Soluble BAFF-R produced by decidual stromal cells plays an inhibitory role in monocytes and macrophages  B.P. Deng, Y. Zhang, Q.J. Wang, X.F. Xu, H. Zhang, Y.M. Yang, H.T. Mao, W.J. Gao, B.F. Song, B.H. Kong, X. Qu  Reproductive BioMedicine Online  Volume 24, Issue 6, Pages 654-663 (June 2012) DOI: 10.1016/j.rbmo.2012.02.024 Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Expression of toll-like receptors (TLR) on decidual stromal cells (DSC). DSC were treated with oestradiol+medroxyprogesterone acetate for 24h or 72h. Flow cytometry was then conducted to assay membrane-bound TLR-2, -3 and -4 expression and 72-h-cultured DSC were stained with (A) fluorescein isothiocyanate-conjugated anti-human TLR-2, (B) phycoerythrin-conjugated anti-human TLR-3 or (C) allophycocyanin-conjugated anti-human TLR-4. Panels show respective anti-TLR staining (black line) and isotope control staining (grey line). The x-axes represent fluorescence intensity, reflecting surface concentration of TLRs. Data are representative samples. (A) TLR-2, 1.29%; (B) TLR-3, 98.7%; (C) TLR-4, 4.33%. Percentages mean % of the DSCs expressed surface TLRs. (D) Analysis of TLR-3 mRNA in 24-h-cultured DSC by reverse-transcription PCR: left side, amplification of β-actin shows equal loading of samples onto the gel; right side, BAFF mRNA was present in three preparations of DSC. Reproductive BioMedicine Online 2012 24, 654-663DOI: (10.1016/j.rbmo.2012.02.024) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 BAFF mRNA and protein expression in decidual stromal cells (DSC). DSC were plated in chamber slides and incubated with oestradiol+medroxyprogesterone acetate for 24h or 72h: (A) negative control in which normal goat IgG was used instead of primary antibody; and (B) immunocytochemistry of BAFF protein expression in DSC after 72h culture (×400, bars=50μm). (C) Analysis of BAFF mRNA in 24-h-cultured DSC by reverse-transcription PCR: upper panel, amplification of β-actin shows equal loading of samples onto the gel; lower panel, BAFF mRNA was present in the positive control (HeLa cells) and four preparations of DSC. Reproductive BioMedicine Online 2012 24, 654-663DOI: (10.1016/j.rbmo.2012.02.024) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 BAFF-R mRNA and protein expression in resting decidual stromal cells (DSC) or polyinosinic:polycytidylic acid (poly(I:C))-treated DSC. (A) DSC after three passages were treated with either vehicle (oestradiol+medroxyprogesterone acetate) or vehicle+25μg/ml poly(I:C) for 72h and concentrations of soluble BAFF-R in the culture supernatants measured by ELISA. (B) DSC were treated with or without 25μg/ml poly(I:C) for 24h and real-time quantitative reverse-transcription PCR data for BAFF-R cDNA were normalized with β-actin in each group and shown as relative gene expression (mean±SEM of 3–5 independent experiments). (C) Cytokine antibody array analysis of BAFF-R protein expression in two groups represented as relative densitometric values. (D) Western blot analysis of BAFF-R protein expression. (E) Differential expression of cell-associated BAFF-R protein in two groups as visualized by immunocytochemistry (×400, bars=50μm). Reproductive BioMedicine Online 2012 24, 654-663DOI: (10.1016/j.rbmo.2012.02.024) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Effect of soluble BAFF-R on CD14+HLA-DR+ monocytes. Monocytes were cultured with the supernatants from resting decidual stromal cells (DSC) or polyinosinic:polycytidylic acid (poly(I:C))-treated DSC with or without 1μg/ml anti-BAFF-R mAb, and these culture systems were mixed with an equal volume of RPMI 1640 supplemented with 15% fetal bovine serum and cultured for 40h. The CD14+HLA-DR+ monocyte ratio was determined by flow cytometry using fluorescein isothiocyanate-conjugated anti-human CD14 and phycoerythrin-Cy5-conjugated anti-human HLA-DR mAbs. Data in A are a representative sample, and data in B are mean±SEM of three independent experiments. Mo=monocytes, CM=cell culture media. Reproductive BioMedicine Online 2012 24, 654-663DOI: (10.1016/j.rbmo.2012.02.024) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions

Figure 5 The effect of soluble BAFF-R on tumour necrosis factor (TNF)α and interleukin (IL) 6 production by monocytes and macrophages. (A,B) Monocytes were cultured with the supernatants from resting decidual stromal cells (DSC) and polyinosinic:polycytidylic acid (poly(I:C))-treated DSC±1μg/ml anti-BAFF-R mAb, and these culture systems were mixed with an equal volume of RPMI 1640 supplemented with 15% fetal bovine serum and cultured for 40h. TNF-α (A) and IL-6 (B) concentrations in the supernatants were assayed by ELISA. (C,D) Purified monocytes were stimulated by 10ng/ml recombinant human BAFF-R with or without 1μg/ml anti-BAFF-R mAb for 40h and TNFα (C) and IL-6 (D) concentrations were determined as above. Data are mean±SEM of 3–5 different experiments. Mo=monocytes, CM=cell culture media. Reproductive BioMedicine Online 2012 24, 654-663DOI: (10.1016/j.rbmo.2012.02.024) Copyright © 2012 Reproductive Healthcare Ltd. Terms and Conditions