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ORIGINAL RESEARCH |
From the 1Department of Obstetrics and Gynecology and the 2Department of Clinical Pharmacology, Turku University Hospital, Turku, Finland.
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ABSTRACT |
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), interleukin (IL)-1ß, and IL-6, transfer across the placenta, and thereby determine whether intra-amniotic inflammatory response, measured from the amniotic fluid, is of maternal or fetal origin.
METHODS: Nineteen placentas from healthy women undergoing elective cesarean delivery at term with intact membranes and without labor, were dually perfused ex vivo in an open circulation system for either 30 minutes or 2 hours. Tumor necrosis factor-, IL-1ß, and IL-6 were added to maternal or fetal circulation in a concentration usually found in chorioamnionitis. As a reference, placentas without added cytokine were also perfused. The concentrations of cytokines were determined by enzyme immunoassays (enzyme-linked immunosorbent assay [ELISA]).
RESULTS: After the addition of the cytokine to the arterial perfusate, the venous concentration on the same side of the placenta increased rapidly and reached a plateau at 10 minutes. No transfer of any cytokine in either direction was detected. Some endogenous release of IL-6 was observed in response to the perfusion.
CONCLUSION: Proinflammatory cytokines do not cross normal term placenta.
LEVEL OF EVIDENCE: II-1
Intrauterine infection/inflammation is usually asymptomatic until the beginning of labor or a preterm rupture of the membranes. At present, the only method to detect an intrauterine inflammation during pregnancy is to sample amniotic fluid by amniocentesis or to sample fetal blood by cordocentesis. Both of these procedures are invasive and are known to have risks. Increased concentration of inflammatory cytokines, particularly interleukin (IL)-6, in the amniotic fluid or in the fetal blood predict a rapid onset of delivery even better than the presence of bacteria.7 Efforts to measure maternal inflammatory response have failed to reflect inflammatory status in utero.
Placental perfusion is mainly used in studying the placental passage of drugs but is also virtually the only viable method of simulating the real-time function of the human placenta. Because it is not possible to differentiate between maternal and fetal immune response, one can study the origin of the immune response in amniotic fluid by studying whether cytokines transfer across placenta. At present, only a few reports on the permeability of a placenta to inflammatory cytokines have been published. Reisenberger et al11 found that IL-8 does not transfer across placenta in either direction. Zaretsky et al12 presented the same findings for tumor necrosis factor alpha (TNF-) and IL-1. They suggested that IL-6, in contrast to other proinflammatory cytokines of the same molecular size, transfers across a placenta in both directions. Thus, they proposed that it serves as a messenger between a mother and the fetus.
Our assumption is that proinflammatory cytokines do not cross a placenta and consequently the inflammatory response detected in amniotic fluid is of fetal origin. We tested this hypothesis by dually perfusing term human placentas ex vivo with TNF-, IL-1ß, and IL-6.
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MATERIALS AND METHODS |
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The perfusion method was modified from that reported by Schneider et al.13 All experiments were carried out in an open (nonrecirculating) system. The open system was chosen to control the content of the cytokines in the arterial influx. If a closed system (recirculation) is used, the perfusate subsequently contains all the substances released from the placenta during perfusion, and this can affect the results.
After delivery, placentas, with the cords clamped, were immediately transported to a perfusion laboratory. Heparinized 0.9% saline solution was then injected into the umbilical artery, a visually good intact cotyledon was chosen, and corresponding distal branches from the chorionic artery and vein were cannulated. The cotyledon was cut from the placenta and placed on a meshed metal plate, with the maternal side upward over a plexiglas cone, into which maternal effluent was allowed to drain. To establish maternal perfusion flow, the maternal (decidual) surface of the placenta was gently pierced with four butterfly needles. The perfusion was carried out at a flow rate of 3 mL/min on the fetal side and 10 mL/min on the maternal side. The perfusate on both sides was a Krebs-Ringer bicarbonate buffer (Sigma Chemical, St. Louis, MO), which contained 30 g/L of bovine albumin (Equitech Bio, Kerrville, TX). The perfusate was maintained at 37°C and equilibrated with 95% oxygen and a 5% carbon dioxide mixture throughout perfusion. All perfusions were initiated within 15 minutes after placental delivery.
During the first 30 minutes of perfusion, the cotyledon was allowed to stabilize with the perfusion environment. After stabilization, the maternal or fetal perfusate was replaced with a perfusate containing TNF-, IL-1ß, or IL-6 (Peprotech, London, UK). The cytokine concentrations used were chosen within the range of increased concentrations detected in maternal and umbilical serum during spontaneous preterm labor or chorionamnionitis.8,14–16 Tumor necrosis factor- was perfused on its own to avoid the possible effect on other cytokines.17 Interleukin-1ß and IL-6 were perfused simultaneously. Antipyrine (80 mg/L; Sigma Chemical), which is known to diffuse passively across the placenta, was used as a reference compound to detect possible failure in each individual perfusion.
After stabilization, 14 placentas were perfused for 30 minutes; 2 being perfused without added cytokines to determine the amount of cytokines the placenta produces or releases during perfusion. Three perfusions with TNF- in both directions (maternal-fetal and fetal-maternal, n = 3 + 3) and 3 perfusions with IL-1ß and IL-6 in both directions (n = 3 + 3) were performed. Samples of perfusates were collected from the fetal venous outflow and from the maternal effluent reservoir at initiation (zero minutes). Thereafter, samples were collected every 5 minutes from the side where cytokine was added and every 10 minutes from the other side. This process was repeated up to 30 minutes. When no cytokine was added, both sides were sampled every 10 minutes. Samples were also collected from the arterial inflow (every 10 minutes), to ensure the initial concentrations of the cytokines were stable. In addition, five placentas were perfused for 2 hours to discern the effects of longer perfusion time. Two of these placentas were perfused with both IL-1ß and IL-6, with cytokines added to the maternal arterial side, and in 3 placentas the same cytokines were added to the fetal artery. The samples were collected every 20 minutes from the venous outflows and the arterial inflow. Interleukin-1ß was not analyzed from the last 2-hour perfusion in the fetal-maternal direction. The collected samples were centrifuged (2,000 rpm, 5 minutes) to remove cellular debris and stored at –20°C until they were analyzed.
A blood gas analysis (pH, Po2, and Pco2; Rapidlab 248, Bayer, UK) was performed of fetal and maternal arterial and fetal venous perfusates to test the viability of placental tissue at the beginning and at the end of each perfusion. For the experiment to be accepted, the values had to be within the physiological range. In addition, the integrity of the cotyledon was continuously assessed by monitoring the perfusion pressure of the fetal artery (Multi-Pen Recorder; Rikadenki, Tokyo, Japan). The volumes of the fetal and maternal effluents were also measured to ensure that no perfusion mismatch occurred.
The measurement of cytokine concentrations was performed by using a PeliKine-compact "sandwich-type" enzyme immunoassay (ELISA) from CLB Sanguin (Amsterdam, The Netherlands), according to the supplier's instructions. The sensitivity of the kit was 3 pg/mL for TNF-, 1.5 pg/mL for IL-1ß, and 0.4 pg/mL for IL-6.
To analyze the antipyrine concentrations in the Krebs solution, antipyrine and the internal standard (ritonavir) were extracted from the samples by undertaking solid phase extraction with Sep-Pak Vac 1 mL (100 mg) tC18 SPE columns (Waters Company, Milford, MA). The antipyrine concentrations were measured with high performance liquid chromatography (HPLC) by using a Perkin Elmer (Shelton, CT) Spheri-5 Cyano 4.6 x 100 mm (5 µm) column and mixtures of acetonitrile and 0.05 mM sodium phosphate buffer, pH 4.24, as eluents. The ultraviolet wavelength was 254 nm, and the run time was 17 minutes. The range of the quantitative analysis method was from 100 ng/mL to 25.0 µg/mL. The average between-batch precision (coefficient of variation [CV]%) was 7.9%.
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RESULTS |
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The arterial concentrations of the cytokines were 15–70 pg/mL for TNF-, 25–100 pg/mL for IL-1ß, and 80–520 pg/mL for IL-6. In the effluents of control placentas with no added cytokine, no production of TNF- or IL-1ß in the maternal or fetal venous side was detected; 2.5–4.5 pg/mL of IL-6 was detected in the maternal effluent of the control placentas and also in the maternal zero-samples from the cytokine perfusions. This indicated a low-level release of IL-6 from the placenta during the experiment. In the fetal venous effluent, no IL-6 was observed.
The results of all the experiments are summarized in Figure 1. The charts of the cytokine perfusions are equiform, independent of the cytokine perfused and the concentration used. In general, there was a minute or no basic production of cytokine from the placenta. After the addition of cytokine to the arterial perfusate, the cytokine concentration increased rapidly in the venous outflow of the same side, reaching a plateau at 10 minutes. There was no passage of cytokine to the collateral side of the placenta. The results did not differ between the 30-minute and the 2-hour perfusions. The plateau in longer perfusions was observed at a time-point of 20 minutes because of the longer interval between samples.
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As expected, when IL-6 was added to the fetal artery, there was a small amount (0.8–38 pg/mL) of IL-6 detected in the maternal venous outflow. This corresponded to the spontaneous release of cytokine from the placenta.18 The level of cytokine concentration on the contralateral side of the placenta varied between the placentas and remained at a stable level throughout individual perfusion. This showed that placentas differ in endogenous cytokine release. Furthermore, in both the 2-hour perfusions of IL-1ß—with cytokine added to maternal artery—a very low concentration (1.6–3.8 pg/mL) of cytokine could occasionally be detected in the fetal outflow. In one perfusion with TNF- added to the maternal artery and in one with IL-1ß added to the fetal artery, there was a single sample from the contralateral venous side that contained a minute amount (4.4 pg/mL TNF- and 2.1 pg/mL IL-1ß) of the studied cytokine.
In general, the amount of cytokine in the venous outflow was close to the one in the artery of the same side of the placenta. In some placentas there was some discrepancy between the arterial and venous concentration, suggesting either production or release of the cytokine or that it was bound in the cotyledon. The correlation of the averages of arterial and corresponding venous concentrations during the steady state of the perfusion are shown in Figure 2. The concentration of the cytokine did not affect the correlation.
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DISCUSSION |
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, IL-1ß, and IL-6 are proteins with a molecular weight of 17.4, 17.3, and 20.9 kDa, respectively. Significant amounts of molecules of this size are highly unlikely to traverse a placenta by diffusion. Instead, the passage across a placenta would need active transport with specific receptors. In this study we found no evidence of the passage of cytokines across placentas. The 30-minute perfusion showed a rapid flow of cytokine from the artery to the vein in the same side of the placenta, with concentration close to the original concentration in the arterial perfusate. No transfer was observed with longer perfusions, where active transport would likely to have been detected. For example, the transport of IgG across a placenta,19 which is thought to involve Fc' receptors on the syncytiotrophoblast, and of alpha-fetoprotein,20 was observed during a 2-hour perfusion.
In some of the perfusions, a low concentration of cytokine was also detected on the other side of the placenta. This was especially observed when perfusing IL-6 from fetal to maternal side, but also in 2-hour perfusions with IL-1ß from maternal to fetal side. Studies by Pierce et al18,21 have shown that even optimal perfusion raises the concentration of IL-6 in effluent over time. In nonoptimal conditions, such as during hypoperfusion or hyperoxia, the concentrations of TNF- and IL-6 are more markedly elevated. Other cytokines, such as monocyte chemotactic peptide-1 (MCP-1), IL-8, and RANTES (regulated on activation, normal T-cell expressed and secreted), are also known to be released from a cotyledon during perfusion.22 One can expect the rise in the concentration of IL-1ß to happen by the same mechanism. When the low level of cytokine concentration was observed on the contralateral side in our experiments, it remained quite stable throughout individual perfusion or was only temporarily detected. No increasing trend was noticed, suggesting the low-level production or release of cytokine from the placenta in response to the cytokine or the perfusion itself. Analyzing cytokine concentrations with ELISA does not allow differentiation between the added perfusate cytokine and the one produced or released in a placenta. To distinguish these two, a cytokine with a radioactive label should be used.
Zaretsky et al12 suggested a bidirectional transfer of IL-6 across the placenta. There are, however, a few points in their methods and conclusions that can be re-evaluated. For the first 60 minutes of their perfusion, they used open circulation, where the perfusate passes placenta only once, and from 60 to 120 minutes the system was changed to closed circulation, in which the perfusate is recirculated from the vein back to the artery of the same side of the placenta. If one compares their results from the open perfusion to ours, they are virtually identical. Only when closed circulation is used do IL-6 concentrations start to rise. This result can be interpreted in 2 different ways. One can conclude, like Zaretsky et al that there is a transport of IL-6 to the other side of a placenta that can be detected after a 1-hour perfusion. Our results, however, do not agree with this interpretation. On the other hand, the use of closed circulation may bias the results. This is because in closed circulation, the content of the perfusate changes after each cycle in the placenta. This is due to the system's not allowing the end-products of metabolic processes and excretory products to be cleared. This leads to the accumulation of substances released from the placenta during perfusion, including IL-6. In turn, these may induce a placenta to produce or release even more IL-6, with the amount increasing with every cycle. The difference between the results obtained by Zaretsky et al and by us can be explained by the use of a different method in perfusate circulation.
The results of our experiments only apply to term placentas. There are no publications on the transfer of cytokines in preterm placentas or placentas with infection. The permeability of the placenta to cytokines may change during gestation, choriodecidual infection, and the onset of labor. Cytokines may also transfer from a maternal compartment to a fetal compartment, or vice versa, by paracellular diffusion through fetal membranes. Membranes weakened by proteases induced by inflammation might be a particular site for possible transfer. In the future, studies of placentas and fetal membranes from different conditions are needed to further understand the process of intrauterine inflammation and its connection with the onset of preterm labor.
Pregnancy is a state of relative immunosuppression, which somewhat increases susceptibility to infections. However, systemic maternal infection, with a marked rise in inflammatory markers in peripheral blood, seldom affects the continuation of a pregnancy. Nevertheless, high intrauterine concentrations of inflammatory markers, especially IL-6, are connected with the rapid onset of labor and also correlate to infant morbidity. According to our results, it seems that a normal term placenta does not permeate inflammatory cytokines, and thus it is believed the inflammatory response in amniotic fluid and in fetal blood is solely of fetal origin. If this also applied to preterm and inflamed placentas, it would support the earlier findings,7 which stated that the onset of preterm delivery is dependent on the fetal immune response, not that of the mother.
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Footnotes |
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Corresponding author: Riikka Aaltonen, MD, the Department of Obstetrics and Gynecology, Turku University Hospital, Kiinamyllynkatu 4-8, FIN-20520 Turku, Finland; e-mail: riikka.aaltonen{at}pp.inet.fi.
doi:10.1097/01.AOG.0000178750.84837.ed
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REFERENCES |
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