Anti-fetal immune response mechanisms may be involved in the pathogenesis of placental abruption

Clinical Immunology 110 (2004) 45 – 54 www.elsevier.com/locate/yclim

Anti-fetal immune response mechanisms may be involved in the pathogenesis of placental abruption A. Steinborn, a,* C. Seidl, b C. Sayehli, a C. Sohn, a E. Seifried, b M. Kaufmann, a and E. Schmitt c a

Department of Obstetrics and Gynecology, University of Frankfurt, Frankfurt, Germany b Institute of Transfusion Medicine and Immunohaematology, Frankfurt, Germany c Institute of Immunology, University of Mainz, Mainz, Germany Received 21 April 2003; accepted with revision 9 September 2003

Abstract Placental abruption is an unpredictable severe complication in pregnancy. In order to investigate the possibility that the activation of the fetal nonadaptive immune system may be involved in the pathogenesis of this disease, IL-6 release from cord blood monocytes was examined by intracellular cytokine staining and flow cytometric analysis. Our results demonstrate that preterm placental abruption (n = 15) in contrast to uncontrollable preterm labor (n = 33) is associated with significantly ( P < 0.001) increased release of IL-6 from the fetal monocytes. The same holds true for rhesus disease (n = 9, P < 0.001) that is characterized by a maternal production of antibodies against the rhesus-D antigen expressed by the fetal erythrocytes. This suggests that during rhesus disease, IL-6 release of monocytes is induced by antibody-mediated cross-linking of these cells to the erythrocytes in the fetal circulation. Hence, this assumption favors the idea that also in case of placental abruption, an increased maternal antibody production against paternal antigens leads to an elevated IL-6 release by the fetal monocytes. To elucidate this potential mechanism, the presence of anti-HLA-antibodies was assessed in the maternal circulation of patients with placental abruption (n = 17) and patients with uncontrollable preterm labor (n = 29). The percentage of women producing anti-paternal HLA-antibodies was significantly ( P < 0.01) increased in the group of women with preterm placental abruption (47%) in comparison to women with uncontrollable preterm labor (14%). Therefore, our results suggest that an increased humoral immune response of the mother against the fetus may be decisively involved in the pathogenesis of placental abruption. D 2003 Elsevier Inc. All rights reserved. Keywords: Pregnancy; Placental abruption; Preeclampsia; Rhesus disease; Monocyte activation; Fetal immune system; Inflammatory cytokines; Reproductive immunology

Introduction Preterm placental abruption is an unpredictable severe complication in pregnancy that is associated with significant perinatal morbidity an mortality. Diagnosis of placental abruption is difficult. It is based on abdominal pain, vaginal bleeding, maternal hemodynamic instability, and evidence by ultrasonographic or other fetal monitoring. As yet, pathogenesis of this suddenly occurring disease is completely unknown. Up to now, extensive application of Doppler ultrasonography simply has provided the basic

* Corresponding author. Department of Obstetrics and Gynecology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany. Fax: +49-69-6301-6317. E-mail address: [email protected] (A. Steinborn). 1521-6616/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2003.09.004

finding that placental abruption shares the existence of an insufficient uteroplacental circulation with other pregnancyspecific diseases, such as spontaneous abortion, intrauterine growth retardation (IUGR), preeclampsia, and Hemolysis-, Elevated Liver enzymes, Low Platelet count (HELLP)syndrome. All these complications occur at different gestational stages and show completely different clinical symptoms. Despite of these differences, analogies in the underlying pathologic processes are observed. The occurrence of all these pregnancy-related disorders is associated with poor placental perfusion, due to inadequate trophoblast invasion in early pregnancy [1,2]. In normal pregnancy, progressive trophoblast invasion transforms the high-resistance uteroplacental spiral arteries into low-resistance blood vessels. This vascular transformation facilitates the increase in the uteroplacental blood flow that occurs between conception and term. Pathologic findings of the uterine arteries,

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such as persistent notching after 24 –26 weeks of gestation or increased resistance indices, predict the occurrence of preeclampsia, intrauterine growth retardation (IUGR), fetal distress, and intrauterine fetal death long before clinical symptoms can be detected [3,4]. In the literature, these abnormal Doppler findings were correlated with changes in placental morphologic characteristics, such as chronic inflammatory reactions and vasoocculsive lesions in the placenta [5]. Meanwhile, it is known that there is a strong systemic maternal inflammatory response beside these striking features in the placenta, especially in case of preeclampsia [6]. The broad systemic activation of cells of the maternal nonadaptive immune system gives rise to the suspicion that also reactions of the adaptive immune system of the mother may be involved in the genesis of these pregnancy-specific diseases. In normal pregnancy, the maternal immune system does not reject the semi-allogenic fetus. A decisive factor for the avoidance of rejection is the restricted HLA-expression of the trophoblast cells. In these cells, the expression of the most polymorphic and strongly antigenic HLA-class I and class II genes is suppressed. Instead, HLA-G, a minimal polymorphic class I gene, is expressed [7]. Membranebound HLA-G molecules have the capacity to inhibit natural killer (NK)-cell-mediated cytolysis as well as antigen-specific CD8+ T-cell-mediated cytolysis [8]. Its soluble forms induce the Fas/FasL-mediated apoptosis of CD8+ T-cells and NK cells [9,10]. Currently, we have demonstrated that soluble HLA-G levels are strongly decreased in the circulation of women with placental abruption in comparison to women with normal pregnancy [11]. These findings indicate that soluble HLA-G molecules have a decisive effect on suppression of fetal allograft rejection processes that may substantially be involved in the pathogenesis of placental abruption and possibly in the pathogenesis of other pregnancy-related diseases associated with insufficient trophoblast invasion. Recently, we have discussed that the occurrence of preeclampsia and HELLP-syndrome may be associated with an increased humoral immune response of the mother against the fetus. This conclusion was made because we had demonstrated that the occurrence of normal spontaneous term labor, but not uncontrollable preterm labor, was associated with increased release of IL-6 from fetal monocytes [12]. Preterm activation of these fetal monocytes was shown to correlate with the occurrence of clinical symptoms seen in patients with preeclampsia or HELLP-syndrome [13]. Monocyte activation can be mediated by cytokine release (IFN-g) from activated T-cells, but fetal T-cell activation was not detectable during the whole course of pregnancy. Alternatively, binding of Ig-coated cells (lymphocytes, endothelial cells) via Fc-receptors plays an important role in monocyte activation leading to a strong IL-6 release from these cells [14,15]. Therefore, our results suggest that fetal monocyte activation may be induced by humoral immune response mechanisms of the

mother against fetal antigens. Such maternal anti-paternal immunoglobulins may cross the placenta and react with the fetal cells in the fetal circulation. Binding of these antibody-loaded fetal cells to monocytes via Fc-receptors may then induce activation and IL-6 release from these cells. The purpose of this study was to examine whether preterm occurrence of placental abruption is also associated with fetal monocyte activation, whether increased amounts of anti-HLA-antibodies are detectable in the maternal circulation in case of placental abruption, and whether these anti-paternal HLA-antibodies are essentially directed against the fetal HLA-antigens.

Patients and methods Patient populations participating in flow cytometric studies of cord blood monocytes The patient sample population consisted of 123 women with singleton gestation who had attended our delivery rooms between January 1998 and June 1999. The study was approved by the Regional Ethics Committee. All women were fully informed of the aim of the study and informed consent was obtained from all participants. All women were stratified into four different groups according to pregnancy complications and outcome. The first group consisted of 66 healthy women delivering term neonates (38 – 42 weeks of gestation) with normal birth weight after the onset of spontaneous term labor (n = 39) or elective cesarean section, because of breech presentation or cephalopelvic disproportion (n = 27). None of these patients showed any clinical signs of pregnancy-associated diseases. The second group consisted of 33 women delivering preterm (25 – 36 weeks of gestation) because of uncontrollable intrauterine activation and suspicion of intra-amniotic infection. The patients had uncontrollable preterm labor accompanied by advanced cervical dilatation. Prolonged rupture of fetal membranes was detected in 14 cases, and 18 women had rising infectious indices (leukocytes > 10 000/Al and C-reactive protein levels > 1 mg/dl). Intra-amniotic infection confirmed by histological examination of the fetal membranes, umbilical cord, and chorionic plate was diagnosed for 17 patients. The third group consisted of 15 women delivering preterm (25 – 37 weeks of gestation) with diagnosis of placental abruption, which was defined as the complete or partial separation of the placenta before delivery. The diagnose was based on the clinical presentation and examination of the delivered placenta by the attendant at delivery. Further pregnancy complications, such as IUGR or preeclampsia, were not diagnosed for these patients. Conspicuously, four patients had elevated liver enzymes (aspartate aminotransferase and alanine aminotransferase >

A. Steinborn et al. / Clinical Immunology 110 (2004) 45–54

30 U/l), but no further clinical symptoms of HELLPsyndrome were present. For one patient, clinical diagnosis of intra-amniotic infection was histologically confirmed. All patients had an emergency delivery because of vaginal bleeding and pathological cardiotocogram (CTG). None of these patients had trauma, a history of substance abuse, or any other inciting event that could explain such a sudden and extensive detachment of the placenta. The fourth group consisted of nine non-hydrophic singleton pregnancies complicated by rhesus alloimmunization (antiRhD-titer > 1:8). All women were undergoing clinically indicated cordocentesis and transfusion because of rising anti-RhD-titers and high or increasing amniotic fluid deviation from linearity of the optical density reading at 450 nm. The mean gestational age and standard deviation at cordocentesis was 27.1 and 5.0 weeks. Severe anemia (fetal hemoglobin level < 10 g/dl) was noted in five (56%) fetal cord blood specimens obtained. All women gave verbal consent. Collection of human umbilical cord blood mononuclear cells Human venous umbilical cord blood was collected into tubes containing ethylenediaminetetraacetic acid (EDTA) from neonates immediately after delivery. The cord was double clamped and the blood was taken from the tucked part of the cord or from the placental side of the clamp. Mononuclear cells (MNC) were separated by Ficoll density gradient centrifugation. Cord blood was collected from four different patient groups described above. Women in the fourth group had a cord blood transfusion because of severe fetal anemia due to rhesus alloimmunization of the mother. Flow cytometric analysis Determination of IL-6 producing monocytes in the cord blood of the different patient groups was done using twocolor flow cytometric analysis. The surface phenotype of MNC was estimated by staining 1  106 cells with 0.5 Ag of FITC-conjugated mAb specific for CD14 expressed on monocytes. Cells were washed twice with PBS supplemented with 0.5% FCS, 5 mM EDTA, and centrifuged. Intracellular staining for IL-6 was done using Cyto stain kits purchased from Pharmingen according to the manufacturer’s instructions. Briefly, cells were fixed and permeabilized by incubating cells in 250-Al Cytofix/Cytoperm solution for 20 min at 4jC and resuspended in 50-Al perm/wash solution containing 0.1 Ag of rat-anti-human IL-6 IgG1 mAb. Cells were incubated at 4jC for 30 min. Negative staining controls were done using an IgG1 isotype control of irrelevant specificity in the same concentration as the anti-IL-6 mAb. Staining was analyzed by flow cytometry (FACScan, Becton Dickinson, Heidelberg, Germany). Analysis was based on both forward and side scatter characteristics. Only monocytes and granulocytes were gated; lymphocytes were elim-

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inated from analysis. The percentage of IL-6 positive monocytes was estimated within four different patient groups. Patient populations participating in HLA-molecular typing and HLA-antibody detection The patient sample population consisted of 106 women with singleton gestation who had attended our delivery rooms between July 1999 and February 2002. The study was approved by the Regional Ethics Committee. All women were fully informed of the aim of the study and informed consent was obtained from all participants. All women were stratified into three different groups according to pregnancy complications and outcome. The first group consisted of 60 healthy women delivering term neonates after the onset of normal spontaneous term labor or elective cesarean section (38 – 42 weeks of gestation). The second group consisted of 29 women delivering preterm (24 –37 weeks of gestation) because of uncontrollable intrauterine activation and suspicion of intra-amniotic infection. The patients had uncontrollable preterm labor accompanied by advanced cervical dilatation. Prolonged rupture of fetal membranes was detected in 10 cases and 15 women had

Table 1 Clinical data and percentage of IL-6 positive monocytes in the cord blood of patients with normal term and preterm delivery

Uncontrollable preterm labor Prolonged PROM Increased infect. indicesa Chorioamnionitisb IUGRc Hypertensiond Proteinuriae Elevated liver enzymesf Thrombocytopeniag IL-6 positive monocytes (%) Median value Range

Normal term delivery, 38 – 42 weeks, n = 66

Preterm intrauterine activation, 25 – 36 weeks, n = 33

Preterm placental abruption, 25 – 37 weeks, n = 15

Rhesus diseases cordocentesis, 23 – 36 weeks, n=9

0/66

33/33

0/15

0/9

0/66 0/66

14/33 18/33

0/15 0/15

0/9 0/9

n.d. 0/66 0/66 0/66 0/66

17/33 0/33 0/33 0/33 0/33

1/15 0/15 0/15 0/15 4/15

n.d. 0/9 0/9 0/9 0/9

0/66

0/33

0/15

0/9

16.1 1.0 – 78.7

3.0 1.0 – 21.9

20.2 51.0 9.0 – 58.0 18.0 – 72.0

PROM: preterm rupture of fetal membranes; IUGR: intrauterine growth retardation; n.d.: not detected. a Leukocytes > 10 000/Al; C-reactive protein levels > 1 mg/dl. b Extensive invasion of the fetal membranes with granulocytes and monocytes accompanied by small necrosis. c Birth weight < 10th percentile for gestational age. d Blood pressure > 140/90 mm Hg in two determinations apart. e >300 mg per 24 h. f Aspartate aminotransferase and alanine aminotransferase > 30 U/l. g Thrombocyte count < 150 000/Al.

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rising infectious indices (leukocytes > 10 000/Al and Creactive protein levels > 1 mg/dl). Intra-amniotic infection confirmed by histological examination of the fetal membranes, umbilical cord, and chorionic plate was diagnosed for six patients. The third group consisted of 17 women delivering preterm (25 – 37 weeks of gestation) with diagnosis of placental abruption, which was defined as described above. One patient had hypertension (blood pressure > 140/ 90 mm Hg) in two determinations 4 h apart, but proteinuria (>300 mg per 24 h) was not diagnosed. Prolonged rupture of fetal membranes was detected for one patient. Further pregnancy complications were not diagnosed for these patients.

All patients had an emergency delivery because of vaginal bleeding and pathological cardiotocogram (CTG). None of these patients had trauma, a history of substance abuse, or any other inciting event that could explain such a sudden and extensive detachment of the placenta. Detection of anti-HLA-class-I and II-specific antibodies in the maternal circulation Anti-HLA-class-I and II antibodies were detected in the ethylenediaminetetraacetic acid (EDTA) plasma obtained from all women in three different groups. Blood

Fig. 1. Expression of IL-6 in cord blood monocytes of different patient populations. In comparison to women delivering preterm because of uncontrollable intrauterine activation (labor, rupture of fetal membranes, cervical insufficiency), the percentage of IL-6-positive monocytes was significantly increased in the group of women delivering preterm because of placental abruption. The percentage of IL-6 positive monocytes was also significantly increased in the group of women with rhesus disease.

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samples were taken at admission into the delivery room, normally within 24 h before delivery, and centrifuged at 1500  g for 10 min at 4jC. The plasma was removed and stored in aliquots at 80jC until assayed. Antibody (IgG) detection was performed using ‘‘PRA-STAT 2 patients per Plate Format’’ qualitative ELISA kits (SangStat Medical Corporation, Menlo Park, CA, USA) according to manufacturer’s instructions. The data were then analyzed using SangStat proprietary software (SOFTSTAT II, SangStat Medical Corporation). HLA-molecular typing HLA-molecular typing was done for 12 patients delivering preterm because of placental abruption (n = 8) or because of uncontrollable intrauterine activation (n = 4) who had a positive anti-HLA-antibody test. Molecular HLA-typing was also done for the neonates delivered by these women. Both maternal blood samples and cord blood samples were collected into tubes containing EDTA and centrifuged at 1500  g for 10 min at 4jC immediately after delivery. The plasma was removed and the buffy coat was separated and stored at 80jC until assayed. DNA extraction from 200-Al buffy coat obtained from both maternal and fetal EDTA-blood samples was performed using Qiagen extraction columns (QIAamp Mini Kit, Qiagen, Hilden, Germany). Mother and fetus were typed for HLA-A, -B, -DRB1 and -DQB1 alleles by HLA locusspecific PCR amplification of genomic DNA and hybridization with sequence-specific oligonucleotides (PCR-SSO)

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(Dynal Reli SSO-typing kit, Dynal Biotech GmbH, Hamburg, Germany). Statistical analysis Statistical comparison of the percentages of IL-6 positive cells among the different patient populations was done using the nonparametric H test of Kruskal and Wallis, which is used for simultaneous comparison of more than two sample populations. Each H test was followed by a Dunn test. P < 0.05 was considered significant. Statistical evaluation of the presence of anti-HLA-antibodies in the maternal circulation of the different patient populations was done using Fisher’s Exact Test. P < 0.05 was considered significant.

Results Placental abruption but not uncontrollable preterm intrauterine activation (labor, rupture of fetal membranes, cervical insufficiency) is associated with fetal monocyte activation In this study, cord blood monocytes obtained from neonates after normal term delivery (spontaneous term labor, elective cesarean section, 37 – 42 weeks of gestation), preterm intrauterine activation (uncontrollable labor, prolonged rupture of fetal membranes, cervical insufficiency, 25 – 36 weeks of gestation), preterm placental abruption (25 – 37

Fig. 2. Flow cytometric analysis of IL-6 expression in CD14-positive cord blood monocytes. The blood was obtained from one representative neonate of each group: after preterm delivery because of uncontrollable intrauterine activation, after preterm delivery because of placental abruption, and after clinically indicated cordocentesis because of rhesus disease. An increased percentage of IL-6 positive monocytes is detectable in case of both placental abruption and Rhesus disease. Negative staining controls were done using IgG1 isotype control of irrelevant specificity at the same concentration as the IL-6 monoclonal antibody and are shown on the left.

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weeks of gestation), and cordocentesis because of rhesus disease (24 – 36 weeks of gestation) were analyzed for intracellular IL-6 staining by two-color flow cytometry. Analysis was performed on cells isolated by FICOLL density gradient centrifugation immediately after delivery. The gate was set for both forward and side scatter and included monocytes and contaminating granulocytes whereas lymphocytes were excluded. Table 1 summarizes further clinical data and the percentage of IL-6 positive monocytes detected in the cord blood obtained from the patients in these four groups. In comparison to cord blood cells obtained from neonates after preterm delivery because of uncontrollable intrauterine activation, a significantly ( P < 0.001) increased percentage of IL-6 positive monocytes was detected in the cord blood of women delivering preterm because of placental abruption (Figs. 1 and 2). There were no significant differences between women that delivered preterm because of placental abruption and those that delivered at term in the absence of any complications. Fig. 3 presents all data concerning IL-6 release from fetal monocytes obtained from preterm- and term-delivered neonates. These results clearly demonstrate that IL-6 release from fetal monocytes increases during course of pregnancy and that maximal IL-6 production occurs at the end of pregnancy in association with the onset of normal spontaneous term labor. Obviously, in case of placental abruption, a comparatively high percentage of fetal monocytes produces increased amounts of IL-6 before term. Such results indicate an upregulation of mechanisms inducing fetal monocyte activation at a rather early stage of pregnancy. In comparison to women that delivered preterm because of

intrauterine activation, significantly ( P < 0.001) increased amounts of IL-6 positive monocytes were also found in the cord blood of patients undergoing clinically indicated cordocentesis because of rhesus disease (Figs. 1 and 2). There were no significant differences in the percentage of IL-6 positive monocytes in the cord blood between neonates delivered preterm because of placental abruption and unborn fetuses of alloimmunized RhD-negative mothers. There were also no significant differences in the percentage of IL-6 positive monocytes in the cord blood between women with rhesus disease and women with normal term delivery. Placental abruption is associated with significantly increased amounts of maternal anti-fetal-HLA-antibodies in the maternal circulation Testing for the presence of antibodies directed against HLA antigens was performed using PRA-STAT-ELISA kits in combination with SOFT-STAT II software. SOFTSTAT II is a computer program designed to efficiently retrieve and analyze the results of the PRA-STAT assay. Panel reactive antibody (PRA) testing allows the qualitative detection of anti-HLA-class-I and II IgG antibodies in the maternal circulation. In most cases, interpretation of the results also allows the definition of antibody specificity. In this study, PRA testing was done with plasma samples obtained from 29 preterm-delivering women because of uncontrollable intrauterine activation (labor, prolonged rupture of fetal membranes, cervical insufficiency, 24– 37 weeks of gestation), 17 preterm-delivering women because of placental abruption (25 – 37 weeks of gestation), and 60

Fig. 3. The percentage of IL-6 positive cord blood monocytes obtained from neonates delivered preterm or at term (w), and from neonates delivered because of placental abruption (x). The percentage of IL-6 positive monocytes in fetal cord blood increases with gestational age, while maximal cytokine expression was detected in association with spontaneous term delivery. Obviously, in case of placental abruption, fetal monocyte activation occurs at earlier gestation than in normal pregnancy.

A. Steinborn et al. / Clinical Immunology 110 (2004) 45–54 Table 2 Clinical data and number (percentage) of anti-HLA-antibody positive patients in normal term and preterm delivery

Uncontrollable preterm labor Prolonged PROM Increased infect. indicesa Chorioamnionitisb IUGRc Hypertensiond Proteinuriae Elevated liver enzymesf Thrombocytopeniag Number (percentage) of anti-HLA-antibody positive patients

Table 3 Detection of anti-HLA-antibodies in the maternal circulation Patient

Clinical diagnosis

Maternal genotype

Fetal genotype

Antibody specificity

1

placental abruption placental abruption placental abruption placental abruption placental abruption placental abruption placental abruption placental abruption uncontrollable preterm labor uncontrollable preterm labor uncontrollable preterm labor uncontrollable preterm labor

A*02 B*08, *18 A*11, *24 B*18, *44 A*01, *03 B*07, *08 A*32 B*38, *40 A*26, *30 B*07, *44 A*02 B*07, *44 A*01, *02 B*27, *38 A*02, *03 B*27, *35 A*02, *30 B*18, *49 A*02 B*08, *15 A*24, *29 B*15, *44 A*02, *03 B*07, *51

A*02, *03 B*18, *51 A*11, *24 B*44, *49 A*03 B*07, *13 A*32, *68 B*38, *45 A*26, *68 B*44, *57 A*02, *24 B*07, *39 A*01, *25 B*38, *58 A*03, *23 B*35, *44 A*30 B*18, *42 A*02 B*08, *40 A*11, *29 B*35, *44 A*2, *24 B*51, *55

Anti-B*51

Normal term delivery, 38 – 42 weeks, n = 60

Preterm intrauterine activation, 24 – 37 weeks, n = 29

Preterm placental abruption, 25 – 37 weeks, n = 17

2

0/60

29/29

0/17

3

0/60 0/60

10/29 15/29

1/17 0/17

4

n.d. 0/60 0/60 0/60 0/60

6/29 0/29 0/29 0/29 0/29

n.d. 0/17 1/17 0/17 0/17

0/60 13/60 (22%)

0/29 4/29 (14%)

0/17 8/17 (47%)

5 6 7 8

PROM: preterm rupture of fetal membranes; IUGR: intrauterine growth retardation; n.d.: not detected. a Leukocytes > 10 000/Al; C-reactive protein levels > 1 mg/dl. b Extensive invasion of the fetal membranes with granulocytes and monocytes accompanied by small necrosis. c Birth weight < 10th percentile for gestational age. d Blood pressure > 140/90 mm Hg in two determinations apart. e >300 mg per 24 h. f Aspartate aminotransferase and alanine aminotransferase > 30 U/l. g Thrombocyte count < 150.000/Al.

term-delivering women after the onset of normal spontaneous term labor or elective cesarean section (38 – 42 weeks of gestation). Further clinical data of the patients in these three groups are given in Table 2. Presence of anti-HLA-antibodies was detected for four patients with uncontrollable intrauterine activation (14%), for eight

Fig. 4. Detection of anti-HLA antibodies in the circulation of patients with preterm and normal term delivery. In comparison to women delivering preterm because of uncontrollable intrauterine activation (labor, rupture of fetal membranes, cervical insufficiency), significantly increased amounts of anti-HLA-antibodies were found in the circulation of women delivering preterm because of placental abruption, but not in comparison to women with normal term delivery.

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9 10 11 12

Anti-B*49 Anti-B*13 Anti-B*45 Anti-B*57 Anti-A*24 Anti-A*25 Anti-B*44 Anti-B*42 Anti-B*40 Anti-A*11 Anti-A*24

patients with placental abruption (47%), and for 13 patients with normal term delivery (22%). Statistical evaluation revealed that the frequency of having a positive anti-HLA-antibody test was significantly increased in the group of patients with placental abruption in comparison to women with uncontrollable intrauterine activation ( P < 0.01), but not in comparison to women with normal term delivery ( P = 0.06) (Fig. 4). In order to examine whether these anti-HLA-antibodies detected in the maternal circulation were directed against paternal HLA-antigens expressed by the fetus, molecular HLA-typing was done for both maternal and fetal leukocytes. Table 3 summarizes the results including the specificity of the anti-HLA-antibodies detected in the maternal circulation. In all cases, the antibody specificity was shown to be directed against a nonmaternal, fetal HLA-A or HLAB locus. Therefore, our results clearly indicate that humoral immune response mechanisms may occur in pregnancy and that these antibodies are directed against paternal HLA class I antigens expressed by the fetus. Increased humoral immune reactions against paternal HLA class II antigens were not detected (data not shown).

Discussion In the present study, we have documented that the occurrence of preterm placental abruption is associated with an intensive activation of monocytes in the fetal circulation. The same phenomenon was observed in case of rhesus

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disease. Rhesus disease is known to be caused by the induction of maternal antibodies against the rhesus-D-antigen expressed on the fetal erythrocytes. These anti-rhesus-D antibodies may cross the placenta and react with the fetal erythrocytes in the fetal circulation. Cross-linking of these antibody-loaded erythrocytes to the fetal monocytes via Fcreceptors expressed on the monocytes may induce IL-6 release from these cells (Fig. 5B). Induction of IL-6 release from monocytes through cross-linking of antibody-loaded cells to monocytes via Fc-receptors is a well-known mechanism and described in the literature [14,15]. Because an increased IL-6 release from fetal monocytes was detectable in both alloimmunized rhesus-D-negative women and women that delivered preterm because of placental abruption, it seems likely that the maternal production of anti-paternal HLA-antibodies may have the same effect on fetal monocyte activation. In this case, cross-linking of fetal monocytes to other fetal cells (leukocytes, endothelial cells) may be mediated by maternal anti-paternal HLA-antibodies, which have reached the fetal circulation (Fig. 5A). Therefore, the phenomenon of monocyte activation in the fetal circulation probably reflects an increased humoral immune response of the mother against antigens expressed by fetal cells but not maternal cells. In case of rhesus disease, anti-rhesus-D antibodies may couple the fetal monocytes to the fetal erythrocytes, while in case of placental abruption, antipaternal-HLA-antibodies may couple the fetal monocytes to other leukocytes or endothelial cells. These assumptions are further confirmed by the fact that significantly increased amounts of anti-HLA-antibodies were detected in the circulation of women with placental abruption, in comparison to women delivering preterm because of uncontrollable intrauterine activation and by the fact that the specificity of these antibodies was shown to be directed against the paternal HLA-antigens expressed by the fetus. Therefore, our findings suggest that an exaggerated maternal humoral immune response of the mother against the fetus may probably be involved in the pathogenesis of placental abruption. Like other pregnancy-related diseases, such as preeclampsia and fetal growth retardation, the occurrence of placental abruption is associated with an insufficient trophoblast invasion in early pregnancy. Yet, there is little evidence that alloimmune stimulation may also be involved in the pathogenesis of these diseases. It is known that severe pregnancyinduced hypertension is a strong risk factor for the occurrence of placental abruption [16]. Moreover, it is reported that systemic immunological deviations toward suppression or decreased activity of the immunological response are seen in normal pregnancies, but these deviations are not seen in preeclampsia, intrauterine growth retardation, and placental abruption [17]. Although such findings provide only a weak indication that the immune system may be involved in the pathogenesis of all these diseases, it is striking that these complications do not only share the common feature of insufficient trophoblast invasion in early pregnancy, but also show similarities concerning immunological reaction pro-

Fig. 5. Mechanism of fetal monocyte activation without participation of fetal T-cell responses. (A) In case of placental abruption and (B) in case of rhesus disease: An increasing cell traffic between mother and fetus or shedding of soluble fetal HLA-antigens at the end of pregnancy may provoke the production of maternal antibodies against paternal HLAantigens expressed by the fetus. These IgG antibodies may cross the placenta and bind to HLA-antigen expressing fetal leukocytes or endothelial cells in the fetal circulation. Cross-linking of these antibodyloaded cells to the fetal monocytes via Fc-receptors may cause IL-6 release from the fetal monocytes. In case of rhesus disease, the RhD-negative mother is producing antibodies against the RhD-antigen expressed by the fetal erythrocytes. These antibodies may also cross the placenta and bind to fetal erythrocytes in the fetal circulation. Cross-linking of these antibodyloaded erythrocytes to fetal monocytes via Fc-receptors may cause monocyte activation by the same mechanism as described above. Therefore, detection of activated fetal monocytes may reflect the induction of a maternal humoral immune response against paternal HLA-antigens.

files in the fetal circulation. Both preeclampsia and placental abruption are associated with preterm activation of the fetal monocytes, indicating the existence of an increased humoral immune response of the mother against the semi-allogenic fetus. Especially, the maternal syndrome of preeclampsia has previously been ascribed to a generalized maternal endothelial cell dysfunction. Further investigations demonstrated

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that endothelial dysfunction is only part of a more generalized intravascular inflammatory reaction involving extensive leukocyte activation as well as platelet activation [18]. That kind of inflammatory response during course of pregnancy was shown to be nonspecific, because effector cells of the nonadaptive immune system such as granulocytes and monocytes are activated [19,20]. Currently, further investigations demonstrated that the activation of cells of the adaptive immune system may also be involved in the pathogenesis of preeclampsia. A study conducted in patients with preeclampsia and normal pregnancy revealed that preeclampsia is associated with an increase in the percentage of maternal CD45RO-positive T-lymphocytes (memory cells) and a decrease in the percentage of CD45RA-positive T-lymphocytes (naive cells) [21]. Such an increase in the percentage of CD45RO-positive memory T-cells indicates that patients with preeclampsia have evidence of previous antigenic exposure. The stimulus responsible for such an activation of cells of the adaptive immune system remains elusive; nevertheless, these results are in good agreement with our proposition that both preeclampsia and placental abruption may be a problem of pathological alloimmune stimulation. Our findings conflict with published data confirming an upregulation of cell-mediated immunity (Th-type-1 reactions) and downregulation of humoral immunity (Th-type2 reactions) in preeclamptic patients [22,23]. However, recently, experimentally induced preeclampsia in mice was shown to be affected by both excessive Th1 and Th2 activation [24]. Such findings propose that there is a general enhancement of immune response mechanisms in pregnant women developing preeclampsia and possibly also in women developing placental abruption. Despite similarities in the pathogenesis of pregnancyrelated diseases associated with insufficient trophoblast invasion, all of these complications are characterized by completely different clinical symptoms. Therefore, the basic cause of all these complications may be deficiencies in tolerance induction involving two different pathways of allorecognition. Both the direct and the indirect pathway contribute to allograft rejection. Although it has been suggested that the direct pathway predominates during early acute rejection and that the indirect pathway provides a continuous supply of alloantigen responsible for chronic rejection, the true relative contribution of each pathway on the overall rejection process is still not entirely known [25]. Recently, we have shown that in comparison to women delivering preterm because of uncontrollable intrauterine activation, significantly increased levels of soluble HLADR (sHLA-DR) molecules are detectable in the circulation of patients with HELLP-syndrome, while strongly decreased sHLA-DR levels were detected in the circulation of patients with fetal growth retardation, preeclampsia, and placental abruption [26]. Such an increase of sHLA-DR levels is reported for acute graft rejection after organ transplantation or severe graft versus host disease occurring

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after bone marrow transplantation [27]. In contrast, complications such as placental abruption and preeclampsia obviously show marked similarities with chronic rejection processes that are also associated with increased production of donor-specific HLA-antibodies [28]. In conclusion, our results support the theory that a humoral immune response of the mother against paternal HLA-antigens is established at the end of normal pregnancy. Obviously, in case of placental abruption, an excessive immune response occurs at a very early stage of pregnancy. Therefore, dysregulation of tolerance inducing mechanisms may be an important factor in the pathogenesis of placental abruption, and probably also in the pathogenesis of other complications in pregnancy associated with insufficient trophoblast invasion.

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