B lymphocytes differentially use the Rel and nuclear factor kappaB1 (NF-kappaB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells

Abstract

Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.

Figure 1

The absence of NF-κB1 accelerates apoptosis of quiescent B cells. (A) B cells turnover more rapidly in nfkb1^−/− mice. The turnover of virgin and mature splenic B cells was determined by BrdU incorporation. Splenic B cells isolated from normal, c-rel^−/−, and nfkb1^−/− mice fed with BrdU for 2, 4, or 7 d were subjected to three-color immunofluorescence staining, after which flow cytometric analysis was used to identify virgin (sIgM⁺sIgD⁻) and mature (sIgM⁺sIgD⁺) B cells and to determine the fraction of these cells that had incorporated BrdU. Examples from the analysis of mice fed BrdU for 4 d are shown. The top row shows two-color FACS^® dot plots (x-axis, staining with R-PE–labeled anti-IgM antibodies; y-axis, staining with biotinylated anti-IgD antibodies plus Tricolor-streptavidin), with the boxed regions used for electronic gating of mature B cells. The middle rows present histograms of the anti-BrdU–labeling intensity (FITC) of these mature B cells. The percentages of BrdU⁺ cells are indicated as are the boundaries used for distinguishing them from BrdU⁻ cells. The bottom panels summarize the kinetics of BrdU labeling for virgin and mature splenic B cells over a 4- and 7-d time course, respectively. The values are arithmetic means plus SD from the analysis of three normal, c-rel^−/−, and nfkb1^−/− mice at each time point. (B) Cell death in culture. Resting splenic B cells from normal (triangles), c-rel^−/− (circles), and nfkb1^−/− (squares) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and flow cytometric analysis of fixed cells stained with PI. At the start of the experiment, >99% of cells of all genotypes were viable. The data represents the mean ±SD of five experiments.

Figure 1

The absence of NF-κB1 accelerates apoptosis of quiescent B cells. (A) B cells turnover more rapidly in nfkb1^−/− mice. The turnover of virgin and mature splenic B cells was determined by BrdU incorporation. Splenic B cells isolated from normal, c-rel^−/−, and nfkb1^−/− mice fed with BrdU for 2, 4, or 7 d were subjected to three-color immunofluorescence staining, after which flow cytometric analysis was used to identify virgin (sIgM⁺sIgD⁻) and mature (sIgM⁺sIgD⁺) B cells and to determine the fraction of these cells that had incorporated BrdU. Examples from the analysis of mice fed BrdU for 4 d are shown. The top row shows two-color FACS^® dot plots (x-axis, staining with R-PE–labeled anti-IgM antibodies; y-axis, staining with biotinylated anti-IgD antibodies plus Tricolor-streptavidin), with the boxed regions used for electronic gating of mature B cells. The middle rows present histograms of the anti-BrdU–labeling intensity (FITC) of these mature B cells. The percentages of BrdU⁺ cells are indicated as are the boundaries used for distinguishing them from BrdU⁻ cells. The bottom panels summarize the kinetics of BrdU labeling for virgin and mature splenic B cells over a 4- and 7-d time course, respectively. The values are arithmetic means plus SD from the analysis of three normal, c-rel^−/−, and nfkb1^−/− mice at each time point. (B) Cell death in culture. Resting splenic B cells from normal (triangles), c-rel^−/− (circles), and nfkb1^−/− (squares) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and flow cytometric analysis of fixed cells stained with PI. At the start of the experiment, >99% of cells of all genotypes were viable. The data represents the mean ±SD of five experiments.

Figure 2

Cell cycle progression is impaired and apoptosis increased in mitogen activated c-rel^−/− and nfkb1^−/− B cells. Purified, resting normal (triangles), c-rel^−/− (circles), or nfkb1^−/− (squares) B cells were stimulated for 72 h with optimal concentrations of anti-IgM antibodies, LPS, or anti-RP antibodies in the absence (open symbols) or presence (closed symbols) of IL-2, IL-4, and IL-5. (A) Cellular proliferation. B cell proliferation was measured at 24-h intervals over a 72-h period by [³H]thymidine incorporation. (B) Cell death. The frequency of apoptotic cells, expressed as a proportion of the total cell number, was determined by flow cytometric analysis of permeabilized cells stained with PI. Greater than 99% of resting B cells were viable at the start of the experiment. (C) Cell cycle analysis. The fraction of viable B cells in the S, G2, or M phases of the cell cycle expressed as a function of the stimulation period was determined by flow cytometric analysis of permeabilized cells stained with PI. Less than 1% of cells were in G1, S, or G2/M before stimulation. All results in this figure represent the mean ±SD from eight experiments.

Figure 3

The cell cycle block and enhanced apoptosis in mitogen-stimulated c-rel^−/− B lymphocytes are due to a cell autonomous defect. Equivalent numbers of resting c-rel^+/+ Ly5.2 (triangles) and c-rel^−/− Ly5.1 (circles) B cells were stimulated separately (open symbols) or in mixed cultures (closed symbols) with anti-IgM or anti-RP antibodies over a period of 72 h. At 24-h intervals, normal and Rel^−/− cells from individual cultures or isolated from the mixed cultures by cell sorting were fixed, stained with PI, and the DNA content determined by flow cytometric analysis. The frequency of viable normal and c-rel^−/− cells in G0 before stimulation was >99%. (A) Levels of apoptosis in B cell populations from the separate or mixed cultures are equivalent. The frequency of apoptotic cells is expressed as a proportion of the total number of B cells of each genotype. (B) c-rel^−/− B cell division is not influenced by normal cells. The percentage of cells in the S plus G2/M phases of the cell cycle correspond to that fraction of the total viable B cell population in each culture. These results are representative of the mean of four independent experiments.

Figure 4

Mitogen stimulation of c-rel^−/− B cells leads to changes in cell size and the expression of certain cell surface markers. Normal and Rel^−/− splenic B cells in the resting state (T 0), treated with anti-IgM, or treated with anti-CD40 antibodies for 24 h were stained with biotinylated monoclonal antibodies specific for class II MHC, B7.2, and the IL-2Rα chain (CD25) and examined by flow cytometry. FSC, the forward light scatter profiles (x-axis: log scale), is a measure of cell size. The vertical lines shown in the class II MHC panels depict the fluorescence intensity of cells stained with a biotinylated isotype-matched control antibody. Data shown are electronically gated to exclude dead cells and are representative of three experiments.

Figure 5

The cell cycle block in mitogen-activated c-rel^−/− and nfkb1^−/− B cells occurs during the early phase of G1. (A) RNA synthesis is inhibited in mitogen-treated c-rel^−/− and nfkb1^−/− B cells. RNA synthesis in normal (triangles), c-rel^−/− (circles), and nfkb1^−/− (squares) B cells stimulated for 72 h with either anti-IgM antibody, LPS, or anti-RP antibody in the absence (open symbols) or presence (closed symbols) of cytokines was measured by [³H]uridine incorporation. These results represent the mean of four experiments ±SD. (B ). The cell cycle block resulting from an absence of Rel prevents B cells entering G1. B cells stimulated over a 72-h period with anti-RP antibodies or anti-RP antibodies plus cytokines were analyzed by simultaneous flow cytometry for DNA and protein content at 24-h intervals. Data is presented as contour plots of DNA content (y-axis) versus protein content (x-axis). The percentage of viable cells in G0 for each series of stimulations is indicated. Dead cells were excluded from analysis by electronic gating on forward and side scatter.

Figure 5

The cell cycle block in mitogen-activated c-rel^−/− and nfkb1^−/− B cells occurs during the early phase of G1. (A) RNA synthesis is inhibited in mitogen-treated c-rel^−/− and nfkb1^−/− B cells. RNA synthesis in normal (triangles), c-rel^−/− (circles), and nfkb1^−/− (squares) B cells stimulated for 72 h with either anti-IgM antibody, LPS, or anti-RP antibody in the absence (open symbols) or presence (closed symbols) of cytokines was measured by [³H]uridine incorporation. These results represent the mean of four experiments ±SD. (B ). The cell cycle block resulting from an absence of Rel prevents B cells entering G1. B cells stimulated over a 72-h period with anti-RP antibodies or anti-RP antibodies plus cytokines were analyzed by simultaneous flow cytometry for DNA and protein content at 24-h intervals. Data is presented as contour plots of DNA content (y-axis) versus protein content (x-axis). The percentage of viable cells in G0 for each series of stimulations is indicated. Dead cells were excluded from analysis by electronic gating on forward and side scatter.

Figure 6

Bcl-2 inhibits apoptosis of mitogen-stimulated c-rel^−/− B cells, but fails to overcome the cell cycle block. (A) Bcl-2 inhibits the enhanced apoptosis of nfkb1^−/− B cells. Quiescent splenic B cells isolated from c-rel^+/+bcl-2⁺ (triangles), c-rel^−/−bcl-2⁺ (circles), and nfkb1^−/−bcl-2⁺ (squares) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and by flow cytometric analysis of fixed cells stained with PI. (B) Proliferation of c-rel^−/−bcl-2⁺ cells is impaired. The proliferation of c-rel^+/+bcl-2⁺ (triangles) and c-rel^−/−bcl-2⁺ (circles) B cells stimulated with either anti-IgM or anti-RP in the absence (open symbols) or presence (closed symbols) of cytokines was monitored at 24-h intervals over a 72-h period by [³H]thymidine incorporation. (C) Mitogen-induced death of c-rel^−/− cells is rescued by bcl-2. The frequency of apoptotic cells in the mitogen-stimulated c-rel^+/+bcl-2⁺ and c-rel^−/−bcl-2⁺ B cell cultures (symbols as for B) was determined by flow cytometric analysis of PI-stained cells. The frequency of apoptotic cells is presented as a proportion of the total cell number. Greater than 99% of resting B cells of both genotypes were viable before mitogen stimulation. (D) Cell cycle progression of mitogen-stimulated c-rel^−/−bcl-2⁺ cells remains blocked. The DNA content of mitogen-stimulated c-rel^+/+bcl-2⁺ and c-rel^−/−bcl-2⁺ B cells (symbols as in B) was determined by flow cytometric analysis of PI-stained cells. The fraction of viable B cells in S, G2, or M is expressed as a function of the stimulation period. Less than 1% of viable cells were in G1, S, or G2/M before stimulation. The results shown represent the mean and SD from four experiments.

Figure 6

Bcl-2 inhibits apoptosis of mitogen-stimulated c-rel^−/− B cells, but fails to overcome the cell cycle block. (A) Bcl-2 inhibits the enhanced apoptosis of nfkb1^−/− B cells. Quiescent splenic B cells isolated from c-rel^+/+bcl-2⁺ (triangles), c-rel^−/−bcl-2⁺ (circles), and nfkb1^−/−bcl-2⁺ (squares) mice were cultured in DMEM/10% FCS without mitogen for a period of 72 h. At 24-h intervals, the frequency of dead cells was determined by trypan blue exclusion and by flow cytometric analysis of fixed cells stained with PI. (B) Proliferation of c-rel^−/−bcl-2⁺ cells is impaired. The proliferation of c-rel^+/+bcl-2⁺ (triangles) and c-rel^−/−bcl-2⁺ (circles) B cells stimulated with either anti-IgM or anti-RP in the absence (open symbols) or presence (closed symbols) of cytokines was monitored at 24-h intervals over a 72-h period by [³H]thymidine incorporation. (C) Mitogen-induced death of c-rel^−/− cells is rescued by bcl-2. The frequency of apoptotic cells in the mitogen-stimulated c-rel^+/+bcl-2⁺ and c-rel^−/−bcl-2⁺ B cell cultures (symbols as for B) was determined by flow cytometric analysis of PI-stained cells. The frequency of apoptotic cells is presented as a proportion of the total cell number. Greater than 99% of resting B cells of both genotypes were viable before mitogen stimulation. (D) Cell cycle progression of mitogen-stimulated c-rel^−/−bcl-2⁺ cells remains blocked. The DNA content of mitogen-stimulated c-rel^+/+bcl-2⁺ and c-rel^−/−bcl-2⁺ B cells (symbols as in B) was determined by flow cytometric analysis of PI-stained cells. The fraction of viable B cells in S, G2, or M is expressed as a function of the stimulation period. Less than 1% of viable cells were in G1, S, or G2/M before stimulation. The results shown represent the mean and SD from four experiments.

Figure 7

c-myc expression is normal in c-rel^−/− B cells. 10-μg samples of total RNA isolated from resting (lanes 1–4) and anti-IgM antibody-stimulated splenic B cells from normal, c-rel^−/−, nfkb1^−/−, and c-rel^−/−bcl-2⁺ mice stimulated for 2 h (lanes 5–8) and 4 h (lanes 9–12) were fractionated on formaldehyde denaturing gels, transferred to nitrocellulose membranes, and sequentially hybridized with radiolabeled murine c-myc cDNA (32) or glyceraldehyde-3-phosphate-dehydrogenase cDNA (33) probes. Filters were exposed for 48-h autoradiography at −70°C. Lanes 1, 5, and 9, normal B cells; 2, 6, and 10, Rel^−/− B cells; 3, 7, and 11, nfkb1^−/− B cells; and 4, 8, and 12, c-rel^−/−bcl-2⁺ B cells.