Modulation of phosphatidylserine epitope expression by BeWo cells during forskolin treatment.

The adenylcyclase activator forskolin induces the human choriocarcinoma line, BeWo, to undergo differentiation and fusion within 48 to 72 h. Using three monoclonal antibodies that differentiate between the anionic phospholipids cardiolipin (CL) and phosphatidylserine (PS) and immunoperoxidase techniques we investigated the expression of PS by BeWo during 48 h of forskolin treatment. We observed that BeWo cells not exposed to forskolin express an epitope of pS that reacts strongly with monoclonal antibody BA3B5C4 (CL+/PS+), whereas following treatment with forskolin there is a decrease in reactivity with BA3B5C4 and a concurrent increased activity with a second PS-reactive monoclonal antibody, 3SB9b (CL-/PS+). A third monoclonal antibody, D11A4 (CL+/PS-), that reacted with all anionic phospholipids except PS did not bind to BeWo cells, whether forskolin treated or not. These observations support previous interpretations using human placenta that during cytotrophoblast differentiation two antigenic forms of PS are expressed. Based on the described relationship of PS with cellular fusion events in other systems and the association of naturally occurring antibodies against PS with pregnancy loss and intrauterine growth retardation in humans, we propose that altered expression of PS during normal placental development and in BeWo after exposure to forskolin may be critical in the cytotrophoblast differentiation process.

Expression of endogenous HIV-1 crossreactive antigens within normal human extravillous trophoblast cells.

Expression of intact endogenous retroviruses by normal placental villous trophoblast and immuno-crossreactivity of villous trophoblast with anti-retroviral antisera have been documented. The nature and/or potential function of these particles/proteins has not yet been fully defined. We previously reported that monoclonal antibodies directed against HIV-1 envelope and gag proteins react with normal human villous trophoblast. In this study, we report that extravillous trophoblast (EVT) from second- and third-trimester tissue are also cross-reactive with anti-HIV-1 gp120/160 and p17/18 antibodies. We document a differential expression of such cross-reactive epitopes between mononuclear EVT and placental bed giant cells. Mononuclear EVT principally displayed reactivity throughout the cytoplasm with little or no difference between cells, whereas placental bed giant cells displayed distinct localization of labeling to limited areas of cytoplasm. This pattern of reactivity apparently correlates with trophoblast morphological differentiation and with our earlier observations concerning villous trophoblast. These data illustrate that retrovirus-associated epitopes are expressed by trophoblast throughout the normal human placenta and that this distribution is related to morphologic differentiation of these cells.

A murine monoclonal antibody (RV3-27) raised against isolated human placental endogenous retroviral particles and reactive with syncytiotrophoblast.

Particles with the characteristic shape of enveloped retroviral particles and maximal specific reverse transcriptase (RTase) activity at buoyant density of 1.15-1.17 g/ml have been isolated from human first-trimester chorionic villous tissue. Murine monoclonal antibodies (mAbs) to these isolated particles were generated. One IgM mAb (RV3-27) showed granular staining of cytoplasmic structures within syncytiotrophoblast by immunohistochemistry. Immunoelectron microscopic studies have demonstrated focal localisation to small submembranous regions of syncytiotrophoblast, as well as reaction with detergent-disrupted isolated placental retroviral-like particles. The RV3-27 mAb did not stain other human tissues in this focal manner, although increased generalised cytoplasmic staining was not uncommon; also, this mAb did not react strongly with the surface or cytoplasm of a variety of human cell lines (including choriocarcinoma cells). Immunoblotting and HPLC analyses have indicated the reactive placental antigen to be a 17-25 kDa protein. It is suggested that the RV3-27 mAb may be reactive with a syncytiotrophoblast antigen encoded by an endogenous retroviral sequence.

Monoclonal antiphospholipid antibody reactivity against human placental trophoblast.

Naturally occurring antibodies against the negatively charged phospholipids cardiolipin (CL) and phosphatidylserine (PS) have been associated with recurrent pregnancy loss. One prevalent hypothesis proposes that antiphospholipid antibody (aPL) mediated pathophysiology is through increased placental thrombosis. In this study we investigated the reactivity of three mouse monoclonal aPLs with term and 26 week human placental preparations. Each monoclonal antibody reacted differently with CL and PS; 3SB9b reacted with PS (CL-/PS+), D11A4 reacted with CL (CL+/PS-) and BA3B5C4 reacted with both CL and PS (CL+/PS+). 3SB9b reacted strongly with the syncytiotrophoblastic layer of both formalin fixed and frozen placental tissue. Sporadic reactivity was observed against the cytotrophoblastic layer. BA3B5C4 reacted strongly and specifically with cytotrophoblastic cells. D11A4 had only weak reactivity in the subtrophoblastic stromal region of the placenta in frozen sections. aPL staining was also observed against extravillous cytotrophoblast. BA3B5C4 stained cytoplasmic structures, whereas 3SB9b stained the plasma membrane region with little cytoplasmic staining. These data suggest that the trophoblastic layer is reactive with aPLs and may potentially be directly damaged through mechanisms unrelated to thrombosis. In addition, the trophoblastic layer directly in contact with the maternal circulation is most reactive with aPLs that are PS+ rather than CL+. The differential reactivity of 3SB9b and BA3B5C4 suggests that the antigenic conformation involving PS on the cytotrophoblast is altered concurrent with fusion into the syncytium.

Ultrastructural characterization of endogenous retroviral particles isolated from normal human placentas.

Human placental endogenous retroviral (ERV) particle isolates were investigated by ultrastructural evaluation. Although retrovirus-like structures in normal human placental tissue sections have been described, the precise nature of these particles has not previously been defined. By direct electron microscopic (EM) observation of placental ERV isolates that display retroviral reverse transcriptase (RTase) activity and a buoyant density consistent with type-C retroviruses (1.17 g/ml on sucrose), we have shown these to contain particles with characteristic retroviral ultrastructural features. Samples were stained with 1.0% phosphotungstic acid (PTA), 0.5% uranyl acetate (UA), and low-angle-shadowed with platinum/palladium. Isolated placental ERV particles have an apparent diameter of approximately 120 nm, are membrane-bound with a short surface fringe, and contain capsid particles (about 90 nm in diameter) within an internal matrix structure. These observations support the view that human placental cells normally express ERV particles.

Organ culture of amniochorionic membrane in vitro.

PROBLEM: The purpose of the study was to develop a novel method of amniochorionic membrane culture aimed at maintaining tissue integrity. METHOD: Amniochorionic membranes were collected from women prior to labor, undergoing elective cesarean section, with no history of infection or pregnancy related complication. Fetal membranes were maintained in culture for up to ten days. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a "house keeping" gene and inflammatory cytokine mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) at various periods of culture. mRNA localization was performed by in situ hybridization. RESULTS: GAPDH gene expression was seen throughout the culture period. Inflammatory cytokine mRNA (IL-1 alpha, IL-1 beta and IL-6) were also detected during culture. Cellular and tissue morphology appeared normal. CONCLUSIONS: The culture technique we propose is a simple organ explant system which maintains the morphology and autocrine/paracrine relationships within this tissue.

Expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) in amniochorionic membranes.

OBJECTIVE: This study was designed to investigate the expression of inflammatory cytokines (interleukin-1 beta and interleukin-6) by fetal membranes in response to infection in vivo and to endotoxin in organ culture. STUDY DESIGN: Amniochorionic membranes were collected from infected and uninfected women and analyzed for cytokine messenger ribonucleic acid and protein. Normal membranes were cultured and exposed to endotoxin. Messenger ribonucleic acid expression was analyzed by reverse transcriptase-polymerase chain reaction. Cellular localization of messenger ribonucleic acid and protein was determined by in situ hybridization and immunocytochemical evaluation, respectively. RESULTS: Messenger ribonucleic acid for interleukin-1 beta appeared to be increased in infected or endotoxin-stimulated amniochorionic membranes, whereas interleukin-6 messenger ribonucleic acid was only observed in infected membranes or after endotoxin stimulation. Interleukin-1 beta messenger ribonucleic acid was localized exclusively to chorionic cells, whereas protein was observed in both chorion and amnion. Interleukin-6 messenger ribonucleic acid and protein were produced in both amniotic and chorionic cells. CONCLUSION: Amniochorionic membranes are a site of inflammatory cytokine production. These findings may have significance in preterm labor or premature rupture of membranes.

The adapter protein LAT enhances fcgamma receptor-mediated signal transduction in myeloid cells.

FcgammaR clustering in monocytes initiates a cascade of signaling events that culminate in biological responses such as phagocytosis, production of inflammatory cytokines, and generation of reactive oxygen species. We have identified and determined the function of the adapter protein linker of activation of T cell (LAT) in FcgammaR-mediated signaling and function. Clustering of FcgammaRs on the human monocytic cell line, THP-1, induces phosphorylation of a major 36-kDa protein which immunoreacts with anti-LAT antisera. Our data indicate that although both the 36-kDa and 38-kDa isoforms of LAT are expressed in THP-1 and U937 human monocytic cells, FcgammaR clustering induces phosphorylation of the 36-kDa isoform only. Co-immunoprecipitation experiments revealed a constitutive association of p36 LAT with both FcgammaRI and FcgammaRIIa immunoprecipitates, and an activation-induced association of LAT with PLCgamma1, Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Transient transfection experiments in COS-7 cells indicated that overexpression of a wild type but not a dominant-negative LAT, that is incapable of binding to p85, enhances phagocytosis by FcgammaRI. Furthermore, bone marrow-derived macrophages from LAT-deficient mice displayed reduced phagocytic efficiency in comparison to the macrophages from wild-type mice. Thus, we conclude that p36 LAT serves to enhance FcgammaR-induced signal transduction in myeloid cells.

Expression of phosphatidylserine-dependent antigens on the surface of differentiating BeWo human choriocarcinoma cells.

PROBLEM: Antiphospholipid antibodies (aPLs) are associated with pregnancy loss, pregnancy-induced hypertension, and intrauterine growth retardation. We have previously reported that phosphatidylserine (PS)-dependent antigens are expressed in formalin-fixed cells concurrent with differentiation in a choriocarcinoma model (BeWo) of cytotrophoblast. That study, however, could not differentiate between cytoplasmic or surface antigen expression. METHOD: Three monoclonal aPLs that differentiate between PS- and cardiolipin (CL)-dependent antigens were reacted with BeWo, with or without forskolin activation, before fixation, and antibody binding was evaluated by immunoperoxidase techniques. RESULTS: Activation with forskolin induced a PS-dependent antigenic determinant on the surface on BeWo cells. CL-reactive monoclonal antibodies did not react with the cell surface, whether forskolin treated or not. CONCLUSION: These observations demonstrate that a PS-dependent antigen is expressed on the surface of a model of differentiating cytotrophoblastic cells and should be accessible in vivo to circulating aPLs.

Monoclonal IgM antiphosphatidylserine antibody reacts against cytoskeleton-like structures in cultured human umbilical cord endothelial cells.

PROBLEM: It has been proposed that antibodies against phospholipid-dependent antigens (aPLs), induce recurrent pregnancy loss and thrombosis through modulation of endothelial cell function, yet aPLs have not been conclusively shown to bind with endothelial cells. METHOD: Using indirect immunofluorescence we investigated the anti-endothelial cell reactivity of three monoclonal antibodies that differentiate between the phospholipids cardiolipin (CL) and phosphatidylserine (PS): BA3B5C4 (CL+/PS+); 3SB9b (CL-/PS+); and D11A4 (CL+/PS-). Cultured umbilical cord endothelial cells were prepared without fixation or with cold acetone fixation. RESULTS: None of the aPLs reacted with endothelial cells prepared without fixation. 3SB9B reacted strongly with cytoskeletal-like components in acetone-fixed cells, whereas BA3B5C4 and D11A4 were unreactive. The cytoskeletal-like binding of 3SB9b was completely blocked by a monoclonal antibody against vimentin, whereas antibodies against tubulin or actin were not inhibitory. Lipid extraction of the cells destroyed the 3SB9b reactive antigen without affecting the reactivity of anti-vimentin. CONCLUSION: These results suggest that phospholipid-dependent antigenic determinants are not expressed on the surface of resting endothelial cells but that a PS-dependent antigenic determinant is associated with endothelial cell intermediate filaments.

Heparin-induced endothelial cell cytoskeletal reorganization: a potential mechanism for vascular relaxation.

We have previously shown that heparin given subcutaneously on a daily basis lowers blood pressure in hypertensive rat models, and that this blood pressure lowering effect is endothelium-dependent. The present study describes the effects of heparin on endothelial cell (EC) apical surface structures and cytoskeletal elements, namely, actin and vimentin as well as EC proliferative activity. The EC line (CRL 1998) was cultured, treated with different concentrations of heparin (0, 50, 100, 500 U/ml) for 4, 24 or 48 hours, and fixed for scanning electron microscopy (SEM), and immunofluorescence microscopy (IFM) studies. Enzyme-linked immunosorbent assays (ELISA) and flow cytometric analysis were performed on EC monolayers treated with different concentrations of heparin for quantitative detection of actin and vimentin. By SEM study the cell surface showed generalized smoothing as a result of blunting of surface microvilli with increasing time of exposure and dosage of heparin. By IFM study, the detectable actin signal within ECs became progressively reduced in both its cellular distribution and the apparent number of cells that remained reactive. By 48 hr/500 U heparin, the actin signal was almost undetectable. Vimentin showed a moderate reduction in the cellular distribution of labeling. Quantitatively, actin was significantly reduced after the 24 hour treatment with a higher dose of heparin (500 U/ml), from a baseline optical density (OD) of 1.12 +/- 0.060 to 0.866 +/- 0.008 (P < 0.0027). After 48 hours of treatment at both 100 U/ml and 500 U/ml heparin, actin was significantly reduced from a baseline OD of 1.347 +/- 0.063 to 1.090 +/- 0.039 (P < 0.0039) and 0.844 +/- 0.074 (P < 0.008), respectively. However, vimentin was significantly reduced only after 48 hours of treatment with a high dose of heparin (500 U/ml), from baseline OD 1.82 +/- 0.052 to 1.41 +/- 0.004 (P < 0.002). The flow cytometric findings were virtually identical to the ELISA data for actin and vimentin. These qualitative and quantitative changes in actin and vimentin are consistent with apparent smoothing and relaxation of the EC's apical surface. Labeling with the cell cycle marker MIB-1 (monoclonal antibody Ki-67), showed a progressive reduction in the observed intensity in heparin treated cells with substantially fewer cells being positive. After a 48 hour treatment with heparin (500 U/ml), most ECs displayed only dim labeling of the nucleolus. This finding is consistent with an antiproliferative effect. Overall, these findings are additive to our previous observations, and demonstrate that heparin causes EC cytoskeletal reorganization which is a potential mechanism for vascular relaxation.

The Fc receptor for IgG expressed in the villus endothelium of human placenta is Fc gamma RIIb2.

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fc gamma receptor (Fc gamma R) expression by immunohistology and immunoblotting. Several pan-Fc gamma RII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical Fc gamma RIIa were not expressed in transport-related endothelium of the placenta. Our inference, that Fc gamma RIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of Fc gamma RIIb. These experiments showed that the Fc gamma RII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.