Objective
To determine whether trophoblast-derived microparticles can induce different inflammatory responses of the peripheral blood mononuclear cells depending upon the state of trophoblast when the microparticles are generated.
Study Design
A trophoblast-derived cell line (ATCC no. CRL-1584) was cultured under normal or hypoxic conditions. Microparticles were isolated from the cell culture supernatants (microparticles from normal trophoblast; microparticles from hypoxic trophoblast). Peripheral blood mononuclear cells were cultured alone or cocultured with either microparticles from normal trophoblast or microparticles from hypoxic trophoblast.
Results
After 48 hours, the peripheral blood mononuclear cells cocultured with microparticles from normal trophoblast released higher concentrations of interleukin-6 than peripheral blood mononuclear cells cultured alone. The peripheral blood mononuclear cells cocultured with microparticles from hypoxic trophoblast showed higher concentration of interleukin-6 and tumor necrosis factor alpha than peripheral blood mononuclear cells cocultured with microparticles from normal trophoblast, after 24 hours and 48 hours.
Conclusion
More intense and rapid inflammatory response of peripheral blood mononuclear cells was observed with microparticles from hypoxic trophoblast than with microparticles from normal trophoblast. This difference might explain the exaggerated systemic inflammatory response as a result of placental hypoxia in preeclampsia.
Microparticles are small membrane vesicles, which originate from blebbing membranes of either activated cells or cells undergoing apoptosis. The syncytiotrophoblast forms the maternal-placental interface, and it has been suggested that microparticles from this cell (syncytiotrophoblast microparticles [STBMs]) may be the factor that disturbs the maternal endothelium and mediates the inflammatory response, which is the characteristic feature in preeclampsia.
However, microparticles also showed immunosuppressive characteristics, and this immunoregulatory function of microparticles has been thought as the explanation of immune tolerance in normal pregnancy. The mechanism of this different action of microparticles (ie, proinflammatory vs immune-regulatory) in preeclampsia and normal pregnancy is not well determined.
Preeclampsia is initiated by reduced uteroplacental blood flow, resulting in placental hypoxia, and the experimental evidences and histologic feature of placenta in preeclampsia supports this theory. During normal placentation, the maternal spiral arteries are invaded by trophoblast, resulting in extensive remodeling of spiral arteries to ensure an adequate blood supply to the placenta and fetus. However, placentas of preeclamptic show shallow trophoblast invasion and defects in spiral artery remodeling, resulting in poor perfusion and ischemia. As a result of placental ischemia, it is believed that placenta release a number of soluble factors into maternal circulation, eliciting characteristic features of preeclampsia. In this context, our hypothesis was that the trophoblast in a different condition (ie, normal vs hypoxic, representing normal pregnancy vs preeclampsia) may shed microparticles with different characteristics, resulting in a different systemic inflammatory response to these microparticles.
To this end, we undertook this study to determine whether trophoblast-derived microparticles can induce different inflammatory response, according to the condition of the trophoblast.
Materials and Methods
Cell culture
A trophoblast-derived cell line (ATCC no. CRL-1584), which has been used as the model for placenta in previous studies, was obtained from the Global Bioresource Center ( http://www.atcc.org ) and cultured in 10% MEM media (modified Eagle’s medium; GIBCO, Langley, OK; with 10% fetal bovine serum and antibiotics). The cultures were maintained at 37°C in an atmosphere of 5% of CO 2 .
Induction of hypoxic cell death
Two million trophoblast cells were cultured either in normal or hypoxic conditions. For normal condition, the cultures were maintained in an atmosphere of 5% of CO 2 . For hypoxic condition, cells were cultured in the presence of 100 μmol/L rotenone (Sigma-Aldrich, St. Louis, MO), which induces chemical hypoxia by disruption of the mitochondrial electron transport chain. To confirm the process of apoptotic cell death after the addition of rotenone, floating cells were collected and adherent cells were detached with trypsin/EDTA, and the cells were pooled with centrifugation (300× g, at room temperature, for 5 minutes), and were analyzed with flow cytometric analysis (FACS) using the FITC Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ). For assessment, cells were resuspended in 100 μL of binding buffer at a concentration of 1 × 10 6 cells/mL, and 5 μL of FITC Annexin V and 5 μL of PI was added. After incubation for 15 minutes at room temperature in the dark, 400 μL of binding buffer was added and the analysis was performed using FACSCalibur (Becton Dickinson, San Jose, CA) and the data were evaluated with CellQuest software (Becton Dickinson). In the presence of rotenone, 15 to 40% of the trophoblast cells were stained with Annexin V after 24 hours of culture, and 50 to 80% of cells were stained with Annexin V after 48 hours of culture, although less than 15% of the trophoblast cells were stained with Annexin V after 24 and 48 hours of culture in normal condition (ie, in the absence of rotenone).
Isolation and quantification of microparticles
After 24 hours of culture in normal or hypoxic condition, the supernatants were collected and microparticles were isolated by differential centrifugation. First, cell culture supernatants were separated from detached cells by 2 centrifugation steps (300× g, 5 minutes and 800× g, 5 minutes). Then, the cell-free supernatants were ultracentrifuged at 100,000× g at 4°C for 90 minutes. After the removal of the supernatants, the pellet was washed twice with phosphate-buffered saline (PBS) and finally suspended in 10% MEM media.
The number of microparticles was counted by FACS, and adjusted to the concentration of trophoblast cells. Trophoblast cells were counted with a hemocytometer and 2 × 10 6 cells/mL of trophoblast cells were used for the experiment. In brief, the number of each microparticles and trophoblast cells were counted for 30 seconds at the “low-flow” modus and the concentration of microparticles was calculated by the following formula, then the concentration of microparticles was adjusted to standard concentration for the following experiment.
The concentration of microparticles = (microparticles count/trophoblast cells count) × (2 × 10 6 ) (counts/mL).
Coculture of PBMCs with microparticles
Peripheral venous blood (40 mL) was taken from healthy nonpregnant women (n = 4). PBMCs were isolated by density gradient centrifugation using the Ficoll method (Sigma-Aldrich HISTOPAQUE-1077). The final pellet was resuspended in 10% MEM, and 2 × 10 6 cells/mL, 0.2 mL aliquots were placed in a 96-well plate.
The PBMCs were cultured alone, or cocultured with microparticles derived from the trophoblast in normal condition (MP_C, 5 × 10 6 /mL), or cocultured with microparticles derived from the trophoblast in hypoxic condition (MP_H, 5 × 10 6 /mL), in the presence or absence of T-cell mitogen, phytohemagglutinin (PHA, 5 μg/mL) up to 48 hours. Each experiment was done in duplicate. After either 24 or 48 hours of culture, cellular proliferation was measured with the Cell Counting Kit-8 (CCK-8, Dojindo, Japan), and the coculture supernatants were obtained and assayed for interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) using the commercial enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, MN).
The institutional review board of the Seoul National University Hospital approved this study. The Seoul National University has a Federal Wide Assurance (FWA) with the Office for Human Research Protections (OHRP) of the Department of Health and Human Services (DHHS) of the United States.
Statistical analysis
Comparisons of continuous variables between groups were performed with the Mann-Whitney U test. A P value < .05 was considered as statistically significant.
Results
PBMCs cocultured with microparticles from trophoblast in normal condition
After 24 hours of coculture, the cytokine (both IL-6 and TNF-α) production and cellular proliferation was not different between PBMCs cultured alone and PBMCs cocultured with MP_C. This was the same in the addition of PHA ( Figures 1 and 2 ). However, after 48 hours of coculture, the supernatant of PBMCs cocultured with MP_C had significantly higher concentrations of IL-6 than that of PBMCs cultured alone ( Figure 1 ). Although the supernatant TNF-α concentration of PBMCs cocultured with MP_C was also higher than that of PBMCs cultured alone, this difference did not reach statistical significance. After 48 hours, the cellular proliferation of PBMCs was also increased when cocultured with MP_C than when cultured alone ( Figure 2 ). In the presence of PHA, the supernatant IL-6 concentration of PBMCs cocultured with MP_C after 48 hours was also higher than that of PBMCs cultured alone, although the cellular proliferation was not increased ( Figures 1 and 2 ).
PBMCs cocultured with MP_C or MP_H
The inflammatory response of PBMCs was compared according to the type of microparticles. The supernatant IL-6 and TNF-α concentration of PBMCs cocultured with MP_H was significantly higher than that of PBMCs cocultured with MP_C, after both 24 and 48 hours of culture ( Figure 3 ). In the presence of PHA, PBMCs cocultured with MP_H also showed more intense proinflammatory response. The cellular proliferation was not different between PBMCs cocultured with MP_C and MP_H ( Figure 4 ).
When cultured alone for 24 or 48 hours, neither MP_C nor MP_H in the absence of PBMCs produced any measurable amounts of IL-6 or TNF-α, both in the absence or presence of PHA (data not shown).
Results
PBMCs cocultured with microparticles from trophoblast in normal condition
After 24 hours of coculture, the cytokine (both IL-6 and TNF-α) production and cellular proliferation was not different between PBMCs cultured alone and PBMCs cocultured with MP_C. This was the same in the addition of PHA ( Figures 1 and 2 ). However, after 48 hours of coculture, the supernatant of PBMCs cocultured with MP_C had significantly higher concentrations of IL-6 than that of PBMCs cultured alone ( Figure 1 ). Although the supernatant TNF-α concentration of PBMCs cocultured with MP_C was also higher than that of PBMCs cultured alone, this difference did not reach statistical significance. After 48 hours, the cellular proliferation of PBMCs was also increased when cocultured with MP_C than when cultured alone ( Figure 2 ). In the presence of PHA, the supernatant IL-6 concentration of PBMCs cocultured with MP_C after 48 hours was also higher than that of PBMCs cultured alone, although the cellular proliferation was not increased ( Figures 1 and 2 ).
