochemistry with Ki-67-antibody. A large number (P , 0.001) of multinucleated syncytia was formed in VMCM. In VMCM, cytotrophoblast cell fusion was completed by 96 h, which coincided with the peak of hCG secretion and initiation of human placental lactogen (hPL) release. Levels of hCG (P , 0.001) and hPL (P , 0.001) secretion from syncytial cells were significantly higher in VMCM than in PMCM or in VFCM. Concentrations of macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) analyzed by ELISA were greater in VMCM than in PMCM or in VFCM, whereas monocyte chemoattractant protein-1 (MCP-1) concentration was high in PMCM. The expression patterns of M-CSF, VEGF, and MCP-1 in villous macrophages and peritoneal macrophages by reverse transcriptase-polymerase chain reaction were similar to their secretion patterns. Thus, villous macrophages have a greater ability to stimulate hCG and hPL secretion than do peritoneal macrophages. This study suggests that macrophages within the villous stroma may stimulate the growth and differentiation of trophoblasts through their secreted substances.
INTRODUCTION
The growth and differentiation of trophoblasts have been studied in in vitro model systems by many investigators in the last two decades. The collective information shows that mononuclear cytotrophoblast cells form a terminally differentiated syncytial phenotype. In vitro, cytotrophoblast cells in serum-free conditions fail to aggregate or fuse and thus remain in a mononuclear state [1]. In contrast, when the cells are cultured in medium supplemented with fetal bovine serum (FBS), they spontaneously fuse to form multinucleated cells that resemble phenotypically mature syncytiotrophoblast cells and concomitantly produce hCG [2]. Douglas and King [3] have reported that keratinocyte growth medium, a growth medium originally formulated for keratinocytes, supplemented with heat-inactivated FBS substantially increases the rate and extent of syncytiotroCorrespondence: H. Katabuchi, Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, Honjo 1–1-1, KumamotoCity 860-8556, Japan. FAX: 81 96 363 5164; e-mail:
[email protected]
1
Received: 27 July 1999. First decision: 15 September 1999. Accepted: 6 December 1999. Q 2000 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
phoblast formation from cytotrophoblast cells, accompanied by an increase in chorionic gonadotropin secretion. Furthermore, recent investigations have demonstrated that cytotrophoblast cells cultured in medium containing serum from pregnant women progress to an advanced stage of trophoblast differentiation [4, 5]. These studies indicate that in addition to the classical endocrine hormones, cytokines, growth factors, and as yet undefined substances can modulate the growth and differentiation of trophoblasts. Cells of the monocyte-macrophage lineage play important roles in the events of reproduction [6]. In addition to their role in immune function, many kinds of cytokines are released from macrophages and contribute to the proliferation and differentiation of cells in the female reproductive organs. In particular, macrophages have been shown to participate in the proliferation of granulosa cells [7, 8] and in the production of steroids from the luteal cells [9]. It is evident that a network of cytokines locally produced by macrophages coordinates to regulate folliculogenesis and steroidogenesis [10]. In chorionic villi, macrophages may also exert specific effects on the trophoblast functions because anatomically the vast majority of the nontrophoblast cells are macrophages with some immature mesenchymal cells and fibroblasts. Recently, recombinant cytokines and growth factors, including macrophage colony-stimulating factor (M-CSF) [11], epidermal growth factor [12], vascular endothelial growth factor (VEGF) [13], and leukemia inhibitory factor [14], have been shown to be expressed in human placenta and/or to stimulate the differentiation of trophoblasts in vitro. However, little attention has been paid to the relationship between trophoblasts and the resident macrophages in chorionic villi, and there is no convincing evidence that trophoblast growth and function are regulated by the cytokines derived from these macrophages. As described above, in the existing experimental systems for trophoblast differentiation, serum appears to play an indispensable role because it contains a myriad of proliferating and differentiating agents, some of which may be cytokines and other substances produced and released by macrophages [2–5]. To provide a better understanding of the role of macrophages in the process of trophoblast proliferation and differentiation, a stronger study can be conducted by using macrophage-conditioned media in place of serum in the development of trophoblasts. In the present study, we used villous macrophage-conditioned medium in primary culture of first-trimester cytotrophoblast cells to investigate the paracrine role of villous macrophages on the growth and differentiation of trophoblast. Functional differentiation of cytotrophoblast cells was examined by measuring the concentrations of hCG and human placental lactogen (hPL) using enzyme immunoassay (EIA) and RIA, respectively. Immunocytochemistry was applied to examine the proliferative activity and differentiation of trophoblasts by using antibodies against a cell cycle marker and hCG. Furthermore, to determine the po-
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MATERIALS AND METHODS
their immunoreactivity to a CD68 monoclonal antibody specific for monocyte-macrophages [17]. Villous stromal fibroblasts were also collected from chorionic villous tissues according to the method of Garcia-Lloret et al. [18]. The cells were identified as fibroblasts on the basis of their characteristic morphological appearance, positive staining for vimentin, and absence of staining for the macrophage marker. The procedure for immunocytochemical evaluation is described later.
Human Cytotrophoblast Cells
Human Peritoneal Macrophages
Tissues of chorionic villi were obtained from 15 normal pregnant women from 6 to 10 wk of gestation undergoing legal termination of pregnancy. Informed consent was obtained from each person. The study was performed according to the guidelines of the Ethics Committee of Kumamoto University School of Medicine. By the combination of a standard enzymatic digestion procedure for first-trimester chorionic villous tissues [15] and a Percoll gradient-purification technique for term placental trophoblasts [2], cytotrophoblast cells were isolated in a multi-stage process. First villous tissue was minced into small pieces. Next, the minced tissue was transferred to 50 ml of calcium- and magnesium-free Hanks’ balanced salt solution (Gibco, Grand Island, NY) containing 0.06 mg/ml Pronase (protease, 85 U/ml; Calbiochem-Novabiochem Corp., La Jolla, CA) and 0.5 mg/ml DNase I (600 U/ml; Sigma Chemical Co., St. Louis, MO) and incubated for 30 min at 378C in a humidified atmosphere of 5% CO2 and 95% air. After supernatant was aspirated, the remaining tissue was redigested two more times according to the same procedure. The resultant cell suspensions were pooled, centrifuged at 1200 3 g for 5 min, and resuspended in 4 ml of Roswell Park Memorial Institute 1640 (RPMI 1640; Gibco) medium. This suspension was layered over a preformed Percoll gradient made from 70%-5% (v:v) Percoll (Sigma). The gradient was centrifuged at 1200 3 g at room temperature for 30 min. The intermediate layer (1.048–1.062 g/ml) containing a uniform population of mononuclear cells was removed, and the cells were resuspended in the medium. The viability of the cells was assessed by trypan blue 0.05% dye exclusion during a 2-min incubation. Cells were plated in 35-mm dishes (Nunc, Roskilde, Denmark) and cultured in medium supplemented with 15 mM (Nacalai Tesque, Kyoto, Japan), 25 U/ml penicillin G (Gibco), and 25 mg/ ml streptomycin (Gibco). Cytotrophoblast cells were recognized by immunocytochemical staining with an anti-cytokeratin monoclonal antibody [2,3] as described later in the Immunocytochemistry section.
Peritoneal ascitic fluid was collected from 16 female patients undergoing laparoscopy for benign conditions. Patients with endometriosis or pelvic inflammatory diseases were not included. Peritoneal macrophages were isolated according to a method described elsewhere [19]. In brief, the ascitic fluid was centrifuged at 1200 3 g for 5 min, and supernatant was aspirated. The pellet was washed with PBS (pH 7.2), suspended with RPMI 1640, and plated in 60mm dishes. After incubation for 1 h at 378C in a humidified atmosphere of 5% CO2 and 95% air, nonadherent cells were washed with PBS. Adherent cells were detached from the dishes by trypsinization.
tential candidates for macrophage-derived cytokines/growth factors involved in the regulation of trophoblast differentiation, M-CSF, VEGF, and monocyte chemoattractant protein-1 (MCP-1) secretion were analyzed from macrophageconditioned media by ELISA, and mRNA expressions of these factors in macrophages were detected by reverse transcription (RT)-polymerase chain reaction (PCR).
Human Chorionic Villous Macrophages and Fibroblasts
Macrophages were isolated from first-trimester human chorionic villous tissues according to the methods described previously [16] with some modifications. Normal villous tissues, collected as described above, were cut into small pieces and incubated with slow agitation for 60 min at 378C in RPMI 1640 containing 85 U/ml of pronase. The cell suspension was centrifuged, and the remaining tissues were redigested in fresh medium containing 85 U/ml of Pronase. The procedure was repeated three times. After the cell suspension was centrifuged, the pellet was resuspended with RPMI 1640 and incubated in 60-mm dishes (Nunc) for 2 h at 378C in 5% CO2 and 95% air. The dishes were washed several times in PBS (pH 7.2) to remove the nonadherent cells. The adherent cells were macrophages as identified by
Macrophage- and Fibroblast-Conditioned Media
The numbers of villous macrophages, peritoneal macrophages, and villous fibroblasts were counted. Cells were plated at 4 3 106 cells/well on 60-mm dishes (Nunc) in serum-free RPMI 1640 medium and cultured for 48 h at 378C in 5% CO2 and 95% air. After 48-h culture, supernatant was aspirated from culture dishes containing villous macrophages, peritoneal macrophages, and villous fibroblasts and stored at 2208C for the incubation study with cytotrophoblast cells. Incubation of Cytotrophoblast Cells in Conditioned Media
Percoll gradient-purified cytotrophoblast cells were plated at 1 3 106 cells in 35-mm dishes (Nunc). Cytotrophoblast cells were incubated in designated media: in serumfree RPMI 1640 medium (SFM), in 10% fetal calf serum (FCS; HyClone Laboratories Inc., Logan, UT) containing RPMI 1640 medium (FCSM), and in villous macrophageconditioned medium (VMCM). In 1 3 103 nuclei of cytotrophoblast cells, numbers of cell aggregations were counted in each group after culture for 24 h, 48 h, 72 h, and 96 h. Cells containing more than four nuclei were classified as multinucleated cells. Five different consecutive cultures were performed. In the second incubation study, cytotrophoblast cells were cultured in five media: SFM, FCSM, VMCM, villous fibroblast-conditioned medium (VFCM), and peritoneal macrophage-conditioned medium (PMCM). The culture medium was removed and replaced by fresh medium every 24 h over 4 days. The culture supernatant was collected at 24 h, 48 h, 72 h, and 96 h and stored at 2208C for the determination of hCG and hPL. Immunocytochemistry
For immunocytochemistry, Percoll gradient-purified trophoblasts were prepared for cell smears by using a cytocentrifuge (Sakura Co. Ltd., Tokyo, Japan) and fixed in 4% periodate-lysine and 2% paraformaldehyde. Isolated macrophages and fibroblasts were fixed in 95% ethanol. After
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TROPHOBLAST DEVELOPMENT BY VILLOUS MACROPHAGES TABLE 1.
Primer pairs used for detection of mRNA for M-CSF, VEGF, and MCP-1.
Factors
Primer
M-CSF
Sense: 59-ATGACAGACAGGTGGAACTGCCAGTGTAGAGG-39 Antisense: 59-TCACACAACTTCAGTAGGTTCAGGTGATGGGC-39 Sense: 59-CTGCTGTCTTGGGTGCATTGG-39 Antisense: 59-CACCGCCTTGGCTTGTCACAT-39 Sense: 59-CAAACTGAAGCTCGCACTCTCGCC-39 Antisense: 59-GCAAAGACCCTCAAAACATCCCAG-39 Sense: 59-GTCTTCACCACCATGGAGAA-39 Antisense: 59-AAGTGGTCGTTGAGGGCAAT-39
VEGF MCP-1 GAPDH
fixation, slides were kept in 2208C until used. The slides were defrosted by being placed in distilled water, and immunocytochemical staining was performed. Mouse anti-human cytokeratin group 56-kDa monoclonal antibody (Immunotech, Marseilles, France; 1:40 dilution) was used for the detection of cytotrophoblast cells. Mouse monoclonal antisera against human CD68 (Dako, Carpenteria, CA; 1: 100 dilution) and human vimentin (Dakopatts, Glostrup, Denmark; 1:20 dilution) were used for fibroblast and macrophage identification. Cytotrophoblast proliferation and differentiation were evaluated by immunostaining with mouse anti-human Ki-67 monoclonal antibody (Dakopatts; 1:50 dilution) and rabbit anti-human hCG-b subunit (hCGb) polyclonal antibody (Dakopatts; 1:100 dilution), respectively. Ki-67 antigen is expressed during S, G2, and M phases of the cell cycle, but not in resting G0 phase [20]; consequently, the antibody detects only proliferating cells. hCG is a marker of syncytial formation along with hPL and pregnancy-specific b1-glycoprotein [2, 21]. CD 68, cytokeratin, vimentin, and Ki-67 were detected by the avidinbiotin peroxidase complex (ABC) method with a Vectastain ABC kit (Vector, Burlingame, CA). For the detection of hCG-b, the peroxidase and anti-peroxidase method was performed with a DAKO PAP kit (Dako). Peroxidase activity was visualized with 3,39-diaminobenzidine (Sigma) as a substrate in Tris-HCL buffer (0.5 mg/ml pH 7.6) containing 0.01% H2O2. Nuclear staining was performed with hematoxylin. Appropriate positive controls were used for each immunoprotein, and negative controls were prepared by replacing the primary antibody with nonimmune antiserum of rabbit. EIA
The concentration of hCG-b was analyzed by the sandwich EIA system using the polyclonal antibody (Meiji Seika Co. Ltd., Tokyo, Japan) as described previously [22]. Briefly, the beads coated with the first antibody were incubated with 50-ml samples in 0.02 M PBS (pH 6.8) containing 0.1% BSA for 3 h at 378C. The beads were washed twice with deionized water and transferred to new assay tubes. Fifty microliters of phosphate citrate buffer containing 0.02% hydrogen peroxide and 2 mg/ml o-phenylene diamine were added for color development. After 30 min, the reaction was stopped by the addition of 1 N sulfuric acid and measured at 492 nm of absorbance.
Size of PCR product (bp)
Ref.
437 bp
[23]
431, 563 and 635 bp
[24]
592 bp
[25]
620 bp
ELISA
Villous fibroblasts, villous macrophages, or peritoneal macrophages were cultured for 48 h in serum-free RPMI 1640 medium, and then the concentrations of M-CSF, VEGF, and MCP-1 were measured in the three culture-conditioned media. A human M-CSF kit (kindly provided by Yoshimoto Pharmaceutical Ind. Ltd., Osaka, Japan), a human VEGF kit (Biopool International Inc., Umea, Sweden), and a human MCP-1 kit (R&D system, Oxon, UK), respectively, were used for each assay according to the manufacturers’ instructions. RT-PCR
Isolated macrophages from chorionic villous tissue and the peritoneal cavity were cultured for 48 h in serum-free RPMI 1640 medium. After being washed with PBS (pH 7.2), cells were scraped and total RNA was extracted using a RNAzol B kit (TEL-TEST, Inc., Friendswood, TX). Total RNA was also extracted from whole chorionic villous tissue. The cDNAs were prepared using a first-strand cDNA kit (Gibco-BRL), and PCR was carried out with cDNA. Synthetic oligonucleotides used for M-CSF, VEGF, and MCP-1 [23–25] are listed in Table 1. The PCR mixtures were subjected to 35–40 cycles of amplification consisting of denaturation at 958C for 1 min, annealing at 608C for 1 min, and elongation at 728C for 1 min. The integrity of the extracted mRNA was tested by amplification of the 620base pair (bp) splice product of the human glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene using primers specific to nucleotides 361–380 and nucleotides 961–980 of the GAPDH DNA. After amplification, DNA fragments were electrophoresed with 2% agarose gels, stained with ethidium bromide, visualized by UV irradiation, and photographed by a Polaroid (Cambridge, MA) camera; and integrity of mRNAs was calculated by computerized analysis. Statistical Analysis
All values are expressed as mean 6 standard error of the mean (SEM). For multiple comparisons, the significance of the difference was calculated by two-way ANOVA. Comparisons between significance in the two experimental groups were made by both Scheffe’s and Bonferroni’s methods using the unpaired Student’s t-test. P , 0.05 was considered statistically significant.
RIA
RESULTS
The concentration of hPL was analyzed with a specific homologous hPL RIA kit (Pharmacia-Upjohn, Uppsala, Sweden). RIA was performed according to the procedure described by the manufacturer. The cross-reactivities with human growth hormone and prolactin were , 0.5% and , 0.06%, respectively.
Proliferation, Aggregation, and Syncytial Formation of Cytotrophoblast Cells
Cells isolated by the Percoll density gradient-purification method showed 98% viability and yielded approximately 94% cytokeratin-positive, vimentin-negative, and CD68-
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FIG. 1. Photomicrographs of trophoblast cellular morphology and immunocytochemistry with anti-hCG-b antibody. After 24 h of plating, cell aggregations were found in VMCM (A) and in FCSM (B). At 48 h, a large cell aggregate was observed in VMCM (C) and in FCSM (D). At 96 h, a syncytia with centrally placed nuclei was formed in VMCM (E) but not in FCSM (F). hCG-b was positively immunostained in the cytoplasm (C–F). Giemsa stain (A, B), hematoxylin stain (C–F). Original magnifications: 320, A, B; 3200, C–F; (published at 68%).
negative mononuclear cells, implying that the cells were highly purified cytotrophoblast cells. To determine the effects of VMCM, FCSM, and SFM on trophoblast proliferation, aggregation, and syncytial formation, the mononuclear cytotrophoblast cells were cultured in these three media for 120 h and examined at 24 h, 48 h, 72 h, and 96 h after plating. When the cells were cultured in VMCM, a number of aggregates were identified interspersed among isolated cytotrophoblast cells at 24 h (Fig. 1A). After 48-h culture, a cluster of the aggregates was noted, but the nuclei appeared to be evenly spaced and not crowded into central portions of large cells (Fig. 1C). The aggregates were observed as multinuclear clusters at 72 h. Syncytial formation first appeared at 96 h in VMCM (Fig. 1E). The syncytia contained 6–12 centrally placed nuclei with no visible intervening membranes after cell fusion was completed (Fig. 1E). The multinuclear aggregates or putative syncytia possessed numerous vesicles in the cytoplasm. In FCSM, morphological changes similar to those in VMCM were evident in the cultured cytotrophoblast cells (Fig. 1, B and D). However, the syncytia formed more
slowly in FCSM than in VMCM. Single cell membranes in the aggregates were still present after 96 h culture (Fig. 1F), and syncytial formation was first observed in FCSM at 120 h (not shown). In SFM, cytotrophoblast cells were not spread, and aggregation was not observed. When cytotrophoblast cells were cultured in VMCM, positive staining of Ki-67 antigen was observed in the dispersed cells or single cells around the aggregates at 24 h (Fig. 2A). After 48-h culture, little or no positive staining was shown in VMCM, suggesting that early proliferation was induced in VMCM and the subsequent process of differentiation began thereafter. On the other hand, in accordance with the previous report [2], FCSM had little effect on trophoblast proliferation as far as observed immunocytochemically by Ki-67 antibody (Fig. 2B). No positive reaction was found in trophoblasts cultured in SFM. The percentage of Ki-67-positive cells per 100 cytotrophoblast cells was calculated. Approximately 27% and 0.9% of cytotrophoblast cells were positive in VMCM and in FCSM, respectively. When the total number of multinucleated cells per 1 3
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FIG. 2. Photomicrographs of immunostaining with Ki-67 antibody in cultured trophoblasts. At 24 h, positive nuclear staining was shown in single cells or single cells around an aggregation in VMCM (A), whereas very weak or no positive staining was observed in FCSM (B). Hematoxylin stain (A, B). Original magnification: 3200, A, B (published at 72%).
103 cytotrophoblast cells was counted, it increased in a time-dependent manner in VMCM and in FCSM (Fig. 3) but not in SFM. VMCM was shown to have a greater effect on aggregate formation than FCSM at each time point (Fig. 3; P , 0.001). An antiserum to hCG-b stained in the cytoplasm of cultured trophoblasts in VMCM and in FCSM at 48 h culture (Fig. 1, C and D). Strong granular immunoreactions were observed in the cytoplasm of syncytial cells at 96 h in VMCM and in FCSM (Fig. 1, E and F). Trophoblasts cultured in SFM exhibited no cytoplasmic hCG-b reactivity at any of the designated times. hCG and hPL Secretion from Cytotrophoblast Cells Cultured in Conditioned Media
The concentrations of hCG and hPL were measured after cytotrophoblast cells were incubated in SFM, FCSM, VMCM, PMCM, and VFCM. In VMCM cultures, the hCG concentration increased with time from 24 h to 96 h (Fig. 4A). In FCSM, it reached peak values at 72 h and then decreased throughout the remaining hours in culture. Values measured in VMCM were approximately 4.5–6 times high-
FIG. 3. Quantitative analysis of trophoblast aggregates per 1000 cytotrophoblast cells in three culture conditions. VMCM induced a greater effect on aggregate formation at 24 h, 48 h, 72 h, and 96 h than did FCSM. Results are expressed as mean 6 SEM (n 5 5). *P , 0.05, **P , 0.001.
er than those in FCSM (Fig. 4A: 24 h, 48 h, 72 h, 96 h; P , 0.001). A parallel increase in hPL secreted into the media during the same time period was shown in VMCM and in FCSM. The hPL concentration in VMCM was approximately 2- to 3-fold higher than that in FCSM over 4 days (Fig. 4B: 48 h, 72 h, P , 0.05; and 96 h, P , 0.001). In SFM, an extremely low concentration of hCG was secreted throughout the culture period, and the concentration of hPL was always below the detection limit (Fig. 4, A and B). When cytotrophoblast cells were cultured in PMCM, the secretion of the two hormones showed a similar pattern of rise with time (Fig. 4, A and B). However, the amplitude of concentrations of both hCG and hPL was significantly lower in PMCM than in VMCM at each time point examined (Fig. 4A: 24 h, 48 h, 72 h, 96 h, P , 0.001; and Fig. 4B: 48 h, 96 h, P , 0.05; 72 h, P , 0.001). After 96h culture, the concentrations of hCG and hPL in VMCM were approximately 5-fold and 1.5-fold higher than those in PMCM. In VFCM, the concentration of hCG was lower than that in FCSM, and the concentration of hPL was below a detectable level throughout the culture time (Fig. 4, A and B). Secretion and Expression of M-CSF, VEGF, and MCP-1 in Villous and Peritoneal Macrophages M-CSF, VEGF, and MCP-1 were all detectable in each culture supernatant when villous macrophages, villous fibroblasts or peritoneal macrophages were cultured in serum-free media for 48 h. As shown in Figure 5, concentrations of both M-CSF and VEGF were significantly higher in VMCM than in PMCM (Fig. 5, A and B; P , 0.05). In contrast, the MCP-1 concentration was lower in VMCM than in PMCM (Fig. 5C; P , 0.05). Levels of secretion of M-CSF, VEGF, and MCP-1 were very low in VFCM (Fig. 5, A–C). M-CSF, VEGF, and MCP-1 mRNAs were all expressed in villous and peritoneal macrophages by RT-PCR (Fig. 6, A–C). When ratios of M-CSF, VEGF, and MCP-1 mRNAs to GAPDH mRNA were calculated by computerized analysis, expression of M-CSF (437 bp) and VEGF (536 bp and 431 bp) was stronger in villous macrophages than in peritoneal macrophages (Fig. 6, A and B). Conversely, MCP-1 (592 bp) expression was stronger in peritoneal macrophages than in villous macrophages (Fig. 6C), showing
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that the expression patterns of these three cytokines coincided with the above-described secretion patterns. The two major bands of VEGF RT-PCR amplification corresponded closely to products expected from 536- and 431-bp transcripts. The VEGF gene is alternatively spliced and gives rise to three transcripts coding for proteins of 189, 165, and 121 amino acids, which, in turn, form the active dimeric factors [24]. The mRNAs for the VEGF 165 (563 bp) and VEGF 121 (431 bp) subunits are the dominant forms expressed, and homo- and heterodimers of the two smaller subunits appear to account for the bioactivity [24]. DISCUSSION
FIG. 4. hCG and hPL production from trophoblasts in five culture conditions. A time-dependent increase of hCG (A) and hPL (B) released from differentiated cytotrophoblast cells occurred in VMCM, and hCG and hPL concentrations were higher than those in FCSM and PMCM. hPL concentration was below the detectable limit in SFM and in VFCM. Values represent the mean 6 SEM of four separate experiments. *P , 0.05, **P , 0.001.
FIG. 5. M-CSF, VEGF, and MCP-1 in three culture conditions. Concentrations of these factors in VFCM, VMCM, and PMCM were evaluated. Estimated mean concentrations of M-CSF (A), VEGF (B), and MCP1 (C) are shown. Data are presented as mean 6 SEM. *P , 0.05, **P , 0.001.
Normal trophoblast of the human placenta secretes two major pregnancy-associated hormones, hCG and hPL. hCG maintains corpus luteum function until about 9th week of pregnancy, at which time the primary site of progesterone synthesis shifts to the placenta [26]. The continued presence of hCG after the luteo-placental shift may suggest that hCG is required throughout pregnancy [27]. hCG-a subunit (hCG-a) and hCG-b mRNAs are localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer [28]. hPL is an important hormone involved in fetal growth [29]. The elucidation of processes regulating the endocrinology of the human materno-fetoplacental unit is vital to understanding the mechanism that controls the maintenance of pregnancy. One of the most common in vitro model systems for studying placental function was originally described by Hall et al. [30]. In 1986, Kliman et al. [2] modified their system to establish a method to isolate and enrich term cytotrophoblast cells by trypsin digestion and subsequent Percoll gradient centrifugation. By use of this method, chorionic villous tissues from the first trimester of pregnancy were prepared in the present study because trophoblasts progress to an advanced stage of differentiation and exhibit a greater level of hCG secretion during early gestation. Consequently, approximately 94% cytotrophoblast cells were purified from early-pregnancy villous tissue and were cultured over 4–5 days. Consistent with the observation of Kliman et al. [2], in the 10% FCS culture, multicellular aggregates were clearly evident within 24–48 h after plating, and syncytial formation progressed in the following culture time. Additionally, the morphological differentiation of trophoblasts was accompanied by hCG and hPL secretion. hCG secre-
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FIG. 6. M-CSF, VEGF, and MCP-1 expression in villous and peritoneal macrophages. Ethidium bromide-stained agarose gels from RT-PCR of RNA extracted from whole chorionic villi, villous macrophages, and peritoneal macrophages. A) M-CSF PCR products. B) VEGF PCR products. C) MCP1 PCR products. D) GAPDH PCR products. Left lane, molecular markers. Lane 1, whole chorionic villi; lane 2, villous macrophages; lane 3, peritoneal macrophages. Ratios of M-CSF, VEGF, and MCP-1 mRNAs to GAPDH mRNA were calculated by computerized analysis.
tion increased with time, reached peak values at 72 h, and declined at 96 h. hPL secretion also increased in a timedependent manner from 72 h to 96 h. Our present in vitro culture system may also mimic an in vivo process in which a multinucleated syncytium is formed via aggregation and fusion of mononucleated cytotrophoblast progenitors and is followed by syncytiotrophoblast secretion of hCG and hPL. The results reported here provide evidence that VMCM exerted marked effects on the proliferation and morphological differentiation of cytotrophoblast cells in comparison with FCSM. By means of immunocytochemistry using Ki67 antibody, a large number of nuclear-positive mononuclear trophoblasts was observed at 24 h of culture in VMCM. Positive cells were scarcely evident in the subsequent culture. This may be due to the fact that most of the cells were in aggregated form and thus failed to proliferate with the progression of syncytial formation. In addition, a greater number of aggregations was observed in VMCM than in FCSM. Moreover, these cells incubated in macrophage-conditioned media released larger amounts of hCG and hPL than those incubated in FCSM. To determine
whether this stimulatory effect of VMCM was specific, trophoblasts were cultured in PMCM. Intriguingly, VMCM showed a greater effect on hCG and hPL secretion than did PMCM. From these results, we hypothesize that villous macrophage-derived specific products can significantly affect secretion of trophoblast-specific hormones in a paracrine manner. In addition to many classical endocrine hormones, a network of interrelated cytokines and their receptors present at the feto-maternal interface may have a regulatory role in the morphological and functional development of human placenta [31, 32]. Potential candidates include M-CSF and VEGF. The physiological relevance of M-CSF and VEGF in the initiation and maintenance of normal pregnancy has been documented in knock-out mice [33–35]. In vitro studies also demonstrated that recombinant M-CSF [36] and VEGF [37] stimulate cytotrophoblast cell proliferation and syncytiotrophoblast formation. In order to explore the hypothesized paracrine regulation of trophoblast differentiation by macrophage-derived cytokines, we measured the concentrations of M-CSF, VEGF, and MCP-1 from villous
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macrophages and peritoneal macrophages. Greater expression as well as secretion of M-CSF and VEGF in villous macrophages than in peritoneal macrophages suggests that M-CSF and VEGF may mediate the proliferation and differentiation of trophoblasts caused by VMCM. In contrast, peritoneal macrophages were greater sources of MCP-1. In this study, the role of MCP-1 in trophoblast development could not be clarified because peritoneal macrophages, being a greater source of MCP-1, had lower effects on trophoblast differentiation and endocrine function. MCP-1, one of the most potent monocyte/macrophage chemoattractants, was present in normal peritoneal fluid and was found to be high in women with pelvic endometriosis [38], and it was expressed in peritoneal macrophages (our unpublished data). Although significantly higher levels of MCP-1 were reported to be released in amniotic fluid and extra-embryonic coelomic fluid [39], the presence and role of MCP-1 in early fetal development could not be clarified until now. These findings indicate that there may be some differences between macrophages of pelvic cavity origin and those of chorionic villous origin. We also examined the cytokine secretion of villous fibroblasts and the stimulatory effects on trophoblast differentiation because fibroblasts are also known candidates for cytokine production in placentae [18]. However, VFCM had a very weak or no stimulatory effect on trophoblast differentiation in serum-free culture: the levels of secretion of M-CSF, VEGF, and MCP-1 from villous fibroblasts were very low. Taken together, villous macrophages may play a leading role in production and secretion of some specific cytokines for the proliferation and differentiation of trophoblasts. This is the first report to reveal the physiological involvement of human villous macrophages in the development of early pregnancy trophoblasts in vitro. Recently, Cervar et al. [40] found that villous macrophage-conditioned media inhibited hCG, hPL, and prostanoid secretion of trophoblasts. The discrepancy between their findings and our present data may be due to a different study protocol or cell conditions because they obtained populations of both trophoblasts and macrophages from term placentae. Although it is not well known which of the cytokines are secreted from villous macrophages in a given stage of pregnancy, it is tempting to speculate that macrophages may regulate their own secretion depending on the physiological needs and pregnancy stage, and that the secreted factors may send signals to trophoblasts to modulate synthesis of the trophoblast-specific hormones hCG and hPL. In conclusion, macrophages may play a central role in the development and maintenance of the human placenta by affecting trophoblast growth and differentiation through their secreted cytokines. Therefore, it seems likely that the necessary signals that regulate trophoblast development lie within the villous core. Further study is underway to define other macrophage-derived cytokines that may coordinate the mechanism of the cytokine network in the human placenta and to clarify the effects of their recombinant proteins on trophoblast functions. ACKNOWLEDGMENTS We would like to thank Dr. T. Ohba, Dr. Y. Nagata, Dr. Y. Suenaga, Dr. T. Ujioka, and Dr. K. Ishikawa in the Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, for their technical assistance in this research. We also thank Dr. M. Nagano and T. Kitano, Department of Public Health, Kumamoto University School of Medicine, for their advice on statistical analysis.
REFERENCES 1. Ringler GE, Strauss III JF. In vitro systems for the study of human placental endocrine function. Endocr Rev 1990; 11:105–123. 2. Kliman HJ, Nestler JE, Sermasi E, Sanger JM, Strauss III JF. Purification, characterization and in vitro differentiation of cytotrophoblasts from human term placentae. Endocrinology 1986; 118:1567–1582. 3. Douglas GC, King BF. Differentiation of human trophoblast cells in vitro as revealed by immunocytochemical staining of desmoplakin and nuclei. J Cell Sci 1990; 96:131–141. 4. Richards RG, Hartman SM, Handwerger S. Human cytotrophoblast cells cultured in maternal serum progress to a differentiated syncytial phenotype expressing both human chorionic gonadotropin and human placental lactogen. Endocrinology 1994; 135:321–329. 5. Henson MC, Shi W, Greene SJ, Reggio BC. Effects of pregnant human, nonpregnant human, and fetal bovine sera on human chorionic gonadotropin, estradiol, and progesterone release by cultured human trophoblast cells. Endocrinology 1996; 137:2067–2074. 6. Katabuchi H, Fukumatsu Y, Araki M, Khan S, Suenaga Y, Matsuura K, Okamura H. Morphodynamics of macrophages in the female genital tract. In: Motta PM (ed.), Recent Advances in Microscopy of Cells, Tissues and Organs. The Female Reproductive System. Rome: Antonio Delfino Editore; 1997: 547–552. 7. Fukumatsu Y, Katabuchi H, Naito M, Takeya M, Takahashi K, Okamura H. Effect of macrophages on proliferation of granulosa cells in the ovary in rats. J Reprod Fertil 1992; 96:241–249. 8. Araki M, Fukumatsu Y, Katabuchi H, Shultz LD, Takahashi K, Okamura H. Follicular development and ovulation in macrophage colonystimulating factor-deficient mice homozygous for the osteopetrosis (op) mutation. Biol Reprod 1996; 54:478–484. 9. Kirsch TM, Friedman AC, Vogel RL, Flickinger GL. Macrophages in corpora lutea of mice: characterization and effects on steroid secretion. Biol Reprod 1981; 25:629–638. 10. Fukumatsu Y, Katabuchi H, Okamura H. Immunohistochemical localization of epidermal growth factor and its effect on granulosa cell proliferation in rat ovary. Endocr J 1995; 42:467–473. 11. Daiter E, Pamper S, Yeung YG, Barad D, Stanley ER, Pollard JW. Expression of human colony-stimulating factor-1 in the human uterus and placenta. J Clin Endocrinol Metab 1992; 74:850–858. 12. Morrish DW, Bhardwaj D, Dabbagh LK, Marusyk H, Siy O. Epidermal growth factor induces differentiation and secretion of human chorionic gonadotropin and placental lactogen in normal human placenta. J Clin Endocrinol Metab 1987; 65:1282–1290. 13. Sharkey AM, Charnock-Jones DS, Boocock CA, Brown KD, Smith SK. Expression of mRNA for vascular endothelial growth in human placenta. J Reprod Fertil 1993; 99:609–615. 14. Nachtigall MJ, Kliman HJ, Feinberg RF, Olive DL, Engin O, Arici A. The effect of leukemia inhibitory factor (LIF) on trophoblast differentiation: a potential role in human implantation. J Clin Endocrinol Metab 1996; 81:801–806. 15. Loke YW, Burland K. Human trophoblast cells cultured in modified medium and supported by extracellular matrix. Placenta 1988; 9: 173–182. 16. Zaccheo D, Pistoia V, Castellucci M, Martinoli C. Isolation and characterization of Hofbauer cells human placental villi. Arch Gynecol Obstet 1989; 246:189–200. 17. Pulford KA, Rigney EM, Micklem KJ, Jones M, Stross WP, Gatter KC, Mason DY. KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections. J Clin Pathol 1989; 42:414–421. 18. Garcia-Lloret MI, Morrish DW, Wegmann TG, Honore L, Turner AR, Guilbert LJ. Demonstration of functional cytokine-placental interactions: CSF-1 and GM-CSF stimulate human cytotrophoblast differentiation and peptide hormone secretion. Exp Cell Res 1994; 214: 46–54. 19. Fukumatsu Y, Katabuchi H, Miyamura S, Matsuura K, Okamura H, Naito M, Takahashi K. Activated macrophages in the peritoneal fluid of women with endometriosis: examination of the intracytoplasmic localization of endogenous peroxidase and interleukin-1. Acta Obstet Gynecol Jpn 1992; 44:529–536. 20. Grerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984; 133: 1710–1715. 21. Katabuchi H, Fukumatsu Y, Araki M, Mizutani H, Ohba T, Okamura H. Localization of chorionic gonadotropin in macrophages of the human chorionic villi. Endocr J 1994; 41:141–153.
TROPHOBLAST DEVELOPMENT BY VILLOUS MACROPHAGES 22. Nishimura R, Kitajima T, Hasegawa K, Takeuchi K, Mochizuki M. Molecular forms of human chorionic gonadotropin in choriocarcinoma serum and urine. Jpn J Cancer Res 1989; 80:968–974. 23. Savarese DMF, Valinski H, Quesenberry P, Savarese T. Expression and function of colony-stimulating factors and their receptors in human prostate carcinoma cell lines. Prostate 1998; 34:80–91. 24. Cullinan-Bove K, Koos RD. Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: rapid stimulation by estrogen correlates with estrogen induced increases in uterine capillary permeability and growth. Endocrinology 1993; 133:829–837. 25. Lehmann MH, Kuhnert H, Muller S, Sigusch HH. Monocyte chemoattractant protein 1 (MCP-1) gene expression in dilated cardiomyopathy. Cytokine 1998; 10:739–746. 26. Goldstein DP. Gestational neoplasms. In: Degroot LJ, Cahill GF, Odell WD, Martini L, Potts JT Jr, Nelson DH, Steinberger E, Winegrad AI (eds.), Endocrinology. Vol 3. New York: Grune and Stratton Inc.; 1979: 1629–1648. 27. Rodway MR, Rao CV. A novel perspective on the role of human chorionic gonadotropin during pregnancy and in gestational trophoblastic disease. Early Pregnancy 1995; 1:176–187. 28. Hoshina M, Hussa R, Pattillo R, Camel HM, Boime I. The role of trophoblast differentiation in the control of the hCG and hPL genes. Adv Exp Med Biol 1984; 176:299–312. 29. Miller WL, Eberhardt NL. Structure and evolution of the growth hormone gene family. Endocr Rev 1983; 4:97–130. 30. Hall CStG, James TE, Goodyer C, Branchaud C, Guyda H, Giroud CJP. Short term tissue culture of human midterm and term placenta: parameters of hormonogenesis. Steroids 1977; 30:569–580. 31. Mitchell MD, Trautman MS, Dudley DJ. Cytokine networking in the placenta. Placenta 1993; 14:249–275. 32. Guilbert L, Robertson SA, Wegmann TG. The trophoblast as an in-
33.
34. 35.
36. 37. 38.
39.
40.
1083
tegral component of a macrophage-cytokine network. Immunol Cell Biol 1993; 71:49–57. Yoshida H, Hayashi S, Kunisada T, Ogawa M, Nishikawa S, Okamura H, Sudo T, Shultz LD, Nishikawa S. The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 1990; 345:442–443. Pollard JW, Hunt JS, Wiktor-Jedrzejczak W, Stanley ER. A pregnancy defect in the osteopetrotic (op/op) mouse demonstrates the requirement for CSF-1 in female fertility. Dev Biol 1991; 148:273–283. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996; 380:435–439. Saito S, Saito M, Enomoto M, Ito A, Motoyoshi K, Nakagawa T, Ichijo M. Human macrophage colony-stimulating factor induces the differentiation of trophoblast. Growth Factors 1993; 9:11–19. Athanassiades A, Hamilton GS, Lala PK. Vascular endothelial growth factor stimulates proliferation but not migration or invasiveness in human extravillous trophoblast. Biol Reprod 1998; 59:643–654. Akoum A, Turcot-Lemay L, Lemay S. Elevated concentration and biologic activity of monocyte chemoattractant protein-1 in the peritoneal fluid of patients with endometriosis. Fertil Steril 1996; 66: 17–23. Denison FC, Riley SC, Wathen NC, Chard T, Calder AA, Kelly RW. Differential concentrations of monocyte chemotactic protein-1 and interleukin-8 within the fluid compartments present during the first trimester of pregnancy. Hum Reprod 1998; 13:2292–2295. Cervar M, Blaschitz A, Dohr G, Desoye G. Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages. Cell Tissue Res 1999; 295:297–305.
