The combined absence of NF-kappa B1 and c-Rel reveals that overlapping roles for these transcription factors in the B cell lineage are restricted to the activation and function of mature cells

Abstract

Transcription factors NF-kappaB1 and c-Rel, individually dispensable during embryogenesis, serve similar, yet distinct, roles in the function of mature hemopoietic cells. Redundancy among Rel/NF-kappaB family members prompted an examination of the combined roles of c-Rel and NF-kappaB1 by using mice that lack both proteins. Embryonic development and the maturation of hemopoietic progenitors were unaffected in nfkb1(-/-)c-rel(-/-) mice. Peripheral T cell populations developed normally, but follicular, marginal zone, and CD5(+) peritoneal B cell populations all were reduced. In culture, a failure of mitogen-stimulated nfkb1(-/-)c-rel(-/-) B cells to proliferate was caused by a cell cycle defect in early G(1) that prevented growth. In vivo, defects in humoral immunity and splenic architecture seen in nfkb1(-/-) and c-rel(-/-) mice were exacerbated in the double mutant mice. These findings demonstrate that in the B lineage overlapping roles for NF-kappaB1 and c-Rel appear to be restricted to regulating the activation and function of mature cells.

Figure 1

The peritoneal CD5⁺ B cell population is diminished in nfkb1^−/− c-rel^−/− mice. Cells isolated from the peritoneal cavity of wild-type, c-rel^−/−, nfkb1^−/−, and nfkb1^−/− c-rel^−/− mice were stained with mAbs for B220 and CD5, then examined by flow cytometry. A typical series of staining profiles is shown, with the proportion of CD5⁺ B cells (boxed region) observed in mice of each genotype expressed as a percentage of the total peritoneal cell population.

Figure 2

Absence of c-Rel does not enhance spontaneous death in culture or turnover of nfkb1^−/− B cells in vivo. (A) Splenic B cell turnover in vivo. Splenocytes isolated from wild-type and mutant mice fed BrdUrd over 5 days were subjected to three-color immunofluorescent staining and flow cytometry to determine the fraction of immature (sIgM^hiIgD^lo) and mature (sIgM^loIgD^hi) B cells incorporating BrdUrd. The kinetics of BrdUrd incorporation by immature and mature wild-type (○), nfkb1^−/− (▵), c-rel^−/− (◊), and nfkb1^−/−c-rel^−/− (□) B cells over a 5-day time course are summarized. Values represent the mean ± SD from four mice per genotype at each time point; the data are representative of two experiments. (B) Survival of quiescent B cells in culture. Resting splenic B cells from wild-type (○), nfkb1^−/− (▵), c-rel^−/− (◊), and nfkb1^−/−c-rel^−/− (□) mice were cultured in DME/10% FCS without mitogens or cytokines for 72 h. At 24-h intervals, the frequency of dead cells was determined by flow cytometric analysis of PI-stained cells. At the start of the experiments, >99% of all cells were viable. The data represent the mean ± SD of five experiments.

Figure 3

nfkb1^−/−c-rel^−/− B cells fail to proliferate in response to mitogenic stimulation. (A) B cell proliferative responses. Purified resting wild-type (filled bars), nfkb1^−/− (hatched bars), c-rel^−/− (empty bars), or c-rel^−/−nfkb1^−/− (stippled bars) splenic B cells were stimulated for 72 h with optimal concentrations of anti-IgM (Fab)₂ antibodies, anti-RP antibodies, LPS, anti-IgM antibodies plus LPS, or anti-RP antibodies plus LPS. Proliferation was measured by [³H]thymidine incorporation. (B) Cell cycle analysis. The fraction of viable cells of each genotype (identification code as above) in the S, G₂, or M phases of the cell cycle after stimulation for 72 h with the indicated mitogens was determined by flow cytometric analysis of permeabilized cells stained with PI. Before stimulation, more than 99% of resting B cells were viable and <1% of cells had entered the cell cycle. (C) Cell death. The frequency of apoptotic cells (genotypic code as for A) expressed as a proportion of the total cell number after 72 h of mitogen stimulation was determined by flow cytometric analysis of permeablized cells stained with PI. Cells with a DNA content <2n were deemed to be apoptotic. All results shown represent the mean ± SD from five experiments.

Figure 4

A combined absence of NF-κB1 and c-Rel prevents mitogen-induced B cell growth. Normal, c-rel^−/−, nfkb1^−/−, and nfkb1^−/−c-rel^−/− splenocytes stimulated in culture with anti-RP antibodies plus LPS for 72 h were stained at 24-h intervals with biotinylated B220, then examined by flow cytometry. Forward light scatter profiles (x axis: linear scale) are a measure of cell size. The data shown were electronically gated to exclude dead cells and are representative of three experiments. Similar results were obtained by using different stimuli (results not shown).

Figure 5

Antibody production and germ-line CH gene expression in nfkb1^−/−c-rel^−/− mice. (A) Serum Ig levels in naive mice. Levels of serum Igs (μg/ml) in naive 8- to 10-week-old litter-matched mice was determined by isotype-specific ELISA. Filled, hatched, empty, and stippled bars correspond to wild-type, nfkb1^−/−, c-rel^−/−, and nfkb1^−/c-rel^−/− mice, respectively. Five mice of each genotype were examined. IgG1 levels were below the level of detection (ND) in nfkb1^−/c-rel^−/− mice. (B) Humoral response to NP-KLH. Five age-matched mice of each genotype were immunized with NP-KLH and serum samples were collected after 21 days. The concentration (μg/ml) of NP-specific IgG1 is indicated for wild-type (filled bars), nfkb1^−/− (hatched bars), and c-rel^−/− (empty bars) mice. Antigen-specific IgG1 was below the levels of detection (ND) in nfkb1^−/c-rel^−/− mice. (C) Germ-line CH transcript expression. RNA isolated from equal numbers of wild-type (lane 1), nfkb1^−/− (lane 2), c-rel^−/− (lane 3), and nfkb1^−/−c-rel^−/− (lane 4), B cells stimulated in culture for 6 h with various mitogen plus cytokine combinations (13) was subjected to semiquantitative reverse transcription–PCR using primers specific for Cγ3, Cγ1, Cɛ, and Cα germ-line RNAs and β-actin mRNA as a control. The PCR products were then fractionated on agarose gels. B cells were stimulated with LPS alone (Cγ3 RNA), LPS plus IL-4 (Cγ1,Cɛ RNA), and LPS plus transforming growth factor β (Cα RNA).

Figure 6

Germinal centers do not form in nfkb1^−/−c-rel^−/− mice. Sections of spleens from wild-type, nfkb1^−/−, c-rel^−/−, and nfkb1^−/−c-rel^−/− mice 14 days postimmunization with NP-KLH were stained with horseradish peroxidase-labeled B220 and biotinylated PNA. Follicles (F) and germinal centers (arrows) are indicated (×100 magnification). The stained sections shown are representative of mice of each genotype.

Figure 7

Roles of Rel/NF-κB proteins during B cell development and activation. The functions of different Rel/NF-κB proteins within the B cell lineage were deduced from the phenotype of single and multiple combinations of null mutations for these proteins.