Search

The purpose of this study was to determine whether pre-B-cell colony-enhancing factor is a secreted cytokine in the human amnion and to study its chemotaxic and antiapoptotic properties.Pre-B-cell colony-enhancing factor secretion was studied from amniotic epithelial-like WISH cells and primary amniotic epithelial cells that were seeded on squares of immobilon-P membrane and stimulated with lipopolysaccharide or tumor necrosis factor-alpha, respectively. The pre-B-cell colony-enhancing factor protein was detected both intracellularly and after secretion, as bound to the membrane, by immunostaining and densitometry. Medium and cell lysates that were obtained from WISH cells that were treated with lipopolysaccharide alone or together with a pre-B-cell colony-enhancing factor antisense oligonucleotide to block pre-B-cell colony-enhancing factor translation were also analyzed for secreted pre-B-cell colony-enhancing factor by Western blotting and densitometry. A chemotaxic effect of pre-B-cell colony-enhancing factor on human neutrophils was compared with the chemoattractants interleukin-8 and N-Formyl-Met-Leu-Phe methyl ester in a rapid fluorescence-based neutrophil migration assay. Apoptosis was induced in primary amniotic epithelial cells and fibroblasts by actinomycin D (1 microg/mL); the antiapoptotic effects of pre-B-cell colony-enhancing factor on early apoptosis were measured by the annexin V assay, and the late effects were determined by measurement of nuclear matrix protein in the media.Treatment of amnion cells that adhered to immobilon-P membrane to induce the secretion of pre-B-cell colony-enhancing factor showed significantly (P.05) more pre-B-cell colony-enhancing factor protein surrounding the cells compared with the controls. Although the addition of lipopolysaccharide to cultured WISH cells caused the secretion of pre-B-cell colony-enhancing factor into the medium, co-treatment with an antisense oligonucleotide to pre-B-cell colony-enhancing factor obliterated it. Analysis of the cell lysates showed no significant change, which suggests that most of the pre-B-cell colony-enhancing factor protein had been secreted. No significant chemotaxic effects of pre-B-cell colony-enhancing factor were observed; however, pre-B-cell colony-enhancing factor treatment (100 ng/mL), together with actinomycin D, cancelled the early induction of apoptosis, although there was a dose-dependent and significant late antiapoptotic effect on primary amnion epithelial cells (P.001) and fibroblasts (P.01).Pre-B-cell colony-enhancing factor is a secreted protein from amniotic epithelial cells. Although it had no chemotaxic effects, it was antiapoptotic for both amniotic epithelial cells and fibroblasts and may protect these cells against apoptosis that is induced by chronic distension, labor, or infection.

Objective: Our purpose was to determine whether pre-B-cell colony-enhancing factor (PBEF) is expressed in the human fetal membranes during normal gestation and parturition in the absence of infection and to show its effects on the expression of interleukin (IL)-6 and IL-8. Study Design: PBEF was immunolocalized in the fetal membranes from early pregnancy, at preterm, and at term. Its expression was quantitated by Northern analysis in separated uninfected amnion, chorion, decidua, and placenta of patients at term before labor and in full-thickness membranes before and after spontaneous labor at preterm and at term. Amnion-like epithelial (WISH) cells and fetal membrane explants were treated with recombinant PBEF (rhPBEF), and the expression of IL-6 and IL-8 was quantitated. Results: PBEF was immunolocalized throughout gestation in the amniotic epithelium and mesenchymal cells as well as the chorionic cytotrophoblast and parietal decidua. Northern analysis showed significantly more (P

Our purpose was to show the effects of pre-B-cell colony-enhancing factor on the genes that are expressed by the human fetal membranes.Explants of fetal membranes (amnion, chorion, and decidua) from three term patients were treated with 100 ng/mL recombinant human pre-B-cell colony-enhancing factor for 4 hours. RNAs were hybridized to gene chips that contained18,000 known genes. One experiment was done in triplicate to assess replication. Data were analyzed to quantitate the signal intensities of each complementary DNA on the array. Confirmation of the results was carried out on tissues from nine other patients by the measurement of the proteins or quantitative real-time reverse transcriptase-polymerase chain reaction.Replication gave92.6% identical results, which showed high method reproducibility. Pre-B-cell colony-enhancing factor treatment caused a significant increase in 103 genes and decrease in 139 genes. Only 8 genes were up-regulated consistently and significantly in all three patients (three key inflammatory cytokines [tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta], four important chemokines [macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, macrophage inflammatory protein-3alpha, and growth-related oncogene-gamma], and prostaglandin-endoperoxide synthase 2). These data were confirmed by the measurement in the media with the use of specific enzyme-linked immunosorbent assays for tumor necrosis factor-alpha, interleukin-6, and interleukin-1beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and macrophage inflammatory protein-3alpha and by quantitative real-time reverse transcriptase-polymerase chain reaction for growth-related oncogene-gamma and prostaglandin-endoperoxide synthase 2.Pre-B-cell colony-enhancing factor appears to be at the proximal end of the pathway to labor initiation and may link sterile distention-induced labor with that of infection-induced labor.