BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway - PubMed (original) (raw)

BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

Andrew Craxton et al. J Exp Med. 2005.

Abstract

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)-induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3-only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector-mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal-regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim(-/-) mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.

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Figures

Figure 1.

Figure 1.

BAFF and APRIL block BCR-induced apoptosis in WEHI-231 B Cells. (A) WEHI-231 cells were treated for 48 h with anti-IgM in the presence or absence of BAFF or APRIL, and apoptotic cells were quantified by FACS using TdT-mediated dUTP nick-end labeling. Results show the means ± SD from three independent experiments. (B) Binding of BAFF to Daudi (left) and WEHI-231 (right) B cell lines was detected by FACS using biotinylated BAFF and streptavidin–PE. Biotinylated BAFF + streptavidin–PE (shaded) and streptavidin–PE only (bold line) traces are shown. Results shown are from one of three experiments. (C) Daudi cells were treated for 48 h with anti-IgM in the presence (open bar) or absence (shaded bar) of BAFF or etoposide (as a positive control), and cell death was quantified by FACS using Mitotracker Red CMXRos. Results shown are from one of two experiments. (D) BAFF-R, TACI, BCMA, and β-actin (as a loading control) were amplified by RT-PCR using gene-specific primers from fourfold serial dilutions of WEHI-231, A20, and splenic B cell cDNA (Table S1, available at

http://www.jem.org/cgi/content/full/jem.20051283/DC1

). PCR products were resolved on NuSieve 3:1 agarose.

Figure 2.

Figure 2.

BAFF and APRIL block BCR-induced cell death upstream of caspase-3 and -9 activation and mitochondrial inner membrane depolarization. (A) WEHI-231 cells were treated for the indicated times with anti-IgM antibodies in the presence or absence of BAFF or APRIL. Immunoblots were probed with anti-PARP, anti–cleaved caspase-3, or anti–caspase-9. n.s., nonspecific. (B) WEHI-231 cells were treated for 24 or 48 h with either medium, anti-IgM, or etoposide (as a positive control). (top right) Percentages of Mitotracker Red CMXRos–low cells are shown. (C) WEHI-231 cells were stimulated for 48 h with the indicated concentrations of BAFF in the presence (□) or absence (▪) of anti-IgM. Δψm was determined by FACS using Mitotracker Red CMXRos. Results shown are the means ± SD from three experiments.

Figure 3.

Figure 3.

BAFF- and APRIL-induced down-regulation of Bim correlates with a blockade of BCR-induced apoptosis. (A) NP-40 lysates from WEHI-231 cells were immunoprecipitated with anti-Bim mAb or IgG2a isotype control. Immune complexes were probed with anti-Bim serum. Positions of Bim isoforms including multiple BimEL species are shown by arrows. (B) WEHI-231 cells were incubated for 0–48 h with anti-IgM, BAFF, or APRIL (as in Fig. 2 A). Western blots were probed with anti-Bim or anti-APRIL serum (loading control). (C) Purified mouse splenic B cells were incubated with anti-IgM and/or BAFF for 24 h. Immunoblots were probed with anti-Bim or anti-actin serum (loading control). The ratio of BimEL to actin was assessed by scanning densitometry. Dividing lines separate images from different parts of the same gel. (D) WEHI-231 cells were treated for the indicated times with anti-IgM, BAFF, or anti-CD40. Immunoblots were probed with anti-Bim, anti–Bcl-xL, anti-Bmf, or anti–NF-κB2 serum. Nonspecific (n.s.) proteins serve as loading controls. (E and F) WEHI-231 cells were incubated with anti-IgM antibodies and/or BAFF as indicated. Bim (E) or Bcl-2 (F) were immunoprecipitated from NP-40 lysates, which were probed with anti–Bcl-2 or anti-Bim, stripped, and reprobed with anti-Bim or anti–Bcl-2, respectively. Nonspecific (n.s.) bands are indicated. The ratio of Bcl-2 to Bim was quantified by scanning densitometry.

Figure 4.

Figure 4.

Down-regulation of Bim inhibits BCR-induced apoptosis in WEHI-231 B cells. (A) Immunoblots were prepared as in Fig. 2 and probed with anti-Bim or anti-p38 MAPK (loading control). BimEL species migrating with different mobilities are indicated by arrows. (B) Stable WEHI-231 cell lines expressing either Bim-specific siRNAs or a control vector were treated for 48 h with the indicated doses of anti-IgM, and Δψm was determined by FACS using Mitotracker Red CMXRos. Results shown are the means ± SD from three independent experiments.

Figure 5.

Figure 5.

BAFF induces Bim phosphorylation via sustained activation of the MEK–ERK pathway. (A) WEHI-231 cells were treated for 24 h with medium, anti-IgM, BAFF, or anti-CD40. Cell lysates were incubated with or without λ protein phosphatase. Immunoblots were probed with anti-Bim serum. (B) WEHI-231 cells were pretreated for 2 h with DMSO vehicle, 5 μM MEK inhibitor U0126 (U), or 5 μM p38 MAPK inhibitor SB203580 (SB) before incubation with BAFF for 24 h. Blots were probed with anti-Bim, anti–phospho-ERK, or anti-actin (loading control). (C and D) WEHI-231 cells were treated for 0–24 h with anti-IgM in the presence or absence of BAFF. Immunoblots were probed with either anti–phospho-ERK, anti–phospho-MEK, anti–phospho–c-Raf (Ser 338), anti-MKP2, anti-Bim, or anti–Bcl-2 serum. In (D), immunoblots for ERK2 (top) or MEK1/2 (bottom) were quantified by scanning densitometry to measure the fold change in each phosphorylated protein.

Figure 6.

Figure 6.

Blockade of BCR-induced apoptosis by chimeric BAFF receptors correlates with changes in Bim mobility and expression. (A) Chimeric BAFF receptors were generated by in-frame fusion of the extracellular and transmembrane domains of mouse CD8α to the cytoplasmic regions of BAFF-R, TACI, and BCMA. (B) Surface levels of chimeric BAFF receptors on clonal WEHI-231 cell lines were quantified by FACS using biotinylated anti-CD8α mAb. Isotype control (open trace) and CD8α cell surface (shaded trace) expression levels are shown. (C and D) Clonal WEHI-231 cell lines expressing chimeric BAFF receptors were incubated for 48 h with the indicated stimuli. Cell death was quantified by trypan blue staining. Results shown are the means ± SD from at least three independent clonal lines. Bim and NF-κB2 were detected by immunoblotting of cell lysates as described in Fig. 3. A dividing line separates images from different parts of the same gel. n.s., nonspecific.

Figure 7.

Figure 7.

Splenic B cell subsets are dysregulated in Bim KO mice and differ in their expression of BH3-only Bcl-2 family members. (A) Splenic B cells were isolated from WT and Bim−/− mice, stained with αIgD–FITC, αCD21–PE, and αIgM-PerCP, and the numbers of T1, T2-FOP, FO, T2-MZP, and MZ B cells were determined. Data shown are the means ± SEM from three mice per phenotype. MZ B cell numbers in WT and Bim KO mice were 1.7 ± 0.2 × 106 and 0.73 ± 0.09 × 106, respectively. P < 0.05 (*) and P < 0.02 (**) were determined using an unpaired t test. (B) Cell lysates of isolated splenic B cells from WT and Bim−/− mice were probed with anti-Bim or anti-actin (loading control). (C) T1, T2, FO, and MZ splenic B cell subsets were purified by FACS cell sorting using αCD21–FITC, αCD23-APC, and αCD24–PE. Spontaneous cell death was quantified at the indicated times by FACS using Mitotracker Red CMXRos. (D) T1, T2, and FO splenic B cell subsets were isolated as described in C and treated for 18 h with the indicated doses of anti-IgM, and cell death was analyzed as described in C. (E) T1, T2, FO, and MZ splenic B cell subsets were purified as described in C. Bim, Bik, Bmf, Noxa, Puma, and β-actin (loading control) were amplified by RT-PCR (Table S1), and PCR products were resolved as in Fig. 1D. (F) Splenic B cell subsets were isolated by FACS cell sorting as described above. Immunoblots were probed with anti-Bim or anti-actin (loading control).

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References

    1. Marsden, V.S., and A. Strasser. 2003. Control of apoptosis in the immune system: Bcl-2, BH3-only proteins and more. Annu. Rev. Immunol. 21:71–105. - PubMed
    1. Strasser, A. 2005. The role of BH3-only proteins in the immune system. Nat. Rev. Immunol. 5:189–200. - PubMed
    1. Chung, J.B., M. Silverman, and J.G. Monroe. 2003. Transitional B cells: step by step towards immune competence. Trends Immunol. 24:343–349. - PubMed
    1. Carsetti, R., G. Kohler, and M.C. Lamers. 1995. Transitional B cells are the target of negative selection in the B cell compartment. J. Exp. Med. 181:2129–2140. - PMC - PubMed
    1. Turner, M., A. Gulbranson-Judge, M.E. Quinn, A.E. Walters, I.C. MacLennan, and V.L. Tybulewicz. 1997. Syk tyrosine kinase is required for the positive selection of immature B cells into the recirculating B cell pool. J. Exp. Med. 186:2013–2021. - PMC - PubMed

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