Paracrine regulation of distinct trophoblast functions in vitro by placental macrophages

Abstract

In view of the accumulating evidence for paracrine mechanisms regulating trophoblast function, we tested the hypothesis that placental macrophages affect trophoblast activity in a paracrine fashion. Trophoblast was isolated from 17 term placentas (-IP). One aliquot of cells was further immunopurified (+IP) using an HLA class I antibody. This increased the proportion of trophoblast (+IP >97%; -IP approximately 70%) as identified by rigorous immunocytochemistry. Most (approximately 70%) non-trophoblast cells in -IP were macrophages. The cells were cultured for 5 days with a daily medium change. In addition, +IP cells from seven placentas were cultured with lipopolysaccharide (LPS)-stimulated or -unstimulated macrophage-conditioned media. The concentrations of lactate, trophoblast-specific hormones, human chorionic gonadotropin-beta (hCG-beta) and human placental lactogen (hPL), of several prostanoids and of endothelin-1 and angiotensin II were determined in the culture media. The accumulated amounts of substances released into the culture media, corrected for the greater proportion of trophoblast in +IP cultures, were on average two- to threefold higher (hCG-beta: 18-fold) in +IP than in -IP, with the exception of endothelin-1,2 (no change), angiotensin II (-70%) and 6-keto-prostaglandin-F1alpha (-40%). [3H]leucine incorporation into the trichloroacetic acid (TCA)-precipitable pool measured on day 5 was twofold higher in +IP than in -IP. Addition of conditioned media reverted these changes. The data demonstrate that placental macrophages in culture affect trophoblast biosynthetic activity in a paracrine fashion. We conclude that macrophages are important regulators of trophoblast activity.

Characterization of Human Villous and Extravillous Trophoblasts Isolated from First Trimester Placenta

“…As shown in Figure 8, we observed that our population of isolated EVCT was 97% pure as analyzed by double labeling using antibodies against cytokeratin 7, a specific marker of trophoblast Tarrade et al cells (Cervar et al, 1999), and hPL. In addition, these EVCT cells expressed c-erbB2 (Fig.…”

“…During this process, the length of enzymatic digestion, the bath of trypsin used, and the absence of agitation are critical steps to monitor. The different immunocytochemical studies performed on these EVCT confirmed the presence of cytokeratin 7, specifically expressed in the trophoblast cells (Cervar et al, 1999). We also checked the absence of fibroblastic contamination by vimentin immunostaining of immunocompetent cells from the decidua by CD45 and of endothelial cells by antiWillebrand factor immunostaining.…”

Insulin control of placental gene expression shifts from mother to foetus over the course of pregnancy

“…Isolation of cells Trophoblasts (TTs) and ECs were isolated from term placentas after uncomplicated pregnancy and vaginal delivery following a standard protocol [12,13]. First-trimester trophoblasts (FTs) were isolated from placental tissue after pregnancy terminations for psychosocial reasons (seventh to tenth week of pregnancy) as described previously [13].…”

“…Characterisation and culture of human trophoblasts and ECs All cell preparations were subjected to rigorous immunocytochemical characterisation [12][13][14]. Trophoblasts were tested for viability by measuring secreted human chorionic gonadotropin hormone (hCG) levels using a commercially available assay (Dade Behring, Deerfield, IL, USA).…”

“…ECs were characterised by internalisation of acetylated LDL (Biomedical Technologies, Stoughton, MA, USA) [12]. They were cultured on 1% (v/v) gelatin-coated plates using Endothelial Basal Medium (Clonetics, Cambrex, Walkersville, MD, USA) supplemented with the EGM-MV BulletKit (Clonetics) until 70% confluency, at which time insulin was added.…”