The Fas-associated death domain protein is required in apoptosis and TLR-induced proliferative responses in B cells

Abstract

The Fas-associated death domain protein (FADD)/Mort1 is a signaling adaptor protein which mediates the activation of caspase 8 during death receptor-induced apoptosis. Disruption of FADD in germ cells results in death receptor-independent embryonic lethality in mice. Previous studies indicated that in addition to its function in apoptosis, FADD is also required in peripheral T cell homeostasis and TCR-induced proliferative responses. In this report, we generated B cell-specific FADD-deficient mice and showed that deletion of FADD at the pro-B cell stage had minor effects on B cell development in the bone marrow, and resulted in increased splenic and lymph node B cell numbers and decreased peritoneal B1 cell numbers. As in T cells, a FADD deficiency inhibited Fas-induced apoptosis in B cells. However, B cell-proliferative responses induced by stimulation of the BCR and CD40 using anti-IgM or anti-CD40 Abs were unaffected by the absence of FADD. Further analyses revealed that FADD-deficient B cells were defective in proliferative responses induced by treatments with dsRNA and LPS which stimulate TLR3 and TLR4, respectively. Therefore, in addition to its apoptotic function, FADD also plays a role in TLR3- and TLR4-induced proliferative responses in B cells.

FIGURE 1

(A) Diagrammatic scheme in generating B cell-specific FADD^-/- mice. The CD19 locus is closely linked to FADD on mouse chromosome 7 with a ~10 centimorgan distance (top). The CD19-Cre allele (48) was crossed to the FADD knockout allele (22) (middle) by mouse mating in order to delete FADD:GFP^flox specifically in B cells in FADD^-/- mice. The promoter of CD19 (arrows), the neomycin-resistant gene (neo), exons of FADD (boxes) and GFP are indicated. Drawings are not to scale. (B) Flow cytometric analysis of FADD:GFP deletion in B cells. Single cell suspensions were prepared from the bone marrow, spleen, lymph nodes, and peripheral blood, and stained for CD19. The indicated percentages of GFP^- cells in the CD19⁺ population of FADD^-/- FADD:GFP^flox CD19-Cre mice were determined by flow cytometry. Cells from FADD^+/- CD19-Cre and FADD^-/- FADD:GFP^flox mice were used as GFP^- and GFP⁺ controls, respectively. Histograms are from one experiment using one mouse of each genotype, and are representative of 14 independent experiments. (C) Total thymocytes and splenocytes (left) or sorted GFP^- B cells (right) from FADD^-/- FADD:GFP^flox CD19-Cre mice were analyzed by western blotting using anti-FADD antibodies. FADD^+/-, FADD^+/- FADD:GFP^flox, FADD^-/- FADD:GFP^flox, FADD^+/- FADD:GFP^flox CD19-Cre mice were used as controls. The absence of FADD:GFP in GFP^- cells were confirmed in three independent experiments.

FIGURE 2

Flow cytometric analyses of the B cell development in the bone marrow in the absence of FADD. (A) Deletion of FADD:GFP in the B lineage in the bone marrow of FADD^-/- FADD:GFP^flox CD19-Cre mice was detected by the presence of the indicated percentages of GFP^- B cells at various developmental stages. FADD^+/- CD19-Cre mice were used as GFP^- controls (dotted histograms). Histograms are from one pair of mice, representative of 14 independent experiments. (B) Pro-B (CD19⁺c-Kit⁺), pre-B (CD19⁺CD25⁺), immature (IgD^-IgM⁺), transitional (CD19⁺AA4.1⁺), and re-circulating follicular (IgD⁺IgM⁺) B cells in the bone marrow of FADD^-/- FADD:GFP^flox CD19-Cre (FADD^-/-) mice were detected by flow cytometry upon staining with the indicated stage-specific markers. Cells from FADD^+/- FADD:GFP^flox CD19-Cre mice (FADD^+/-) were used as controls. Data shown are representative of 10 pairs of mice (age 2 to 6 month) analyzed.

FIGURE 3

Analysis of the peripheral lymphoid system in B cell-specific FADD^-/- mice. (A) Total (left) and CD19⁺ B (right) cell numbers in the spleen and lymph nodes from FADD^-/- FADD:GFP^flox CD19-Cre mice (FADD^-/-) were compared to that of control FADD^+/- FADD:GFP^flox CD19-Cre mice (FADD^+/-). Error bars indicate standard deviation from analysis of 7 mice of each indicated genotype. (B) Representative dot plots from flow cytometric analyses of CD3⁺ and CD19⁺ B cells in 15 mice of each genotype indicated. (C) Immunohistological analysis of cryosections of the spleen and lymph nodes. B cells were stained with anti-B220 antibodies (blue) and T cells were stained with anti-CD4 antibodies (brown). Data shown is representative of analysis of 5 mice of each indicated genotype.

FIGURE 4

Flow cytometric analysis of B cell development in the spleen and peritoneal cavity in the B cell-specific FADD^-/- mice. FADD^+/- FADD:GFP^flox CD19-Cre mice (FADD^+/-) were used as controls. Spleen cells were stained for IgD and IgM (A), or for CD21 and CD23 (B), and peritoneal cells were stained for IgM and Mac-1 (C). Percentages of splenic transitional (T1 and T2), follicular (FO), marginal zone (MZ), and peritoneal B cells are indicated. Flow cytometric plots are representative of analysis of 5 mice of each indicated genotype.

FIGURE 5

Apoptosis and serum Ig analyses. (A) FADD:GFP, FADD^+/-, and FADD^-/- B cells were sorted from FADD^-/- FADD:GFP^flox, FADD^+/- FADD:GFP^flox CD19-Cre and FADD^-/- FADD:GFP^flox CD19-Cre mice, respectively. Apoptosis in these cells was induced by stimulation with sFasL for 16 h, and measured by PI staining and flow cytometry. Error bars indicate standard deviation of triplicates from one experiment, and is representative of seven independent experiments using mice of different ages. (B) Sera were collected from 5 mice of the FADD^+/- FADD:GFP^flox CD19-Cre (open circle) and FADD^-/- FADD:GFP^flox CD19-Cre (filled diamond) genotypes, aged from 2 to 10 months. Ig levels were determined by ELISA. Bars indicate mean values for each Ig assay.

FIGURE 6

Analysis of B cell proliferation. FADD^+/- (filled bars) and FADD^-/- B cells (open bars) were sorted for the GFP^- population from FADD^+/- FADD:GFP^flox CD19-Cre and FADD^-/- FADD:GFP^flox CD19-Cre mice, and stimulated in triplicates with anti-IgM Abs (A), anti-CD40 Abs (B), poly (I:C) (C), LPS (D), CpG-containing DNA (E), or nonspecific, control oligo DNA (F) at the concentrations indicated. Proliferation was indicated by the amount of [³H] thymidine incorporated (shown as counts per min on Y axis). Error bars indicate standard deviation of triplicates. Data shown are from one experiment and represent three to five independent experiments. (G) Expression of TLR3 expression was determined by western blotting. (H) Expression of TLR4 was determined by flow cytometry.

FIGURE 7

Cell division, survival and death analyses. (A) Purified FADD^+/- and FADD^-/- splenic and lymph node B cells were labeled with CFSE, and stimulated with anti-IgM Abs (10 μg/ml; top) or LPS (10 μg/ml, bottom), and division of B cells was determined by flow cytometry at 72, 96, and 120 h post stimulation. Percentages of divided cells were indicated. (B) To determine survival capability, sorted FADD^-/- and FADD^+/- control B cells were cultured in complete medium for two days, and at indicated times cells were analyzed by flow cytometry for viablility as indicated by PI exclusion. Cell death in mutant and control B cells stimulated with LPS (1 μg/ml) were determined similarly by flow cytometry. (C) Cell death and division were analyzed by two-color flow cytometry by 7-AAD staining of CFSE-labeled B cells stimulated with LPS for 72 h. Data shown are from one experiment using one pair of mice, and are representative of 3 independent experiments.

FIGURE 8

Analysis of activation marker upregulation and intracellular signaling in B cells. (A) Induction of CD54, CD86 and MHC class II was analyzed by staining with indicated antibodies at 0 (thin lines) and 16 h (thick lines) after LPS stimulation of sorted FADD^-/- mutant and FADD^+/- control B cells. (B) Total proteins from FADD^+/- and FADD^-/- B cells stimulated with LPS (10 μg/ml) for the indicated times were analyzed for NF-κB activation by detecting degradation of IκB. To analyze Erk activation, the same nitrocellulose membrane was re-probed with antibodies specific for phosphorylated (p)-Erk1/2, and probed with anti-Erk1/2 antibodies after stripping. The membrane was probed for the fourth time with anti-p-Jnk antibodies after the second stripping. A separate membrane with samples collected at the indicated times was probed with anti-p-Akt antibodies to detect Akt phopshorylation induced by LPS stimulation (10 μg/ml). FADD^+/- B cells were used as control. Data shown are representative of at least three independent experiments.