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Neutrophil oxygen radical production in pre-eclampsia with HELLP syndrome
European Journal of Obstetrics & Gynecology and Reproductive Biology 99 (2001) 213±218
Neutrophil oxygen radical production in pre-eclampsia with HELLP syndrome Petra L.M. Zusterzeela, Geert J.A. Wantenb, Wilbert H.M. Petersb, Hans M.W.M. Merkusa, Eric A.P. Steegersa,* a
Departments of Obstetrics and Gynaecology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b Departments of Gastroenterology, University Medical Centre Nijmegen, Nijmegen, The Netherlands Received 22 November 2000; received in revised form 5 March 2001; accepted 15 March 2001
Abstract Objective: To determine whether severe pre-eclampsia complicated by hemolysis, elevated liver enzymes, low platelets (HELLP) -syndrome alters neutrophil oxygen radical production. Materials and Methods: Neutrophils were obtained from 10 healthy non-pregnant, 9 normal pregnant and 9 women with severe preeclampsia with concurrently HELLP syndrome. Oxygen radical production was evaluated using luminol-enhanced chemiluminescence and measured by cytochrome C reduction. Furthermore we incubated sera from cases and controls with isolated healthy neutrophils and measured their capacity to generate oxygen radicals. Results: Unstimulated neutrophil oxygen radical production was signi®cantly lower in severe pre-eclamptics compared with healthy nonpregnant and pregnant subjects, whereas phorbol ester-induced oxygen radical production did not differ among categories. Cytochrome C reduction of unstimulated neutrophils showed similar results. Healthy neutrophils incubated with sera from pre-eclamptics enhanced the oxygen radical production signi®cantly more than neutrophils incubated with sera from the healthy subjects. Conclusions: Severe pre-eclampsia is characterised by decreased unstimulated neutrophil oxygen radical production. This may be the result of an exhausted cellular response due to stimulation by a factor present in the serum of these patients. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemiluminescence; Neutrophil; Oxygen radical; Pre-eclampsia; HELLP
1. Introduction Although pre-eclampsia complicates up to 5% of pregnancies and is a major cause of both maternal and perinatal morbidity and mortality, the aetiology of this condition remains unclear. Endothelial damage and dysfunction may play an important role in the pathofysiology of preeclampsia [1]. Neutrophil activation can result in vascular damage by their ability to produce reactive oxygen species [2]. Activated neutrophils attach to the endothelium and subsequently release contents of their granules, such as elastase and other proteases, which are capable of destroying the integrity of endothelial cells. The oxygen radicals generated by activated neutrophils can result in membrane lipid peroxidation, and subsequent increased vascular permeability and reactivity [2]. It is unclear what triggers the endothelial disturbance in pre-eclampsia, but neutrophil activity * Corresponding author. Tel.: 31-24-3619449; fax: 31-24-3619036. E-mail address: [email protected] (E.A.P. Steegers).
may play a role in endothelial damage in this disorder. Neutrophil adhesion to the vascular endothelium is dependent on the association between adhesion molecules present on the surface of endothelial cells. Increased expression of such adhesion molecules was shown in pre-eclampsia [3,4] in addition to increased plasma levels of granular components, such as elastase, lactoferrin, endothelin-1, and gravidin [5±7]. Most studies, however, used indirect techniques to show neutrophil activation. Neutrophils produce oxygen radicals in response to stimulation, and direct measurement of oxygen radical production is a good marker for neutrophil activation [8]. To study the role of neutrophils in the pathogenesis of preeclampsia and hemolysis, elevated liver enzymes, low platelets (HELLP) -syndrome [9], we measured the oxygen radical production from neutrophils of non-pregnant and normotensive pregnant women and women with severe preeclampsia complicated by HELLP syndrome, using both luminol-enhanced chemiluminescence with and without an external phorbol ester phorbol 12-myristate 13-acetate
0301-2115/01/$ ± see front matter # 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 1 ) 0 0 3 7 7 - 3
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(PMA) stimulus [10]. Furthermore we measured superoxide anion production with a cytochrome C reduction assay. To determine whether there is a humoral factor in serum from women with pre-eclampsia, which alters neutrophil oxygen radical production, we also studied the activation of neutrophils from a healthy female blood donor in response to serum (10% v/v) of all subjects. 2. Material and methods 2.1. Subjects The subjects were Dutch women cared for at the University Medical Centre Nijmegen, The Netherlands, from September 1998 to February 1999. Nine primigravidas with severe pre-eclampsia complicated by HELLP syndrome constituted the study population. Severe pre-eclampsia was de®ned as persistent diastolic blood pressure of greater than 110 mm Hg (Korotkoff V) on two or more consecutive occasions in a previously normotensive woman after 20 weeks of pregnancy, with proteinuria of greater than 3 g per 24 h. HELLP syndrome was de®ned as the simultaneous occurrence of a platelet count <10 109 /l, serum lactate dehydrogenase (LDH) concentrations greater than 600 IU/l, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations greater than 70 IU/l [11]. All pre-eclamptic women were severely ill and admitted to the intensive care unit. Six of the pre-eclamptic patients were treated with methyldopa (Aldomet#; Merck Sharp & Dohme BV, Haarlem, The Netherlands) and intravenously administered magnesiumsulfate, one with ketanserin hydrogentartrate (Ketensin#; Pharmacia & Upjohn, Woerden, The Netherlands) and intravenously administered magnesiumsulfate, and two were treated with methyldopa only. Nine normotensive healthy primigravidas, randomly recruited from women visiting the outpatient clinic, comprised a control group. All controls were normotensive without proteinuria. Subjects were matched for maternal and gestational age. In addition, blood was obtained from 10 healthy non-pregnant subjects of comparable age with an uncomplicated obstetrical history. Subjects with a history of renal or vascular disease, diabetes, any autoimmune disorder, or taking any kind of medication other than vitamin- or iron supplements were excluded from the study. Informed consent was obtained from all cases and controls before inclusion, and the local ethical committee approved the study. Blood samples were collected at a progressed stage of the disease for the patients with pre-eclampsia, and during a routine prenatal visit for the pregnant controls. Both patients and pregnant controls were not in labour at the time of blood sampling. 2.2. Procedures and assays Blood samples, anticoagulated with lithium-heparin (for cell isolation) or without anticoagulants (for serum
preparation) were collected from each subject. Chemiluminescence measurements in freshly diluted heparinised blood were always simultaneously performed in matched pairs of pre-eclamptic, normotensive, and non-pregnant subjects. Blood collected in corvac vacutainer tubes was centrifuged at 3000 g for 10 min and serum was stored until assessment at 208C. Upon use, the serum was diluted in phosphate buffered saline (PBS) and added to the neutrophils at a ®nal concentration of 10% (v/v). Chemicals were from Sigma (St. Louis, MO, USA), unless stated otherwise. 2.2.1. Isolation of neutrophils Isolation of neutrophils was performed as described previously [12,13]. Blood diluted 1:1 with PBS containing 0.4% (w/v) trisodium citrate (pH 7.4), was placed on Percoll (speci®c density 1.076 g/ml; Pharmacia Biotech, Uppsala, Sweden) and centrifuged (700 g for 20 min at 258C). The pellet was suspended in 50 ml of ice-cold isotonic lysis solution (containing 155 mmol/l NH4Cl, 10 mmol/l KHCO3, and 0.1 mmol/l EDTA pH 7.4) for 10 min. After centrifugation (400 g for 5 min at 48C) the remaining erythrocytes were lysed on ice in fresh lysis solution for another 5 min. Cells were then washed and resuspended to a ®nal concentration of 2 106 cells/ml and kept at room temperature. Cytospin preparations showed more than 97% granulocytes and viability greater than 99%. 2.2.2. Chemiluminescence Luminol-enhanced chemiluminescence was measured at 378C for 120 min in 96-well microplates with an automated LB96V Microlumat Plus luminometer (EG & G Berthold, Bad Wildberg, Germany), as described previously [13]. To each well containing 200 ml of neutrophil suspension (2 106 cell/ml), 20 ml of a 1:20 dilution of luminol stock in hanks' balanced salt solution (HBSS) (Life Technology, Breda, The Netherlands) supplemented with 0.5% (w/v) human serum albumin (Behring, Westwood, MA, USA) was added. In addition to basal measurements, also 20 ml of PMA; 0.1 mg/ml was added as a stimulus. Chemiluminescence output was monitored every 60 s for 2 h and the peak value (representing the maximum rate of radical production) and integral over this period (as a measure for overall radical production) were expressed as relative light units (RLU). To also monitor the effect of oxygen radical production in the early phase of the burst, radical production rates were measured immediately after induction with luminol and stimulation with a membrane permeable stimulus (PMA), in the presence or absence of serum (10% v/v). Data were analysed with Winglow (EG & G Berthold) software. The activation of neutrophils from a healthy female blood donor in response to serum of patients and controls was measured as follows: to 100 ml of neutrophil suspension (2 106 cells/ml) 100 ml of serum (10% v/v) was added, immediately followed by 20 ml of a 1:20 dilution of luminol stock in HBSS medium.
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2.2.3. Superoxide production Superoxide production was determined at 378C as the superoxide dismutase inhibitable reduction of ferricytochrome C on a thermostated spectrophotometer (PerkinElmer, Norwalk, CT, USA), as described previously [14]. Measurements were expressed as the maximum rate of cytochrome C reduction in nmol/min per 106 neutrophils at 550 nm, using 21.1 mmol/l cm as molar extinction coef®cient of cytochrome C [13]. 2.3. Statistical analysis Variations between groups were indicated using Kruskal± Wallis one-way ANOVA and Wilcoxon's signed rank test, when appropriate. A probability level of P < 0.05 (twotailed) was considered to represent statistical signi®cance. All statistical analyses were performed using the Astute Statistical Add In for Microsoft Excel (1993 UDD Software, The University of Leeds, UK). 3. Results Maternal age, gestational age, and parity at sampling did not differ signi®cantly between the women with pre-eclampsia and HELLP syndrome and the healthy pregnant women.
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The diastolic blood pressure was signi®cantly higher in the pre-eclamptic women than in the healthy pregnant women (P < 0:01). Table 1 presents these data for the three study groups. In women with pre-eclampsia complicated by HELLP syndrome the oxygen radical production of unstimulated neutrophils was lower than in healthy pregnant women (both P < 0:05 for maximum rate of radical production and overall radical production) as well as in non-pregnant controls (both P < 0:01 for maximum rate of radical production and overall radical production). The response of unstimulated pregnancy neutrophils was signi®cantly reduced over nonpregnant controls (P < 0:05 for maximum rate and P < 0:01 for overall radical production). We did not ®nd any differences between pre-eclamptic women who were treated with (n 7) or without (n 2) magnesiumsulfate. Neutrophils stimulated with PMA showed no differences in oxygen radical production between non-pregnant, healthy pregnant and pre-eclamptic women. The results are summarised in Table 2. In addition, superoxide production, as measured with a cytochrome C reduction assay, was signi®cantly lower in pre-eclamptic women compared with non-pregnant (P < 0:05), but not with healthy pregnant women (P 0:21). Superoxide production showed a tendency to be lower in healthy pregnant women compared with non-pregnant women, but this difference did not reach
Table 1 Case characteristicsa
Maternal age (year) Diastolic blood pressure (mm Hg) Gestational age (week days) Nulliparous (%) Hemoglobin (g/dl) Hematocrit (l/l) Platelet count (109/l) Serum AST (IU/l) Serum ALT (IU/l) Serum LDH (IU/l) Urinary protein (g/l) a b
PE with HELLP (n 9)
Pregnancy (n 9)
Non-pregnant state (n 10)
29 (28±32) 110 (110±130)b 301 (292 to 321) 8 (89%) 13.0 (10.1±14.0) 0.35 (0.28±0.40) 53 (33±90) 128 (79±1230) 121 (72±985) 825 (760±1380) 7.2 (3.2±23.5)
31 (29±34) 75 (65±80) 311 (295 to 324) 7 (78%) 11.9 (11.2±12.9) 0.34 (0.32±0.46) ND ND ND ND 0.3
30 (27±35) 80 (70±85) ± ± 11.4 (11.0±13.2) 0.36 (0.31±0.50) ND ND ND ND ND
Values are given as median (interquartile range), or percentage; PE: pre-eclampsia; ND: not determined. Kruskal±Wallis one-way ANOVA, pre-eclamptic women vs. healthy pregnant and non-pregnant women P < 0.01.
Table 2 Neutrophil oxygen radical productiona Unstimulated
Non-pregnant (n 10) Pregnant (n 9) PE with HELLP (n 9) a
PMA-stimulated
Overall production (RLU 103)
Maximum rate (RLU)
Overall production (RLU 106)
Maximum rate (RLU 103)
198 (162±237) 156 (79±199)b 68 (50±87)c,d
49 (39±67) 33 (21±47)b 17 (9±30)c,d
138 (122±198) 139 (125±197) 146 (127±161)
99 (81±130) 104 (85±123) 103 (87±126)
Values are given as median (interquartile range); RLU: relative light units; PE: pre-eclampsia. Wilcoxon's signed rank test, normal pregnant women compared to non-pregnant women. P < 0.05. c Wilcoxon's signed rank test, pre-eclamptic women compared to normal pregnant women. P < 0.05. d Wilcoxon's signed rank test, pre-eclamptic women compared to non-pregnant women. P < 0.001. b
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Table 3 Ratios of neutrophil oxygen radical production in response to addition of serum (10% v/v)a Unstimulated
Non-pregnant (n 10) Pregnant (n 9) PE with HELLP (n 9)
PMA-stimulated
Overall production (RLU 103)
Maximum rate (RLU)
Overall production (RLU 106)
Maximum rate (RLU 103)
297 (277±436) 301 (259±371) 540 (416±667)
122 (97±168) 126 (122±165) 236 (154±278)b,c
524 (396±574) 535 (493±625) 530 (466±581)
290 (246±339) 347 (287±394) 339 (299±348)
a
Values are given as median (interquartile range); RLU: relative light units; PE: pre-eclampsia. Wilcoxon's signed rank test, pre-eclamptic women compared to healthy pregnant women. P < 0.05. c Wilcoxon's signed rank test, pre-eclamptic women compared to non-pregnant women. P < 0.01. b
statistical signi®cance. In non-pregnant, pregnant, and preeclamptic women, the median values and interquartile ranges for the production of superoxide by PMA-stimulated neutrophils were 9.2 (7.3±9.6) nmol/min per 106 cells, 7.2 (6.0±7.5) nmol/min per 106 cells, and 5.8 (5.2±6.2) nmol/ min per 106 cells, respectively. Serum from pre-eclamptic women, when added to neutrophils isolated from blood of a healthy female blood donor did increase radical production more than serum from either healthy pregnant or non-pregnant women. In this test system, serum from pre-eclamptic women showed a higher maximum rate of oxygen radical production compared to serum from healthy pregnant (P < 0:05) or non-pregnant (P < 0:01) women. Again, no differences between groups were found when the neutrophils were stimulated by PMA. The results of these experiments are listed in Table 3. 4. Discussion A number of studies indicate that inappropriate activation of neutrophils may be implied in endothelial damage in preeclampsia [1,3]. Activated neutrophils release granule constituents and oxygen free radicals. Oxygen radicals can initiate peroxidation and may lead to lysis and other damage of endothelial cells. In the present study we measured the production of oxygen radicals of unstimulated (basal) and PMA stimulated neutrophils using luminol-ampli®ed chemiluminescence and a superoxide dismutase inhibitable reduction of cytochrome C. We also evaluated the effect of serum from pre-eclamptic, normal pregnant and nonpregnant women on isolated healthy neutrophils on their capacity to generate oxygen radicals. Our data on basal neutrophil responses suggest that oxygen radical production is diminished in the normal pregnant group, and to a much higher extent in the pre-eclamptic group, whereas stimulated neutrophil oxygen radical production (respiratory burst) remains unaltered. However, the response to the external stimulus PMA was not different between neutrophils from patients and controls. Because PMA is receptor-independent and directly activates protein kinase [10], this indicates that another pathway may be involved, e.g. a receptor-mediated process. Although, consistent with
several studies on direct measurement of superoxide production in normal pregnancy [15±18], these results do con¯ict with other studies on oxygen radical production in both normal pregnancy and pre-eclampsia. Crouch et al. using lucigenin-enhanced chemiluminescence with formylmethionyl-leucyl-phenylalanine (FMLP) or zymosan as a stimulus showed a signi®cant lower granulocyte and monocyte oxygen radical production in healthy pregnant women at 30±34 weeks compared to non-pregnant controls, but revealed no differences between groups without an external stimulus [15]. Selvaraj et al. however, using polystyreneinduced luminol-enhanced chemiluminescence showed a trend for higher neutrophil oxygen radical production in the third trimester of normal pregnancy as compared to nonpregnant controls [18]. Studying both stimulus-induced luminol-enhanced chemiluminescence and measurement of NADPH-oxidase activity, Miller and Russell showed that chemiluminescence was increased whereas superoxide anion production was reduced in pregnancy [19]. Tsukimori et al. reported that pre-eclampsia was characterised by enhanced superoxide anion production of neutrophils stimulated with FMLP, a receptor-mediated agonist [20]. However, these results could not be reproduced by another study from the same research group [21]. Furthermore, they did not ®nd any differences in superoxide production of neutrophils when PMA, an agonist that bypasses the receptors and directly activates protein kinase, was used as a stimulus [20]. Another study on oxygen radical production in pre-eclampsia by Sacks et al., showed a trend of increasing oxygen radical production in both unstimulated and FMLP stimulated peripheral granulocytes and monocytes in both normal pregnancy and pre-eclampsia during the course of pregnancy, but they could not demonstrate a signi®cant difference between these groups [22]. Recently, Crocker et al. measured lower oxygen radical production in neutrophils from healthy pregnant women as compared to nonpregnant women in response to FMLP and serum-activated zymosan using lucigen-induced chemiluminescence, but measurements in pre-eclampsia did not show a similar reduction as in normal pregnancy [17]. These apparently con¯icting results may be caused by differences in the severity of the disease. Our pre-eclamptic cases were all severely ill and the disease was complicated by HELLP
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syndrome, whereas in other studies also mild and moderate cases of pre-eclampsia were included [20,22]. Differences in the methodologies used might also contribute to con¯icting results. Some reports discuss methodological problems related to measuring neutrophil activation with luminol-ampli®ed chemiluminescence, because luminol may inhibit the release of superoxide under certain circumstance [23]. Furthermore, luminol may also detect the intracellular oxidase activity, since it is able to transverse biological membranes [22]. Therefore, we cannot rule out that differences in cellular myeloperoxidase could have contributed to our results. It is also important to realise that the neutrophil isolation techniques may lead to neutrophil activation, thereby causing artefacts [24]. Kuijpers et al. showed that beside the density gradient neither the incubation, the washing steps, nor the lysis step during the puri®cation procedure altered antigen expression signi®cantly. The density gradient contributed only to the upregulation from molecules already expressed on directly ®xed neutrophils [12]. Since all samples were treated in exactly similar way, above-mentioned effects cannot explain the differences found between pre-eclamptic women and controls. Also variability in treatment with magnesiumsulfate does not seem to in¯uence neutrophil oxygen radical production. We also demonstrated that neutrophils from a healthy female blood donor without an external stimulus showed more activation in response to serum of pre-eclamptic women than to serum of healthy pregnant or non-pregnant women. Because pre-eclampsia and HELLP syndrome are associated with enhanced complement activation [25], we performed this experiments by using serum with and without complement inactivation (serum heated at 568C for 30 min), but no signi®cant differences in neutrophil activation were found. This suggests that the complement system may not be responsible for the differences found. The serum-effect could be explained, at least in part, by assuming that humoral factors in the serum stimulated the neutrophils. We therefore hypothesise that neutrophils from patients with pre-eclampsia are activated in an early phase of pre-eclampsia, possibly due to a circulating factor in the serum, and become exhausted at a progressed stage of the disease. A serumenhancing effect on neutrophil activation in pre-eclampsia was shown previously by Tsukimori et al. [20]. In addition, Clark et al. demonstrated an increase in adhesion of preeclampsia-derived neutrophils to the endothelium and an increase in neutrophil locomotory responses when such cells are incubated with serum from women with pre-eclampsia [26,27]. Early supplementation with antioxidants, such as Vitamins C and E, which were proven to be bene®cial in the prevention of pre-eclampsia in women at increased risk for this disease [28], or N-acetylcysteine, an antioxidant and precursor of the endogenous antioxidant glutathione [29], may be effective in decreasing oxidative stress by stabilizing or even recover the process of endothelial damage.
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In conclusion, our study indicates a possible role for neutrophils in the pathofysiology of pre-eclampsia. We hypothesise that the decreased oxygen radical production of neutrophils from women with severe pre-eclampsia complicated by HELLP syndrome compared with those of normal pregnant and non-pregnant controls, might be due to overstimulation by a factor present in the serum of these patients. Acknowledgements We thank Mrs. Annie van Schaik and Mrs. Hennie Roelofs from the Department of Gastroenterology for their technical assistance.
References [1] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Pre-eclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200±4. [2] Harlan JM. Neutrophil-mediated vascular injury. Acta Med Scand 1987;715(Suppl 1):123±9. [3] Haller, H, Ziegler EM, Homuth V, et al. Endothelial adhesion molecules and leukocyte integrins in pre-eclamptic patients. Hypertension 1997;29:291±6. [4] Lyall F, Greer IA, Boswell F, Macara LM, Walker JJ, Kingdom JC. 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;101:485±7. [5] Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA. Neutrophil activation in pregnancy-induced hypertension. Br J Obstet Gynaecol 1989;96:978±82. [6] Crocker IP, Wellings RP, Hayman RG, Fletcher J, Baker PN. The role of the endogenous anti-inflammatory compound gravidin in preeclampsia. Am J Obstet Gynecol 1998;179:1305±11. [7] Chen, CK, Lee CN, Shyu MK, et al. Fibronectin levels in normal pregnancy and pre-eclampsia. J Formos Med Assoc 1994;93:921±4. [8] Borregaard N. The human neutrophil. Function and dysfunction. Eur J Haematol 1988;41:401±13. [9] Weinstein L. Pre-eclampsia/eclampsia with hemolysis, elevated liver enzymes, and thrombocytopenia. Obstet Gynecol 1985;66:657± 60. [10] Rossi F. The O2 forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 1986;853:65±89. [11] Sibai BM. The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets): much ado about nothing? Am J Obstet Gynecol 1990;162:311±3116. [12] Kuijpers TW, Tool AT, van der Schoot CE, et al. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood 1991;78:1105±11. [13] Wanten GJA, Naber AH, Kruimel JW, Tool AT, Roos D, Jansen JBPJ. Influence of structurally different lipid emulsions on human neutrophil oxygen radical production. Eur J Clin Invest 1999;29:357±63. [14] Kessels GC, Krause KH, Verhoeven AJ. Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C. Biochem J 1993;292:781±5.
218
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[15] Crouch SP, Crocker IP, Fletcher J. The effect of pregnancy on polymorphonuclear leukocyte function. J Immunol 1995;155:5436± 43. [16] Cotton DJ, Seligmann B, O'Brien WF, Gallin JI. Selective defect in human neutrophil superoxide anion generation elicited by the chemoattractant N-formylmethionylleucyl-phenylalanine in pregnancy. J Infect Dis 1983;148:194±9. [17] Crocker IP, Wellings RP, Fletcher J, Baker PN. Neutrophil function in women with pre-eclampsia. Br J Obstet Gynaecol 1999;106:822±8. [18] Selvaraj RJ, Sbarra AJ, Thomas GB, Cetrulo CL, Mitchell GW. A microtechnique for studying chemiluminescence response of phagocytes using whole blood and its application to the evaluation of phagocytes in pregnancy. J Reticuloendothel Soc 1982;31:3±16. [19] Miller C, Russell AS. Polymorphonuclear leukocyte response to stimulation in vitro during pregnancy. Inflammation 1986;10:215±22. [20] Tsukimori K, Maeda H, Ishida K, Nagata H, Koyanagi T, Nakano H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. Obstet Gynecol 1993;81:536±40. [21] Amagase H, Ishida K, Koyanagi T, Tsukimori K, Nakano H. Neutrophil degranulation activity in pre-eclampsia. Hypertens Pregnancy 1997;16:203±12. [22] Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and pre-eclampsia both produce inflammatory changes in peripheral
[23] [24] [25]
[26] [27] [28] [29]
blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998;179:80±6. Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3±14. Sacks GP, Studena K, Sargent K, Redman CW. CD11b expression on circulating neutrophils in pre-eclampsia. Clinical Science 1997;93:187±8. Haeger M, Unander M, Norder Hansson B, Tylman M, Bentsson A. Complement neutrophil, and macrophage activation in women with severe pre-eclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets. Obstet Gynecol 1992;79:19±26. Clark CJ, Chettibi S, Young JD, Greer IA, Lyall F. Locomotion of human neutrophils in response to plasma and serum of women with pre-eclampsia. Hypertens Pregnancy 1996;15:229±40. Clark CJ, Boswell F, Greer IA, Lyall F. Treatment of endothelial cells with serum from women with pre-eclampsia: effect on neutrophil adhesion. J Soc Gynecol Investig 1997;4:27±33. Chappell LC, Seed PT, Briley AL, et al. Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial. Lancet 1999;354:810±6. Roes EM, Raijmakers MTM, Zusterzeel PLM, Knapen MFCM, Peters WHM, Steegers EAP. Deficient detoxifying capacity in the pathophysiology of pre-eclampsia. Med Hypotheses 2000;55:415±8.
Neutrophil oxygen radical production in pre-eclampsia with HELLP syndrome Petra L.M. Zusterzeela, Geert J.A. Wantenb, Wilbert H.M. Petersb, Hans M.W.M. Merkusa, Eric A.P. Steegersa,* a
Departments of Obstetrics and Gynaecology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b Departments of Gastroenterology, University Medical Centre Nijmegen, Nijmegen, The Netherlands Received 22 November 2000; received in revised form 5 March 2001; accepted 15 March 2001
Abstract Objective: To determine whether severe pre-eclampsia complicated by hemolysis, elevated liver enzymes, low platelets (HELLP) -syndrome alters neutrophil oxygen radical production. Materials and Methods: Neutrophils were obtained from 10 healthy non-pregnant, 9 normal pregnant and 9 women with severe preeclampsia with concurrently HELLP syndrome. Oxygen radical production was evaluated using luminol-enhanced chemiluminescence and measured by cytochrome C reduction. Furthermore we incubated sera from cases and controls with isolated healthy neutrophils and measured their capacity to generate oxygen radicals. Results: Unstimulated neutrophil oxygen radical production was signi®cantly lower in severe pre-eclamptics compared with healthy nonpregnant and pregnant subjects, whereas phorbol ester-induced oxygen radical production did not differ among categories. Cytochrome C reduction of unstimulated neutrophils showed similar results. Healthy neutrophils incubated with sera from pre-eclamptics enhanced the oxygen radical production signi®cantly more than neutrophils incubated with sera from the healthy subjects. Conclusions: Severe pre-eclampsia is characterised by decreased unstimulated neutrophil oxygen radical production. This may be the result of an exhausted cellular response due to stimulation by a factor present in the serum of these patients. # 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Chemiluminescence; Neutrophil; Oxygen radical; Pre-eclampsia; HELLP
1. Introduction Although pre-eclampsia complicates up to 5% of pregnancies and is a major cause of both maternal and perinatal morbidity and mortality, the aetiology of this condition remains unclear. Endothelial damage and dysfunction may play an important role in the pathofysiology of preeclampsia [1]. Neutrophil activation can result in vascular damage by their ability to produce reactive oxygen species [2]. Activated neutrophils attach to the endothelium and subsequently release contents of their granules, such as elastase and other proteases, which are capable of destroying the integrity of endothelial cells. The oxygen radicals generated by activated neutrophils can result in membrane lipid peroxidation, and subsequent increased vascular permeability and reactivity [2]. It is unclear what triggers the endothelial disturbance in pre-eclampsia, but neutrophil activity * Corresponding author. Tel.: 31-24-3619449; fax: 31-24-3619036. E-mail address: [email protected] (E.A.P. Steegers).
may play a role in endothelial damage in this disorder. Neutrophil adhesion to the vascular endothelium is dependent on the association between adhesion molecules present on the surface of endothelial cells. Increased expression of such adhesion molecules was shown in pre-eclampsia [3,4] in addition to increased plasma levels of granular components, such as elastase, lactoferrin, endothelin-1, and gravidin [5±7]. Most studies, however, used indirect techniques to show neutrophil activation. Neutrophils produce oxygen radicals in response to stimulation, and direct measurement of oxygen radical production is a good marker for neutrophil activation [8]. To study the role of neutrophils in the pathogenesis of preeclampsia and hemolysis, elevated liver enzymes, low platelets (HELLP) -syndrome [9], we measured the oxygen radical production from neutrophils of non-pregnant and normotensive pregnant women and women with severe preeclampsia complicated by HELLP syndrome, using both luminol-enhanced chemiluminescence with and without an external phorbol ester phorbol 12-myristate 13-acetate
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(PMA) stimulus [10]. Furthermore we measured superoxide anion production with a cytochrome C reduction assay. To determine whether there is a humoral factor in serum from women with pre-eclampsia, which alters neutrophil oxygen radical production, we also studied the activation of neutrophils from a healthy female blood donor in response to serum (10% v/v) of all subjects. 2. Material and methods 2.1. Subjects The subjects were Dutch women cared for at the University Medical Centre Nijmegen, The Netherlands, from September 1998 to February 1999. Nine primigravidas with severe pre-eclampsia complicated by HELLP syndrome constituted the study population. Severe pre-eclampsia was de®ned as persistent diastolic blood pressure of greater than 110 mm Hg (Korotkoff V) on two or more consecutive occasions in a previously normotensive woman after 20 weeks of pregnancy, with proteinuria of greater than 3 g per 24 h. HELLP syndrome was de®ned as the simultaneous occurrence of a platelet count <10 109 /l, serum lactate dehydrogenase (LDH) concentrations greater than 600 IU/l, and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations greater than 70 IU/l [11]. All pre-eclamptic women were severely ill and admitted to the intensive care unit. Six of the pre-eclamptic patients were treated with methyldopa (Aldomet#; Merck Sharp & Dohme BV, Haarlem, The Netherlands) and intravenously administered magnesiumsulfate, one with ketanserin hydrogentartrate (Ketensin#; Pharmacia & Upjohn, Woerden, The Netherlands) and intravenously administered magnesiumsulfate, and two were treated with methyldopa only. Nine normotensive healthy primigravidas, randomly recruited from women visiting the outpatient clinic, comprised a control group. All controls were normotensive without proteinuria. Subjects were matched for maternal and gestational age. In addition, blood was obtained from 10 healthy non-pregnant subjects of comparable age with an uncomplicated obstetrical history. Subjects with a history of renal or vascular disease, diabetes, any autoimmune disorder, or taking any kind of medication other than vitamin- or iron supplements were excluded from the study. Informed consent was obtained from all cases and controls before inclusion, and the local ethical committee approved the study. Blood samples were collected at a progressed stage of the disease for the patients with pre-eclampsia, and during a routine prenatal visit for the pregnant controls. Both patients and pregnant controls were not in labour at the time of blood sampling. 2.2. Procedures and assays Blood samples, anticoagulated with lithium-heparin (for cell isolation) or without anticoagulants (for serum
preparation) were collected from each subject. Chemiluminescence measurements in freshly diluted heparinised blood were always simultaneously performed in matched pairs of pre-eclamptic, normotensive, and non-pregnant subjects. Blood collected in corvac vacutainer tubes was centrifuged at 3000 g for 10 min and serum was stored until assessment at 208C. Upon use, the serum was diluted in phosphate buffered saline (PBS) and added to the neutrophils at a ®nal concentration of 10% (v/v). Chemicals were from Sigma (St. Louis, MO, USA), unless stated otherwise. 2.2.1. Isolation of neutrophils Isolation of neutrophils was performed as described previously [12,13]. Blood diluted 1:1 with PBS containing 0.4% (w/v) trisodium citrate (pH 7.4), was placed on Percoll (speci®c density 1.076 g/ml; Pharmacia Biotech, Uppsala, Sweden) and centrifuged (700 g for 20 min at 258C). The pellet was suspended in 50 ml of ice-cold isotonic lysis solution (containing 155 mmol/l NH4Cl, 10 mmol/l KHCO3, and 0.1 mmol/l EDTA pH 7.4) for 10 min. After centrifugation (400 g for 5 min at 48C) the remaining erythrocytes were lysed on ice in fresh lysis solution for another 5 min. Cells were then washed and resuspended to a ®nal concentration of 2 106 cells/ml and kept at room temperature. Cytospin preparations showed more than 97% granulocytes and viability greater than 99%. 2.2.2. Chemiluminescence Luminol-enhanced chemiluminescence was measured at 378C for 120 min in 96-well microplates with an automated LB96V Microlumat Plus luminometer (EG & G Berthold, Bad Wildberg, Germany), as described previously [13]. To each well containing 200 ml of neutrophil suspension (2 106 cell/ml), 20 ml of a 1:20 dilution of luminol stock in hanks' balanced salt solution (HBSS) (Life Technology, Breda, The Netherlands) supplemented with 0.5% (w/v) human serum albumin (Behring, Westwood, MA, USA) was added. In addition to basal measurements, also 20 ml of PMA; 0.1 mg/ml was added as a stimulus. Chemiluminescence output was monitored every 60 s for 2 h and the peak value (representing the maximum rate of radical production) and integral over this period (as a measure for overall radical production) were expressed as relative light units (RLU). To also monitor the effect of oxygen radical production in the early phase of the burst, radical production rates were measured immediately after induction with luminol and stimulation with a membrane permeable stimulus (PMA), in the presence or absence of serum (10% v/v). Data were analysed with Winglow (EG & G Berthold) software. The activation of neutrophils from a healthy female blood donor in response to serum of patients and controls was measured as follows: to 100 ml of neutrophil suspension (2 106 cells/ml) 100 ml of serum (10% v/v) was added, immediately followed by 20 ml of a 1:20 dilution of luminol stock in HBSS medium.
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2.2.3. Superoxide production Superoxide production was determined at 378C as the superoxide dismutase inhibitable reduction of ferricytochrome C on a thermostated spectrophotometer (PerkinElmer, Norwalk, CT, USA), as described previously [14]. Measurements were expressed as the maximum rate of cytochrome C reduction in nmol/min per 106 neutrophils at 550 nm, using 21.1 mmol/l cm as molar extinction coef®cient of cytochrome C [13]. 2.3. Statistical analysis Variations between groups were indicated using Kruskal± Wallis one-way ANOVA and Wilcoxon's signed rank test, when appropriate. A probability level of P < 0.05 (twotailed) was considered to represent statistical signi®cance. All statistical analyses were performed using the Astute Statistical Add In for Microsoft Excel (1993 UDD Software, The University of Leeds, UK). 3. Results Maternal age, gestational age, and parity at sampling did not differ signi®cantly between the women with pre-eclampsia and HELLP syndrome and the healthy pregnant women.
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The diastolic blood pressure was signi®cantly higher in the pre-eclamptic women than in the healthy pregnant women (P < 0:01). Table 1 presents these data for the three study groups. In women with pre-eclampsia complicated by HELLP syndrome the oxygen radical production of unstimulated neutrophils was lower than in healthy pregnant women (both P < 0:05 for maximum rate of radical production and overall radical production) as well as in non-pregnant controls (both P < 0:01 for maximum rate of radical production and overall radical production). The response of unstimulated pregnancy neutrophils was signi®cantly reduced over nonpregnant controls (P < 0:05 for maximum rate and P < 0:01 for overall radical production). We did not ®nd any differences between pre-eclamptic women who were treated with (n 7) or without (n 2) magnesiumsulfate. Neutrophils stimulated with PMA showed no differences in oxygen radical production between non-pregnant, healthy pregnant and pre-eclamptic women. The results are summarised in Table 2. In addition, superoxide production, as measured with a cytochrome C reduction assay, was signi®cantly lower in pre-eclamptic women compared with non-pregnant (P < 0:05), but not with healthy pregnant women (P 0:21). Superoxide production showed a tendency to be lower in healthy pregnant women compared with non-pregnant women, but this difference did not reach
Table 1 Case characteristicsa
Maternal age (year) Diastolic blood pressure (mm Hg) Gestational age (week days) Nulliparous (%) Hemoglobin (g/dl) Hematocrit (l/l) Platelet count (109/l) Serum AST (IU/l) Serum ALT (IU/l) Serum LDH (IU/l) Urinary protein (g/l) a b
PE with HELLP (n 9)
Pregnancy (n 9)
Non-pregnant state (n 10)
29 (28±32) 110 (110±130)b 301 (292 to 321) 8 (89%) 13.0 (10.1±14.0) 0.35 (0.28±0.40) 53 (33±90) 128 (79±1230) 121 (72±985) 825 (760±1380) 7.2 (3.2±23.5)
31 (29±34) 75 (65±80) 311 (295 to 324) 7 (78%) 11.9 (11.2±12.9) 0.34 (0.32±0.46) ND ND ND ND 0.3
30 (27±35) 80 (70±85) ± ± 11.4 (11.0±13.2) 0.36 (0.31±0.50) ND ND ND ND ND
Values are given as median (interquartile range), or percentage; PE: pre-eclampsia; ND: not determined. Kruskal±Wallis one-way ANOVA, pre-eclamptic women vs. healthy pregnant and non-pregnant women P < 0.01.
Table 2 Neutrophil oxygen radical productiona Unstimulated
Non-pregnant (n 10) Pregnant (n 9) PE with HELLP (n 9) a
PMA-stimulated
Overall production (RLU 103)
Maximum rate (RLU)
Overall production (RLU 106)
Maximum rate (RLU 103)
198 (162±237) 156 (79±199)b 68 (50±87)c,d
49 (39±67) 33 (21±47)b 17 (9±30)c,d
138 (122±198) 139 (125±197) 146 (127±161)
99 (81±130) 104 (85±123) 103 (87±126)
Values are given as median (interquartile range); RLU: relative light units; PE: pre-eclampsia. Wilcoxon's signed rank test, normal pregnant women compared to non-pregnant women. P < 0.05. c Wilcoxon's signed rank test, pre-eclamptic women compared to normal pregnant women. P < 0.05. d Wilcoxon's signed rank test, pre-eclamptic women compared to non-pregnant women. P < 0.001. b
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Table 3 Ratios of neutrophil oxygen radical production in response to addition of serum (10% v/v)a Unstimulated
Non-pregnant (n 10) Pregnant (n 9) PE with HELLP (n 9)
PMA-stimulated
Overall production (RLU 103)
Maximum rate (RLU)
Overall production (RLU 106)
Maximum rate (RLU 103)
297 (277±436) 301 (259±371) 540 (416±667)
122 (97±168) 126 (122±165) 236 (154±278)b,c
524 (396±574) 535 (493±625) 530 (466±581)
290 (246±339) 347 (287±394) 339 (299±348)
a
Values are given as median (interquartile range); RLU: relative light units; PE: pre-eclampsia. Wilcoxon's signed rank test, pre-eclamptic women compared to healthy pregnant women. P < 0.05. c Wilcoxon's signed rank test, pre-eclamptic women compared to non-pregnant women. P < 0.01. b
statistical signi®cance. In non-pregnant, pregnant, and preeclamptic women, the median values and interquartile ranges for the production of superoxide by PMA-stimulated neutrophils were 9.2 (7.3±9.6) nmol/min per 106 cells, 7.2 (6.0±7.5) nmol/min per 106 cells, and 5.8 (5.2±6.2) nmol/ min per 106 cells, respectively. Serum from pre-eclamptic women, when added to neutrophils isolated from blood of a healthy female blood donor did increase radical production more than serum from either healthy pregnant or non-pregnant women. In this test system, serum from pre-eclamptic women showed a higher maximum rate of oxygen radical production compared to serum from healthy pregnant (P < 0:05) or non-pregnant (P < 0:01) women. Again, no differences between groups were found when the neutrophils were stimulated by PMA. The results of these experiments are listed in Table 3. 4. Discussion A number of studies indicate that inappropriate activation of neutrophils may be implied in endothelial damage in preeclampsia [1,3]. Activated neutrophils release granule constituents and oxygen free radicals. Oxygen radicals can initiate peroxidation and may lead to lysis and other damage of endothelial cells. In the present study we measured the production of oxygen radicals of unstimulated (basal) and PMA stimulated neutrophils using luminol-ampli®ed chemiluminescence and a superoxide dismutase inhibitable reduction of cytochrome C. We also evaluated the effect of serum from pre-eclamptic, normal pregnant and nonpregnant women on isolated healthy neutrophils on their capacity to generate oxygen radicals. Our data on basal neutrophil responses suggest that oxygen radical production is diminished in the normal pregnant group, and to a much higher extent in the pre-eclamptic group, whereas stimulated neutrophil oxygen radical production (respiratory burst) remains unaltered. However, the response to the external stimulus PMA was not different between neutrophils from patients and controls. Because PMA is receptor-independent and directly activates protein kinase [10], this indicates that another pathway may be involved, e.g. a receptor-mediated process. Although, consistent with
several studies on direct measurement of superoxide production in normal pregnancy [15±18], these results do con¯ict with other studies on oxygen radical production in both normal pregnancy and pre-eclampsia. Crouch et al. using lucigenin-enhanced chemiluminescence with formylmethionyl-leucyl-phenylalanine (FMLP) or zymosan as a stimulus showed a signi®cant lower granulocyte and monocyte oxygen radical production in healthy pregnant women at 30±34 weeks compared to non-pregnant controls, but revealed no differences between groups without an external stimulus [15]. Selvaraj et al. however, using polystyreneinduced luminol-enhanced chemiluminescence showed a trend for higher neutrophil oxygen radical production in the third trimester of normal pregnancy as compared to nonpregnant controls [18]. Studying both stimulus-induced luminol-enhanced chemiluminescence and measurement of NADPH-oxidase activity, Miller and Russell showed that chemiluminescence was increased whereas superoxide anion production was reduced in pregnancy [19]. Tsukimori et al. reported that pre-eclampsia was characterised by enhanced superoxide anion production of neutrophils stimulated with FMLP, a receptor-mediated agonist [20]. However, these results could not be reproduced by another study from the same research group [21]. Furthermore, they did not ®nd any differences in superoxide production of neutrophils when PMA, an agonist that bypasses the receptors and directly activates protein kinase, was used as a stimulus [20]. Another study on oxygen radical production in pre-eclampsia by Sacks et al., showed a trend of increasing oxygen radical production in both unstimulated and FMLP stimulated peripheral granulocytes and monocytes in both normal pregnancy and pre-eclampsia during the course of pregnancy, but they could not demonstrate a signi®cant difference between these groups [22]. Recently, Crocker et al. measured lower oxygen radical production in neutrophils from healthy pregnant women as compared to nonpregnant women in response to FMLP and serum-activated zymosan using lucigen-induced chemiluminescence, but measurements in pre-eclampsia did not show a similar reduction as in normal pregnancy [17]. These apparently con¯icting results may be caused by differences in the severity of the disease. Our pre-eclamptic cases were all severely ill and the disease was complicated by HELLP
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syndrome, whereas in other studies also mild and moderate cases of pre-eclampsia were included [20,22]. Differences in the methodologies used might also contribute to con¯icting results. Some reports discuss methodological problems related to measuring neutrophil activation with luminol-ampli®ed chemiluminescence, because luminol may inhibit the release of superoxide under certain circumstance [23]. Furthermore, luminol may also detect the intracellular oxidase activity, since it is able to transverse biological membranes [22]. Therefore, we cannot rule out that differences in cellular myeloperoxidase could have contributed to our results. It is also important to realise that the neutrophil isolation techniques may lead to neutrophil activation, thereby causing artefacts [24]. Kuijpers et al. showed that beside the density gradient neither the incubation, the washing steps, nor the lysis step during the puri®cation procedure altered antigen expression signi®cantly. The density gradient contributed only to the upregulation from molecules already expressed on directly ®xed neutrophils [12]. Since all samples were treated in exactly similar way, above-mentioned effects cannot explain the differences found between pre-eclamptic women and controls. Also variability in treatment with magnesiumsulfate does not seem to in¯uence neutrophil oxygen radical production. We also demonstrated that neutrophils from a healthy female blood donor without an external stimulus showed more activation in response to serum of pre-eclamptic women than to serum of healthy pregnant or non-pregnant women. Because pre-eclampsia and HELLP syndrome are associated with enhanced complement activation [25], we performed this experiments by using serum with and without complement inactivation (serum heated at 568C for 30 min), but no signi®cant differences in neutrophil activation were found. This suggests that the complement system may not be responsible for the differences found. The serum-effect could be explained, at least in part, by assuming that humoral factors in the serum stimulated the neutrophils. We therefore hypothesise that neutrophils from patients with pre-eclampsia are activated in an early phase of pre-eclampsia, possibly due to a circulating factor in the serum, and become exhausted at a progressed stage of the disease. A serumenhancing effect on neutrophil activation in pre-eclampsia was shown previously by Tsukimori et al. [20]. In addition, Clark et al. demonstrated an increase in adhesion of preeclampsia-derived neutrophils to the endothelium and an increase in neutrophil locomotory responses when such cells are incubated with serum from women with pre-eclampsia [26,27]. Early supplementation with antioxidants, such as Vitamins C and E, which were proven to be bene®cial in the prevention of pre-eclampsia in women at increased risk for this disease [28], or N-acetylcysteine, an antioxidant and precursor of the endogenous antioxidant glutathione [29], may be effective in decreasing oxidative stress by stabilizing or even recover the process of endothelial damage.
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In conclusion, our study indicates a possible role for neutrophils in the pathofysiology of pre-eclampsia. We hypothesise that the decreased oxygen radical production of neutrophils from women with severe pre-eclampsia complicated by HELLP syndrome compared with those of normal pregnant and non-pregnant controls, might be due to overstimulation by a factor present in the serum of these patients. Acknowledgements We thank Mrs. Annie van Schaik and Mrs. Hennie Roelofs from the Department of Gastroenterology for their technical assistance.
References [1] Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Pre-eclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200±4. [2] Harlan JM. Neutrophil-mediated vascular injury. Acta Med Scand 1987;715(Suppl 1):123±9. [3] Haller, H, Ziegler EM, Homuth V, et al. Endothelial adhesion molecules and leukocyte integrins in pre-eclamptic patients. Hypertension 1997;29:291±6. [4] Lyall F, Greer IA, Boswell F, Macara LM, Walker JJ, Kingdom JC. 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;101:485±7. [5] Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA. Neutrophil activation in pregnancy-induced hypertension. Br J Obstet Gynaecol 1989;96:978±82. [6] Crocker IP, Wellings RP, Hayman RG, Fletcher J, Baker PN. The role of the endogenous anti-inflammatory compound gravidin in preeclampsia. Am J Obstet Gynecol 1998;179:1305±11. [7] Chen, CK, Lee CN, Shyu MK, et al. Fibronectin levels in normal pregnancy and pre-eclampsia. J Formos Med Assoc 1994;93:921±4. [8] Borregaard N. The human neutrophil. Function and dysfunction. Eur J Haematol 1988;41:401±13. [9] Weinstein L. Pre-eclampsia/eclampsia with hemolysis, elevated liver enzymes, and thrombocytopenia. Obstet Gynecol 1985;66:657± 60. [10] Rossi F. The O2 forming NADPH oxidase of the phagocytes: nature, mechanisms of activation and function. Biochim Biophys Acta 1986;853:65±89. [11] Sibai BM. The HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets): much ado about nothing? Am J Obstet Gynecol 1990;162:311±3116. [12] Kuijpers TW, Tool AT, van der Schoot CE, et al. Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation. Blood 1991;78:1105±11. [13] Wanten GJA, Naber AH, Kruimel JW, Tool AT, Roos D, Jansen JBPJ. Influence of structurally different lipid emulsions on human neutrophil oxygen radical production. Eur J Clin Invest 1999;29:357±63. [14] Kessels GC, Krause KH, Verhoeven AJ. Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C. Biochem J 1993;292:781±5.
218
P.L.M. Zusterzeel et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 99 (2001) 213±218
[15] Crouch SP, Crocker IP, Fletcher J. The effect of pregnancy on polymorphonuclear leukocyte function. J Immunol 1995;155:5436± 43. [16] Cotton DJ, Seligmann B, O'Brien WF, Gallin JI. Selective defect in human neutrophil superoxide anion generation elicited by the chemoattractant N-formylmethionylleucyl-phenylalanine in pregnancy. J Infect Dis 1983;148:194±9. [17] Crocker IP, Wellings RP, Fletcher J, Baker PN. Neutrophil function in women with pre-eclampsia. Br J Obstet Gynaecol 1999;106:822±8. [18] Selvaraj RJ, Sbarra AJ, Thomas GB, Cetrulo CL, Mitchell GW. A microtechnique for studying chemiluminescence response of phagocytes using whole blood and its application to the evaluation of phagocytes in pregnancy. J Reticuloendothel Soc 1982;31:3±16. [19] Miller C, Russell AS. Polymorphonuclear leukocyte response to stimulation in vitro during pregnancy. Inflammation 1986;10:215±22. [20] Tsukimori K, Maeda H, Ishida K, Nagata H, Koyanagi T, Nakano H. The superoxide generation of neutrophils in normal and preeclamptic pregnancies. Obstet Gynecol 1993;81:536±40. [21] Amagase H, Ishida K, Koyanagi T, Tsukimori K, Nakano H. Neutrophil degranulation activity in pre-eclampsia. Hypertens Pregnancy 1997;16:203±12. [22] Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and pre-eclampsia both produce inflammatory changes in peripheral
[23] [24] [25]
[26] [27] [28] [29]
blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998;179:80±6. Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3±14. Sacks GP, Studena K, Sargent K, Redman CW. CD11b expression on circulating neutrophils in pre-eclampsia. Clinical Science 1997;93:187±8. Haeger M, Unander M, Norder Hansson B, Tylman M, Bentsson A. Complement neutrophil, and macrophage activation in women with severe pre-eclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets. Obstet Gynecol 1992;79:19±26. Clark CJ, Chettibi S, Young JD, Greer IA, Lyall F. Locomotion of human neutrophils in response to plasma and serum of women with pre-eclampsia. Hypertens Pregnancy 1996;15:229±40. Clark CJ, Boswell F, Greer IA, Lyall F. Treatment of endothelial cells with serum from women with pre-eclampsia: effect on neutrophil adhesion. J Soc Gynecol Investig 1997;4:27±33. Chappell LC, Seed PT, Briley AL, et al. Effect of antioxidants on the occurrence of pre-eclampsia in women at increased risk: a randomised trial. Lancet 1999;354:810±6. Roes EM, Raijmakers MTM, Zusterzeel PLM, Knapen MFCM, Peters WHM, Steegers EAP. Deficient detoxifying capacity in the pathophysiology of pre-eclampsia. Med Hypotheses 2000;55:415±8.