the cytokine interleukin-6 increases expression of ...

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HYPERTENSION IN PREGNANCY, 16(3), 403-415 (1997)

THE CYTOKINE INTERLEUKIN-6 INCREASES EXPRESSION OF THE CELL ADHESION MOLECULES E-SELECTIN AND VCAM-1 ON ENDOTHELIAL CELLS IN VITRO: A ROLE IN PREECLAMPSIA? Fiona Lyall,' Ph.D., Fiona Boswel1,Z B.Sc., Anne Young: Catherine J. Clark,2 Ph.D., and Ian A. Greer: M.D. 'Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow, G3 8SJ, UK *Department of Obstetrics and Gynecology, Queen Elizabeth Building, Royal Infirmary, Glasgow, G31 2ER, UK

ABSTRACT Objective: To determine whether the previously reported increases in Interleukin-6 and cell adhesion molecules in preeclampsia may be linked, the effects of Interleukin-6 on endothelial cell expression of cell adhesion molecules, E-Selectin and vascular cell adhesion molecule (VCAM), were studied in vitro. Methods: Endothelial cells were prepared from umbilical veins and used on the first passage. VCAM and E-Selectin mRNA were assessed by Northern analysis following incubation of the cells with Interleukin-6 (0, 10,100,500, or 1000 Units/mL) for 4 h at 37°C. VCAM-1 and E-Selectin proteins were assessed by immunocytochemistry following incubation of the cells with Interleukin-6 (500 Units/mL) for 0, 4, 8, or 24 h at 37°C. Results: Interleukin-6 resulted in a dose- and time-dependent increase in mRNA and protein levels of both E-Selectin and protein.

Address correspondence to: Dr. Fiona Lyall, Maternal and Fetal Medicine Section, Institute of Medical Genetics, Yorkhill, Glasgow, G3 SSJ, UK. Fax: 0141 357 4277; E-mail GQTAOl @udcf.gla.ac.uk.

403 Copyright 0 1997 by Marcel Dekker, lnc


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Conclusions: These results suggest that Interleukind may, at least in part, be responsible for the increase in E-Selectin and VCAM-1 that occur in this disorder. Key Words: E-Selectin; Endothelial cells; Interleukin-6; Preeclampsia; VCAM.

Although preeclampsia complicates up to 15% of pregnancies, the etiology of this condition remains unclear. However, endothelial damage and dysfunction appear to be common to all its pathological features (1,2). There is a disturbance of normal endothelial function with deficiency of antithrombotic and vasodilator factors such as prostacyclin (3) and an excess of prothrombotic products such as von Willebrand factor (4),and increased vasoconstrictor products such as the endothelium-dependant vasoconstrictor peptide endothelin (43). Serum concentrations of thrombomodulin are also increased in preeclampsia, providing further evidence of endothelial damage in this condition (6). It is unclear what triggers this endothelial disturbance in preeclampsia, but neutrophil activation can result in similar vascular damage in the nonpregnant state (7). We have previously reported that concentrations of neutrophil elastase, a specific marker for neutrophil activation in-vivo, are elevated in the peripheral circulation of women with preeclampsia (8) and fetal growth restriction (9), and that this activation is confined to the maternal circulation (10). Furthermore, we have found increased numbers of elastase-positive neutrophils in the decidua of the placenta in preeclampsia (1I), the site where the characteristic vascular lesion, acute atherosis, is seen. We also reported a significant correlation between plasma neutrophil elastase and von Willebrand factor, a marker of endothelial damage, suggesting that neutrophil activity may contribute to the endothelial damage seen in this condition (4). Although these data indicate clearly that neutrophil activation occurs in preeclampsia, the mechanism underlying this activation remains to be elucidated. The attachment and extravasation of leucocytes is controlled by expression of cell surface adhesion molecules on both the circulating cells and the vascular endothelium (12). The major endothelial adhesion molecules include E-Selectin, intercellular adhesion molecule 1 (ICAM- l), and the closely related vascular endothelial cell adhesion molecules 1 and 2 (VCAM-1 and VCAM-2). We recently showed that circulating concentrations of the cell adhesion molecules VCAM-1 and E-Selectin are elevated in the maternal circulation of women with preeclampsia (13J4). Increased expression of adhesion molecules by the endothelium could, in part, be responsible for the neutrophil activation that occurs in preeclampsia. Pro-inflammatory cytokines could also be responsible for many features of endothelial dysfunction (15). They could trigger neutrophil activation, expression of von Willebrand factor, and cell adhesion molecules on the endothelium with


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resultant vascular damage. In support of this, we and others have recently shown a selective increase in the pro-inflammatory cytokine Interleukin-6 ([ILI-6) in the maternal circulation in preeclampsia (16,17). Although [ILI-6 and VCAhUE-Selectin are increased in preeclampsia, there are no published data on the effect of IL-6 on cell adhesion molecules expression. Because such a link may be involved in the pathology of preeclampsia, the aim of this study was to determine whether [ILI-6 could increase expression of E-Selectin and VCAM-1/2 on endothelial cells in vitro. If so, this would suggest one possible mechanism through which cell adhesion molecule expression is stimulated in this disorder.

MATERIALS AND METHODS Cell Culture Umbilical cords were obtained from the delivery suite, transported to the laboratory in sterile 0.9% NaC1, and used within 48 h of delivery. There was no difference in the yield, viability, or growth rates of cells obtained from cords up to 48 h. The umbilical vein was cannulated and then flushed with serum-free growth medium (Dulbecco’s Modification of Eagle’s medium with 4.5 g/L dextrose, ICN Inc., Thame, Oxfordshire, UK) containing 2.5 p&mL Fungizone (amphotericin B) (Gibco BRL, Paisley, UK), 100 IU/mL-100 kg/mL penicillin-streptomycin (Gibco BRL), and 2 mmoVL glutamine (ICN Inc.). A solution of 0.1% type I1 collagenase (Sigma, Poole, Dorset, UK) was then introduced, the vessel was clamped off at the bottom, wrapped in clingfilm, and incubated at 37°C for 15 min. The cord was rubbed gently to detach the cells and then the cells were flushed out of the vein with serum-free medium. The cell suspension was then spun at 1000 rpm at 4°C for 5 min, the supernatant decanted, the pellet was washed once more in serum-free medium, and then the pellets of separate cords were combined and resuspended (6 mL120 cm length of cord) in growth medium (serum-free medium containing heatinactivated 10% fetal bovine serum [ICN Inc.] and heat-inactivated 10% donor horse serum [ICN Inc.]). The cells were grown in 6-well clusters (Costar Corp., Cambridge, MA) at a density of 3 mL cell suspensiotdwell. All plastics were coated with 2% gelatin (Sigma). Cells were maintained at 37°C in a 5% C0,/95% air humidified atmosphere. When the cells reached confluence (usually 5-7 days) they were trypsinized from the plates using standard techniques, combined, and then either split 1:3 into 6-well clusters for experiments using Northern analysis or split 1:3 and grown in 8-well Lab-Tek@chamber slides (Nunc. Inc., Naperville, IL) in preparation for immunocytochemistry. This split meant that the cells were 30-50% confluent when they were plated and took approximately 2 days to reach confluence on the 8-well Lab-Tek@dishes and 5 days to reach confluence on the 6-well clusters. On the first passage, the medium was supplemented with SOp,g/mL endothelial cell growth supplement (Sigma, UK).


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Experimental Protocol: Northern Analysis When the cells reached confluence the medium was replaced with fresh growth medium (horse serum omitted) containing recombinant human [ILI-6 (Genzyme, West Mallin, Kent, UK) 0, 10, 100, 500, or 1000 Units/mL. The Interleukin-6 was added from lyophilized powder reconstituted in 0.1% bovine serum albumin to give a stock solution of 200,000 Units/mL. This was stored in working aliquots at -70°C. Cells were incubated with [ILI-6 for 4 h at 37°C. This time point was chosen since pilot experiments showed maximal induction at this point (not shown). At the end of the incubation, total RNA was extracted using the RNAzol'" B method (Biogenesis, UK) according to the manufacturer's instructions. Integrity of RNA was confirmed by the presence of intact 18s and 28s bands on agarose gels, and free of protein and DNA by having a ratio (optical density at 260 nm/280 nm) of > 1.9. RNA samples were stored in 10 pg batches in two volumes of ethanol and one-tenth volume of 3 m o m sodium acetate, pH 5.2 at -70°C.

Northern Analysis RNA (10 pg) was dissolved in 10 pL of H,O and 10 pL of the following solution was then added: deionized formamide, 1250 pL; 37% formaldehyde, 400 pL; lox MOPS (0.2 m o m 3-[N-Morpholino] propane-sulphonic acid; 0.05 m o m Na acetate pH 7.0; 0.01 m o m Na,EDTA), 250 FL, and heated at 65°C for 10 min. Samples were chilled on ice and 1 pL of 1 mg/mL ethidium bromide and 2 pL of 50% glycerol containing 0.1 mg/mL bromophenol blue was added. Samples were separated on 1.2% agarose gels containing 6% formaldehyde and 20 m m o m MOPS. Gels were electrophoresed at 60 V for 3-4 h. RNA was transferred from the gels to nylon membranes (Hybond-N, Amersham International, Buckinghamshire, UK) in 20x SSC (3 m o m NaC1; 0.3 mol/L Na, citrate, pH 7.0) and fixed to the membrane by UV irradiation. Membranes were prehybridized for 4 h in 10 mL of the following mixture at 65°C: 5x SSC; 5x Denhardt's solution (50x Denhardt's is 1% polyvinylpyrrolidone, 1% Ficoll400 [Pharmacia, Uppsala, Sweden], and 1% bovine serum albumin); 0.5% sodium dodecyl sulphate, and 100 pg/mL boiled salmon sperm DNA. Filters were then hybridized in the same buffer overnight with the appropriate 32P-labeled (oligolabeling kit, Pharmacia) cell adhesion molecule cDNA. The VCAM-2 cDNA (1.9 Kb cDNA) was a gift from Dr. Dave Simmons, International Cancer Research Fund, Oxford, UK and the E-Selectin cDNA (1.2 Kb insert) was a gift from Dr. Michael Bevilacqua, Howard Hughes Medical Institute, CA. Filters were washed to a final stringency of 0 . 5 SSC, ~ 0.1% sodium dodecyl sulfate (SDS) at 65°C. Autoradiography was performed with Kodak X-omat film at -70°C for 3 days. The bands on the autoradiographs were quantified with a scanning densitometer (Hoefer Scientific Instruments, purchased from Biotech Instruments Ltd.) and arbitrary densitometry units were recorded. The densitometer was calibrated once for each of the three VCAM-1 experiments and once for the three E-Selectin experiments as follows. The background on the film was given a



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densitometric unit of 0 (bottom of the chart paper) and a value of 10 was given to the maximum peak obtained from the three experiments (adjusted to be the top of the chart paper). The mean and SD of the arbitrary densitometry units were calculated for each adhesion molecule and compared using the Student’s ?-test.

Experimental Protocol: Immunocytochemistry When the cells reached confluence they were incubated with [ILI-6 (500 Units/mL) for 0, 4, 8, or 24 h. At the end of the incubation the medium was removed from the cells, the wells were gently rinsed with phosphate buffered saline (pH 7.4), and then the cells were fixed in ice-cold acetone for 10 min. at 4°C. The wells were allowed to air dry for 5 min, the upper chambers were peeled away, and then the slides were processed for immunocytochemistry as follows. Slides were incubated in 0.5% hydrogen peroxide in methanol to block endogenous peroxidase activity. They were then washed in tris buffered saline (TBS) buffer ( 5 mmoVL tris/ HCI pH 7.4; 0.9% NaCl) then blocked with 20% normal goat serum (NGS) for 30 min at room temperature. Next, the slides were incubated overnight at 4°C with primary antibody diluted in 2% NGS in TBS. The cell adhesion molecule antibodies were obtained from R&D Systems (Abingdon, UK). These were monoclonal mouse anti-human IgG, subclass ( a ) E-Selectin (1:250 final dilution) and ( b ) VCAM-1 (1500 final dilution). Negative controls were slides incubated with 2% NGS in TBS or with a mouse monoclonal IgG, Aspergillus niger glucose oxidase (DAKO Ltd., High Wycombe, UK), an enzyme that is neither present or inducible in mammalian tissues. Slides were next washed in TBS then incubated for 1 h at room temperature with 1:200 biotinylated goat anti-mouse immunoglobulins (DAKO Ltd.) in 2% NGS/5% normal human serum in TBS. After washing in TBS, slides were incubated for 30 min at room temperature with 1:400 peroxidaseconjugated streptavidin (DAKO Ltd.) in TBS, then washed again in TBS. Finally, immunoreactive cell adhesion molecules, which appear as a brown end-product, were localized by incubating sections in 1 mg/mL 3,3’-diaminobenzidine tetrahydrochloride in 0.02% hydrogen peroxide in TBS. The slides were counterstained in Harris hematoxylin. Slides were examined by an investigator blinded to their identity and scored for staining (0, +, ++, or +++). Statistical analysis was performed using the chi-squared test. To assess [ILI-6 receptor expression on the cells, an anti-human [ILI-6 receptor antibody (R & D Systems) was used at a dilution of 1:lOOO. Immunostaining was detected using the ABC method with a Vectastain kit (Vector Laboratories, Peterborough, UK) according to the manufacturer’s instructions.

RESULTS Northern Analysis Northern analysis revealed that unstimulated endothelial cells expressed little or no VCAM-2 mRNA. However, in response to the addition of [ILI-6 (0,10, 100,

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0

10

100

500

1000

[IL]-6 Units/ml

[IL-61

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1000 8.5+1.1

Figure I . Northern analysis of VCAM-2 mRNA expression in human umbilical vein endothelial cells following addition of increasing concentrations of [ILI-6. (a) Photograph of gel showing even loading of RNA (10 pgflane) and intact 28s and 18s rRNA bands. RNA samples were separated on 1.2% agarose gels. (b) RNA was transferred from the gels to nylon membranes and fixed to the membrane by UV irradiation. Membranes were prehybridized then hybridized overnight with 32Plabeled VCAM-2 cDNA. Filters were washed to a final stringency of 0 . 5 ~SSC at 65°C. Autoradiography was performed with Kodak X-omat film at -70°C for 3 days. (c) Bands on the autoradiographs were quantified with a scanning densitometer and an arbitrary densitometry unit was obtained for each band. The mean and SD of densitometry units are shown for three separate experiments. Values were compared with the Student’s t-test. * = P < 0.05,lO units vs. 0 units [LL]-6; ** P < 0.05, 100 units vs. 10 units [ILJ-6; *** P < 0.05, 500 units vs. 100 units [ILI-6.



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500, and 1000 Units/mL) for 4 h, there was a dose-dependent increase in VCAM-2 mRNA, which reached maximal levels at 1000 Units/mL. Shown is an autoradiograph representative of three separate experiments [Fig. I(b)]. Also shown [Fig. l(a)] is the gel from the experiment showing equal loading of the RNA (10 pg/lane) and intact 28s and 18s ribosomal RNA bands. The mean I+_ SD densitometry units are shown for three separate experiments [Fig. l(c)]. Northern analysis also revealed that unstimulated endothelial cells did not express E-Selectin mRNA. However, in response to the addition of [ILI-6 (0, 10, 100, 500, and 1000 Units/mL) for 4 h, there was a dose-dependent increase in E-Selectin mRNA, which reached maximal levels at 500 Units/mL. Shown is an autoradiograph representative of three separate experiments [Fig. 2(b)]. Also shown [Fig. 2(a)] is the gel from the experiment showing equal loading of the RNA (10 pg/lane) and intact 28s and 18s ribosomal RNA bands. The mean ? SD densitometry units are shown for three separate experiments [Fig. 2(c)].

Immunocytochemistry Before performing the immunocytochemistry, we first established that endothelial cells expressed [ILJ-6receptors. To show that receptor expression was not an effect of the cells being in culture, we also performed immunocytochemistry for the receptor on cryosections of a term human placenta. As shown in Fig. 3, both endothelial cells in vitro and endothelial cells in the vessels of the placenta stained positively with the [ILI-6 receptor antibody. Fig. 4 shows a representative immunostaining result for VCAM-1 protein on human umbilical vein endothelial cells (HUVECs) following incubation with [ILI-6 (500 Units/mL). VCAM-1 protein was undetectable on HUVECs in the absence of [ILI-6 treatment [Fig. 4(a)1, however, following the addition of [IL]-6 to the cells there was significant increase in VCAM immunostaining at 4 h [Fig. 4(b)], which remained significantly elevated at 8 h [Fig. 4(c)] and then declined toward basal levels by 24 h [Fig. 4(d)]. Fig. 5 shows a representative immunostaining result for E-Selectin protein on endothelial cells following incubation with [ILI-6 (500 Units/mL). E-Selectin protein was undetectable on endothelial cells in the absence of [ILJ-6 treatment [Fig. 5(a)], however, following the addition of [ILI-6 to the cells there was significant increase in E-Selectin immunostaining at 4 h [Fig. 5(b)], which remained significantly elevated at 8 h [Fig. 5(c)] and then declined toward basal levels by 24 h [Fig. 5(d)]. Table 1 summarizes the data for three experiments for VCAM-1 and E-Selectin immunostaining.

DISCUSSION Although the etiology of preeclampsia still remains unclear, the evidence accumulated to date supports a model whereby abnormal trophoblast invasion of

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(C)

[IL-61 Units/ml Densitometry units

0

o

500

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10 *

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**

4.i+o.i a.5rto.7 g.i+i.i 9.7ko.5

Figure 2. Northern analysis of E-Selectin mRNA expression in human umbilical vein endothelial cells following addition of increasing concentrations of [a]-6. (a) Photograph of gel showing even loading of RNA (10 pg/lane) and intact 28s and 18s rRNA bands. RNA samples were separated on 1.2% agarose gels. (b) RNA was transferred from the gels to nylon membranes and fixed to the membrane by UV irradiation. Membranes were prehybndized then hybridized overnight with 3*Plabeled E-Selectin cDNA. Filters were washed to a final stringency of 0 . 5 SSC ~ at 65°C. Autoradiography was performed with Kodak X-omat film at -70°C for 3 days. (c) Bands on the autoradiographs were quantified with a scanning densitometer and an arbitrary densitometry unit was obtained for each band. The mean and SD of densitometry units are shown for three separate experiments. Values were compared with the Student’s ?-test. * = P < 0.05, 10 units vs. 0 units [ILI-6; ** P < 0.05, 100 units vs. 10 units [ILI-6.



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Figure 3. Demonstration that endothelial cells express [ILI-6 receptors both in vivo and in vitro. Immunolocalization of rILl-6 receptor protein on human umbilical vein endothelial cells in vitro (b) and on a term human placenta cryosection (d). Plates (a) and (c) show no in vitro and in vivo immunostaining respectively when the primary antibody was omitted. lmmunostaining was performed as described in the Methods section.

maternal spiral arteries (18) results in poor placental perfusion. Poor placental perfusion might in turn result in production of one or more factors by the placenta that activates or injures endothelial cells (19-21). These factors remain to be identified but may also be linked directly or indirectly to the neutrophil activation that occurs in this disease (8). and to our recent demonstration that circulating concentrations of the cell adhesion molecules VCAM-I and E-Selectin are elevated in the maternal circulation of women with preeclampsia (13,14). Increased expression of adhesion molecules by the endothelium could, in part, be responsible for this neutrophil activation. Pro-inflammatory cytokines may play a role in endothelial activation (15) and can directly or indirectly, via the endothelium, increase neutrophil activation. Cytokmes are also emerging as excellent candidates for physiologic “adhesion triggers” (12). Thus, cytokmes, by activating endothelial cells, may in part be responsible for the endothelial dysfunction and resultant vascular damage that have been described in preeclampsia. In support of this, we and others have recently shown a selective increase in the pro-inflammatory cytokine [ILI-6 in the maternal circulation in preeclampsia (1516). We have also shown that [ILI-6 concentrations correlate with cell adhesion molecule concentrations (16). [ILI-6 plays a major role in the mediation of inflammatory and immune responses and is produced by a number of cell types including activated endothelial cells, monocytes, and macro-


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Figure 4. Immunolocalization of VCAM-I protein on endothelial cells following incubation with [ILI-6 for 0,4,8, or 24 h. [IL]-6 (500 Unitslrnl) was incubated with endothelial cells for (a) 0 h (b) 4 h (c) 8 h or (d) 24 h. Immunolocalization of cell adhesion molecules was performed as described in the Methods section.

phages (22). The effects of [ILI-6 on cells are numerous and varied and include induction of cell proliferation (23). The association noted above between [ILI-6 and cell adhesion molecules led to the present study. Since the E-Selectin and VCAM-1 Northern blots were performed on different days with different batches of "PCTP, we cannot comment on the intensity of the VCAM-1 and E-Selectin bands compared to each other, or whether one might be induced to a higher level than another. Regardless of this, in this study we have shown that [ILI-6 can induce the expression of both E-Selectin and VCAM-2 mRNA on endothelial cells in vitro. This induction was both time and dose dependent. Furthermore, both E-Selectin and VCAM-1 protein were also induced on the cells following [ILI-6 treatment. although the immunocytochemical staining can only be assessed in a semiquantitative manner, the results were nevertheless unambiguous since the cells did not express these adhesion molecules prior to treatment. VCAM-1, a member of the immunoglobulin superfamily of cell adhesion molecules, is a 90 kDa cell surface glycoprotein. VCAM-1 has 7 immunoglobulin domains and is expressed on a number of cells including endothelium, macrophages kidney epithelia, dendritic cells, myoblasts, and bone marrow fibroblasts. An alternatively spliced form of VCAM-1 (VCAM- 2) lacks domain 4 of the 7 immunoglobulin domains, and its expression is limited to endothelial cells (24). In this study we used a VCAM-1 antibody and a VCAM-2 cDNA. The VCAM-2

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Figure 5. lrnrnunolocalization of E-Selectin protein on endothelial cells following incubation with rIL1-6 for0,4. 8, or 24 h. [IL]-6(500 UnitsirnL) was incubated with endothelial cells for (a) 0 h (b)4 h (c) 8 h or (d) 24 h. Irnrnunolocalization of cell adhesion molecules was performed as described in the Methods section.

cDNA recognizes the same sequence as VCAM-I. Interestingly, only one transcript was apparent with the VCAM-2 cDNA. However, Hession et al. (25) were unable to distinguish between both the 6- and 7-domain forms of VCAM on cytokine-stimulated umbilical vein endothelial cells by Northern analysis when using a probe that could recognize both forms equally well. Indeed, discrimination between the two forms necessitated the use of the reverse transcriptase-polymerase chain reaction. They also showed that the 7-domain is the major form produced by endothelial cells. Furthermore, both forms of VCAM support adhesion, indicating that domain 4 is not essential for the binding of leucocytes to VCAM-1.

Table 1. Immunolocalization of Cell Adhesion Molecules E-Selectin and VCAM on Endothelial Cells TIME TREATED WITH [IL]-6 (H) NO. EXPERIMENTS

0

Intensity of E-selectin immunostaining (-, +, ++. or + + + ) Intensity of VCAM- I imrnunostaining (-, +. + + , o r + + + )

-

3

-

4 3

8 3

++ + + ++

++

24 3 -

+++ +

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E-Selectin and VCAM-1 are expressed on a number of activated cells, including activated endothelial cells, and the expression of VCAM-1 and E-Selectin on cells is regulated, at least in part, by changes in cytokine concentrations. Since the common pathological feature of preeclampsia is endothelial damage and dysfunction, one likely source of the circulating adhesion molecules is the endothelium, and these data therefore suggest one possible mechanism through which cell adhesion molecule expression is stimulated by increased concentrations of [ILI-6. In summary, the presence of increased concentrations of [ILI-6 in the maternal circulation of pregnancies complicated by preeclampsia may contribute to endothelial damage and dysfunction seen in this disorder, such as expression of endothelial adhesion molecules resulting in leucocyte attraction to the endothelium.

ACKNOWLEDGMENT This work was supported by a grant from Action Research and the Scottish Hospitals Endowments Research Trust.

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10. Greer IA, Dawes J, Johnston TA, Calder AA. Neutrophil activation is confined to the maternal circulation in pregnancy-induced hypertension. Obstet Gynecol 1991;78: 28-32. 11. Butterworth B, Greer IA, Liston WA, Haddad NG, Johnston TA. Immunocytochemical localisation of neutrophil elastase in term placenta decidua and myometrium in pregnancy-induced hypertension. Br J Obstet Gynaecol 1991;98:929-33. 12. Harlan JM, Liu DY, eds. Adhesion: Its Role in Inflammatory Disease. London: Freeman, 1992. 13. Lyall F, Greer IA, Boswell F, Macara LM, Walker JJ, Kingdom JCP. The cell adhesion molecule VCAM-1 is selectively elevated in serum in pre-eclampsia: does this indicate the mechanism of leucocyte activation? Br J Obstet Gynaecol 1994;lOl: 485-7. 14. Lyall F, Greer IA. [Letter]. Br J Obstet Gynaecol 1994;102:173-4. 15. Anderson AO. The role of cells and cytokines in immunity and inflammation. In: Oppenheim JJ, Shevach EM, eds. Immunophysiology. Oxford: Oxford University Press, 1990:14-45. 16. Greer IA, Lyall F, Perera T, Boswell F, Macara LM. Increased concentrations of cytokines Interleukin-6 and Interleukin-1 receptor antagonist in plasma of women with pre-eclampsia: a mechanism for endothelial dysfunction? Br J Obstet Gynaecol 1994;84:937-40. 17. Vince GS, Starkey PM, Austuglen R, Kwiatkowski D, Redman CWG. Interleulun-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br J Obstet Gynaecol 1995;102:20-5. 18. Sheppard BL, Bonnar J. An ultrastructural study of uteroplacental spiral arteries in hypertensive and normotensive pregnancy and fetal growth retardation. Br J Obstet Gynaecol 1981;88:695-705. 19. Roberts JM, Taylor RN, Goldfien A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4: 700-8. 20. Roberts JM, Redman CWG. Preeclampsia: more than pregnancy-induced hypertension. Lancet 1993;341:1447-54. 21. Lyall F, Greer IA. Pre-eclampsia: a multifaceted vascular disorder of pregnancy. Rec Advan in Obstet Gynaecol 1994;18:3-21. 22. Wong GG, Clark SC. Multiple actions of interleukin 6 within a cytolune network. Immunol Today 1988;9:137-9. 23. Tosato G, Pike SE. A rnonocyte-derived B-cell growth factor identified is IFN-beta-2 BSF-2RL-6. Ann N Y Acad Sci 1989;557:181-91. 24. Cybulsky MI, Fries JWU, Williams AJ, Sultan P, Davis VM, Gimbrone MA Jr., Collins T. Alternative splicing of human VCAM-1 in activated endothelium. Am J Pathol 1991;138:815-20. 25. Hession C, Tizard R, Vassallo C, Schiffer SB, Goff D, Moy P, et al. Cloning of an alternative form of vascular cell adhesion molecule-1 (VCAM1). J Biol Chem 1991; 266:6682-6685.