János Rigó

Circulating cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia determined by multiplex suspension array

BackgroundPreeclampsia is a severe complication of pregnancy characterized by an excessive maternal systemic inflammatory response with activation of both the innate and adaptive arms of the immune system. Cytokines, chemokines and adhesion molecules are central to innate and adaptive immune processes. The purpose of this study was to determine circulating levels of cytokines, chemokines and adhesion molecules in normal pregnancy and preeclampsia in a comprehensive manner, and to investigate their relationship to the clinical features and laboratory parameters of the study participants, including markers of overall inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) and endothelial injury (fibronectin), oxidative stress (malondialdehyde) and trophoblast debris (cell-free fetal DNA).ResultsSerum levels of interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, interferon-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were measured in 60 preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women by multiplex suspension array and ELISA. In normal pregnancy, the relative abundance of circulating IL-18 over IL-12p70 and the relative deficiency of the bioactive IL-12p70 in relation to IL-12p40 might favour Th2-type immunity. Although decreased IL-1ra, TNF-alpha and MCP-1 concentrations of healthy pregnant relative to non-pregnant women reflect anti-inflammatory changes in circulating cytokine profile, their decreased serum IL-10 and increased IP-10 levels might drive pro-inflammatory responses. In addition to a shift towards Th1-type immunity (expressed by the increased IL-2/IL-4 and IFN-gamma/IL-4 ratios), circulating levels of the pro-inflammatory cytokines IL-6 and TNF-alpha, the chemokines IL-8, IP-10 and MCP-1, as well as the adhesion molecules ICAM-1 and VCAM-1, were raised in preeclampsia compared with healthy pregnancy, resulting in an overall pro-inflammatory systemic environment. Increased IP-10, MCP-1, ICAM-1 and VCAM-1 concentrations of preeclamptic patients showed significant correlations with blood pressure values, renal and liver function parameters, as well as with CRP, malondialdehyde, von Willebrand factor antigen and fibronectin levels.ConclusionsAccording to our findings, preeclampsia was associated with an overall pro-inflammatory systemic environment. Elevated amounts of pro-inflammatory cytokines, chemokines and adhesion molecules in the maternal circulation might play a central role in the excessive systemic inflammatory response, as well as in the generalized endothelial dysfunction characteristics of the maternal syndrome of preeclampsia.

Activation of the complement system in normal pregnancy and preeclampsia.

The purpose of this study was to explore the role of the complement system in normal human pregnancy and preeclampsia in a comprehensive manner, measuring circulating levels of complement proteins, their activation fragments and regulatory factors, as well as those of C-reactive protein (CRP). Sixty preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Circulating levels of complement components and CRP were determined with ELISA, radial immunodiffusion and particle enhanced immunoturbidimetric assay. Levels of CRP, C4d, C3a, SC5b9, C3, C9 and factor H antigen were significantly higher, while those of C1-inhibitor were significantly lower in healthy pregnant than non-pregnant women. In addition, preeclamptic patients had significantly higher CRP, C4d, C3a, SC5b9 levels and significantly lower C3 concentrations as compared to healthy pregnant women. Their CRP, C4d, C3a, SC5b9, C4, C3, C9 and factor H antigen levels were significantly higher, while C1-inhibitor concentrations were significantly lower compared with healthy non-pregnant women. However, no significant difference was found in Bb and C4b-binding protein levels among the three study groups. Preeclamptic patients with fetal growth restriction had significantly higher plasma SC5b9 levels than those without IUGR. There was a relative deficiency of C1-inhibitor and C4b-binding protein, and a relative abundance of factor H both in normal pregnancy and preeclampsia. Activation of the classical or lectin pathway (C4d) showed significant positive correlation to C3 activation (C3a) both in healthy pregnant women and preeclamptic patients. However, the correlation between C3 and terminal pathway activation was dominating only in patients with preeclampsia, but not in healthy pregnant women. In conclusion, the complement system is activated through the classical and/or lectin pathways with increased terminal complex formation in the third trimester of normal human pregnancy, and further in preeclampsia, as shown by the elevated amounts of activation markers in the systemic circulation. Excessive activation of the terminal pathway is associated with fetal growth restriction in preeclamptic women. However, additional studies are required to determine the cause and consequence of systemic complement activation in this pregnancy-specific disorder.

Increased Prevalence of IL-17-Producing Peripheral Blood Lymphocytes in Pre-eclampsia

Citation 
Toldi G, Rigó J Jr, Stenczer B, Vásárhelyi B, Molvarec A. Increased prevalence of IL‐17‐producing peripheral blood lymphocytes in pre‐eclampsia. Am J Reprod Immunol 2011; 66: 223–229

Serum leptin levels in relation to circulating cytokines, chemokines, adhesion molecules and angiogenic factors in normal pregnancy and preeclampsia.

ObjectiveIn this study, we determined circulating levels of C-reactive protein, several cytokines, chemokines, adhesion molecules and angiogenic factors along with those of leptin in healthy non-pregnant and pregnant women and preeclamptic patients, and investigated whether serum leptin levels were related to the clinical characteristics and measured laboratory parameters of the study participants.MethodsSixty preeclamptic patients, 60 healthy pregnant women and 59 healthy non-pregnant women were involved in this case-control study. Levels of leptin and transforming growth factor (TGF)-beta1 in maternal sera were assessed by ELISA. Serum levels of interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p40, IL-12p70, IL-18, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interferon-gamma-inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were determined by multiplex suspension array. Serum C-reactive protein (CRP) concentrations were measured by an autoanalyzer. Serum total soluble fms-like tyrosine kinase-1 (sFlt-1) and biologically active placental growth factor (PlGF) levels were determined by electrochemiluminescence immunoassay. For statistical analyses, non-parametric methods were applied.ResultsThere were significant differences in most of the measured laboratory parameters among the three study groups except for serum IL-1beta and TGF-beta1 levels. Serum leptin levels were significantly higher in preeclamptic patients and healthy pregnant women than in healthy non-pregnant women. Additionally, preeclamptic patients had significantly higher leptin levels as compared to healthy pregnant women. Serum leptin levels were independently associated with BMI in healthy non-pregnant women. In healthy pregnant women, both BMI and serum CRP concentrations showed significant positive linear association with leptin levels. There were significant positive correlations between serum leptin concentrations of healthy pregnant women and systolic blood pressure, as well as serum levels of IP-10, while their serum leptin levels correlated inversely with fetal birth weight. In preeclamptic patients, a significant positive correlation was observed between serum concentrations of leptin and IP-10. Furthermore, elevated serum leptin level and sFlt-1/PlGF ratio had an additive (joint) effect in the risk of preeclampsia, as shown by the substantially higher odds ratios of their combination than of either alone.ConclusionsSimultaneous measurement of leptin with several inflammatory molecules and angiogenic factors in this study enabled us to investigate their relationship, which can help to understand the role of circulating leptin in normal pregnancy and preeclampsia.

Oxytocin activates mitogen-activated protein kinase and up-regulates cyclooxygenase-2 and prostaglandin production in human myometrial cells☆☆☆★

OBJECTIVE The objective of our study was to test the hypothesis that oxytocin promotes prostaglandin production by up-regulating cyclooxygenase-2 via activation of mitogen-activated protein kinase cascade in human myometrial cells. STUDY DESIGN Confluent cultures of human myometrial cells obtained from uterine specimens of premenopausal women undergoing hysterectomy were serum starved for 48 hours before oxytocin stimulation. Prostacyclin levels, as 6-keto-prostaglandin F(1) (alpha), were measured by radioimmunoassay, and the cellular cyclooxygenase-2 protein content was determined by Western blot. Mitogen-activated protein kinase activity was assessed by measuring the phosphorylation of myelin basic protein. RESULTS In a time- and dose-dependent manner oxytocin promoted prostacyclin production in human myometrial cells. Maximal responses were observed after 8 hours of stimulation at a dose of 100 nmol/L. This effect was mainly due to the expression of cyclooxygenase-2 protein. Within 5 minutes oxytocin significantly stimulated mitogen-activated protein kinase, as compared with the expression in untreated controls. The maximal increase in enzyme activity (2.5-fold) was obtained at 45 minutes. A selective inhibitor of mitogen-activated protein kinase activation (PD98059), as well as herbimycin, a tyrosine kinase inhibitor, and the transcriptional blocker actinomycin D, suppressed oxytocin-induced cyclooxygenase-2 expression and prostacyclin production. The stimulatory action of oxytocin was also sensitive to inhibition by pertussis toxin but appeared to be independent of protein kinase C activation. CONCLUSION Our data indicate a largely unrecognized signal transduction mechanism for oxytocin, involving G-protein-coupled activation of mitogen-activated protein kinase and cyclooxygenase-2 gene expression, leading to increased prostaglandin production in human myometrial cells. This signaling pathway complements the rapid activation of the phosphoinositide cycle and may be responsible for sustained release of prostaglandins in uterine tissues, promoting labor and parturition.

Placental protein 13 (galectin-13) has decreased placental expression but increased shedding and maternal serum concentrations in patients presenting with preterm pre-eclampsia and HELLP syndrome

Placental protein 13 (PP13) is a galectin expressed by the syncytiotrophoblast. Women who subsequently develop preterm pre-eclampsia have low first trimester maternal serum PP13 concentrations. This study revealed that third trimester maternal serum PP13 concentration increased with gestational age in normal pregnancies (p < 0.0001), and it was significantly higher in women presenting with preterm pre-eclampsia (p = 0.02) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome (p = 0.01) than in preterm controls. Conversely, placental PP13 mRNA (p = 0.03) and protein, as well as cytoplasmic PP13 staining of the syncytiotrophoblast (p < 0.05) was decreased in these pathological pregnancies compared to controls. No differences in placental expression and serum concentrations of PP13 were found at term between patients with pre-eclampsia and control women. In contrast, the immunoreactivity of the syncytiotrophoblast microvillous membrane was stronger in both term and preterm pre-eclampsia and HELLP syndrome than in controls. Moreover, large syncytial cytoplasm protrusions, membrane blebs and shed microparticles strongly stained for PP13 in pre-eclampsia and HELLP syndrome. In conclusion, parallel to its decreased placental expression, an augmented membrane shedding of PP13 contributes to the increased third trimester maternal serum PP13 concentrations in women with preterm pre-eclampsia and HELLP syndrome.

Increased serum heat-shock protein 70 levels reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia

It has been previously reported that serum levels of 70-kDa heat-shock protein (Hsp70) are elevated in preeclampsia. The aim of the present study was to examine whether increased serum Hsp70 levels are related to clinical characteristics and standard laboratory parameters of preeclamptic patients, as well as to markers of inflammation (C-reactive protein), endothelial activation (von Willebrand factor antigen) or endothelial injury (fibronectin), trophoblast debris (cell-free fetal DNA) and oxidative stress (malondialdehyde). Sixty-seven preeclamptic patients and 70 normotensive, healthy pregnant women were involved in this case-control study. Serum Hsp70 levels were measured with enzyme-linked immunosorbent assay (ELISA). Standard laboratory parameters (clinical chemistry) and C-reactive protein (CRP) levels were determined by an autoanalyzer using the manufacturer’s kits. Plasma von Willebrand factor antigen (VWF:Ag) levels were quantified by ELISA, and plasma fibronectin concentration by nephelometry. The amount of cell-free fetal DNA in maternal plasma was determined by quantitative real-time polymerase chain reaction analysis of the sex-determining region Y gene. Plasma malondialdehyde levels were measured by the thiobarbituric acid-based colorimetric assay. Serum Hsp70 levels were increased in preeclampsia. Furthermore, serum levels of blood urea nitrogen, creatinine, bilirubin and CRP, serum alanine aminotransferase and lactate dehydrogenase (LDH) activities, as well as plasma levels of VWF:Ag, fibronectin, cell-free fetal DNA and malondialdehyde were also significantly higher in preeclamptic patients than in normotensive, healthy pregnant women. In preeclamptic patients, serum Hsp70 levels showed significant correlations with serum CRP levels (Spearman R = 0.32, p = 0.010), serum aspartate aminotransferase (R = 0.32, p = 0.008) and LDH activities (R = 0.50, p < 0.001), as well as with plasma malondialdehyde levels (R = 0.25, p = 0.043). However, there was no other relationship between serum Hsp70 levels and clinical characteristics (age, parity, body mass index, blood pressure, gestational age, fetal birth weight) and laboratory parameters of preeclamptic patients, including markers of endothelial activation or injury and trophoblast debris. In conclusion, increased serum Hsp70 levels seem to reflect systemic inflammation, oxidative stress and hepatocellular injury in preeclampsia. Nevertheless, further studies are required to determine whether circulating Hsp70 plays a causative role in the pathogenesis of the disease.

Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue.

RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for β globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives’ yields for various size real-time PCR amplicons. Longer amplicons (above ∼225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.

Decreased number of FoxP3+ regulatory T cells in preeclampsia

Systemic inflammation is characteristic for preeclampsia (PE). A hypothesis for immune dysregulation is that the function of regulatory T cells (CD4+FoxP3+, Tregs) inhibiting the activation of lymphocytes is impaired. We investigated the proportion of Tregs and their cellular network in preeclamptic women. Fifteen preeclamptic and 17 healthy pregnant women were enrolled in the 32nd gestational week (median age 29 (range 22–45) and 32 (range 26–38) years, respectively). PE was diagnosed according to international criteria at a median of 30 gestational weeks (range 21–31). Peripheral blood was taken and blood mononuclear cells were isolated. Flow cytometry was used to determine the proportion of regulatory (CD4+FoxP3+) T cells, lymphoid and myeloid dendritic cells, natural killer and natural killer T cells, naive and memory and activated CD4+ and CD8+cells. The proportion of Tregs and that of naive CD4+CD45RA+ cells was lower in preeclamptic than in control women (p = 0.025, p = 0.04, respectively). The proportion of other investigated cell types did not differ. Low Treg numbers may support the notion that PE shares similar features to autoimmune disorders. Low Treg numbers are not reflected in the proportion of activated lymphocytes, at least in this stage of pregnancy. This does not exclude, however, the functional alterations of these cell types.

Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia.

The purpose of this study was to determine whether increased serum soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased placental growth factor (PlGF) levels in pre-eclampsia are related to the clinical features and laboratory parameters of the patients, including markers of inflammation, endothelial activation and injury, oxidative stress and trophoblast debris. A total of 54 pre-eclamptic patients, 58 healthy pregnant and 52 healthy non-pregnant women were involved in this case–control study. Serum sFlt-1 and PlGF levels were measured by electrochemiluminescence immunoassay. Serum levels of sFlt-1 and PlGF were significantly higher in pre-eclamptic patients and healthy pregnant women than in healthy non-pregnant women. In addition, pre-eclamptic patients had significantly higher sFlt-1 levels and significantly lower PlGF concentrations compared with healthy pregnant women. According to the subgroup analyses, sFlt-1 levels were significantly higher in severely pre-eclamptic patients than in those with mild pre-eclampsia, whereas pre-eclamptic patients with fetal growth restriction or preterm onset of the disease had significantly lower PlGF concentrations compared with those without intrauterine growth restriction or with a disease onset at term. In the pre-eclamptic group, there were significant positive correlations between serum sFlt-1 levels and systolic and diastolic blood pressure, serum levels of blood urea nitrogen and creatinine, as well as plasma levels of von Willebrand factor antigen, fibronectin and cell-free fetal DNA. Furthermore, serum PlGF concentrations of pre-eclamptic patients showed significant positive correlations with gestational age at disease onset and delivery, as well as with fetal birth weight, and significant inverse correlations with levels of blood urea nitrogen, creatinine and fibronectin. In conclusion, increased serum sFlt-1 and decreased PlGF levels are associated with blood pressure, renal and endothelial dysfunction, trophoblast deportation, as well as with a shorter duration of pregnancy, fetal growth restriction, the severity and preterm onset of the disease in pre-eclampsia. These findings indicate the central role of an angiogenic imbalance in the pathogenesis of this pregnancy-specific disorder.