CD38 expression in early B-cell precursors contributes to extracellular signal-regulated kinase-mediated apoptosis

Abstract

CD38 is a 45,000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B-cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B-cell development. Pre-pro-B, pro-B, pre-B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B-cell maturation. To assess the role of CD38 during B-cell maturation, CD38-deficient mice were analysed. CD38(-/-) mice showed a significant increase in both the frequency of B-lineage cells and the absolute numbers of pre-pro-B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross-linking in Ba/F3 cells promoted apoptosis and marked extracellular signal-regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B-cell precursors from bone marrow. These data demonstrate that B-cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK-associated regulation of the B-lineage differentiation pathway.

Keywords: CD38; bone marrow; extracellular signal-regulated kinase; mouse B-cell development.

Figure 1

Expression of CD38 on bone marrow (BM) B-cell precursors. (a) Bone marrow cells from C57BL/6 mice were stained with: anti-CD19, anti-CD45R, anti-IgM, anti-CD43, and anti-CD38 antibodies. Pre-pro-B (B220^– CD43⁻ CD19⁻), pro-B (B220^– CD43⁻ CD19^–), pre-B (B220^– CD43^– CD19^– IgM⁻), and immature B (B220^– CD43⁻ CD19^– IgM^–) cell fractions were analysed through flow cytometry. (b) CD38 expression within the pre-pro-B, pro-B, pre-B, and immature B-cell populations from murine BM. Values shown in mean and standard deviation (SD). (c) Median fluorescence intensity (MFI) of CD38 expression in different populations of BM B cells. (d) Analysis of granularity and CD38 expression of pro-B cells from murine BM, representative figure from four independent experiments.

Figure 2

CD38 deficient mice show increased numbers of Pre-Pro-B cells. Cell frequencies (a, c) and absolute numbers (b, d) of pre-pro-B, pro-B, pre-B and immature B cells from the bone marrow (BM) of wild-type (WT) and CD38^−/− mice. The average ± SD from 10 animals is shown.

Figure 3

CD157 expression on pre-pro-B, pro-B, pre-B and immature B cells from the bone marrow (BM) of C57BL/6 and CD38^−/− mice. (a) Expression of CD157 within the pre-pro-B, pro-B, pre-B and immature B cell populations from murine BM, [wild-type (WT) or CD38^−/− mice]. (b) Mean fluorescence intensity (MFI) of CD38 expression in different bone marrow B-cell populations. The average ± SD from five independent experiments is shown. (c) Analysis of granularity and CD157 expression in pro-B cells from murine BM.

Figure 4

NIM-R5 induces extracellular signal-regulated kinase ( ERK) phosphorylation in the CD38-transfected pro-B-cell line Ba/F3. (a) CD38-transfected (right histogram) or mock-transfected (left histogram) Ba/F3 cells (10⁶) were stained with the monoclonal antibody NIM-R5 followed by a goat anti-rat IgG2a-phycoerythrin-labelled antibody. The cell suspensions were analysed through FACS. (b) Ba/F3 cells stably transfected with murine CD38 or mock-transfected cells (10⁷ cells/condition) were stimulated with anti-CD38 (NIM-R5) for 0, 1, 5 and 10 min at 37°. The cells were lysed for 30 min at 4° in a buffer containing 1% nonidet-40 and protease inhibitors, and 10⁶ cell equivalents were resolved through SDS–PAGE and WB for phospho-ERK (upper), total ERK (lower). (c) CD38-transfected Ba/F3 cells were left untreated or were treated with rat IgG, anti-CD38 (NIM-R5) or PMA for 10 min at 37°. The cell lysates were resolved through SDS–PAGE and WB for phospho-ERK (upper) and total ERK (lower). (d) CD38-transfected Ba/F3 cells (10⁶) were stained with the monoclonal antibody NIM-R8 (anti-CD44) followed by a goat anti-rat IgG2a-phycoerythrin-labelled antibody. The cell suspensions were analysed through FACS.

Figure 5

The extracellular signal-regulated kinase (ERK) pathway inhibitor PD98059 interferes with the CD38-induced apoptosis in CD38-transfected Ba/F3 cells. (a) CD38-transfected Ba/F3 cells (10⁶) were pre-incubated for 30 min with either DMSO (upper panel) or the inhibitor PD98059 (20 µm, lower panel). The cells were stimulated with anti-CD38 (NIMR5), rat IgG or left untreated for 4 hr at 37° in the constant presence of vehicle or inhibitor. After stimulation, the cells were transferred to FACS tubes and Annexin-V/propidium iodide staining was performed as described in the Materials and methods. The cells were resuspended in 300 µl binding buffer and immediately captured using a FACSCalibur cytometer (Becton Dickinson). (b) Graph of percentages of Annexin-V/propidium iodide-positive cells. The averages ± SD from three independent experiments is shown. The groups were compared using unpaired Student's t-test and one-way analysis of variance, and P-values ≤ 0·05 were considered significant. PD98059 (−): absence of inhibitor; PD98059 (–): presence of inhibitor. Anti-CD44 was used as control (IgG2a).

Figure 6

NIM-R5 induces apoptosis in B-cell precursors from bone marrow. (a) Purified B-cell precursors (Pre-pro-B, pro-B, pre-B and immature B) were stimulated for 12 hr in the presence 50 µg/ml anti-CD38 monoclonal antibody (NIM-R5) or an isotype control (rat IgG) antibody. Cells were then stained with annexin-V FITC and propidium iodide (AnV–PI) and analysed by FACS. AnV–IP– Percentage averages ± SD of three independent experiments are shown. (b) Representative dot plots of apoptosis of pre-pro-B cells and (c) immature B cells.

Figure 7

Extracellular signal-regulated kinase (ERK) is phosphorylated after CD38 ligation in total bone marrow B cells. Total bone marrow cells were stimulated with (a) PMA (0·2 µg/ml) as control for activation, (b) anti-CD38 (50 µg/ml) and an isotype control (rat IgG, 50 µg/ml) for 5, 10 or 20 min. After stimulation, pERK expression was measured in B cells using flow cytometry (c); mean fluorescence intensity (MFI) averages ± SD of three independent experiments are shown. (d) Western blot analysis and (e) fold increase of pERK.

Figure 8

Extracellular signal-regulated kinase (ERK) is phosphorylated after CD38 ligation in B-cell precursors from bone marrow. Purified B-cell precursors (Pre-pro-B, pro-B, pre-B and immature B) were stimulated with PMA (0·2 µg/ml) as control for activation and anti-CD38 (50 µg/ml) or an isotype control (rat IgG) antibody for 5 and 15 min. After stimulation, pERK were measured using flow cytometry.