BMP-6 inhibits growth of mature human B cells; induction of Smad phosphorylation and upregulation of Id1

Abstract

Background: Bone morphogenetic proteins (BMPs) belong to the TGF-beta superfamily and are secreted proteins with pleiotropic roles in many different cell types. A potential role of BMP-6 in the immune system has been implied by various studies of malignant and rheumatoid diseases. In the present study, we explored the role of BMP-6 in normal human peripheral blood B cells.

Results: The B cells were found to express BMP type I and type II receptors and BMP-6 rapidly induced phosphorylation of Smad1/5/8. Furthermore, Smad-phosphorylation was followed by upregulation of Id1 mRNA and Id1 protein, whereas Id2 and Id3 expression was not affected. Furthermore, we found that BMP-6 had an antiproliferative effect both in naive (CD19+CD27-) and memory B cells (CD19+CD27+) stimulated with anti-IgM alone or the combined action of anti-IgM and CD40L. Additionally, BMP-6 induced cell death in activated memory B cells. Importantly, the antiproliferative effect of BMP-6 in B-cells was completely neutralized by the natural antagonist, noggin. Furthermore, B cells were demonstrated to upregulate BMP-6 mRNA upon stimulation with anti-IgM.

Conclusion: In mature human B cells, BMP-6 inhibited cell growth, and rapidly induced phosphorylation of Smad1/5/8 followed by an upregulation of Id1.

Figure 1

BMP-6 inhibits proliferation of human B cells. A) CD19⁺ B cells were isolated from peripheral blood and stimulated in triplicates with anti-IgM (37.5 μg/ml) or anti-IgM and CD40 ligand (CD40L; 10 ng/ml) in the presence or absence of BMP-6 for 72 hours. DNA synthesis was measured by [3H]-thymidine incorporation for the last 18 hours. Data are given as relative proliferation obtained by normalizing the mean counts per minute (cpm) for each stimulation to the mean cpm obtained for anti-IgM stimulated cells ± SEM. (mean cpm = 25 352 for anti-IgM stimulated cells, *p ≤ 0.0002 (n = 8), **p ≤ 0.023 (n = 6). B) Dose dependent inhibition of BMP-6 of anti-IgM induced DNA-synthesis of CD19⁺ B cells (relative proliferation ± SEM, n = 3) and C) the Burkitt lymphoma cell line Ramos (relative proliferation ± SEM, n = 3). Ramos cells were cultured for 72 hours and [3H]-thymidine were added for the last 4 hours. D) Noggin (5 μg/ml) and BMP-6 (0,25 or 1 μg/ml) were preincubated for 1 h at 37°C and then added to the CD19⁺ B cells in the presence of anti-IgM(37.5 μg/ml). Cells were cultured for 72 h and DNA synthesis was measured by ³H-thymidine incorporation. One representative of three separate experiments is shown (mean cpm ± SD of triplicates). E) Highly purified CD19⁺CD27^- or CD19⁺CD27⁺ cells were obtained by cell sorting of CD19⁺ cells and stimulated with anti-IgM in the presence or absence of BMP-6, as indicated for 72 hours. DNA synthesis was measuredby [3H]-thymidine incorporation. Data are given as relative proliferation obtained by normalizing the mean cpm for each stimulation to the mean cpm obtained for anti-IgM stimulated cells (mean cpm = 18 221 for CD19⁺CD27^- naïve B cells, mean cpm = 8 930 for CD19⁺CD27⁺ memory B cells, n = 5; * p ≤ 0,004, **p ≤ 0.001).

Figure 2

BMP-6 induces cell death in B cells. CD19⁺CD27^- naïve B cells or CD19⁺CD27⁺ memory B cells were cultured for 48 h with BMP-6 (1 μg/ml) with or without anti-IgM (37.5 μg/ml). Cell death (PI⁺ cells) was then measured by flow cytometry analysis. Data are shown as mean percentage PI⁺ cells from five independent donors (± SEM; p ≤ 0,003).

Figure 3

Ramos cells were cultured in the presence or absense of BMP-6 (1 μg/ml) for 48 h before analysis of cell death by PI staining. Data are shown as mean percentage PI⁺ cells (± SEM, n = 3, p ≤ 0,001).

Figure 4

CD19⁺ B cells express BMP type I and type II receptors. Total cell lysates were prepared from the myeloid cell line HL60, the cell line Ramos or CD19⁺ B cells isolated from peripheral blood and analyzed by western blot for expression of BMP type I and type II receptors. One representative experiment of four is shown.

Figure 5

BMP-6-induced phosphorylation of Smad1/5/8 in CD19⁺ B cells and Ramos, but not in HL60 cells. CD19⁺ B cells, or the cell lines Ramos or HL60

Figure 6

were cultured in X-vivo 15 over night before treatment with BMP-6 for 30 minutes, or for the indicated time points before total cell lysates were prepared. The amount of phosphorylated Smad 1/5/8 was determined by western-blot analysis. The membranes were reprobed for Smad1. One representative experiment of three is shown.

Figure 7

BMP-6 induced upregulation of Id1 at the mRNA and protein level. CD19⁺ B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points. Total RNA was extracted and Id1, Id2 or Id3-expression was analysed by real-time RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2^-average ΔΔC_T – relative to Id1, Id2 or Id3 expression in the cell-line Ramos). One representative of three independent experiments is shown.

Figure 8

Id protein expression was determined by western-blot analysis. CD19⁺ B cells were cultured in X-vivo 15 over night before treatment with BMP-6 for the indicated time points and cell lysates were prepared. One representative experiment of three is shown. HeLa cells were used as a positive control for Id1 protein detection.

Figure 9

Relative protein expression of Id1, Id2 and Id3. Quantifications of Id1, Id2 and Id3 protein levels were performed using β-actin as normalization and expressed as mean ± SEM (Id1: n = 4, *p ≤ 0.020; Id2 and Id3: n = 3).

Figure 10

Anti-IgM rapidly upregulates BMP-6 mRNA expression in human B-cells. CD19-positive B cells were cultured in X-vivo 15 over night and stimulated with anti-IgM for the indicated time periods, before total RNA was extracted. The expression of BMP-6 mRNA by realtime RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2^-average ΔΔC_T – relative to BMP-6 expression in the cell-line Ramos. One representative of five independent experiments is shown.

Figure 11

Fetal calf serum and human AB serum upregulates BMP-6 mRNA expression in human B-cells. CD19-positive B cells were cultured in X-vivo 15 over night and with fetal calf serum or human AB serum at the indicated dilutions for four hours, before total RNA was extracted. The expression of BMP-6 mRNA by realtime RT-PCR; values are normalised to the expression level of PGK1 mRNA and expressed as relative quantification (2^-average ΔΔC_T – relative to BMP-6 expression in the cell-line Ramos. One representative of three independent experiments is shown.