Supernatants from co-cultured endothelial cells and ...

Human Reproduction vol.14 no.4 pp.919–924, 1999

Supernatants from co-cultured endothelial cells and syncytiotrophoblast microvillous membranes activate peripheral blood leukocytes in vitro

Peter von Dadelszen1, Georgina Hurst and Christopher W.G.Redman Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK 1To

whom correspondence should be addressed at: Division of Maternal-Fetal Medicine, Department of Obstetrics and Gynaecology, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5

There is evidence for both endothelial cell and peripheral blood leukocyte (PBL) activation in pre-eclampsia. Syncytiotrophoblast microvillous membranes (STBM) are shed in greater quantities from the placenta in pre-eclampsia, disrupt cultured endothelial cells in vitro and may be the immediate cause of the maternal syndrome. The aim of this study was to determine if endothelial cells co-cultured with STBM release factors that can activate PBL in vitro. Flow cytometry was used to measure changes in intracellular free ionized calcium ([Ca2F]i), pH (pHi) and reactive oxygen species (iROS) as indices of leukocyte activation. PBL from male non-pregnant donors was exposed to supernatants from human umbilical vein endothelial cells (HUVEC) cultured with STBM. The time course of changes in [Ca2F]i, pHi and iROS was determined and compared with appropriate control measurements. The test supernatants caused significant activation of granulocytes and monocytes in terms of increases in [Ca2F]i and falls in pHi and release of iROS. Lymphocytes responded only with respect to increases in iROS. The results define a possible mechanism for the activation of PBL in pre-eclampsia, as being secondary to endothelial cell activation caused by circulating STBM shed in excess amounts from the placenta. Key words: activation/endothelium/leukocyte/pre-eclampsia/ syncytiotrophoblast

Introduction It is generally accepted that the features of the maternal syndrome of pre-eclampsia are caused by maternal endothelial cell activation (Roberts et al., 1989) directly or indirectly dependent on the presence of the placenta (Redman, 1991). We have recently shown that this is one part of a more generalized process that involves graduated activation of peripheral blood leukocytes (PBL), accompanied by changes in surface expression of activation markers, intracellular reactive oxygen species (iROS) (Sacks et al., 1998) and increases in intracellular free calcium (von Dadelszen et al., 1997). The three major subtypes of leukocytes are all involved. © European Society of Human Reproduction and Embryology

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 (TNFα) (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 (Smarason et al., 1993), that similar activity can be ´ demonstrated in the plasma of pre-eclamptic women (Smarason 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 preeclampsia 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).

Materials and methods Materials Heparin was obtained from Leo (High Wycombe, UK), dextran from Fisons (Loughborough, UK) and trypsin, EDTA, and soybean trypsin inhibitor from Gibco (Paisley, UK). Endotoxin-free phosphate buffered saline (PBS-E), bovine serum albumin (BSA), M199 medium, Hank’s buffered saline solution (HBSS), HEPES buffer, propidium iodide, nigericin, streptomycin, kanamycin, fetal calf serum, endothelial cell

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growth supplement (from bovine neural tissue), gelatin, trypsin, dimethylformamide, and MnCl2 were all supplied by Sigma-Aldrich Company, Poole, Dorset, UK. Fluo-3/AM ester, carboxySNARF-1 and dihydrochlorofluorescein diacetate were purchased from Molecular Probes (Eugene, OR, USA) and anhydrous dimethylsulphoxide (DMSO) from Aldrich (Gillingham, Dorset, UK). Normal human serum (NHS) was obtained from healthy adult donors, both male and female. Tissue culture flasks and 96-well plates were purchased from Nunc (Copenhagen, Denmark). Milli-Q-UF ultrafiltered water system and 0.45 µm filters were supplied by Millipore (Watford, UK). Syncytiotrophoblast microvillous membranes ´ These were prepared using a modified method (Smarason et al., 1993) of Smith et al. (1974). The placentae were taken from women with singleton pregnancies at elective Caesarean sections for nonurgent 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 (Smarason 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 (1.53104 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 (2– 3 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 20–30 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 PBSE 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 ([Ca21]i), a change in intracellular pH (pHi) and

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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 [Ca21]i, carboxySNARF-1 (c SNARF-1) for pHi and dihydrochlorofluorescein diacetate (H2DCFDA) 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 3104 events at 33104/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 H2DCFDA 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 [Ca21]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 [Ca21]i. However, as the fluorescence emission intensity of Fluo-3 is directly proportional to [Ca21] over a wide range of Ca21 concentrations (Minta et al., 1989), changes were measured as a ratio of fluorescence intensity relative to time zero (T0).

PBL activation by HUVEC/STBM co-culture supernatants

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 naıve 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 Mann–Whitney 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 5.82–7.78 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 [Ca21]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.

Figure 1. The figure illustrates how the areas under the response curves were determined. In this example the intracellular pH (pHi) response of monocytes to medium conditioned by cultured human umbilical vein endothelial cells (HUVEC) is shown. The final measurement is negative because of the fall of the pH below the baseline. AUC: area under curve; pHi : intracellular pH. Intracellular reactive oxygen species PBL prepared from six male volunteers were suspended in 3 ml HBSS/HEPES and incubated for 15 min at 37°C with H2DCF-DA/ dimethylformamide at a final concentration of 5 µM. Samples were not centrifuged to avoid activation of leukocytes by shear forces. Leukocyte populations were identified by size and granularity as already described and separately analysed. Baseline fluorescence emission was determined for 30 s at 530 nm, after which the culture supernatant was added, the dead space cleared and emission data acquired continuously for a duration determined by the computing capacity of the flow cytometer – on average about 30 s. A peak response was always obtained with the signal returning to baseline by the end of the run. The modal intensity on a log green fluorescence scale in the rectangular gates assigned to each of the three subsets of leukocytes was measured at baseline and maximum after stimulation. The results were compared using the Wilcoxon test for paired samples. In all iROS and pHi experiments a probability of P , 0.05 was used as the threshold for statistical significance.

Results Intracellular free calcium ions Lymphocytes remained stable after exposure to all the potential stimuli (Table Ia). In contrast, both monocytes and granulocytes (Figure 2 and Table Ia) showed a biphasic response to the HUVEC/STBM conditioned medium, with an initial peak followed by a secondary wave of Ca21 release. Neither cell type showed significant responses to the control media. No effect, caused by the order in which the samples were tested, was detected. Intracellular pH A similar pattern was observed in the measurements of [pH]i with no changes in lymphocytes (Table Ib) but significant changes in monocytes and granulocytes exposed to HUVEC conditioned media with STBM (Figure 3 and Table Ib). Following exposure to HUVEC conditioned medium without STBM the [pH]i of monocytes tended to rise for the first 5–6 min. In contrast, the HUVEC/STBM conditioned medium 921


Table Ia. Responses of peripheral blood leukocytes to various conditioned and control media. Intracellular free calcium. See text for experimental details and abbreviations Source of conditioned media

Intracellular free calcium release Area under the curve [median (range)] Granulocytes

HUVEC with STBM (n 5 10) HUVEC without STBM (n 5 10) STBM in buffer (n 5 13) STBM in normal human serum (n 5 8) Buffer alone (n 5 6) Normal human serum alone (n 5 7)

3831 2674 2374 2593 2239 2559

Monocytes 5967)a

(2498, (1187, (1975, (1609, (2089, (2207,

3826) 2724) 2808) 2991) 3231)

Lymphocytes 9380)a

4112 (2340, 2012 (1449, 2398) 2021 (1505, 3100) 2533 (894, 5080) 1879 (1433, 2708) 2056 (1824, 2851)

2204 2565 2320 2086 2294 2226

(1755, (1953, (1712, (1361, (2180, (1656,

3106) 3162) 2788) 2447) 2578) 2458)

aP , 0.01; Mann–Whitney and Wilcoxon tests (see text for details); comparisons of cells exposed to conditioned media from HUVEC with STBM and to various control media.

Table Ib. Intracellular pH, analysed in terms of areas under the response curves Source of conditioned media

HUVEC with STBM (n 5 6) HUVEC without STBM (n 5 6)

Intracellular pH change Area under the curve [median (range)] Granulocytes

Monocytes

Lymphocytes

–415 (–833, 44)a –23 (–505, 128)

–148 (–594, –41)a 140 (0, 248)

–126 (–291, 409) –133 (–291, 409)

aP , 0.05; Wilcoxon test; paired comparisons of cells exposed to conditioned media from HUVEC with or without STBM.

Table Ic. Oxidative responses, analysed in terms of peak responses Source of conditioned media

HUVEC with STBM (n 5 6) HUVEC without STBM (n 5 6)

Oxidative burst ratio (peak:basal) fluorescence intensity [median (range)] Granulocytes

Monocytes

Lymphocytes

1.56 (1.52, 2.13)a 1.12 (1.02, 1.33)

2.98 (2.33, 5.00)a 2.55 (1.72, 3.15)

2.57 (1.79, 3.28)a 1.60 (1.25, 2.03)

aP , 0.05; Wilcoxon test; paired comparisons of cells exposed to conditioned media from HUVEC with and without STBM.

caused a fall in the [pH]i which was sustained for the 20 min period of observation. Intracellular reactive oxygen species The median analysis time was 40 s (range 19.5–150). The median times to maximal response did not differ for control or test samples and ranged from 1.76–5.33 s for individual experiments. In six separate experiments the ratio of peak to basal fluorescence was significantly higher when cells were treated with supernatants from HUVEC exposed to STBM than with control conditioned media. All leukocytes were significantly stimulated (Table Ic). Discussion Release of STBM from the placental surface into the maternal circulation occurs in normal pregnancy but is significantly exaggerated in pre-eclampsia (Knight et al., 1998). Interaction between STBM and endothelial cells in vitro leads to disruption ´ of the endothelial monolayer (Smarason et al., 1993) and could account for the endothelial cell dysfunction which is a central part of the pathogenesis of the end-stages of the disease. 922

Recent reports from our group (von Dadelszen et al., 1997; Sacks et al., 1998) and several from others (for example, Barden et al., 1997) indicate that peripheral blood leukocytes are also generally activated in pre-eclampsia. Whether endothelial or leukocyte activation is the primary process is unclear because each could initiate the other in a secondary fashion. These results are consistent with the concept that the innate maternal immune system is activated in pre-eclampsia (Schuiling et al., 1997; Sacks et al., 1998). In this report we show that when STBM disrupt endothelial cells in culture, they stimulate the production, in addition, of soluble factors that activate leukocytes, especially granulocytes and monocytes, as demonstrated by three different measures. 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 [Ca21]i activates calcium dependent enzymes that stimulate different cell responses. Measurements of [Ca21]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 [Ca21]i was significantly increased in

PBL activation by HUVEC/STBM co-culture supernatants

Figure 2. Time courses of the responses of granulocytes (a, upper panel) or monocytes (b, lower panel) in terms of excitation of loaded Fluo-3, measured flow cytometrically, and caused by increases in intracellular free calcium ions after exposure to conditioned media from cultured human umbilical vein endothelial cells. –j– HUVEC cultured with syncytiotrophoblast microvesicles; –m– HUVEC cultured on their own (n 5 10). For other control data and statistical analyses see Table I. STBM 5 syncytiotrophoblast microvillous membranes.

all three subsets of leukocytes – lymphocytes, granulocytes and monocytes – from women with pre-eclampsia compared with the three control groups. In this study, [Ca21]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

Figure 3. Time courses of the responses of granulocytes (a, upper panel) or monocytes (b, lower panel) in terms of intracellular pH (pHi) measured flow cytometrically after exposure to conditioned media from cultured human umbilical vein endothelial cells. –j– HUVEC cultured with syncytiotrophoblast microvesicles; –m– HUVEC cultured on their own (n 5 6). For other control data and statistical analyses see Table I.

compensatory activation of the Na1/H1 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 [Ca21]. Therefore the observed changes in [Ca21]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 923


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 preeclampsia (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.

Acknowledgements We gratefully acknowledge the statistical advice of Dr Pat Yudkin. Dr von Dadelszen was supported by a grant from Action Research and by a Girdlers’ Fellowship, and wishes to acknowledge the support of LAM. Ms Hurst is a Rhodes Scholar.

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