Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK
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Abstract |
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Key words: activation/endothelium/leukocyte/pre-eclampsia/syncytiotrophoblast
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Introduction |
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Endothelial cells can be activated in several different ways potentially relevant to the origins of pre-eclampsia and several factors such as lipoproteins or lipid peroxides (Hubel et al., 1996) or tumour necrosis factor alpha (TNF
) (Vince et al., 1995
) have been implicated. We have given evidence for an alternative mechanism, namely deported syncytiotrophoblast microvillous membrane (STBM) fragments shed from the placental surface in greater amounts in pre-eclampsia (Knight et al., 1998
); and we suggest that these comprise one end of the spectrum of trophoblast deportation (Chua et al., 1991
). This group has previously shown that, in vitro, such fragments specifically activate and disrupt cultured human endothelial cells (Smárason et al., 1993
), that similar activity can be demonstrated in the plasma of pre-eclamptic women (Smárason et al., 1996
) and that STBM affect endothelial dependent relaxation of perfused small human arteries ex vivo (Cockell et al., 1997
).
Endothelial cells can activate leukocytes (Zimmerman et al., 1992) or vice versa (Mantovani and Dejana, 1989
) and, as yet, it is not clear which comes first in pre-eclampsia. Hence several possible mechanisms of the activation of PBL in pre-eclampsia can be considered, all dependent on the syncytiotrophoblast microvillous surface membrane which is the placental surface in contact with maternal blood. Factors intrinsic to the syncytiotrophoblast may activate PBL in transit through the intervillous space or in the peripheral circulation if they are released. A third possibility is that deported STBM could disrupt maternal endothelium which would then cause a secondary activation of PBL.
The last mechanism is investigated in this study. STBM prepared from normal placentae were incubated with cultured human umbilical vein endothelial cells (HUVEC). The supernatants were tested for their effects on PBL using flow cytometric techniques and found to cause immediate activation. In addition to intracellular free calcium, changes in intracellular pH and iROS were measured, because previous reports have indicated that they accompany granulocyte, monocyte and lymphocyte activation (Busa and Nuccitelli, 1984; Azuma et al., 1996
; Brumell et al., 1996
; Sacks et al., 1998
).
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Materials and methods |
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Syncytiotrophoblast microvillous membranes
These were prepared using a modified method (Smárason et al., 1993) of Smith et al. (1974). The placentae were taken from women with singleton pregnancies at elective Caesarean sections for non-urgent reasons such as a previous Caesarean section. None of the women had pre-eclampsia. The pelleted preparation was divided into aliquots and stored at 70°C in PBS-E until use. Aliquots from 10 normal placentae were pooled at a protein concentration of 1 mg/ml, which was used in all the experiments described below.
Human umbilical vein endothelial cells
These were prepared by the method of Jaffe et al. (1973) with some modifications, as previously described (Smárason et al., 1993). Cells in the primary culture were grown to confluence in M199 medium supplemented with 20% (v/v) heat-inactivated fetal calf serum and endothelial cell growth supplement (30 mg/ml), heparin (90 mg/ml), kanamycin (100 mg/ml), penicillin (50 U/ml) and streptomycin (50 mg/ml) in 25 cm2 tissue culture flasks (Nunc) coated with 1% gelatin. Confluent cells were detached with trypsin/EDTA, then trypsin inhibitor was added to the cell suspension and the cells were washed in M199 medium. HUVEC (1.5x104 cells/well) were added to gelatin-coated 96-well plates in 100 µl of assay medium M199 medium containing 40% (v/v) heat-inactivated pooled human serum and endothelial cell growth supplement (60 mg/ml), heparin (180 mg/ml), kanamycin (200 mg/ml), penicillin (100 U/ml) and streptomycin (100 mg/ml)]. After the cells attached to the wells (23 h) medium or STBM (10 µg protein/well) were added to the cell monolayer. Following 48 h incubation at 37°C in 5% CO2 in air, the supernatants were aspirated and stored at 20°C.
Peripheral blood leukocytes
Polypropylene or polystyrene tubes were used throughout to minimize monocyte adhesion. All centrifugation steps were completed without braking. A total of 2030 ml of venous blood was added immediately to 6% dextran in PBS-E, containing 10 mU/ml of heparin, topped up to 50 ml total volume with PBS-E and allowed to sediment spontaneously for 30 min at 20°C. The supernatant containing PBL was divided into four aliquots and each was diluted to 50 ml with PBS-E and centrifuged (400 g, 10 min, 20°C). The pellets were resuspended in 2 ml PBS-E and the red cells lysed in 48 ml of lysis buffer. This was prepared from 8.29 g NH4Cl and 1 g KHCO3 dissolved in 1 l of double distilled, ultrafiltered water produced by the Milli-Q-UF system. The solution was then filtered through a 0.45 µm filter in 48 ml aliquots. After 7 min, the tubes were centrifuged (400 g, 10 min, 4°C). The PBL pellets were resuspended in PBS-E (20 ml/tube), washed for 5 min at 4°C, and suspended at 107 cells/ml in PBS-E with 20 mM glucose and 0.2% BSA (PGE/BSA). The leukocytes from one individual contributed one data point only to each set of experiments. All samples were taken from a single pool of 13 donors.
Leukocyte responses to endothelial cell culture supernatants
Three indices of activation were used, namely increased free ionized intracellular calcium ([Ca2+]i), a change in intracellular pH (pHi) and an increase in intracellular reactive oxygen species (iROS). Each was estimated flow cytometrically using different fluorophores loaded into the leukocytes: Fluo-3/AM ester for [Ca2+]i, carboxySNARF-1 (c SNARF-1) for pHi and dihydrochlorofluorescein diacetate (H2DCF-DA) for intracellular reactive oxygen species, using modifications of methods previously described (Rabinovitch and June, 1994; Sacks et al., 1998
).
Flow cytometry
Samples were analysed in a Coulter Elite® flow cytometer (Coulter Electronics Inc., Hileah, FL, USA) (excitation wavelength: 488 nm; emission wavelength: 530 nm [Fluo-3 and H2DCF-DA]; emission wavelengths: 575 and 675 nM [cSNARF-1]) with an added time zero module (Cytek®; Cytek Development Inc., Fremont, CA, USA) to allow the immediate analysis of acute cellular responses to reagents added to a sample. Four x104 events at 3x104/s were recorded unless otherwise stated. Monocytes, lymphocytes and granulocytes were separately identified by their size and granularity and placed in rectangular analysis gates in which the changes in fluorescence intensities were individually assessed. These gates were confirmed by cell type-specific surface antigen expression analysis using fluorescein isothiocyanate-conjugated mouse anti-human antibodies (Serotec, Kidlington, UK). Lymphocytes were identified by their expression of CD3 (UCHT 1), monocytes by CD14 (UCHM 1), and granulocytes by CD15 (Km-93) (data not shown). Granulocyte subsets could not be identified confidently by size and granularity characteristics alone. Data were saved in listmode for retrospective analysis.
Leukocyte preparations were loaded in the dark with the appropriate fluorophore, after which a 1 ml aliquot was placed in the time-zero module maintained at a temperature of 20°C when Fluo-3 or H2DCF-DA were used, and at 37°C when cSNARF-1 was used. In the last instance, 5 min was allowed for temperature equilibration. Baseline fluorescence was measured for all three PBL populations (see below). Through the sideport of the time-zero module, 3 ml of test or control sample was injected in a single bolus. Simultaneously the dead space in the tubing from the time-zero module was cleared.
Measurements were made of the fluorescence emission responses for variable lengths of time (see below) at the appropriate wavelength(s). Cell viability was measured flow cytometrically. One µl propidium iodide (PI) stock solution (10 mg/ml double distilled water) was added per ml of cell suspension, gently mixed and incubated at room temperature for 5 min. The cell suspension was analysed flow cytometrically for the percentage of PI-positive (that is dead) cells.
Test and control media
For the [Ca2+]i experiments, conditioned media from HUVEC cultured on their own and with added STBM (always from the same preparation) formed the central comparison of the study. To exclude a direct effect due to STBM, these were pre-incubated (100 µg protein/ml) in PBS-E with or without 10% NHS on their own. Two further controls of PBS-E without STBM and with or without 10% NHS were also included. As there was no measurable difference ascribable to STBM alone, in the subsequent pHi and iROS experiments, the protocol was simplified so that the conditioned media comprised solely HUVEC culture supernatants with or without added STBM.
Intracellular free calcium ion
The requirements of rapid and repetitive measurements after stimulating the PBL with test supernatants precluded absolute measurement of [Ca2+]i. However, as the fluorescence emission intensity of Fluo-3 is directly proportional to [Ca2+] over a wide range of Ca2+ concentrations (Minta et al., 1989), changes were measured as a ratio of fluorescence intensity relative to time zero (T0).
It was not possible to measure the test supernatants and all the control samples in a single experiment because the duration would have been too long, with problems arising from leakage of Fluo-3 after cell loading. However the paired cultured supernatants from HUVEC with and without added STBM were always analysed together in the same experiment.
A total of 107 PBL/ml PGE/BSA from normal male donors were loaded with 1 µM Fluo-3/AM as described. Male cells were used exclusively to ensure that the immune system would be naïve to STBM exposure, and to avoid any influence of the menstrual cycle on leukocyte function (Bain and England, 1975). STBM activity is present in the circulation of normally pregnant women (Knight et al., 1998
), therefore they could not be used as donors. Fluo-3/AM ester in anhydrous DMSO was added to a final concentration of 1 µM. After incubation at 37°C for 30 min, the cell suspensions were flooded with HBSS and centrifuged (400 g, 20°C, 5 min). The pellets were resuspended in 6 ml HBSS, providing additional sample volume if needed. Three baseline determinations of fluorescence emission intensity were made with 50 µl aliquots from the initial 1 ml suspension of PBL loaded with Fluo-3/AM ester, after which 3 ml of test medium, conditioned or not, were added to the sample (T0). Thereafter data were acquired every 30 s for 6 min, every minute for the next 4 min, and every 5 min until the experiment was ended after 40 min.
The area under the time-response curve was measured (Prism 2.01) for a run time of 40 min and the results analysed non-parametrically. Because the HUVEC conditioned media were tested in matched pairs, with or without added STBM, the Wilcoxon test for paired data was used. For all other comparisons the MannWhitney test was used. As there were multiple comparisons, P < 0.01 was considered statistically significant.
Intracellular pH
The pH-sensitive fluophore cSNARF-1 was used as previously described (Rabinovitch and June, 1994). cSNARF-1 exhibits large shifts in pH-dependent fluorescence, the emission wavelength lengthening with increasing pH. By collecting emissions at both 575 and 675 nM and determining the ratio of the intensities, the pH can be determined by reference to a standard curve. There are much smaller shifts in absorption maxima with pH so that fluorescence can be stimulated with either argon or krypton lasers. PBL, prepared from six male volunteers, were resuspended at 107 cells/ml PGE/BSA. Seven standard solutions with pH ranging from 5.827.78 were made by mixing two buffers in varying proportions. The buffers contained 20 mM NaCl, 1 mM MgCl2, 1 mM CaCl2 and 10 mM glucose and either 135 mM KH2PO4 or 135 mM K2HPO4. Ten aliquots of PBL suspension were incubated with 2 µM cSNARF-1/AM ester at 37°C for 30 min and centrifuged (400 g, 7 min, 37°C). Seven of the aliquots were used to generate a standard curve for the specified range of pH. Each was resuspended in one of the standard buffers, with nigericin at a final concentration of 2 µg/ml, and incubated for 5 min at 37°C before flow cytometric analysis. The remaining three aliquots were resuspended in 1 ml of HBSS/HEPES for pH analysis before and after stimulation with the conditioned media. As stated, these comprised HUVEC culture supernatants with or without added STBM. The order of analysis was systematically varied to ensure that delay in processing did not bias the results.
Baseline and stimulated measurements were taken, the latter at the same intervals as for the [Ca2+]i measurements, but the experiments were curtailed earlier at 20 min.
The area under the time-response curve was measured (see Figure 1) using Prism 2.01 and the differences between the two supernatants compared non-parametrically, using the Wilcoxon test.
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Results |
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Discussion |
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Intracellular calcium regulates the responses of excitable cells. Concentrations in the cytoplasm are tightly controlled and depend on calcium membrane channels, calcium pumps and release or uptake of calcium from membrane surfaces. A rise of [Ca2+]i activates calcium dependent enzymes that stimulate different cell responses. Measurements of [Ca2+]i have been used to study leukocyte responses in vitro and ex vivo. In our previous report (von Dadelszen et al., 19978) we showed that median basal [Ca2+]i was significantly increased in all three subsets of leukocytes lymphocytes, granulocytes and monocytes from women with pre-eclampsia compared with the three control groups. In this study, [Ca2+]i of lymphocytes were not affected by the test supernatants. This could be because the time course of response is slower than was tested or that lymphocytes are involved secondary to activation of phagocytic leukocytes. Changes in intracellular pH also accompany cell activation. The pH response is generally biphasic, with an early transient acidification followed by a more prolonged rise in the pH. There is evidence that an influx of calcium, such as that stimulated by chemoattractants, causes acidification of the cytoplasm of granulocytes (Busa and Nuccitelli, 1984
) and monocytes (Azuma et al., 1996
). The subsequent increase in the pH is inhibited by amiloride, its analogues or by sodium free conditions, demonstrating a compensatory activation of the Na+/H+ antiporter (Busa and Nuccitelli, 1984
). In our experiments only granulocytes and monocytes demonstrated changes which were within physiological limits. A biphasic response was not seen nor had the pH recovered to basal levels by the end of the experiment at 20 min. Thus the response was different from that reported with chemoattractants such as formylmethionyllencylphenylalanine (fMLP) (Busa and Nuccitelli, 1984
). In addition, acidification reduces the fluorescence emission intensity of Fluo-3 at any given [Ca2+]. Therefore the observed changes in [Ca2+]i in these experiments were not spuriously elevated by cytosolic alkalinization following exposure to HUVEC/STBM conditioned medium. We do not know what this implies because we have not identified what factor or factors cause the observed changes.
Phagocytes (granulocytes and monocytes) produce and secrete ROS (such as H2O2, O2, and OH) as part of their non-specific immune defence mechanism (Himmelfarb et al., 1992
). But iROS are not necessarily secreted, and as products of oxygen metabolism are markers of intracellular metabolic activity (Seeds et al., 1985
). Thus, although lymphocytes are not phagocytic, they also produce iROS (Rabesandratana et al., 1992
) which signify activation (Goldstone et al., 1995
). This was the only measure indicating that lymphocytes could also be stimulated by the culture supernatants, but is consistent with our previous report of activation of peripheral blood lymphocytes, measured in the same way, in women with pre-eclampsia (Sacks et al., 1998
).
These experiments bring together three strands of current thinking on the pathogenesis of pre-eclampsia concerning the involvement of the placenta, of the maternal endothelium and of maternal peripheral blood leukocytes. An in-vitro model of one possible pathogenic sequence has been devised, namely that excessive shedding of syncytiotrophoblast microvesicles in pre-eclampsia causes endothelial cell dysfunction which leads to leukocyte activation. We have not excluded an alternative sequence which would depend on demonstrating that STBM could activate leukocytes directly leading to production of soluble factors that could affect endothelial function. Nor have we yet identified what factor on STBM disrupts endothelium or what are the products of that process that activate leukocytes. These questions are the subjects of ongoing investigations in our laboratories.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 9, 1998; accepted on December 31, 1998.