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ORIGINAL RESEARCH |
From the Departments of Pediatric Research and Obstetrics and Gynecology, Institute of Immunology, The National Hospital, University of Oslo, Oslo, Norway; Department of Immunology and Transfusion Medicine, Nordland Central Hospital, Bodø, and University of Tromsø, Tromsø, Norway; Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands; and Department of Immunology and Transfusion Medicine, Institute of Laboratory Medicine, Norwegian University of Science and Technology, Trondheim, Norway.
Address reprint requests to: Jan Roar Mellembakken, MD, Department of Obstetrics and Gynecology, National Hospital, 0027 Oslo, Norway, E-mail: jan.mellembakken{at}rikshospitalet.no
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Abstract |
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Methods: We measured native complement components and activation products in plasma by enzyme immunoassays in 19 women with uncomplicated pregnancies, 15 with preeclampsia before cesarean deliveries, and 16 nonpregnant women. Neutrophil activation was measured by specific enzyme immunoassays for myeloperoxidase and lactoferrin.
Results: Myeloperoxidase was significantly higher in women with preeclampsia (197 µg/L, 95% confidence interval [CI] 94, 646) than in women with uncomplicated pregnancies (124 µg/L, 95% CI 70, 289; P = .009), whereas lactoferrin did not differ between groups. C4 was decreased in preeclamptic women (0.16 g/L, 95% CI 0.07, 0.48) compared with women with uncomplicated pregnancies (0.21, 95% CI 0.10, 0.30, P < .001). There were no differences for the other native complement components. There was a significant decrease in C1rs-C1 inhibitor, 13 AU/mL (95% CI 9, 34) versus 19 (95% CI 13, 38) (P 
.001) in normal pregnant women compared with nonpregnant women. There also was an increase in C3, C4, C9 (data not shown), C4bp, 132% (95% CI 94%, 161%) versus 91% (95% CI 57%, 128%); C3bc (7.4 AU/mL, 95% CI 4.2, 10.7) versus 4.8 AU/mL (95% CI 3.2, 7.3) and C4bc (8.6 AU/mL, 95% CI 5.7, 14.0) versus 3.5 AU/mL (95% CI 2.2, 6.7) in normal pregnant women compared with nonpregnant women (P .001).
Conclusion: Neutrophil activation in preeclampsia was shown by systemic increases in myeloperoxidase. Except for a decrease in C4, systemic complement activation could not be detected in preeclampsia.
Some studies found an association between systemic complement activation and preeclampsia,1,2 whereas others found neutrophil activation.3,4 Ischemia, as manifested in the uteroplacental circulation by increased frequency of placental infarcts,5 might induce inflammatory events that activate neutrophils and the complement system,6 and complement deposits have been detected in placentas from women with preeclamptic pregnancies.7 Some complement activation products are potent neutrophil activators. Activation products from neutrophils and the complement system might cause endothelial dysfunction, which is central to the etiology of preeclampsia.8 The influence of the complement system in preeclampsia has been addressed in several studies, with conflicting results.1,9 Reduced concentrations of C4b-binding protein (C4bp) were found in one study of preeclampsia10 and might have been caused by increased C4 activation because C4bp is consumed when binding C4b, the larger activation fragment of C4. The aim of the present study was to determine whether there was systemic complement and neutrophil activation in preeclampsia.
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Materials and Methods |
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All initial blood samples were collected before cesareans and before any intravenous (IV) fluid had been given. Samples were drawn into sterile vacutainers (Becton Dickinson, Plymouth, UK) using ethylenedia-minetetra-acetic acid (EDTA) as anticoagulant. The tubes were immersed immediately in melting ice, and polybrene was added after sampling to a final concentration of 0.5 g/L to avoid in vitro C4 activation.11 The tubes were centrifuged within 5 minutes of collection (1500g and 4C for 5 minutes), and plasma was stored at -80C until analyzed in one batch. Myeloperoxidase and lactoferrin were measured by specific enzyme immunoassays.12,13
Concentration of C1 inhibitor antigen was measured by single radial immunodiffusion (NOR-Partigen; Behringwerke A/G, Marburg, Germany) according to manufacturer’s instructions. C1 inhibitor function was measured using a chromogenic substrate assay as described.14 Concentrations of C3 and C4 antigen were measured in the laboratory of the Institute of Immunology and Rheumatology, Oslo, Norway, using standard nephelometric technique. Antigen concentration of C4bp was measured using enzyme immunoassays as described.15 Normal human serum was used as reference that defined 100%. All assays for complement activation products were enzyme immunoassays based on neoepitope-specific monoclonal antibodies to activation products. Complement proteins C1r and C1s in complexes with C1 inhibitor (C1rs-C1 inhibitor), a specific measure of activation of the alternative pathway, were measured using the KOK-12 monoclonal antibody that is specific for a neoepitope in C1 inhibitor when it was in complex with protease.16 Human serum activated with heat-aggregated immunoglobulin (Ig)G was used as standard and defined to contain 1000 arbitrary units (AU)/mL. C4bc (ie, the sum of C4 activation fragments C4b, iC4b, and C4c) was measured as described.11 The same standard was used as for the C1rs-C1 inhibitor assay, defined to contain 1000 AU/mL. The C3bc (ie, the sum of C3b, iC3b, and C3c) assay was developed in our laboratory and done mainly as described.17 Standard was normal human serum, which was activated with zymosan and defined to contain 1000 AU/mL. C9 was measured by enzyme immunoassays as described.18 Normal human plasma was used as standard defining 100%. The Statistical Package for the Social Sciences software (SPSS Inc., Chicago, IL) was used for statistical analysis. The Kruskal-Wallis one-way analysis of variance with additional formulas for multiple comparisons was used to compare groups. P < .01 was considered significant.
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Results |
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. Table 2 gives the results of neutrophil and complement activation before cesareans. Myeloperoxidase was significantly higher in women with pre-eclampsia compared with women with uncomplicated pregnancies, whereas the concentration of lactoferrin was similar between those groups.
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Discussion |
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Myeloperoxidase and lactoferrin are released from neutrophils during activation. Myeloperoxidase is essentially neutrophil specific, whereas lactoferrin also can be released from other cells. In our study, myeloperoxidase, but not lactoferrin, was a sensitive marker of neutrophil activation. Previous studies did not find any differences in plasma lactoferrin between women with preeclamptic compared with uncomplicated pregnancies.20,21
Although there was no systemic complement activation in women with severe preeclampsia, the decrease in C1rs-C1 inhibitor and C4 might be from low-grade activation and consumption in the classical pathway, but C4bp prevents the system from further activation. There are conflicting reports on whether the complement system is activated in preeclampsia. Our data might be biased to some degree by the difference of 4 weeks in gestational age between the two pregnancy groups. However, that is unlikely because there is only a 5–10% increase in complement components from the second to third trimesters.22 Our study was small, which increases the risk of drawing false-negative conclusions. Earlier studies of complement factors in pre-eclampsia measured native factors such as C3, C4, factor B, and hemolytic activity (CH50). No differences were found, but those tests were relatively insensitive as measures of activation.9 Armstrong et al23 measured the complement activation product C3d and found no differences between normal and preeclamptic pregnant women, which agrees with our data. However, two studies that showed increased C5 activation included many women with the hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome, a variant of preeclampsia with hemolysis, liver affliction, and platelet consumption.1,2 That syndrome might not have the same pathogenetic pathway as preeclampsia. Complement activation might happen only locally in uteroplacental circulation, as for joints in rheumatoid arthritis. That belief is strengthened by the fact that complement deposits are found in the placenta in preeclampsia.7
There were more native complement factors and activation products in normal pregnant women compared with nonpregnant controls. The increase in native complement factors was probably from increased synthesis as part of the normal acute-phase reaction of pregnancy. With normal or increased turnover of components, the corresponding activation products also could be increased even without disease activation.
The increase in C4 in normal pregnancies was not seen in preeclampsia. The latter group had C4 concentrations comparable to those of nonpregnant women. We cannot exclude the possibility that the relatively low C4 values in preeclampsia compared with normal pregnancies indicates a low-grade classical pathway activation with consumption of C4. Increased complement activation products have been shown for C3c24 and C3d22,24 in uncomplicated pregnancies. For the native factors the results are conflicting, which might be attributable to old methods.
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Footnotes |
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Received July 10, 2000. Received in revised form October 30, 2000. Accepted November 22, 2000.
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References |
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2. Haeger M, Unander M, Norder-Hansson B, Tylman M, Bengtsson A. Complement, neutrophil and macrophage activation in women with severe preeclampsia and the syndrome of hemolysis, elevated liver enzymes and low platelet count. Obstet Gynecol 1992;79:19–26.
3. Greer IA, Haddad NG, Dawes J, Johnstone FD, Calder AA. Neutrophil activation in pregnancy-induced hypertension. Br J Obstet Gynaecol 1989;96:978–82.[Medline]
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5. Brosens I, Renaer M. On the pathogenesis of placental infarcts in preeclampsia. J Obstet Gynaecol Br Commonw 1972;79:794–9.[Medline]
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7. Tedesco F, Radillo O, Candussi G, Nazzaro A, Mollnes TE, Pecorari D. Immunohistochemical detection of terminal complement complex and S protein in normal and pre-eclamptic placentae. Clin Exp Immunol 1990;80:236–40.[Medline]
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14. Nielsen EW, Johansen HT, Straume B, Mollnes TE. Effect of time, temperature and additives on a functional assay of C1 inhibitor. J Immunol Methods 1994;173:245–51.[Medline]
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16. Fure H, Nielsen EW, Hack CE, Mollnes TE. A neoepitope-based enzyme immunoassay for quantification of C1-inhibitor in complex with C1r and C1s. Scand J Immunol 1997;46:553–7.[Medline]
17. Garred P, Mollnes TE, Lea T. Quantification in enzyme-linked immunosorbent assay of a C3 neoepitope expressed on activated human complement factor C3. Scand J Immunol 1988;27:329–35.[Medline]
18. Mollnes TE, Tschopp J. A unique epitope exposed in native complement component C9 and hidden in the terminal SC5b9 complex enables selective detection and quantification of non-activated C9. J Immunol Methods 1987;100:215–21.[Medline]
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20. Crocker IP, Wellings RP, Fletcher J, Baker PN. Neutrophil function in women with pre-eclampsia. Br J Obstet Gynaecol 1999;106: 822–8.[Medline]
21. Prieto JA, Panyutich AV, Heine RP. Neutrophil activation in preeclampsia. Are defensins and lactoferrin elevated in preeclamptic patients? J Reprod Med 1997;42:29–32.[Medline]
22. Schena FP, Manno C, Selvaggi L, Loverro G, Bettocchi S, Bonomo L. Behaviour of immune complexes and the complement system in normal pregnancy and pre-eclampsia. J Clin Lab Immunol 1982;7: 21–6.[Medline]
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24. Teisner B, Hau J, Tucker M, Lahood J, Grudzinkas JG. Circulating C3, C4, and C3 split products (C3c and C3d) during normal pregnancy. Am J Reprod Immunol 1982;2:309–11.
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