The Salivary Scavenger and Agglutinin (SALSA) in Healthy and Complicated Pregnancy

Pre-eclampsia is a leading cause of maternal and perinatal morbidity and mortality worldwide. The etiology is not clear, but an immune attack towards components of placenta or fetus has been indicated. This involves activation of the complement system in the placenta. We have previously described the presence of the complement-regulating protein salivary scavenger and agglutinin (SALSA) in amniotic fluid. In this study we investigated the potential role of SALSA in pregnancy by analyzing its presence in amniotic fluid and placental tissue during healthy and complicated pregnancies. SALSA levels in amniotic fluid increased during pregnancy. Before 20 weeks of gestation the levels were slightly higher in patients who later developed pre-eclampsia than in gestation age-matched controls. In the placenta of pre-eclamptic patients syncytial damage is often followed by the formation of fibrinoid structures. SALSA was found clustered into these fibrinoid structures in partial co-localization with complement C1q and fibronectin. In vitro analysis showed direct protein binding of SALSA to fibronectin. SALSA binds also to fibrin/fibrinogen but did not interfere with the blood clotting process in vitro. Thus, in addition to antimicrobial defense and epithelial differentiation, the data presented here suggest that SALSA, together with fibronectin and C1q, may be involved in the containment of injured placental structures into fibrinoids.Peer reviewe

Keratins in the human trophoblast

Besides microfilaments and microtubules, intermediate filaments are major components of the cytoskeleton. In epithelial cells intermediate filaments are formed by heterodimers of specific keratins, whose expression pattern highly depends on the type of epithelium and differentiation degree of the cell. During the process of blastocyst implantation and subsequent development of the human placenta a very specialized epithelium appears at the feto-maternal interface. Arising from the trophectoderm of the blastocyst, the epithelium-like layer surrounding the early embryoblast, different trophoblast subtypes differentiate. They either develop into polar cells fulfilling real epithelial functions, or apolar tumor-like cells invading the maternal uterine wall to adapt the maternal tissue to progressing pregnancy. Thus, the whole trophoblast population, with all its subtypes, can be considered as an epithelial compartment and hence expresses keratin filaments. However, differentiation of trophoblast into different phenotypes may be linked to remodeling of the cytoskeletal composition, depending on spatiotemporal requirements of the respective cells. Here, we focus on the keratin composition of different trophoblast subtypes, how these keratins are used in trophoblast research and what is k

Reduced Placental CD24 in Preterm Preeclampsia Is an Indicator for a Failure of Immune Tolerance

Introduction: CD24 is a mucin-like glycoprotein expressed at the surface of hematopoietic and tumor cells and was recently shown to be expressed in the first trimester placenta. As it was postulated as an immune suppressor, CD24 may contribute to maternal immune tolerance to the growing fetus. Preeclampsia (PE), a major pregnancy complication, is linked to reduced immune tolerance. Here, we explored the expression of CD24 in PE placenta in preterm and term cases. Methods: Placentas were derived from first and early second trimester social terminations (N = 43), and third trimester normal term delivery (N = 67), preterm PE (N = 18), and preterm delivery (PTD) (N = 6). CD24 expression was determined by quantitative polymerase chain reaction (qPCR) and Western blotting. A smaller cohort included 3–5 subjects each of term and early PE, and term and preterm delivery controls analyzed by immunohistochemistry. Results: A higher expression (2.27-fold) of CD24 mRNA was determined in the normal term delivery compared to first and early second trimester cases. The mRNA of preterm PE cases was only higher by 1.31-fold compared to first and early second trimester, while in the age-matched PTD group had a fold increase of 5.72, four times higher compared to preterm PE. The delta cycle threshold (ΔCt) of CD24 mRNA expression in the preterm PE group was inversely correlated with gestational age (r = 0.737) and fetal size (r = 0.623), while correlation of any other group with these parameters was negligible. Western blot analysis revealed that the presence of CD24 protein in placental lysate of preterm PE was significantly reduced compared to term delivery controls (p = 0.026). In immunohistochemistry, there was a reduction of CD24 staining in villous trophoblast in preterm PE cases compared to gestational age-matched PTD cases (p = 0.042). Staining of PE cases at term was approximately twice higher compared to preterm PE cases (p = 0.025) but not different from normal term delivery controls. Conclusion: While higher CD24 mRNA expression levels were determined for normal term delivery compared to earlier pregnancy stages, this expression level was found to be lower in preterm PE cases, and could be said to be linked to reduced immune tolerance in preeclampsia

Metalloprotease Dependent Release of Placenta Derived Fractalkine

The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine

Targets for SALSA in human placenta.

<p>To determine further binding sites for SALSA in the placenta, SALSA containing AF was added as an overlay to frozen placental sections prior to detection of SALSA by fluorescence immunohistochemistry. The addition of the SALSA containing overlay (A) revealed a strong binding of SALSA to numerous additional structures in the placenta which were not observed without the overlay (B). SALSA binds to the endothelium of most capillaries (Ca) and larger vessels (Ve) as well as to most of the syncytiotrophoblast layer (A). Without overlay SALSA staining is much more sporadic or lacking in these structures (B). The strong binding after <i>ex vivo</i> addition of SALSA suggests that the focal staining observed in the absence of overlay (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147867#pone.0147867.g002" target="_blank">Fig 2</a>) is a result of limited availability of SALSA rather than the lack of interacting tissue components. 400× magnifications.</p

Immunohistochemical analysis of SALSA in paraffin-embedded healthy first trimester decidua.

<p>SALSA staining (A), (C) and (E) was compared to the endothelial marker CD34 (B) and (D) and the epithelial marker cytokeratin 7 to identify uterine glands and extravillous trophoblasts (F). In the 1^st trimester decidua, SALSA was still found abundantly. However less fibrinoid structures were observed (A) and (C). Instead, SALSA was found to co-localize with CD34 (B) and (D). The SALSA staining pattern of the endothelium is scattered, and found in both small capillaries and larger vessels. Thus, it may be related to the activation state of the endothelium. SALSA was not found to co-localize with cytokeratin 7, and is thus not likely to be produced in the uterine glands (E) and (F). 200× magnifications.</p

Analysis of the effect of SALSA on coagulation.

<p>A pool of citrated plasma was added to SALSA-coated wells (1–5 μg/ml) and coagulation was initiated by adding BC Thrombin reagent at time point 0. The coagulation was followed by absorbance at 405 nm and compared to coagulation in wells without SALSA coating. We did not observe an effect of SASLA on coagulation. Displayed are averages ± SD’s from 5 different wells. A similar result was obtained when soluble SALSA (1–5 μg/ml) was added to the plasma prior to the coagulation test (data not shown).</p

Immunofluorescence microscopy detection of SALSA localization in human placenta.

<p>Paraffin-embedded tissue from healthy (panels A-D) and pre-eclamptic (PE) (panels E-F) placentas. Sections were stained with an anti-SALSA antibody (Hyb 213–06) and Alexa 546-conjugated goat anti-mouse IgG. Red: SALSA, blue: DAPI. * denotes the center of the villus structures. M denotes the intervillous space/maternal tissue. Panels A to F show a widespread but focal staining of SALSA in the human placenta. Based on the morphology and localization of the placental structures SALSA appears to be present in fibrinoid formations, especially in relation to a disruption of the syncytiotrophoblast layer. (A) shows the expression of SALSA found intracellularly in the syncytiotrophoblast layer of some, but not all, villi (white arrowhead). SALSA was observed abundantly in fibrinoid structures at the edge of the villi. A breach of the syncytiotrophoblast layer accompanied by formation of SALSA-positive fibrinoid in the intervillous space can be observed (white arrowhead, panel B). (C) shows SALSA in a necrotic villus with fibrinoid formation. (D) shows a villus with disrupted syncytiotrophoblast layer (white arrowhead). Here the disruption may have led to the influx of maternal blood, and SALSA-positive fibrinoid can be observed separating the syncytiotrophoblast layer from the underlying basement membrane. The fibrinoid may deposit all the way around the exposed villus structure, thus forming a ring structure. Syncytial damage and fibrinoid formation is observed more frequently in PE. (E) and (F) show examples of a necrotic villus and a ring formation in PE placentas. 200× magnifications.</p