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Cytokine mapping of sera from women with preeclampsia and normal pregnancies
Journal of Reproductive Immunology 70 (2006) 83–91
Cytokine mapping of sera from women with preeclampsia and normal pregnancies Yvonne Jonsson a,∗ , Marie Rub`er a , Leif Matthiesen b , G¨oran Berg b , Katri Nieminen b , Surendra Sharma c , Jan Ernerudh a , Christina Ekerfelt a a
Unit of Autoimmunity and Immune Regulation, Department of Molecular and Clinical Medicine, Division of Clinical Immunology, Faculty of Health Sciences, University Hospital, Link¨oping, Sweden b Department of Clinical and Molecular Medicine, Division of Obstetrics and Gynecology, Faculty of Health and Sciences, University Hospital, Link¨oping, Sweden c Departments of Pediatrics and Pathology, Brown University and Women and Infants’ Hospital of Rhode Island, Providence, RI, USA Received 23 June 2005; received in revised form 14 September 2005; accepted 28 October 2005
Abstract Introduction: Preeclampsia is a pregnancy-specific syndrome. The immune system in preeclampsia is changed with an increased innate activity and there is a hypothesis of a shift towards Th1-type immunity. The aim of this study was to determine a spectrum of soluble immunological factors denoting different aspects of immune activation in third trimester sera from women with preeclampsia (N = 15) and compare with levels in sera from normal pregnant women (N = 15). Material and methods: IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-l␣, MIP-1, MCP-1, eotaxin and RANTES were measured in serum using multiplex bead arrays. The levels of soluble CD14 and soluble IL-4 receptor were measured by enzyme-linked immunoassay (ELISA). Results: Preeclamptic women had significantly increased levels of circulating IL-6 (p = 0.002), IL-8 (p = 0.003) and soluble IL-4R (p = 0.037), compared to women with normal pregnancies. Conclusion: This study supports the hypothesis of increased inflammatory responses in preeclampsia, illustrated by the increased levels of IL-6 and IL-8. The finding of increased levels of soluble IL-4 ∗ Corresponding author at: Autoimmunity and Immune Regulation, Pathology Building Level 10, Faculty of Health and Sciences, SE-581 85 Link¨oping, Sweden. Tel.: +46 13 22 73 57. E-mail address: [email protected] (Y. Jonsson).
0165-0378/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2005.10.007
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receptor is an intriguing finding with several interpretations, which may partly support the hypothesis of a Th1 shift in preeclampsia. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cytokines; Serum; Multiple bead array; Preeclampsia
1. Introduction Preeclampsia is a pregnancy-specific syndrome that occurs in the later half of the pregnancy. The symptoms and signs include high blood pressure, proteinuria and general edema (ACOG Committee on Obstetric Practice, 2002) and the syndrome is clinically harmful to both the mother and the fetus. The precise etiology of the disease is still unknown, although the placenta plays a major part, as reviewed by Redman and Sargent (2005) and Robillard (2002). The immune maladaptation hypothesis (Dekker et al., 1998; Dekker and Sibai, 1999) suggests that preeclampsia is due to an inappropriate regulation of the normally Th2 deviated maternal immune responses, leading to a shift towards harmful Th1 immunity (Taylor, 1997; Saito et al., 1999; Darmochwal-Kolarz et al., 2002; Saito and Sakai, 2003). A generalized maternal inflammatory response has been shown to occur during preeclampsia together with increased levels of TNF-␣, IL-6 and IL-8, as reviewed by Redman and Sargent (2003). The significance of such immunological changes in the pathogenesis of preeclampsia is, however, still uncertain, and the immune aberrations per se need to be further elucidated. The nature and amounts of the immunomodulatory milieu present in serum may impart insights on the in vivo regulation of preeclampsia-associated conditions. Thus, mapping of the systemic immune balance by simultaneously measuring cytokines, chemokines and other inflammatory markers in serum from women with preeclampsia and from women with normal pregnancies would be of interest. Multiplex bead array systems allow simultaneous detection of a large number of molecules of interest. The available kits are designed to analyze levels of the classically studied immune markers, as well as newer ones. The purpose of this study was to examine and compare the in vivo network of cytokines, chemokines and other inflammatory markers present in serum from women with preeclamptic and normal pregnancies, utilizing a multiplex bead array system. Both the classically studied markers (interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, interferon (IFN)-␥, tumor necrosis factor (TNF)-␣, granulocyte macrophage colony stimulating factor (GM-CSF) and soluble CD14 (sCD14) as well as markers less defined or novel, in preeclampsia (IL-5, IL-13, IL-15, IL-17, IFN-␣, macrophage inflammatory protein-␣ (MIP-1␣), MIP-1, monocyte chemotactic protein-1 (MCP-1), eotaxin, RANTES (regulated on activation, normal T-cellexpressed and secreted) and soluble IL-4 receptor (sIL-4R)) were measured in serum from women with preeclampsia and compared with normal pregnancies.
2. Material and methods Blood samples were drawn from 15 women with moderate or severe preeclampsia and from 15 women with normal pregnancies (controls). Blood samples were centrifuged
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Table 1 Data on the participating women Diagnosis
Mean age at sampling (range)
Gestational age (completed weeks) at blood sampling (range)
Number of parous women
Normal pregnancy (N = 15) Moderate preeclampsiaa (N = 8) Severe preeclampsiaa (N = 7) All preeclamptic women (N = 15)
30 (25–42) 29 (22–32) 29 (26–32) 29 (22–32)
33 (29–37) 37 (33–40) 32 (29–34) 34 (29–40)
2 1 1 2
a Moderate and severe preeclampsia are defined according to WHO criteria. Moderate preeclampsia = BP > 140/90 and proteinuria less than 5 g/24 h. Severe preeclampsia = BP > 160/110 and/or proteinuria of more than 5 g/24 h and/or subjective symptoms such as visual impairment, headache, abdominal pain.
within 1 h after sampling and sera were then immediately frozen and stored at −70 ◦ C until use. Moderate and severe preeclampsia was defined according to WHO criteria. Moderate preeclampsia: blood pressure >140/90 and proteinuria less than 5 g/24 h; severe preeclampsia: blood pressure >160/110 and/or proteinuria of more than 5 g/24 h and/or subjective symptoms such as visual impairment, headache and abdominal pain. Blood samples from preeclamptic women were taken after diagnosis and admission to hospital, whereas blood samples from healthy women were taken at the Antenatal Care Clinic at the pregnancy week matching a preeclamptic patient ±2 gestational weeks. Four patients received glucocorticoid treatment (2× 7.5 mg within 24 h) prior to the time of blood sample collection. The time between treatment and sampling varied between 1 and 11 days. Data on the participating women are shown in Table 1. 2.1. Multiplex bead array analysis The levels of IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-1␣, MIP-1, MCP-1, eotaxin and RANTES were analyzed in sera according to the manufacturer, except that two extra standard points were added to the standard curve by two additional serial dilutions. The multiplex kits used were LCH0001, LHC0005 and LHC0007 (BioSource, Camarillo, CA, USA), and the amounts of cytokines/chemokines analyzed using the Luminex 100 instrument (Luminex Corporation, Austin, Texas, USA) and the STarStation software (V1.1, Applied Cytometry Systems, Sheffield, UK). Due to insufficient sample volumes only 13 serum samples from women with preeclampsia and 14 serum samples from normal pregnant women were analyzed for IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, eotaxin and RANTES. 2.2. ELISA The levels of soluble CD14 (sCD14) and soluble IL-4 receptor (sIL-4R) in sera were determined by Quantikine ELISA-kits (R&D Systems, Minneapolis, MN, USA). The plates were read at 450 nm with wavelength correction made at 600 nm. Each sCD14 sample was diluted 200 and 400 times in the calibrator diluent RD5P (supplied by manufacturer) and analyzed in a single well for each dilution. Otherwise, analysis was performed according to manufacturer; including the analysis of sIL-4R levels in duplicates in undiluted serum
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samples. Due to insufficient sample volumes, only 13 serum samples from women with preeclampsia and 14 serum samples from normal pregnant women were analyzed for sCD14 and SIL-4R. 2.3. Statistics Data analysis was performed with StatView 5.0.1 for Windows. The Mann–Whitney U-test was used to compare data from women with preeclampsia and normal pregnancies. Since non-parametric statistical tests were used, which compare rankings between samples, and not mean values of the data, cytokine levels below detection limit were given the value of zero. For the group of classically studied immuno-markers during preeclampsia (i.e., IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IFN-␥, TNF-␣, GM-CSF and sCD14), p < 0.0045 was considered to be significant after Bonferroni-correction. For the not so extensively studied or undefined immunomarkers present during preeclampsia (i.e., IL-5, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, Eotaxin, RANTES and sIL-4R), p < 0.05 was considered significant. The latter group has not the same strict significance demands as the first group, since we wanted to extensively map markers in preparation for later studies. 2.4. Ethics The Regional Ethics Committee at the University Hospital of Link¨oping approved this study and informed consent was given from all participants in the study.
3. Results The entire set of data is summarized in Table 2. Women with preeclampsia showed significantly higher serum concentrations of the innate pro-inflammatory cytokine IL-6 (p = 0.002) and elevated levels of the pro-inflammatory chemokine IL-8 (p = 0.003) compared with women with normal pregnancies (Fig. 1a and b). Preeclamptic women also showed higher serum concentrations of the soluble IL-4 receptor (p = 0.037) than women with normal pregnancies (Fig. 1c). For all the other factors tested, i.e., IL-, IL-2, IL-4, IL-5, IL-10, IL-12 p40, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-1␣, MIP-1, MCP-1, RANTES, eotaxin and sCD14, there were no significant differences between women with preeclampsia compared to normal pregnancies. IL-13 was not detectable in any of the samples. When comparing within the group of preeclamptic patients, no significant differences in the levels of immunological factors were found between women with severe or moderate preeclampsia.
4. Discussion Our results show substantially increased levels of IL-6 and IL-8, supporting the hypothesis of increased inflammatory responses in preeclampsia. In addition, we found increased
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Table 2 Serum levels (pg/ml) of different immunological factors from women with preeclampsia and normal pregnancies Factor
Preeclampsia Median
IL-1 IL-2 IL-4 sIL-4R IL-5 IL-6 IL-8 IL-10 IL-12 p40 IL-13 IL-15 IL-17 IFN-␣ IFN-␥ TNF-␣ GM-CSF MIP-1␣ MIP-I MCP-1 RANTES Eotaxin SCD14
0.0 83 0.0 139 0.0 13 26 0.0 27 0.0 0.0 0.0 0.0 0.0 32 0.0 45 145 506 1838 64 1.5 × l06
Normal pregnancy Range 0.0–20 0.0–169 0.0–13 93–327 0.0–6.0 0.0–127 0.0–362 0.0–95 3.4–55 0.0–0.0 0.0–0.0 0.0–0.0 0.0–4.0 0.0–10 0.0–236 0.0–746 33–112 19–694 167–1001 1180–2791 33–157 1.2 × 106 –2.5 × 106
Median
Range
0.0 39 0.0 105 0.0 3.3 0.0 0.0 18 0.0 0.0 0.0 0.0 0.0 9.6 0.0 37 118 415 1928 48 1.5 × 106
0.0–221 0.0–155 0.0–23 82–162 0.0–7.3 0.0–296 0.0–36 0.0–4.5 0.0–52 0.0–0.0 0.0–66 0.0–3.6 0.0–65 0.0–20 0.0–423 0.0–0.0 22–150 22–348 77–1537 983–5669 17–100 1.0 × 106 –2.5 ×106
p-Values
0.984 0.330 0.171 0.037 0.984 0.002 0.003 0.373 0.160 ND 0.752 0.528 0.716 0.724 0.071 0.756 0.254 0.167 0.174 0.662 0.174 0.700
ND = not detectable. p-values refer to comparisons of the two groups by the Mann–Whitney U-test.
levels of sIL-4R in sera from women with preeclampsia compared with normal pregnancies. The finding for IL-6 is in line with previous reports on elevated plasma levels of IL-6 in women with preeclampsia compared with normal pregnancies (Vince et al., 1995; Conrad et al., 1998; Madazli et al., 2003). In contrast, Al-Othman et al. (2001) found no difference in IL-6 levels in serum between preeclampsia and normal pregnancy as analyzed by ELISA. One explanation for this divergence could be that the serum samples in the study of Al-Othman et al., 2001) were frozen and stored at −20 ◦ C instead of −70 ◦ C, which may have affected the result. Although IL-6 is predominantly regarded as a pro-inflammatory cytokine, it shows also a number of other effects due to its pleiotropic nature (Curfs et al., 1997), including inhibition of the IL-6 inducing inflammatory cytokines IL-1 and TNF-␣, stimulation of antibody production from B cells and activation and differentiation of T-cells (Borish and Steinke, 2003). Thus, due to its pleiotropic nature, it is hard to speculate on the clinical effects of elevated IL-6 levels in preeclampsia. The preeclamptic women in our study showed also significantly elevated levels of the chemokine IL-8 compared to women with normal pregnancies (p = 0.003). IL-8 is an inflammatory chemokine locally produced in response to tissue injury, leading mainly to recruitment and activation of neutrophils (Mukaida et al., 1998). Our finding of increased levels of serum IL-8 is in line with others (Velzing-Aarts et al., 2002; Kocyigit et al., 2004), which further support the role of IL-8 in inflammatory responses in preeclampsia.
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Fig. 1. Serum concentrations of the pro-inflammatory cytokine IL-6 (A), the chemokine IL-8 (B) and the soluble IL-4 receptor (IL-4 sR) (C) from women with preeclampsia or normal pregnancies, p-values from comparisons with the Mann–Whitney U-test are shown. Median is indicated.
The finding of increased sIL-4R in women with preeclampsia was somewhat surprising, since sIL-4R has been suggested as a surrogate marker for levels of the cytokine IL-4. This suggestion was based on studies on mice spleen cells, where sIL-4R was shown to reflect IL-4 levels (Chilton and Fernandez-Botran, 1993), and on observations that proteolytic cleavage of sIL-4R was different from IL-4 (Jung et al., 1999), allowing sIL-4R to remain longer in the circulation. Since IL-4 itself is known to be difficult to detect (Bullens et al., 1998; Ekerfelt et al., 2002), as also illustrated in the present study with detectable IL-4 levels only in a few normal pregnancies, the strategy to use sIL-4R as a surrogate marker for IL-4 would be useful since IL-4 is one of the classical Th2 cytokines. The current finding of increased sIL-4R levels in preeclampsia may have three possible explanations; the first is that the hypothesis of decreased IL-4 in preeclampsia could be questioned; secondly, the
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sIL-4R might not reflect IL-4 levels; or, thirdly, there might exist a negative correlation between IL-4 levels and sIL-4 R levels as reported by Protonotariou et al. (2003). Previous studies in human in vitro models (Garrone et al., 1991), as well as an in vivo murine model (Gessner et al., 1994), have shown that sIL-4R can neutralize unbound IL-4, suggesting that sIL-4R plays a role in IL-4 regulation. Speculatively, since sIL-4R is believed to persist longer in the circulation than IL-4 (Jung et al., 1999), the increased level of sIL-4R in preeclampsia may reflect a current decrease of IL-4 secretion, i.e., sIL-4R was shed from Th2 cells upon IL-4 secretion earlier in pregnancy and, after neutralization of all circulating IL-4, only the excess amount of sIL-4R could still be detected. It has, however, to be kept in mind that the finding of increased sIL-4R in preeclampsia must be interpreted with caution since it fulfilled only the lower demand on significance levels used for the screening of novel immuno-markers. Thus, this finding needs to be confirmed in further studies. When considering the findings on classically studied immunological markers during preeclampsia (i.e., IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IFN-␥, TNF-␣, GMCSF and sCD14), it is puzzling that we did not find any other differences than for the levels of IL-6 and IL-8. As discussed above, IL-6 can inhibit IL-1 and TNF-␣ (Borish and Steinke, 2003), which could be one explanation for a lack of differences for the latter two cytokines between women with preeclampsia and normal pregnancies. Alternatively, as reviewed by Conrad and Benyo (1997), many, but not all, researchers found increased plasma levels of TNF-␣ from preeclamptic women when measuring by ELISA. The lack of consistency may be due to the relatively short half-life of the cytokine as well as possible transient and episodic release, which may make the plasma levels vary considerably. Alternatively, the endothelium in some preeclamptic women might be more sensitive to activation by cytokines, so that ‘normal’ levels become injurious. The findings of increased levels of IL-6 and IL-8 suggest that there exists a proinflammatory response in preeclampsia, although this response by itself cannot explain the lack of signs of a shift in the Th1/Th2 balance. We expected to find increased levels of IFN-␥, which is the major Th1-type cytokine in serum from women with preeclampsia, and also increased levels of IL-2, as a reflection of T-cell activation. Furthermore, we expected that women with normal pregnancies would have had higher levels of the Th2type cytokine IL-4 and the anti-inflammatory cytokine IL-10. All these cytokines were successfully detected, but we failed to record the expected differences between preeclampsia and normal pregnancy. This may reflect the true conditions in vivo, but could on the other hand be explained speculatively by the known paracrine action of T-cell cytokines, i.e., secreted cytokines are rapidly bound to receptors on neighbouring cells. Excessive levels in preeclampsia or normal pregnancy may thus be captured already at the site of secretion, resulting in similar serum levels in both groups. We have previously reported decreased levels of IL-10 in preeclampsia, recorded in in vitro studies on peripheral blood mononuclear cells (Jonsson et al., 2005), which may indicate that such studies are better suited for T-cell cytokines. On the other hand, in vitro conditions are artificial, and may affect cytokine responses. Thus, further studies on T-cell responses are needed to elucidate the role of T-cell responses in preeclampsia. As for the group of less defined immunological markers during preeclampsia (i.e., IL-5, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, eotaxin, RANTES and sIL-4R), we were unable to find any significant differences in serum levels between women with
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preeclampsia and normal pregnancies, except for the finding on sIL-4R. In the case of IL13, we were even unable to detect it in our samples. However, these markers were analyzed with the aim of mapping their presence in preparation for later studies; although differences were not found, further studies need to be done to be able to exclude their part in the pathology of preeclampsia. In conclusion, this study indicates an increase of the pro-inflammatory cytokines IL-6 and IL-8 in sera from women with preeclampsia compared with normal pregnancy, supporting the hypothesis that preeclampsia is associated with increased inflammatory responses. Surprisingly, we found also increased levels of sIL-4R, suggested to reflect IL-4 levels, in serum from preeclamptic women. Taken together, our data indicate a disturbance in the systemic immune balance during preeclampsia, but whether this is a cause or a consequence of preeclampsia remains to be elucidated.
Acknowledgements The authors thank all midwifes at the Clinic of Obstetrics and Antenatal Care in Link¨oping and Motala for help with sample collection. We would like also to thank Petra Cassel for excellent technical assistance. This study was supported by financial grants from ¨ The County Council of Ostergotland and The Health Research Council in the South-East of Sweden (FORSS).
References ACOG Committee on Obstetric Practice, 2002. Practice bulletin #33: diagnosis and management of preeclampsia and eclampsia. Obstetr. Gynecol. 99, 159-167. Al-Othman, S., Omu, A.E., Diejomaoh, F.M., Al-Yatama, M., Al-Qattan, F., 2001. Differential levels of interleukin 6 in maternal and cord sera and placenta in women with pre-eclampsia. Gynecol. Obstetr. Invest. 52, 60–65. Borish, L.C., Steinke, J.W., 2003. Cytokines and chemokines. J. Allergy Clin. Immunol. 11l, S460–S475. Bullens, D.M., Kasran, A., Peng, X., Lorre, K., Ceuppens, J.L., 1998. Effects of anti-IL-4 receptor monoclonal antibody on in vitro T-cell cytokine levels: IL-4 production by T-cells from non-atopic donors. Clin. Exp. Immunol. 113, 320–326. Chilton, P.M., Fernandez-Botran, R., 1993. Production of soluble IL-4 receptors by murine spleen cells is regulated by T-cell activation and IL-4. J. Immunol. 151, 5907–5917. Conrad, K.P., Benyo, D.F., 1997. Placental cytokines and the pathogenesis of preeclampsia. Am. J. Reprod. Immunol. 37, 240–249. Conrad, K.P., Mies, T.M., Benyo, D.F., 1998. Circulating levels of immunoreaciive cytokines in women with preeclampsia. Am. J. Reprod. Immunol. 40, 102–111. Curfs, J.H., Meis, J.F., Hoogkamp-Korstanje, J.A., 1997. A primer on cytokines: sources, receptors, effects, and inducers. Clin. Microbial. Rev. 10, 742–780. Darmochwal-Kolarz, D., Rolinski, J., Leszczynska-Goarzelak, B., Oleszczuk, J., 2002. The expressions of intracellular cytokines in the lymphocytes of preeclamplic patients. Am. J. Reprod. Immunol. 48, 381–386. Dekker, G.A., Robillard, P.Y., Hulsey, T.C., 1998. Immune maladaptaiion in the etiology of preeclampsia: a review of corroborative epidemiologic studies. Obstetr. Gynecol. Surv. 53, 377–382. Dekker, G.A., Sibai, B.M., 1999. The immunology of preeclampsia. Semin. Perinatol. 23, 24–33. Ekerfelt, C., Ernerudh, J., Jenmalm, M.C., 2002. Detection of spontaneous and antigen-induced human interleukin4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR. J. Immunol. Meth. 260, 55–67.
Y. Jonsson et al. / Journal of Reproductive Immunology 70 (2006) 83–91
91
Garrone, P., Djossou, O., Galizzi, J.P., Banchereau, J., 1991. A recombinant extracellular domain of the human interleukin 4 receptor inhibits the biological effects of interleukin 4 on T and B lymphocytes. Eur. J. Immunol. 21, 1365–1369. Gessner, A., Schroppel, K., Will, A., Enssle, K.H., Lauffer, L., Rollinghoff, M., 1994. Recombinant soluble interleukin-4 (IL-4) receptor acts as an antagonist of IL-4 in murine cutaneous Leishmaniasis. Infect. Immunol. 62, 4112–4117. Jung, T., Wagner, K., Neumann, C., Heusser, C.H., 1999. Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro. Eur. J. Immunol. 29, 864–871. Jonsson, Y., Matthiesen, L., Berg, G., Ernerudh, J., Nieminen, K., Ekerfelt, C., 2005. Indications of an altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and IL-10 from blood mononuclear cells and in blood basophil counts compared with normal pregnancy. J. Reprod. Immunol. 66, 69–84. Kocyigit, Y., Atamer, Y., Atamer, A., Tuzcu, A., Akkus, Z., 2004. Changes in serum levels of leptin, cytokines and lipoprotein in pre-eclamptic and normotensive pregnant women. Gynecol. Endocrinol. 19, 267–273. Madazli, R., Aydin, S., Uludag, S., Vildan, O., Tolun, N., 2003. Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels. Acta Obstetr. Gynecol. Scand. 82, 797–802. Mukaida, N., Harada, A., Matsushima, K., 1998. Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine Growth Factor Rev. 9, 9–23. Protonotariou, E., Malamitsi-Puchner, A., Rizos, D., Sarandakou, A., Makrakis, E., Salamolekis, E., 2003. Alterations in Thl/Th2 cytokine concentrations in early neonatal life. J. Matern. Fetal. Neonatal. Med. 14, 407–410. Redman, C.W., Sargent, I.L., 2005. Latest advances in understanding preeclampsia. Science 308, 1592–1594. Redman, C.W.G., Sargent, I.L., 2003. Pre-eclampsia, the placenta and the maternal systemic inflammatory response—a review. Placenta 24, S21–S27. Robillard, P.Y., 2002. Interest in preeclampsia for researchers in reproduction. J. Reprod. Immunol. 53, 279–287. Saito, S., Sakai, M., 2003. Thl/Th2 balance in preeclampsia. J. Reprod. Immunol. 59, 161–173. Saito, S., Umekage, H., Sakamoto, Y., Sakai, M., Tanebe, K., Sasaki, Y., Morikawa, H., 1999. Increased T-helper1-type immunity and decreased T-helper-2-type immunity in patients with preeclampsia. Am. J. Reprod. Immunol. 41, 297–306. Taylor, R.N., 1997. Review: immunobiology of preeclampsia. Am. J. Reprod. Immunol. 37, 79–86. Velzing-Aarts, F.V., Muskiet, F.A., van der Dijs, F.P., Duits, A.J., 2002. High serum interleukin-8 levels in afroCaribbean women with pre-eclampsia. Relations with tumor necrosis factor-alpha, duffy negative phenotype and von Willebrand factor. Am. J. Reprod. Immunol. 48, 319–322. Vince, G.S., Starkey, P.M., Austgulen, R., Kwiatkowski, D., Redman, C.W., 1995. Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br. J. Obstetr. Gynaecol. 102, 20–25.
Cytokine mapping of sera from women with preeclampsia and normal pregnancies Yvonne Jonsson a,∗ , Marie Rub`er a , Leif Matthiesen b , G¨oran Berg b , Katri Nieminen b , Surendra Sharma c , Jan Ernerudh a , Christina Ekerfelt a a
Unit of Autoimmunity and Immune Regulation, Department of Molecular and Clinical Medicine, Division of Clinical Immunology, Faculty of Health Sciences, University Hospital, Link¨oping, Sweden b Department of Clinical and Molecular Medicine, Division of Obstetrics and Gynecology, Faculty of Health and Sciences, University Hospital, Link¨oping, Sweden c Departments of Pediatrics and Pathology, Brown University and Women and Infants’ Hospital of Rhode Island, Providence, RI, USA Received 23 June 2005; received in revised form 14 September 2005; accepted 28 October 2005
Abstract Introduction: Preeclampsia is a pregnancy-specific syndrome. The immune system in preeclampsia is changed with an increased innate activity and there is a hypothesis of a shift towards Th1-type immunity. The aim of this study was to determine a spectrum of soluble immunological factors denoting different aspects of immune activation in third trimester sera from women with preeclampsia (N = 15) and compare with levels in sera from normal pregnant women (N = 15). Material and methods: IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-l␣, MIP-1, MCP-1, eotaxin and RANTES were measured in serum using multiplex bead arrays. The levels of soluble CD14 and soluble IL-4 receptor were measured by enzyme-linked immunoassay (ELISA). Results: Preeclamptic women had significantly increased levels of circulating IL-6 (p = 0.002), IL-8 (p = 0.003) and soluble IL-4R (p = 0.037), compared to women with normal pregnancies. Conclusion: This study supports the hypothesis of increased inflammatory responses in preeclampsia, illustrated by the increased levels of IL-6 and IL-8. The finding of increased levels of soluble IL-4 ∗ Corresponding author at: Autoimmunity and Immune Regulation, Pathology Building Level 10, Faculty of Health and Sciences, SE-581 85 Link¨oping, Sweden. Tel.: +46 13 22 73 57. E-mail address: [email protected] (Y. Jonsson).
0165-0378/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jri.2005.10.007
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receptor is an intriguing finding with several interpretations, which may partly support the hypothesis of a Th1 shift in preeclampsia. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Cytokines; Serum; Multiple bead array; Preeclampsia
1. Introduction Preeclampsia is a pregnancy-specific syndrome that occurs in the later half of the pregnancy. The symptoms and signs include high blood pressure, proteinuria and general edema (ACOG Committee on Obstetric Practice, 2002) and the syndrome is clinically harmful to both the mother and the fetus. The precise etiology of the disease is still unknown, although the placenta plays a major part, as reviewed by Redman and Sargent (2005) and Robillard (2002). The immune maladaptation hypothesis (Dekker et al., 1998; Dekker and Sibai, 1999) suggests that preeclampsia is due to an inappropriate regulation of the normally Th2 deviated maternal immune responses, leading to a shift towards harmful Th1 immunity (Taylor, 1997; Saito et al., 1999; Darmochwal-Kolarz et al., 2002; Saito and Sakai, 2003). A generalized maternal inflammatory response has been shown to occur during preeclampsia together with increased levels of TNF-␣, IL-6 and IL-8, as reviewed by Redman and Sargent (2003). The significance of such immunological changes in the pathogenesis of preeclampsia is, however, still uncertain, and the immune aberrations per se need to be further elucidated. The nature and amounts of the immunomodulatory milieu present in serum may impart insights on the in vivo regulation of preeclampsia-associated conditions. Thus, mapping of the systemic immune balance by simultaneously measuring cytokines, chemokines and other inflammatory markers in serum from women with preeclampsia and from women with normal pregnancies would be of interest. Multiplex bead array systems allow simultaneous detection of a large number of molecules of interest. The available kits are designed to analyze levels of the classically studied immune markers, as well as newer ones. The purpose of this study was to examine and compare the in vivo network of cytokines, chemokines and other inflammatory markers present in serum from women with preeclamptic and normal pregnancies, utilizing a multiplex bead array system. Both the classically studied markers (interleukin (IL)-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, interferon (IFN)-␥, tumor necrosis factor (TNF)-␣, granulocyte macrophage colony stimulating factor (GM-CSF) and soluble CD14 (sCD14) as well as markers less defined or novel, in preeclampsia (IL-5, IL-13, IL-15, IL-17, IFN-␣, macrophage inflammatory protein-␣ (MIP-1␣), MIP-1, monocyte chemotactic protein-1 (MCP-1), eotaxin, RANTES (regulated on activation, normal T-cellexpressed and secreted) and soluble IL-4 receptor (sIL-4R)) were measured in serum from women with preeclampsia and compared with normal pregnancies.
2. Material and methods Blood samples were drawn from 15 women with moderate or severe preeclampsia and from 15 women with normal pregnancies (controls). Blood samples were centrifuged
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Table 1 Data on the participating women Diagnosis
Mean age at sampling (range)
Gestational age (completed weeks) at blood sampling (range)
Number of parous women
Normal pregnancy (N = 15) Moderate preeclampsiaa (N = 8) Severe preeclampsiaa (N = 7) All preeclamptic women (N = 15)
30 (25–42) 29 (22–32) 29 (26–32) 29 (22–32)
33 (29–37) 37 (33–40) 32 (29–34) 34 (29–40)
2 1 1 2
a Moderate and severe preeclampsia are defined according to WHO criteria. Moderate preeclampsia = BP > 140/90 and proteinuria less than 5 g/24 h. Severe preeclampsia = BP > 160/110 and/or proteinuria of more than 5 g/24 h and/or subjective symptoms such as visual impairment, headache, abdominal pain.
within 1 h after sampling and sera were then immediately frozen and stored at −70 ◦ C until use. Moderate and severe preeclampsia was defined according to WHO criteria. Moderate preeclampsia: blood pressure >140/90 and proteinuria less than 5 g/24 h; severe preeclampsia: blood pressure >160/110 and/or proteinuria of more than 5 g/24 h and/or subjective symptoms such as visual impairment, headache and abdominal pain. Blood samples from preeclamptic women were taken after diagnosis and admission to hospital, whereas blood samples from healthy women were taken at the Antenatal Care Clinic at the pregnancy week matching a preeclamptic patient ±2 gestational weeks. Four patients received glucocorticoid treatment (2× 7.5 mg within 24 h) prior to the time of blood sample collection. The time between treatment and sampling varied between 1 and 11 days. Data on the participating women are shown in Table 1. 2.1. Multiplex bead array analysis The levels of IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-1␣, MIP-1, MCP-1, eotaxin and RANTES were analyzed in sera according to the manufacturer, except that two extra standard points were added to the standard curve by two additional serial dilutions. The multiplex kits used were LCH0001, LHC0005 and LHC0007 (BioSource, Camarillo, CA, USA), and the amounts of cytokines/chemokines analyzed using the Luminex 100 instrument (Luminex Corporation, Austin, Texas, USA) and the STarStation software (V1.1, Applied Cytometry Systems, Sheffield, UK). Due to insufficient sample volumes only 13 serum samples from women with preeclampsia and 14 serum samples from normal pregnant women were analyzed for IL-12 p40, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, eotaxin and RANTES. 2.2. ELISA The levels of soluble CD14 (sCD14) and soluble IL-4 receptor (sIL-4R) in sera were determined by Quantikine ELISA-kits (R&D Systems, Minneapolis, MN, USA). The plates were read at 450 nm with wavelength correction made at 600 nm. Each sCD14 sample was diluted 200 and 400 times in the calibrator diluent RD5P (supplied by manufacturer) and analyzed in a single well for each dilution. Otherwise, analysis was performed according to manufacturer; including the analysis of sIL-4R levels in duplicates in undiluted serum
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samples. Due to insufficient sample volumes, only 13 serum samples from women with preeclampsia and 14 serum samples from normal pregnant women were analyzed for sCD14 and SIL-4R. 2.3. Statistics Data analysis was performed with StatView 5.0.1 for Windows. The Mann–Whitney U-test was used to compare data from women with preeclampsia and normal pregnancies. Since non-parametric statistical tests were used, which compare rankings between samples, and not mean values of the data, cytokine levels below detection limit were given the value of zero. For the group of classically studied immuno-markers during preeclampsia (i.e., IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IFN-␥, TNF-␣, GM-CSF and sCD14), p < 0.0045 was considered to be significant after Bonferroni-correction. For the not so extensively studied or undefined immunomarkers present during preeclampsia (i.e., IL-5, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, Eotaxin, RANTES and sIL-4R), p < 0.05 was considered significant. The latter group has not the same strict significance demands as the first group, since we wanted to extensively map markers in preparation for later studies. 2.4. Ethics The Regional Ethics Committee at the University Hospital of Link¨oping approved this study and informed consent was given from all participants in the study.
3. Results The entire set of data is summarized in Table 2. Women with preeclampsia showed significantly higher serum concentrations of the innate pro-inflammatory cytokine IL-6 (p = 0.002) and elevated levels of the pro-inflammatory chemokine IL-8 (p = 0.003) compared with women with normal pregnancies (Fig. 1a and b). Preeclamptic women also showed higher serum concentrations of the soluble IL-4 receptor (p = 0.037) than women with normal pregnancies (Fig. 1c). For all the other factors tested, i.e., IL-, IL-2, IL-4, IL-5, IL-10, IL-12 p40, IL-15, IL-17, IFN-␣, IFN-␥, TNF-␣, GM-CSF, MIP-1␣, MIP-1, MCP-1, RANTES, eotaxin and sCD14, there were no significant differences between women with preeclampsia compared to normal pregnancies. IL-13 was not detectable in any of the samples. When comparing within the group of preeclamptic patients, no significant differences in the levels of immunological factors were found between women with severe or moderate preeclampsia.
4. Discussion Our results show substantially increased levels of IL-6 and IL-8, supporting the hypothesis of increased inflammatory responses in preeclampsia. In addition, we found increased
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Table 2 Serum levels (pg/ml) of different immunological factors from women with preeclampsia and normal pregnancies Factor
Preeclampsia Median
IL-1 IL-2 IL-4 sIL-4R IL-5 IL-6 IL-8 IL-10 IL-12 p40 IL-13 IL-15 IL-17 IFN-␣ IFN-␥ TNF-␣ GM-CSF MIP-1␣ MIP-I MCP-1 RANTES Eotaxin SCD14
0.0 83 0.0 139 0.0 13 26 0.0 27 0.0 0.0 0.0 0.0 0.0 32 0.0 45 145 506 1838 64 1.5 × l06
Normal pregnancy Range 0.0–20 0.0–169 0.0–13 93–327 0.0–6.0 0.0–127 0.0–362 0.0–95 3.4–55 0.0–0.0 0.0–0.0 0.0–0.0 0.0–4.0 0.0–10 0.0–236 0.0–746 33–112 19–694 167–1001 1180–2791 33–157 1.2 × 106 –2.5 × 106
Median
Range
0.0 39 0.0 105 0.0 3.3 0.0 0.0 18 0.0 0.0 0.0 0.0 0.0 9.6 0.0 37 118 415 1928 48 1.5 × 106
0.0–221 0.0–155 0.0–23 82–162 0.0–7.3 0.0–296 0.0–36 0.0–4.5 0.0–52 0.0–0.0 0.0–66 0.0–3.6 0.0–65 0.0–20 0.0–423 0.0–0.0 22–150 22–348 77–1537 983–5669 17–100 1.0 × 106 –2.5 ×106
p-Values
0.984 0.330 0.171 0.037 0.984 0.002 0.003 0.373 0.160 ND 0.752 0.528 0.716 0.724 0.071 0.756 0.254 0.167 0.174 0.662 0.174 0.700
ND = not detectable. p-values refer to comparisons of the two groups by the Mann–Whitney U-test.
levels of sIL-4R in sera from women with preeclampsia compared with normal pregnancies. The finding for IL-6 is in line with previous reports on elevated plasma levels of IL-6 in women with preeclampsia compared with normal pregnancies (Vince et al., 1995; Conrad et al., 1998; Madazli et al., 2003). In contrast, Al-Othman et al. (2001) found no difference in IL-6 levels in serum between preeclampsia and normal pregnancy as analyzed by ELISA. One explanation for this divergence could be that the serum samples in the study of Al-Othman et al., 2001) were frozen and stored at −20 ◦ C instead of −70 ◦ C, which may have affected the result. Although IL-6 is predominantly regarded as a pro-inflammatory cytokine, it shows also a number of other effects due to its pleiotropic nature (Curfs et al., 1997), including inhibition of the IL-6 inducing inflammatory cytokines IL-1 and TNF-␣, stimulation of antibody production from B cells and activation and differentiation of T-cells (Borish and Steinke, 2003). Thus, due to its pleiotropic nature, it is hard to speculate on the clinical effects of elevated IL-6 levels in preeclampsia. The preeclamptic women in our study showed also significantly elevated levels of the chemokine IL-8 compared to women with normal pregnancies (p = 0.003). IL-8 is an inflammatory chemokine locally produced in response to tissue injury, leading mainly to recruitment and activation of neutrophils (Mukaida et al., 1998). Our finding of increased levels of serum IL-8 is in line with others (Velzing-Aarts et al., 2002; Kocyigit et al., 2004), which further support the role of IL-8 in inflammatory responses in preeclampsia.
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Fig. 1. Serum concentrations of the pro-inflammatory cytokine IL-6 (A), the chemokine IL-8 (B) and the soluble IL-4 receptor (IL-4 sR) (C) from women with preeclampsia or normal pregnancies, p-values from comparisons with the Mann–Whitney U-test are shown. Median is indicated.
The finding of increased sIL-4R in women with preeclampsia was somewhat surprising, since sIL-4R has been suggested as a surrogate marker for levels of the cytokine IL-4. This suggestion was based on studies on mice spleen cells, where sIL-4R was shown to reflect IL-4 levels (Chilton and Fernandez-Botran, 1993), and on observations that proteolytic cleavage of sIL-4R was different from IL-4 (Jung et al., 1999), allowing sIL-4R to remain longer in the circulation. Since IL-4 itself is known to be difficult to detect (Bullens et al., 1998; Ekerfelt et al., 2002), as also illustrated in the present study with detectable IL-4 levels only in a few normal pregnancies, the strategy to use sIL-4R as a surrogate marker for IL-4 would be useful since IL-4 is one of the classical Th2 cytokines. The current finding of increased sIL-4R levels in preeclampsia may have three possible explanations; the first is that the hypothesis of decreased IL-4 in preeclampsia could be questioned; secondly, the
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sIL-4R might not reflect IL-4 levels; or, thirdly, there might exist a negative correlation between IL-4 levels and sIL-4 R levels as reported by Protonotariou et al. (2003). Previous studies in human in vitro models (Garrone et al., 1991), as well as an in vivo murine model (Gessner et al., 1994), have shown that sIL-4R can neutralize unbound IL-4, suggesting that sIL-4R plays a role in IL-4 regulation. Speculatively, since sIL-4R is believed to persist longer in the circulation than IL-4 (Jung et al., 1999), the increased level of sIL-4R in preeclampsia may reflect a current decrease of IL-4 secretion, i.e., sIL-4R was shed from Th2 cells upon IL-4 secretion earlier in pregnancy and, after neutralization of all circulating IL-4, only the excess amount of sIL-4R could still be detected. It has, however, to be kept in mind that the finding of increased sIL-4R in preeclampsia must be interpreted with caution since it fulfilled only the lower demand on significance levels used for the screening of novel immuno-markers. Thus, this finding needs to be confirmed in further studies. When considering the findings on classically studied immunological markers during preeclampsia (i.e., IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 p40, IFN-␥, TNF-␣, GMCSF and sCD14), it is puzzling that we did not find any other differences than for the levels of IL-6 and IL-8. As discussed above, IL-6 can inhibit IL-1 and TNF-␣ (Borish and Steinke, 2003), which could be one explanation for a lack of differences for the latter two cytokines between women with preeclampsia and normal pregnancies. Alternatively, as reviewed by Conrad and Benyo (1997), many, but not all, researchers found increased plasma levels of TNF-␣ from preeclamptic women when measuring by ELISA. The lack of consistency may be due to the relatively short half-life of the cytokine as well as possible transient and episodic release, which may make the plasma levels vary considerably. Alternatively, the endothelium in some preeclamptic women might be more sensitive to activation by cytokines, so that ‘normal’ levels become injurious. The findings of increased levels of IL-6 and IL-8 suggest that there exists a proinflammatory response in preeclampsia, although this response by itself cannot explain the lack of signs of a shift in the Th1/Th2 balance. We expected to find increased levels of IFN-␥, which is the major Th1-type cytokine in serum from women with preeclampsia, and also increased levels of IL-2, as a reflection of T-cell activation. Furthermore, we expected that women with normal pregnancies would have had higher levels of the Th2type cytokine IL-4 and the anti-inflammatory cytokine IL-10. All these cytokines were successfully detected, but we failed to record the expected differences between preeclampsia and normal pregnancy. This may reflect the true conditions in vivo, but could on the other hand be explained speculatively by the known paracrine action of T-cell cytokines, i.e., secreted cytokines are rapidly bound to receptors on neighbouring cells. Excessive levels in preeclampsia or normal pregnancy may thus be captured already at the site of secretion, resulting in similar serum levels in both groups. We have previously reported decreased levels of IL-10 in preeclampsia, recorded in in vitro studies on peripheral blood mononuclear cells (Jonsson et al., 2005), which may indicate that such studies are better suited for T-cell cytokines. On the other hand, in vitro conditions are artificial, and may affect cytokine responses. Thus, further studies on T-cell responses are needed to elucidate the role of T-cell responses in preeclampsia. As for the group of less defined immunological markers during preeclampsia (i.e., IL-5, IL-13, IL-15, IL-17, IFN-␣, MIP-1␣, MIP-1, MCP-1, eotaxin, RANTES and sIL-4R), we were unable to find any significant differences in serum levels between women with
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preeclampsia and normal pregnancies, except for the finding on sIL-4R. In the case of IL13, we were even unable to detect it in our samples. However, these markers were analyzed with the aim of mapping their presence in preparation for later studies; although differences were not found, further studies need to be done to be able to exclude their part in the pathology of preeclampsia. In conclusion, this study indicates an increase of the pro-inflammatory cytokines IL-6 and IL-8 in sera from women with preeclampsia compared with normal pregnancy, supporting the hypothesis that preeclampsia is associated with increased inflammatory responses. Surprisingly, we found also increased levels of sIL-4R, suggested to reflect IL-4 levels, in serum from preeclamptic women. Taken together, our data indicate a disturbance in the systemic immune balance during preeclampsia, but whether this is a cause or a consequence of preeclampsia remains to be elucidated.
Acknowledgements The authors thank all midwifes at the Clinic of Obstetrics and Antenatal Care in Link¨oping and Motala for help with sample collection. We would like also to thank Petra Cassel for excellent technical assistance. This study was supported by financial grants from ¨ The County Council of Ostergotland and The Health Research Council in the South-East of Sweden (FORSS).
References ACOG Committee on Obstetric Practice, 2002. Practice bulletin #33: diagnosis and management of preeclampsia and eclampsia. Obstetr. Gynecol. 99, 159-167. Al-Othman, S., Omu, A.E., Diejomaoh, F.M., Al-Yatama, M., Al-Qattan, F., 2001. Differential levels of interleukin 6 in maternal and cord sera and placenta in women with pre-eclampsia. Gynecol. Obstetr. Invest. 52, 60–65. Borish, L.C., Steinke, J.W., 2003. Cytokines and chemokines. J. Allergy Clin. Immunol. 11l, S460–S475. Bullens, D.M., Kasran, A., Peng, X., Lorre, K., Ceuppens, J.L., 1998. Effects of anti-IL-4 receptor monoclonal antibody on in vitro T-cell cytokine levels: IL-4 production by T-cells from non-atopic donors. Clin. Exp. Immunol. 113, 320–326. Chilton, P.M., Fernandez-Botran, R., 1993. Production of soluble IL-4 receptors by murine spleen cells is regulated by T-cell activation and IL-4. J. Immunol. 151, 5907–5917. Conrad, K.P., Benyo, D.F., 1997. Placental cytokines and the pathogenesis of preeclampsia. Am. J. Reprod. Immunol. 37, 240–249. Conrad, K.P., Mies, T.M., Benyo, D.F., 1998. Circulating levels of immunoreaciive cytokines in women with preeclampsia. Am. J. Reprod. Immunol. 40, 102–111. Curfs, J.H., Meis, J.F., Hoogkamp-Korstanje, J.A., 1997. A primer on cytokines: sources, receptors, effects, and inducers. Clin. Microbial. Rev. 10, 742–780. Darmochwal-Kolarz, D., Rolinski, J., Leszczynska-Goarzelak, B., Oleszczuk, J., 2002. The expressions of intracellular cytokines in the lymphocytes of preeclamplic patients. Am. J. Reprod. Immunol. 48, 381–386. Dekker, G.A., Robillard, P.Y., Hulsey, T.C., 1998. Immune maladaptaiion in the etiology of preeclampsia: a review of corroborative epidemiologic studies. Obstetr. Gynecol. Surv. 53, 377–382. Dekker, G.A., Sibai, B.M., 1999. The immunology of preeclampsia. Semin. Perinatol. 23, 24–33. Ekerfelt, C., Ernerudh, J., Jenmalm, M.C., 2002. Detection of spontaneous and antigen-induced human interleukin4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR. J. Immunol. Meth. 260, 55–67.
Y. Jonsson et al. / Journal of Reproductive Immunology 70 (2006) 83–91
91
Garrone, P., Djossou, O., Galizzi, J.P., Banchereau, J., 1991. A recombinant extracellular domain of the human interleukin 4 receptor inhibits the biological effects of interleukin 4 on T and B lymphocytes. Eur. J. Immunol. 21, 1365–1369. Gessner, A., Schroppel, K., Will, A., Enssle, K.H., Lauffer, L., Rollinghoff, M., 1994. Recombinant soluble interleukin-4 (IL-4) receptor acts as an antagonist of IL-4 in murine cutaneous Leishmaniasis. Infect. Immunol. 62, 4112–4117. Jung, T., Wagner, K., Neumann, C., Heusser, C.H., 1999. Enhancement of human IL-4 activity by soluble IL-4 receptors in vitro. Eur. J. Immunol. 29, 864–871. Jonsson, Y., Matthiesen, L., Berg, G., Ernerudh, J., Nieminen, K., Ekerfelt, C., 2005. Indications of an altered immune balance in preeclampsia: a decrease in in vitro secretion of IL-5 and IL-10 from blood mononuclear cells and in blood basophil counts compared with normal pregnancy. J. Reprod. Immunol. 66, 69–84. Kocyigit, Y., Atamer, Y., Atamer, A., Tuzcu, A., Akkus, Z., 2004. Changes in serum levels of leptin, cytokines and lipoprotein in pre-eclamptic and normotensive pregnant women. Gynecol. Endocrinol. 19, 267–273. Madazli, R., Aydin, S., Uludag, S., Vildan, O., Tolun, N., 2003. Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels. Acta Obstetr. Gynecol. Scand. 82, 797–802. Mukaida, N., Harada, A., Matsushima, K., 1998. Interleukin-8 (IL-8) and monocyte chemotactic and activating factor (MCAF/MCP-1), chemokines essentially involved in inflammatory and immune reactions. Cytokine Growth Factor Rev. 9, 9–23. Protonotariou, E., Malamitsi-Puchner, A., Rizos, D., Sarandakou, A., Makrakis, E., Salamolekis, E., 2003. Alterations in Thl/Th2 cytokine concentrations in early neonatal life. J. Matern. Fetal. Neonatal. Med. 14, 407–410. Redman, C.W., Sargent, I.L., 2005. Latest advances in understanding preeclampsia. Science 308, 1592–1594. Redman, C.W.G., Sargent, I.L., 2003. Pre-eclampsia, the placenta and the maternal systemic inflammatory response—a review. Placenta 24, S21–S27. Robillard, P.Y., 2002. Interest in preeclampsia for researchers in reproduction. J. Reprod. Immunol. 53, 279–287. Saito, S., Sakai, M., 2003. Thl/Th2 balance in preeclampsia. J. Reprod. Immunol. 59, 161–173. Saito, S., Umekage, H., Sakamoto, Y., Sakai, M., Tanebe, K., Sasaki, Y., Morikawa, H., 1999. Increased T-helper1-type immunity and decreased T-helper-2-type immunity in patients with preeclampsia. Am. J. Reprod. Immunol. 41, 297–306. Taylor, R.N., 1997. Review: immunobiology of preeclampsia. Am. J. Reprod. Immunol. 37, 79–86. Velzing-Aarts, F.V., Muskiet, F.A., van der Dijs, F.P., Duits, A.J., 2002. High serum interleukin-8 levels in afroCaribbean women with pre-eclampsia. Relations with tumor necrosis factor-alpha, duffy negative phenotype and von Willebrand factor. Am. J. Reprod. Immunol. 48, 319–322. Vince, G.S., Starkey, P.M., Austgulen, R., Kwiatkowski, D., Redman, C.W., 1995. Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br. J. Obstetr. Gynaecol. 102, 20–25.