Objective
During an uncomplicated pregnancy the conceptus is a semiallogeneic entity in which rejection is prevented by suppression of the maternal immune system. We hypothesized that this suppression is disturbed in patients with preeclampsia and that a maternal immune response to fetal (foreign/paternal) antigens in the fetal-maternal interface may be responsible for local inflammation, with subsequent endothelial dysfunction and systemic disease.
Study Design
Blood samples were obtained from 14 women with preeclampsia (cases), 14 gestational-age and parity-matched women with uncomplicated pregnancies (controls), and their partners. We determined the partner-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) and the CTLpf directed to unrelated partners with uncomplicated pregnancies. We measured the CTLpf in peripheral blood mononuclear cells (PBMCs) from cases and controls using limited-dilution assays. In addition, proliferation was tested in a mixed-lymphocyte culture (MLR).
Results
The partner-specific CTLpf was significantly higher in cases compared with controls (median, 183 [15-338] vs 67 [9-232] per million PBMCs, P = .02). In contrast, in women with uncomplicated pregnancies, the partner-specific CTLpf was down-regulated compared with the CTLpf directed to an unrelated partner who fathered uncomplicated pregnancies ( P = .02). No difference was found in partner-specific MLR response between cases and controls.
Conclusion
These results suggest that women with preeclampsia have a higher cytotoxic T-cell response to paternal antigens compared with pregnant controls. This insufficiently suppressed immune response may eventually lead to the development of preeclampsia.
Preeclampsia is a common, pregnancy-specific syndrome defined by clinical findings of high blood pressure and proteinuria. Although the outcome is often good, preeclampsia can be devastating and life threatening for both mother and neonate. Although its full etiology is still unknown, research over the last decades suggests its origin in abnormal placentation. Abnormal placentation is characterized by failure of the spiral arteries to remodel, with subsequently decreased placenta perfusion that ultimately results to systemic effects.
Immunological and inflammatory responses are proposed to play a major role in the pathophysiology of this process. Normal pregnancy is generally considered as a Th2 cell–driven phenomenon, characterized by a large production of placental factors that strongly favors an immunosuppressive activity, potentially preventing the rejection of the fetus. Predominant Th1-type immunity in pregnancy is associated with abnormal placentation and clinically in spontaneous abortion.
In line with this hypothesis is that abnormal placentation described in women with pregnancies that are complicated by preeclampsia might be the result of perturbed maternal immune tolerance to the fetus. The fetus is a semiallograft, and half of its histocompatibility antigens are of paternal origin. This could result in a stronger maternal alloimmune reaction with the ability potentially to result in abnormal placentation with finally activation of maternal vascular endothelium. Pregnancy complicated by preeclampsia might be comparable with rejection in transplant studies; the inherited paternal antigens of the fetus are supposed the transplant.
Therefore, to study the mechanism of disturbed immune suppression in women with preeclampsia, analogous to transplant rejection studies, we studied the immune response measuring cytotoxic T-cell precursor frequencies (CTLpf) and mixed lymphocyte reactions (MLRs) in women with preeclampsia (cases) and women with uncomplicated pregnancies (controls). Peripheral blood mononuclear cells (PBMCs) from their partners acted as stimulators of these responses to evaluate the allogeneic impact of class I and class II human leukocyte antigen (HLA) mismatches between donor (fetal/paternal antigens) and graft recipient (mother).
To study the influence of paternal factors, we also used stimulator-PBMCs from partners who also fathered a pregnancy complicated by preeclampsia (case-partner) or from an unrelated partner who fathered at least 2 uncomplicated pregnancies (second control–partner).
Materials and Methods
Patient groups
For this study women and their partners were recruited from the obstetric department of the Erasmus Medical Center Rotterdam (Rotterdam, The Netherlands). Women who developed preeclampsia during their pregnancy (cases, n = 14) and control subjects (n = 14) were selected and followed up during the remainder of their pregnancy. The study protocol was approved by the local ethics review board. Data were analyzed anonymously.
Preeclampsia was defined as blood pressure greater than 140 mm Hg systolic or 90 mm Hg diastolic measured on at least 2 occasions in women normotensive before 20 weeks of gestation and proteinuria (≥2+ [1 g/L] on a voided specimen or ≥1+ [0.3 g/L] on a catheterized specimen) according to the International Society for the Study of Hypertension in Pregnancy.
Women with uncomplicated pregnancies were controls and selected according to the following criteria: no rise in blood pressure, no hypertension or proteinuria, similar age (±5 years), similar parity, no biological relationship, and a delivery date as close as possible to the delivery dates of the case group. Women who suffered from chronic hypertension, renal disease, diabetes, collagen vascular diseases, cancer, or thrombosis before their pregnancy were excluded from the study.
Peripheral blood was drawn from the women and their partners. An additional control group of partners was used who fathered 2 or more healthy pregnancies (second control–partner).
Laboratory data
PBMC sampling
PBMCs of patients and their partners were isolated using a Ficoll-Paque (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient, collected, washed, and stored at –140°C until use.
Limiting dilution analysis
limiting dilution cultures were set up as described previously. In brief, 12 replicates of graded number responder PBMCs were titrated in 7 step double dilutions starting from 5 × 10 4 to 781 PBMCs/well and stimulated with irradiated (40 Gy) partner or control PBMC (5 × 10 4 cells/well) in 200 μL culture medium containing recombinant interleukin (IL)-2 (20 U/well, 12.2 ng/mL IL-2; Chiron BV, Amsterdam, The Netherlands). Additionally, 12 wells contained stimulator cells alone.
After 7 days of culture, each well was individually tested for cytolytic activity against 5 × 10 3 Europium-diethylentriamene pentaacetate–labeled target cells (T-cell blasts, cultured with phytohemagglutinin [PHA; Murex Biotech Ltd, Kent, United Kingdom] and recombinant IL-2 [Chiron]). After 4 hours of incubation, the plates were centrifuged and 20 μL of the supernatant was harvested. Fluorescence of the released Europium was measured in a time-resolved fluorometer and was expressed in counts per second. The mean counts per second of the wells in which only stimulator cells were present, were considered as background.
Experimental wells were scored positive if the counts in that well exceeded the mean plus 3 times SD of the wells in which only stimulator cells were present. For each cell concentration, the number of negative wells was determined and used to calculate the frequency with a computer program designed by Strijbosch et al. The CTLpf was expressed as the number of cytotoxic T-lymphocyte precursors per 10 6 PBMCs.
Mixed lymphocyte culture (MLC) and reaction
MLCs were set up with 100 μL of a 5 × 10 4 responder PBMC suspension in culture medium added in triplicate wells in a round-bottom 96 well plate to 100 μL of (a) 5 × 10 4 irradiated (40 Gy) PBMCs from the partner or control PBMCs or culture medium alone. PHA (2 μg/mL of purified PHA-HA16; Murex Biotech) was used to check the viability of the cells. After 7 days (for PHA, 3 days), proliferation was measured by incorporation of 3 H-thymidine added during the last 8 hours of culture.
The stimulation index (SI) was calculated by the ratio of the counts per minute obtained in the presence of antigen to the counts per minute in the absence of antigen. Only the results of viable cells (SI ≥50 after PHA stimulation) from cases, partners, and controls were analyzed in the described results. For some patients not enough cells were available to perform all tests.
Scheme in vitro stimulation
The T-cell response of the cases was measured against their own partners, against a partner from another case (case-partner), and against an unrelated partner who fathered at least 2 uncomplicated pregnancies (second control–partner). The controls were tested against their own partner, against an unrelated partner who fathered at least 2 uncomplicated pregnancies (second control–partner), and against a partner who fathered a pregnancy complicated by preeclampsia (case-partner).
Statistical analysis
The significance of differences between women with preeclampsia (cases) and women with uncomplicated pregnancies (controls) were analyzed using the Mann Whitney U test and Kruskal-Wallis test as appropriate (1-way analysis of variance for multiple comparisons of nonparametric data). The significance of the difference between the response to different stimulator cells was analyzed using the paired Wilcoxon signed rank test. Two-sided P < .05 was considered significant.
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