Signaling pathways for B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) in human placenta

Abstract

The tumor necrosis superfamily (TNFSF) contains two soluble ligands that are involved in B lymphocyte development, BAFF (B cell activating factor, BlyS, TALL-1, CD257, TNFSF13B) and APRIL (a proliferation inducing ligand, CD256, TNFSF13). These two ligands signal through three receptors: the exclusive BAFF receptor (BAFF-R, CD268, TNFRSF17) and two receptors that recognize both BAFF and APRIL, TACI (transmembrane-activator-1 and calcium-modulator- and cyclophilin ligand-interactor CD267, TNFRSF13B) and BCMA (B cell maturation antigen, CD269, TNFRSF13C). All but BAFF-R are known to be synthesized in term placentas. In this study, expression of the ligands and receptors were distinguished in two embryologically discrete subpopulations of placental cells, villous cytotrophoblast (vCTB) cells and mesenchymal cells (MCs). Real-Time PCR showed that vCTB cells contain low levels of BAFF and APRIL transcripts whereas MCs contain high levels. Both Real-Time PCR and immunohistochemistry identified BAFF-R and BCMA mRNA and proteins in vCTB cells but essentially no TACI. By contrast, MCs contained readily detectable levels of all three receptors. These results illustrating potential autocrine and paracrine pathways for BAFF and APRIL signaling in human placentas suggest that lineage-specific regulation of placental cell viability, differentiation and/or other activities may be novel functions of these proteins.

Figure 1

Immunocytochemical identification of trophoblast cells in the vCTB cell preparations used in the experiments. A: Cartoon showing locations of vCTB and syncytiotrophoblast (sTB) cells and MCs in human term placental villus. B: Identification of cytokeratin-7-positive trophoblast cells. The percentage of trophoblast cells in the vCTB cell preparations was established by centrifuging the cells onto glass slides using a Shandon Cytospin (Shandon, Pittsburgh, PA) and immunostaining for cytokeratin-7 to reveal trophoblast cells. The cells were counterstained with Mayer’s hematoxylin. The inset shows failure of binding of the isotype control mAb. Original magnifications, ×200.

Figure 2

Analysis of CD19 mRNA in cytotrophoblast cells (CTB) and mesenchymal cells (MC) by semiquantitative RT-PCR. Upper panel: The CD19 dimer is present in the positive control cells, Raji and L721.221, but absent in three preparations of CTB cells and matching MCs. Lower panel: Amplification of β-actin showed equal loading of samples onto the gel.

Figure 3

Analysis of BAFF (A) and APRIL (B) mRNA in three preparations of purified vCTB cells and matching MCs from the same placentas by real-time PCR. HeLa cells were used as calibrators.

Figure 4

Analysis of BAFF-R in the three preparations of purified vCTB cells and matching MCs from the same placentas. A: BAFF-R mRNAs in vCTB cells and MCs using 721.221 cells as calibrators in real-time PCR experiments. B: BAFF-R proteins in placental villi identified by immunohistochemistry. Solid arrows point to positive mesenchymal cells; open arrowheads point to syncytiotrophoblast. The inset shows that no signals were obtained when isotype-specific mouse IgG was substituted for anti-BAFF-R. Original magnifications, ×200.

Figure 5

Analysis of TACI and BCMA mRNAs in the three preparations of purified vCTB cells and matching MCs from the same placentas. A: TACI mRNAs in vCTB cells and MCs using 721.221 cells as calibrators in real-time PCR experiments. B: BCMA mRNAs in vCTB cells and MCs using 721.221 cells as calibrators in real-time PCR experiments.

Figure 6

Immunohistochemical identification of TACI and BCMA in human term placentas. A: Anti-TACI immunostaining of term human placenta. Inset, isotype-matched negative control. B: Anti-BCMA immunostaining of human term placenta. Inset, negative control. Open arrowheads mark syncytiotrophoblast; solid arrows mark MCs. Original magnifications, ×200.

Figure 7

Cartoon illustrating the finding that MCs in term placental villi are major sites of BAFF and APRIL ligand synthesis and that both vCTB cells and MCs may receive BAFF/APRIL signals. BAFF and APRIL may target in an autocrine manner to three receptors, BAFF-R, TACI, and BCMA, on MC and in a paracrine manner to two receptors, BAFF-R and BCMA, on vCTB cells.