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Clinical Chemistry 53: 42-52, 2007; 10.1373/clinchem.2007.074401
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(Clinical Chemistry. 2007;53:42-52.)
© 2007 American Association for Clinical Chemistry, Inc.

Proteomics and Protein Markers

Innate Immune Response by Ficolin Binding in Apoptotic Placenta Is Associated with the Clinical Syndrome of Preeclampsia

Chi Chiu Wang1,2,a, Ka Wing Yim1, Terence C.W. Poon3, Kwong Wai Choy1,2, Ching Yan Chu1, Wai Ting Lui1, Tze Kin Lau1,2, Michael S. Rogers1 and Tse Ngong Leung1

1 Li Ka Shing Institute of Health Sciences, 2 Department of Obstetrics and Gynaecology; and 3 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, SAR.

aAddress correspondence to this author at: 1st Floor, Block E, Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong, SAR. Fax 852-2632-2810; e-mail ccwang{at}cuhk.edu.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Unidentified circulating factors derived from placenta are thought to be responsible for the exaggerated systemic inflammation leading to preeclampsia. Our aim was to identify the circulating factors present in preeclampsia and to investigate their relationship to the underlying systemic immune response responsible for the associated clinical manifestations.

Methods: We obtained blood samples from pregnant women with and without preeclampsia and performed comparative proteomic analyses to identify the abnormal circulating factors by 2-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight for protein separation and identification. In placentas from preeclamptic pregnancies, we evaluated the potential role of the candidate proteins identified by Western and immunohistochemical analysis. We also used proinflammatory cytokine antibody arrays to investigate local and systemic immune responses.

Results: We found that ficolins, the pattern-recognition proteins involved in the lectin-complement pathway, were differentially expressed in plasma from preeclamptic pregnancies. Ficolins were present in low concentrations in plasma but at high concentrations in the placenta, particularly in syncytiotrophoblasts undergoing apoptosis. The binding of ficolins in apoptotic trophoblasts induced innate immunity through local and systemic cytokine activation and correlated with the clinical manifestation of preeclampsia.

Conclusions: We identified specific in vivo circulating factors derived from the placenta that are responsible for the local immune recognition and systemic inflammatory response in the development of clinical manifestations of preeclampsia. These findings may have predictive value and also therapeutic applications to lessen adverse clinical outcomes of preeclampsia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Preeclampsia, a complication of the second half of pregnancy, labor, or early puerperium, is characterized by hypertension, proteinuria, and other systemic disturbances (1). This condition has variable modes of clinical presentation and rates of disease progression (2) and poses potential dangers to both mother and baby. Eclampsia, characterized by generalized seizures, is one of the consequences of the disease.

No measures are currently available to predict or prevent this clinical syndrome (3). Histological examination of the placental bed from preeclamptic pregnancies suggests incomplete trophoblastic invasion of uterine spiral arteries as a primary pathology. The resulting poorly perfused fetoplacental unit releases circulating factors that damage maternal vascular endothelium, leading to multisystem dysfunction in susceptible individuals (4). Several candidate factors have been suggested, but none of them have yet been proven to be causative in vivo (1)(5). Recent work has revealed an unusual maternal immune recognition of the fetus (2). The underlying causes of this exaggerated systemic inflammation are still unknown.

We aimed to identify the circulating factors in preeclamptic women, and to characterize the candidate proteins in preeclamptic placenta. We also attempted to link these proteins to the underlying systemic immune response that is responsible for the clinical manifestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
patients
We recruited pregnant women who received antenatal check-ups between September 2003 and December 2004 at the Department of Obstetrics and Gynaecology at the Prince of Wales Hospital, the teaching hospital of the Chinese University of Hong Kong. All women provided written informed consent. Study approval was obtained from the Research Ethics Committee of the University. Only singlet pregnancies were included. The gestational age was confirmed by early ultrasound examination.

We considered preeclampsia to be present, according to The International Society for the Study of Hypertension in Pregnancy, when there was a sustained rise in diastolic blood pressure to ≥110 mmHg on a single occasion, or ≥90 mmHg on ≥2 occasions at least 4 h apart, along with the presence of significant proteinuria (>0.3 g/day or dipstick ≥2+ in 2 clean-catch midstream urine samples) in pregnant women with no history of preexisting or chronic hypertension. We collected 10 mL of maternal blood from the antecubital vein of preeclamptic pregnant women and from pregnant controls who had no medical and antenatal complications. The preeclamptic and control women were matched for fetal gestational age at the time of blood sampling. Women with preeclampsia were managed by the attending physician according to the departmental clinical protocol. All placentas were collected at delivery and vigorously washed in sterile, ice-cold phosphate buffered saline (10 mmol/L PBS: 137 mmol/L NaCl, 10 mmol/L phosphate, 2.7 mmol/L KCl, pH 7.4) solution.

comparative proteomic analysis
We performed 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE)1 analysis, in duplicate, on albumin-depleted plasma. In the 1st-dimension separation, proteins were separated by isoelectric point by isoelectric focusing in a nonlinear isoelectric focusing strip (Bio-Rad) with a voltage gradient for 15 h. In the 2nd-dimension separation, proteins were separated by molecular weight on a precast PAGE gel (Bio-Rad) assembled in a running cassette of a PROTEAN II XL Vertical Electrophoresis Cell (Bio-Rad) with a constant current of 16 mA for stacking, 150 V for protein separation, and then 10 mA for damping. Silver staining (Amersham) was used to visualize the plasma proteins separated in 2D-PAGE. The gel images were acquired by a GS-700 Imaging Densitometer with Quantity One software (Bio-Rad). Protein spots were quantified with Discovery Series PDQuest 2D Analysis Software. Quantities of matched spots were normalized with the sum of spot quantities between the 1st and 3rd quartile and analyzed by Significance Analysis of Microarrays (Stanford University). This program used a moderated t statistic, whereby a constant was added to the denominator of the t-statistic between samples from normal and preeclamptic pregnancies. In the Significance Analysis of Microarrays analysis, "2 classed, unpaired data" were selected as the data type, and 5000 permutations were performed. Each protein was assigned a score on the basis of its change in terms of protein expression relative to the SD of repeated measurements for that protein. The false significant discovery rate was set to zero to avoid the identification of falsely significant proteomic features caused by multiple comparisons.

We manually excised protein spots with a blade and subjected them to in-gel overnight trypsin digestion with 20 mg/L trypsin at 37 °C. We spotted 3 µL concentrated peptides on a metallic plate covered by {alpha}-cyan-4-hydroxy cinnamic acid (Fluka) in 5 mmol/L ammonium dihydrogen phosphate solution (Fluka). The peptide mass and intensity were then measured with a 4700 Proteomics Analyzer (Applied Biosystems) consisting of matrix-assisted laser desorption/ionization time of flight mass spectrometer and tandem mass spectrometer. The experimental peptide mass list was compared with the theoretical mass list for each entry in the database. To confirm identification, the most intense peptides from the peptide mass lists were subjected to further fragmentation. Peptide mass and fragment mass spectrums unique to the peptide were generated, and respective database searches performed using Profound (http://prowl.rockefeller.edu) and Mascot (http://www.matrixscience.com) [see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/context/vol53/issue1].

immunoassay
We used sandwich ELISA methods to measure H-ficolin, L-ficolin, and mannose-binding lectin (MBL) concentrations in plasma samples. The microtiter plate was coated with 100 µL of 25 mg/L of the polysaccharide from Aerococcus viridans (Fukuoka Red Cross Blood Centre) for H-ficolin, 100 µL of 0.5 g/L polyclonal anti-L-ficolin (clone 2F5, Tokai University) for L-ficolin, or 100 µL of 0.5 g/L monoclonal anti-MBL (clone 3E7, Tokai University) for MBL quantification. We incubated 80 µL of the antigen standards (Tokai University) or plasma samples at 37 °C for 1 h and then washed them 3 times with washing buffer (10mM phosphate buffered saline, 5 g/L Tween 20). We added 100 µL of mouse monoclonal anti-H-ficolin (clone 4H5, HyCult Biotechnology b.v.), rabbit polyclonal anti-L-ficolin (Tokai University), or DIG-labeled anti-MBL, at 500-fold dilution, in blocking buffer. One hundred microliters of antimouse Ig-peroxidase conjugate (Dako) for H-ficolin, antirabbit Ig-peroxidase conjugate (Roche Diagnostic) for L-ficolin, or anti-DIG-Ig-peroxidase conjugate (Roche Diagnostic) for MBL, at 500-fold dilution, was then applied and incubated at 37 °C for 1 h. We washed the microtiter plate 4 times and applied 100 µL of BM Blue substrate (Roche Diagnostic). The plate was incubated at room temperature for 30 min, and reaction was stopped with 100 µL of 1 mol/L H2SO4 solution. Absorbance was read with a SPECTRA Rainbow microplate reader (Tecan) at 450nm with a reference wavelength of 690 nm.

western blot analysis
We prepared total protein extracts from placental tissue lysates with lysis buffer (50 mmol/L Tris-HCl, 0.3 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol and 1 mmol/L phenylmethylsulphonyl fluoride). Cell extracts with equal amount of total protein (100 µg) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto a nitrocellulose membrane (Amersham). The membranes were blocked overnight with ovalbumin (Sigma) saturated with tris-based saline (TBS) (50 mmol/L Tris pH8.0 and 150 mmol/L NaCl) and then incubated at 37 °C for 1 h with primary antibody, either monoclonal anti-H-ficolin at 1 mg/L (clone 4H5) from HyCult Biotechnology or rabbit polyclonal anti-L-ficolin at 2 mg/L (clone 2F5) from Tokai University. The membranes were then washed 3 times with TBS followed by incubating with either antimouse horseradish peroxidase secondary antibody (Dako, P0161) or antirabbit IgG horseradish peroxidase secondary antibody (Amersham; NA 934V), at 1:1000 dilution, in TBS for 30 min at room temperature, and developed with an ECL Kit (Amersham) according to the manufacturer’s protocol. The expressed protein band was imaged with the Quantity One® quantification software (Bio-Rad) and normalized with ß-actin expression.

immunohistochemistry
We performed immunohistochemical staining on formalin-fixed and paraffin-embedded placental tissues. The epitope site was retrieved by heating placental sections in a microwave with 10 mmol/L citrate buffer with 0.5 g/L Tween 20, pH 6.0. After blocking the endogenous peroxidase with 30 g/L hydrogen peroxide, for ficolin staining alone, sections were incubated in TBS with normal sheep serum, with either H-ficolin monoclonal antibody (clone 4H5) from HyCult Biotechnology or rabbit polyclonal anti-L-ficolin antibody (clone 2F5) from Tokai University. For ficolin and Fas double staining, sections were incubated in TBS-T with normal sheep serum with either monoclonal antibodies to Fas (B-10) and to H-ficolin (clone 4H5) or to rabbit polyclonal L-ficolin antibody (clone 2F5). We then incubated the sections with secondary antibodies (MACH3 mouse and rabbit probe, Biocare Medical Kit). Ficolin staining was developed with 3, 3'-diaminobenzidine substrate to produce a brown color. Fas staining was revealed by Fast Red, which produced a cherry pink color. Negative controls lacked primary antibodies.

cytokine antibody array
To analyze the cytokine profiles in paired placenta and plasma samples from preeclamptic and nonpreeclamptic pregnancies at the same gestational age, we used a Human Cytokine Antibody Array (RayBiotech Inc) according to the manufacturer’s protocol. Supernatants from placental tissue lysates and plasma samples were diluted 10-fold with blocking buffer before incubation with the array membranes. Biotin-conjugated antibody mixture and horseradish peroxidase-conjugated streptavidin in blocking buffer were added to the membranes after washing with washing buffer. Detection buffer was added to start the detection reaction in the dark. Membranes were exposed to Kodak x-omat AR film, and the developed images were analyzed by a GS-700 Imaging Densitometer (Bio-Rad) and quantified by PDQuest 2D Analysis Software (Bio-Rad).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
differential plasma ficolin concentrations in preeclamptic pregnancies
We undertook comparative proteomic analysis to identify circulating factors in preeclamptic women. Among the proteins tested, we observed only 2 significantly different protein spots in the plasma samples of established preeclamptic pregnancies (Fig. 1 , Table 1 ). These 2 proteins were present in significantly lower concentrations in the preeclamptic plasma samples than in the gestation-age–matched control samples (n = 11 vs 11; Fig. 2A ) The mean decreases in protein spot intensity from preeclamptic plasma were 1.3-fold for Spot 1 and 3.2-fold for Spot 2 [see Table 1 in the online Data Supplement]. By tryptic peptide fingerprinting analysis, we confirmed that the identities of the 2 peptides were Hakata antigen and hucolin, known as H-ficolin and L-ficolin, respectively (Fig. 2B , see Fig. 1 in the online Data Supplement).


Figure 1
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  		Figure 1. 2D-PAGE of normal and preeclamptic plasma.

Plasma proteins from a pair of gestation-age matched normal (left) and preeclamptic (right) pregnancies were separated and visualized. Silver stained 2D-PAGEs after protein separation are shown, and 2 differential protein spots are highlighted with arrows. Spot 1: isoelectric point = 6.04, Mr = 33.1kDa; Spot 2: isoelectric point = 6.25, Mr = 34.3kDa.


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  		Table 1. Demographic characteristics of study participants.


Figure 2
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  		Figure 2. Comparative proteomic analysis in preeclampsia.

(A), two differential protein spots. Eleven pairs of plasma samples from normal (left) and preeclamptic (right) pregnancies, at the same gestational age, were compared (Table 1Up ). Spot 1 and Spot 2 from duplicate 2D-PAGE are shown. The quantity of Spot 1 and Spot 2 were significantly lower in the preeclamptic group (see Table 1Up in the online Data Supplement). (B), H-ficolin and L-ficolin. Spot 1 and Spot 2 were subjected to peptide mass and fragment mass analysis by collision-induced dissociation to obtain amino acid sequence information. Mass spectra were searched against Profound and Mascot databases (see Fig. 1Up in the online Data Supplement). The matched peptide sequences with identical homology to the precursor ions are shown. Spot 1 peptide sequences: H-ficolin (FCN3), accession no. CA 14774, individual ions score >32; Spot 2 peptide sequences: L-ficolin (FCN2), accession no. gi 1669349, individual ions score >47. (C), low ficolin concentrations in preeclamptic plasma. Twenty established preeclamptic pregnancies from another cohort were compared with 45 normal pregnancies by ELISA analysis. There was no significant correlation between gestational age and ficolins/MBL concentrations. Plasma concentrations of H-ficolin and L-ficolin were significantly decreased in preeclamptic plasma (P <0.01, Mann–Whitney U), but there was no difference in MBL.

increased ficolins in physiological immunity during normal pregnancies
Ficolins are pattern-recognition lectins involved in the lectin-complement pathway; they play an important role in innate immunity (6). Ficolins, as well as MBLs, bind to cell-surface carbohydrates and activate MBL-associated serine proteases to remove pathogens by phagocytosis (7). H-ficolin, L-ficolin, and MBLs were found to be maintained at very low concentrations (~3–5 mg/L) in healthy adults (8), but there is no previous report concerning lectin activity in normal pregnancies or pregnancies complicated by preeclampsia. We detected a 4- to 5-fold increase in the concentrations of both H-ficolin and L-ficolin in maternal plasma of normal pregnancies (n = 45; Fig. 2CUp ) compared with concentrations in healthy nonpregnant persons (9). Human pregnancy is considered a unique immune challenge in that the mother has to suppress rejection responses against the fetus while maintaining her resistance to infection. The increase in circulating ficolins may reflect this physiological immunity during pregnancy.

plasma ficolin deprivation during preeclampsia
We confirmed that maternal plasma concentrations of H-ficolin and L-ficolin were significantly lower in preeclamptic pregnancies (n = 20) than in uncomplicated pregnancies (Fig. 2CUp ), with the concentrations in preeclamptic pregnancies remaining as low as in nonpregnant women. These differences suggest the presence of an underlying immune abnormality in preeclampsia. The maternal plasma concentration of MBL in preeclamptic pregnancies, however, was not significantly different from normal pregnancies (Fig. 2CUp ). Deficiency of MBL, but not ficolin, has been related to pregnancy loss in recurrent miscarriages (9)(10), suggesting a differential immunological manifestation between recurrent miscarriage and preeclampsia. The low circulating ficolin concentrations suggested an underlying mechanism of ficolins in preeclampsia.

specific binding of ficolins to preeclamptic placentas
During pregnancy, proper interaction between invasive trophoblasts and maternal lymphocytes is responsible for normal immune recognition (11). Maternal natural killer cells and macrophages facilitate deep trophoblastic invasion into myometrial segments and promote extensive spiral artery remodeling. To understand the role of ficolins in the underlying immune recognition in preeclampsia, we revealed high concentrations of H-ficolin and L-ficolin in placental lysate from established preeclamptic pregnancies (n = 5; Fig. 3A ). We demonstrated localization of H-ficolin and L-ficolin in multinucleate syncytiotrophoblasts in placental sections from established preeclamptic pregnancies (n = 5; Fig. 3B ). In preeclampsia, it is well known that trophoblast invasion is inhibited, the maternal spiral arteries are poorly remodeled, and the capacity of the uteroplacental circulation is decreased (12). The syncytiotrophoblasts covering the trophoblastic microvilli provide direct contact with maternal blood in the expanding intravillous space of the placenta. In our study, the reactivity of ficolins in the syncytiotrophoblasts suggested its specific interaction with the maternal interface in preeclamptic pregnancies.


Figure 3
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  		Figure 3. Specific binding of ficolins in preeclamptic trophoblasts.

(A). high ficolin concentrations in preeclamptic placenta. Gestation-age matched placental tissues from 2 established preeclamptic pregnancies and 1 normal pregnancy were analyzed by Western blotting with antibodies to H-ficolin, L-ficolin, or ß-actin; or without primary antibody as negative control. (B), increased ficolins in preeclamptic syncytiotrophoblasts. H-Ficolin and L-ficolin immunostaining in representative placental sections from normal (left) and preeclamptic (right) pregnancies at the same gestational age are shown. High reactivity with strong ficolin staining can be observed in the multinucleate syncytiotrophoblasts from preeclamptic placenta (arrows). Scale bar: 50µm. (C), specific binding of ficolins to apoptotic trophoblasts in preeclampsia. Increased apoptosis can be observed in the syncytiotrophoblasts in preeclamptic placenta by immunostaining with antibody to apoptotic protein marker Fas. Strong H-ficolin and L-ficolin protein coexpression with Fas in the apoptotic syncytiotrophoblasts was indicated by double immunostaining (arrows). Scale bar: 50 µm.

immune recognition of ficolins in apoptotic trophoblast in preeclampsia
Ficolins recognize and bind to apoptotic cells, activate complement via the lectin pathway, and participate in the clearance of apoptotic cells (13). Because of poorly established uteroplacental circulation in preeclampsia, the ischemic placenta is prone to syncytiotrophoblastic apoptosis and clearance (14). Fas was found to be important for Fas/Fas ligand-mediated death signals in placental tissue (15). To further investigate the potential role of ficolins in the preeclamptic trophoblasts, we demonstrated the strong expression of apoptotic protein marker Fas protein, and also the coexpression of both ficolins and Fas in the syncytiotrophoblasts from established preeclamptic pregnancies (n = 5; Fig. 3CUp ). These confirmed immune recognition and binding of ficolins to the trophoblast cells undergoing apoptosis in preeclampsia, suggesting the underlying mechanism of ficolin-mediated immune response in the preeclamptic placenta. Liver and lung were found to be the major sources of ficolins in humans, and ficolins can bind to the epithelial surfaces for proper immunity in the organs (16). Recruitment of circulating ficolins to the preeclamptic placenta may cause the depletion of circulating ficolins in preeclampsia, but an in vitro model will be required to provide evidence of this effect.

placental innate immune responses in preeclampsia
Ficolins can share specificity for N-acetylglucosamine, binding it through their fibrinogen-like domain to trigger the activation of complement and subsequent inflammatory response for innate immunity (17). To clarify the immune responses mediated by ficolins in the placenta during preeclamptic pregnancy, we found that chemokines/cytokines involved in innate immunity, including monocyte chemoattractant proteins 1/2/3 (MCP1/2/3), monokine induced by gamma-interferon (MIG), macrophage inflammatory proteins 1{delta}/3{alpha} (MIP1{delta}/3{alpha}), tumor necrosis factor {alpha} (TNF{alpha}) and thymus expressed chemokine (TECK) were activated, whereas interferon/cytokines involved in adaptive immunity, including interferon {gamma} (IFN{gamma}), interleukin 13/4/5 (IL13/4/5), transforming growth factor ß1/ß3 (TGFß1/ß3) and TNFß, were suppressed in preeclamptic placenta (n = 10 vs 10; Fig. 4A ). Adhesion molecules/growth factors, including ciliary neurotropic factor (CNTF), epidermal growth factor (EGF), intercellular adhesion molecule (ICAM1), insulin-like growth factor binding proteins (IGFBP4), and bone morphogenetics protein (BMP6), were also stimulated in the preeclamptic placenta (Fig. 4A ). Increased IL12/6/8 and decreased IL10 were also detected, but no significant differences were found. Despite the absence of clinical infection in any of the pregnancies studied, in the placenta of preeclamptic pregnancies compared to those of normal pregnancies the concentrations of the activated innate immune chemokines/cytokines were significantly increased, the suppressed adaptive immune interferon/cytokines were significantly decreased, and the other activated adhesion molecules/growth factors were significantly increased, (Fig. 4B , left panels). The distinct patterns of immune cytokines found in placenta from preeclampsia indicated that differential inflammatory responses were triggered during normal (adaptive immune response) and preeclamptic (innate immune response) pregnancies. Activation of the innate immune cytokines in the placenta from preeclamptic women supported our hypothesis that ficolin-mediated immune recognition is responsible for the innate immunity in the development of preeclampsia.


Figure 4
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  		Figure 4. Innate immune responses in preeclampsia.

(A), innate immune activation and adaptive immune suppression in preeclamptic placenta. Placental lysates from normal (left) and preeclamptic (right) pregnancies, at the same gestational age, are compared. Representative immunoreactivity of Cytokine Antibody Array VI and VII are shown. High reactivity of proinflammatory chemokines/cytokines in innate immunity can be observed in preeclamptic placenta (arrows); and high reactivity of proinflammatory interferon/cytokines in adaptive immunity can be observed in nonpreeclamptic placenta (arrows). Other adhesion molecules/growth factors are also increased in preeclamptic placenta (arrows). The left-most 4 spots in the first row and the right-most 2 spots in the last row are positive controls, while the left-most 4 spots in the second row are negative controls. Each cytokine is provided in duplicate vertically. (B), placental and systemic innate immunity in preeclampsia. Matched placental lysate (left) and plasma (right) samples from normal (open columns) and preeclamptic (solid columns) pregnancies are analyzed (n = 10 vs 10). Chemokines/cytokines in innate immunity: MCP1/2/3, MIG, MIP1{delta}/3{alpha}, TNF{alpha}, and TECK; interferon/cytokines in adaptive immunity: IFN{gamma}, IL13/4/5, TGFß1/ß3 and TNFß; and adhesion molecules/growth factors: CNTF, EGF, ICAM1, IGFBP4, and BMP6. Mean (SD) are shown. *, P <0.05; {dagger}, P <0.01 (Wilcoxon Signed Ranks).

systemic innate immunity induces systemic endothelial dysfunction in preeclampsia
To further evaluate the systemic inflammatory responses in preeclamptic pregnancy, we compared the proinflammatory cytokines in plasma samples from preeclamptic and nonpreeclamptic women. Not all the differentially expressed placental immune cytokines showed significant differences in plasma samples (Fig. 4BUp , right panels). In the peripheral circulation of preeclamptic pregnancies compared with non-preeclamptic pregnancies, MIG and MIP1{delta}/3{alpha} (innate immunity) were significantly increased, TGFß1/ß3 and TNFß (adaptive immunity) were significantly decreased, and other molecules (CNTF, EGF, IGFBP4, and BMP6) were significantly increased (n = 10 vs 10; Fig. 4BUp ). Not only were these placental cytokines significantly correlated with their plasma cytokines, they were also significantly correlated with the maximum diastolic blood pressure of the established preeclamptic pregnancies (Fig. 5 ). Increased plasma MIG and MIP1{delta}/3{alpha} (innate immunity, Fig. 5A ) and CNTF, EGF, IGFBP4, and BMP6 (other responses, Fig. 5C ), and decreased plasma TGFß1/ß3 and TNFß (adaptive immunity, Fig. 5B ) were associated with severity of the clinical syndrome in preeclampsia. In particular, chemokines MIG and MIP1 act as chemoattractants toward monocytes, lymphocytes, and natural killer cells, which are important in establishing innate immunity in human pregnancy (18). These results confirmed the involvement of local and systemic innate immunity in the clinical syndrome of preeclampsia and also suggested that other cytokines/growth factors participate in the inflammatory response in preeclampsia.


Figure 5
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  		Figure 5. Systemic innate immunity and clinical syndrome of preeclampsia.

Cytokine profiles of plasma samples from cytokine antibody arrays are plotted against maximum diastolic blood pressure of the pregnancies with established clinical preeclampsia (n = 10). Differential reactivities of proinflammatory cytokines in preeclamptic samples are dotted. (A), innate immune chemokines/cytokines; (B), adaptive immune interferon/cytokines; (C), adhesion molecules/growth factors. Cytokine data (x axis) are presented as quantity unit; blood pressure data (y axis) are presented as mmHg. Association of circulating immune cytokines and blood pressure was tested by partial correlation with gestational age in blood collection as the control variable. Correlation coefficient r and P values are indicated.

A systemic inflammatory response occurs in preeclamptic pregnancies and, to a lesser extent, in normal pregnancies (19). Many parts of the inflammatory network are involved (inflammatory leukocytes, endothelium, clotting cascade, platelets, and acute-phase reactants), yielding minor systemic changes that have previously been considered to be part of the physiology of normal pregnancy. These features are intensified in the 3rd trimester, more so in preeclampsia, and could contribute to some of its maternal disturbances. For unknown reasons, systemic inflammatory responses are more excessive in preeclampsia than in normal pregnancy (20). In patients with established preeclampsia, we identified the specific binding of ficolins in apoptotic trophoblasts in the placenta and showed that this could induce placental innate immunity through local cytokine activation, resulting in a systemic innate immune response. We identified circulating factors from the placenta that are responsible for the local immune recognition and systemic inflammatory response in the development of clinical manifestations in preeclampsia.

new innate view of preeclampsia
Host responses to inflammatory processes depend on innate immunity, mechanisms that recognize and respond to the presence of a pathogen in the acute phase, followed by adaptive immunity, mediated by clonal selection of specific lymphocytes and leading to long-term protection from disease (21). In pregnancy, suppression of adaptive immunity may activate the maternal innate immune system (18), increasing stimulation of monocytes and granulocytes from the 1st trimester onward (22) and playing an important role in overall maternal immune defense. Excessive innate activation, however, can lead to more severe clinical presentations of some infections and to preeclampsia. In preeclamptic pregnancies, intravascular monocyte and granulocyte activation is excessive; unlike most disease states, severe preeclampsia may involve increased concentrations of IL-12, a principle cytokine of monocytes regulating the innate immune response (23). Additional cytokine abnormalities have been found in preeclamptic plasma (24). The results from this study provided the evidence, and also the basis for, the innate view of preeclamptic pregnancies.

These results provide new insight into the development of preeclampsia. From weeks 6–18 of gestation, the placenta achieves increasing access to the maternal blood supply by extensive remodeling of maternal spiral arteries through extravillous cytotrophoblast invasion (25). In preeclampsia, poor placentation occurs when invading trophoblasts fail to gain full access to the maternal uterine lining. Trophoblast signaling to maternal immune cells is weak and fails to stimulate normal immune collaboration, and a lower adaptive immune response may result. Inhibition of trophoblast invasion causes poor spiral arterial remodeling, and deficient uteroplacental circulation, leading to placental hypoxia with generation of free oxygen radicals and lipid peroxidation products. Enhanced trophoblastic apoptosis induces placental and systemic innate immunity through specific ficolin binding. The innate immune response can activate the lectin-complement pathway to remove trophoblast debris by phagocytosis. As a result, the maternal circulation may contain syncytiotrophoblast membrane microparticles, cytokeratin fragments, soluble RNA and DNA of fetal origin, and even cytotrophoblast cells (19). The activated leukocytes and their products (cytokines, chemokines, proteases, and free radicals) initiate widespread endothelial dysfunction, which underlies the clinical syndrome of preeclampsia.

Poor placentation is likely established before 20 weeks, before clinical signs appear (25). Specific ficolin binding to the dysfunctional placenta may therefore occur before clinical preeclampsia develops. Our current study detected only changes of ficolins in maternal circulation at clinical presentation. Circulating ficolins at an earlier stage may be serological markers for disease prediction. To test this hypothesis, measurements of longitudinal plasma changes of ficolin concentrations from early pregnancies are required. Interventions to prevent the development of preeclampsia have had limited success (26). Our study provides further understanding of the pathogenesis of preeclampsia and a new framework for potential clinical intervention targeting the innate immunity in preeclampsia to improve maternal and perinatal outcomes.


   Acknowledgments
 
The authors thank Prof. Dennis Lo and Rossa Chiu for assistance in sample collection, Irene Ang and Ronald Pang for assistance in proteomic studies; Prof. Misao Matsushita, Dr. Mitsushi Tsujimura, and Dr. Hiroshi Shiraki for H-ficolin, L-ficolin, MBL antibodies/antigens and the polysaccharide from Aerococcus viridans; Dr. Raymond Li for preeclamptic placental tissues; HBT for clone 4H5 HM2089; and Prof. Larry Baum for critical review. The project was partially funded by the Li Ka Shing Institute of Health Sciences Grant (Medicine/PGDC), the Special Equipment Grant 2003/04 (Medicine/FM/AC/80/5), Direct Grant for Research 2004/05 (Medicine/2004.1.073), and the Hong Kong Obstetrical & Gynaecological Trust Fund 2005 (2005/RonaldWang). The work has been partially presented at the XIX International Congress of Clinical Chemistry, Orlando, Florida, July, 2005; the Royal College of Obstetrics and Gynaecology, Blair Bell Research Society Meeting, Liverpool Women’s Hospital, London, United Kingdom, June, 2005; the Hong Kong Society of Medical Genetics, 3rd Hong Kong Medical Genetics Conference, April, 2005; the XV World Congress of the International Society for the Study of Hypertension in Pregnancy, Lisbon, Portugal, July, 2006; and the AIMS AACB Scientific Conference, Hobart, Tasmania, October 2006.


   Footnotes
 
1 Nonstandard abbreviations: 2D-PAGE, 2-dimension polyacrylamide gel electrophoresis; MBL, mannose-binding lectin; TBS, tris-based saline; MCP, monocyte chemoattractant protein; MIG, monokine induced by gamma-interferon; MIP, macrophage inflammatory protein; TNF, tumor necrosis factor; TECK, thymus expressed chemokine; IFN, interferons; IL, interleukin; TGF, transforming growth factor; CNTF, ciliary neurotropic factor; EGF, epidermal growth factor; ICAM, intercellular adhesion molecule; IGFBP, insulin-like growth factor binding proteins; BMP, bone morphogenetics protein.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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