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The Journal of Immunology, 2003, 171: 3963-3969.
Copyright © 2003 by The American Association of Immunologists

Regulation of Developing B Cell Survival by RelA-Containing NF-{kappa}B Complexes 1

Maria Prendes, Ye Zheng and Amer A. Beg2

Department of Biological Sciences, Columbia University, New York, NY 10027


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Mice deficient in the RelA (p65) subunit of NF-{kappa}B die during embryonic development. Fetal liver (FL) hemopoietic precursors from these mice were used to generate RelA-deficient lymphocytes by adoptive transfer into lethally irradiated mature lymphocyte-deficient recombination-activating gene-1-/- mice. Strikingly, RelA-/- lymphocyte generation was greatly diminished compared with that of RelA+/+ lymphocytes. The most dramatic reduction was noticed in the numbers of developing B cells, which were considerably increased when RelA-/- FL cells that were also TNFR1 deficient were used. The role of RelA was further investigated in FL-derived developing B cells in vitro. Our results show that RelA is a major component of constitutive and TNF-{alpha}-induced {kappa}B site-binding activity in developing B cells, and provide evidence for a direct role of TNF-{alpha} in killing RelA-/- B cells. The absence of RelA significantly reduced mRNA expression of the antiapoptotic genes cellular FLICE-inhibitory protein and Bcl-2. Retroviral transduction of RelA-/- B cells with either cFLIP or Bcl-2 significantly reduced TNF-{alpha} killing. Together, these results indicate that RelA plays a crucial role in regulating developing B cell survival by inhibiting TNF-{alpha} cytotoxicity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Progenitors of B and T lymphocytes originate in the bone marrow from hemopoietic stem cells. Both B and T cells express Ag-specific receptors that are generated by V-D-J rearrangement events during development. Commitment to the B cell lineage is initiated in progenitor (pro) B cells, which have not undergone rearrangement of Ig genes. Successful rearrangement of Ig H chain (HC) 3 results in generation of precursor (pre) B cells. Pre-B cells express the pre-B cell receptor (pre-BCR), which comprises the rearranged HC and the V-preB and {lambda}5 molecules. Significantly, signals transmitted by the pre-BCR are required for B cell survival and further development (also see below). Thus, mice deficient in the recombination-activating genes (Rag-1 or Rag-2), which cannot undergo rearrangement of Ig genes, do not progress beyond the pro-B cell stage (1). L chain rearrangement in pre-B cells transforms them into immature and finally into mature B cells. Both immature and mature B cells express cell surface Ig molecules (IgM) in a complex with key signaling molecules that together make the BCR.

A key feature of lymphocyte development is the high rate of apoptosis (programmed cell death), which can take place during several distinct developmental stages. Thus, pro-B cells that do not undergo productive rearrangement of Ig HC are removed by apoptosis (2). It is thought that antiapoptotic members of the Bcl-2 family of proteins, including Bcl-2 and Bcl-xL, play an important role in regulating B cell survival (3). Notably, induction of Bcl-xL expression correlates with expression of the pre-BCR (4). Bcl-xL may therefore be responsible for survival of B cells that have undergone successful rearrangement of Ig HCs (4, 5). In addition, apoptosis plays an important role in removal of lymphocytes that recognize self Ags, a process known as negative selection. Negative selection of both B and T cells is thought to help eliminate autoreactive lymphocytes, and thus prevent development of autoimmunity. Elimination of autoreactive lymphocytes occurs in the bone marrow and thymus, and in the periphery. Significantly, removal of autoreactive lymphocytes is thought to be conducted by members of the TNFR superfamily, in particular Fas and TNFR1 (6, 7). Unlike Fas-deficient mice, TNF-{alpha}- or TNFR1-deficient mice do not display autoimmune pathology. However, absence of both TNFR1 and Fas significantly enhances autoimmune pathology in mice (8), suggesting a possibly redundant role for these two molecules in preventing autoimmunity. Whether TNF-{alpha}-induced cytotoxic pathways also affect survival of developing lymphocytes is presently not clear.

The NF-{kappa}B transcription factors play an important role in regulating inflammatory, immune, and survival pathways (9, 10). Members of this family include several distinct subunits, including cRel, RelB, p52, and perhaps the most ubiquitous RelA (p65) and p50 proteins (9, 10). Although these NF-{kappa}B subunits may form virtually any homo- or heterodimer, the common complexes present in lymphocytes constitute heterodimers of p50/RelA and p50/cRel subunits. Dimeric NF-{kappa}B proteins typically reside in the cytoplasm in a complex with inhibitory I-{kappa}B proteins. Treatment of cells with proinflammatory cytokines (TNF-{alpha}), bacterial products (LPS), or engagement of Ag receptors present on B and T cells (BCR and TCR) results in activation of NF-{kappa}B, through phosphorylation-triggered degradation of I-{kappa}B proteins. This process is initiated by I-{kappa}B kinases (IKK{alpha} and IKK{beta}) (10, 11), and allows nuclear translocation of NF-{kappa}B and activation of expression of target genes (10).

RelA subunit knockout mice die during embryogenesis because of apoptosis of hepatocytes (12, 13). Importantly, hepatocyte apoptosis and embryonic lethality in RelA-/- mice can be prevented in RelA-/-TNF-{alpha}-/- (14) or RelA-/-TNFR1-/- mice (15). Studies with p50-/- and cRel-/- mice have shown that NF-{kappa}B activation following BCR engagement in mature B cells is important for mediating proliferative and survival responses (16, 17, 18, 19, 20) and for development of marginal zone B cells (21). Interestingly, cRel was found to be specifically important for preventing Ag receptor-induced, but not Fas-induced cell death (19). Our recent studies of CD4 T cells deficient in p50 and cRel NF-{kappa}B proteins have identified an important role for these proteins in regulating mature T cell survival (22). In addition, studies with mice lacking p50 + RelA or IKK{beta} have shown impaired lymphocyte generation (23, 24). Significantly, the generation of T cells in IKK{beta}-/- mice can be rescued in a TNFR1-deficient background (24). However, TNF-{alpha}-dependent mechanisms responsible for impaired lymphocyte generation and the precise role of NF-{kappa}B in this process are not known.

In this study, we have studied the role of the NF-{kappa}B RelA subunit in lymphocyte generation. Our in vivo and in vitro results indicate that RelA plays a key role in regulating developing B cell survival, and provide evidence for a direct role of TNF-{alpha} in killing RelA-/- developing B cells. The absence of RelA significantly reduces {kappa}B site-binding activity and mRNA expression of cellular FLICE-inhibitory protein (cFLIP) and Bcl-2. Retroviral transduction of RelA-/- B cells with either cFLIP or Bcl-2 significantly reduces TNF-{alpha} killing. These results indicate that RelA plays a key role in regulating developing B cell survival by inhibiting TNF-{alpha}-induced cell death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Adoptive transfer experiments

TNFR1-/- and Rag-1-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Fetal liver (FL) adoptive transfer experiments were performed, as previously described (25). Briefly, 1 x 106 viable FL cells were injected i.v. into lethally irradiated Rag-1-/- mice (2 doses of 800 and 400 rad, 3 h apart). Mice were sacrificed 6–8 wk after transplantation. Spleen and bone marrow cells were analyzed by FACS. The studies shown in this work represent results from three independent experiments. All experiments with mice were carried out in accordance with institutional guidelines.

Generation of B cells from FL precursors

FL from mouse embryos were obtained on day 13 of development. Single-cell suspensions were cultured in the presence of 5 ng/ml IL-7 in Opti-MEM medium containing 10% FBS and 50 µM 2-ME. On day 8 of culture, nonadherent cells were removed and analyzed by FACS. For TNF-{alpha} treatments, mouse rTNF-{alpha} was added to RelA+/+ and RelA-/- cultures at a final concentration of 10 ng/ml. Following this incubation, the B cells were stained with propidium iodide (PI), and FACS analysis was performed to determine the percentage of cell death.

FACS

Single-cell suspensions of B cells were first incubated with Fc-Block (anti-mouse CD16/CD32), followed by staining with biotin-conjugated mAb (pre-BCR), followed by staining with CyChrome-conjugated streptavidin, PE-conjugated mAb (B220, CD19), and FITC-conjugated mAb. To determine the surface expression of TNFR1, the cells were preincubated with Fc-Block, followed by incubation with a purified TNFR1 Ab. The cells were washed and incubated with a biotin-conjugated cocktail of anti-IgG secondary Abs. After washing, the cells were incubated with PE-conjugated streptavidin. Stained cells were fixed in 4% paraformaldehyde, and analyzed using a BD Biosciences (San Jose, CA) flow cytometer. For intracellular staining, the cells were first fixed in 2% paraformaldehyde in PBS for 1 h at 4°C, permeabilized in 0.2% Tween 20 in PBS, washed, and stained, as described above. All Abs and streptavidin conjugates used were from BD PharMingen (San Diego, CA).

EMSA, Northern blotting, and RNase protection assay (RPA)

Nuclear extracts from in vitro developed B cells were made, as described previously (26). The {kappa}B site oligonucleotide probe was from the mouse MHC class I promoter. As control, we show binding to the constitutive Oct-1 transcription factor site (5-TGTCGAATGCAAATCACTAGAA-3). A total of 5 µg of nuclear extracts was incubated with the 32P-labeled probes for 15 min and loaded on Tris-base/glycine/EDTA gels. The gels were quantified by using a PhosphorImager Storm 860 from Molecular Dynamics (Sunnyvale, CA) and the computer software ImageQuaNT.

On day 9, B cells were left untreated or treated with TNF-{alpha} (10 ng/ml) for 2 or 6 h. Total RNA was extracted using TRIzol reagent (Life Technologies-BRL, Gaithersburg, MD), according to the manufacturer’s instructions, and resuspended in 20 µl deionized water. Total RNA (10 µg) was used per sample for Northern blot analysis with mouse I-{kappa}B{alpha} or {beta}-actin 32P-labeled probes.

The same RNA was also used for RPA. Antisense RNA probes for Bcl-xL, Bcl-2, and cFLIP were prepared using the T7 promoter in pBluescript. The probes were labeled with [32P]UTP using Ambion (Austin, TX) RPA kit. RPA analysis was conducted according to manufacturer’s recommendations (Ambion).

Retroviral infection of developing B cells

The mouse stem cell virus 2.2-internal ribosomal entry site-GFP 3M (MIG) retroviral vector (27) was used for experiments shown in this work (kindly provided by L. Van Parijs, Massachusetts Institute of Technology (Cambridge, MA)). This vector allows simultaneous expression of a gene of interest (Bcl-2 or cFLIP) and green fluorescence protein (GFP). Retrovirus stocks were generated using the ecotropic packaging cell line BOSC23 (28). MIG vectors were cotransfected with PCLEco (encoding retroviral proteins), and supernatants were collected after 48 h. B cells from RelA-/- embryos cultured with IL-7 were infected on days 6 and 7. A total of 500 µl of viral supernatant was used per well in a total volume of 1 ml of B cell culture medium and polybrene at a final concentration of 10 µg/ml. Retroviral infection was performed using centrifugal enhancement (centrifuged for 1 h at 1000 x g at 30°C). FACS analysis and TNF-{alpha} treatments were performed on day 9 of culture, as described in the text.

To measure TNF-{alpha}-specific apoptosis, cells were stained with PI, and the percentage of infected living cells (GFP+ PI-) in untreated samples (Pu) and TNF-{alpha}-treated samples (Pt) was determined. The TNF-{alpha}-specific apoptosis rate was calculated according to the following formula: (1 - (Pt/Pu)) x 100.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Impaired RelA-/- B lymphocyte generation in Rag-1-/- mice

Although absence of RelA results in embryonic lethality, RelA-/- lymphocytes can be obtained after transfer of CD45.2 RelA-/- FL cells into irradiated congenic C57BL/6 CD45.1 recipient mice (23, 25) (CD45.2 and CD45.1 are different isoforms that can be distinguished from each other by Abs). However, one of the problems encountered following this procedure was the presence of a small, but significant number of radioresistant recipient CD45.1 T cells (~15% of total T cells; data not shown) (25). We wanted to use a system in which RelA-/- lymphocytes were generated in the absence of contaminating recipient T cells. To this end, we used Rag-1-deficient mice, which completely lack mature T and B lymphocytes (1).

Rag-1-/- mice were subjected to a lethal dose of 1200 rad of gamma-radiation to completely destroy hemopoiesis (using CD45.1 mice, we have found that this dose eliminates all recipient mouse B cells (B220+ cells); and all B220+ cells detected originate from donor hemopoietic precursors). The studies described in this work are based on three independent experiments in which mice were examined 6–8 wk after adoptive transfer. As expected, injection of RelA+/+ FL cells into irradiated Rag-1-/- mice resulted in reconstitution of splenic B cells (Fig. 1) and T cells (data not shown). In contrast, the numbers of RelA-/- lymphocytes generated in Rag-1-/- mice were greatly reduced. Thus, Rag-1-/- mice injected with RelA-/- FL cells had 10 times less splenic B lymphocytes than RelA+/+ FL-injected Rag-1-/- mice (Fig. 1). FACS analysis of bone marrow (BM) cells from Rag-1-/- mice injected with RelA-/- FL cells revealed an even more significant reduction in B220+IgM- developing B cells (~40-fold less than in RelA+/+ FL-injected mice) (Fig. 1). In addition, RelA-/- FL-injected Rag-1-/- mice typically had atrophied thymi, while RelA+/+ FL-injected Rag-1-/- mice had normal thymi. However, the RelA-/- splenic T cell population was not as significantly reduced (2-fold less than RelA+/+) as the splenic B cell population (data not shown). We have therefore primarily focused on RelA function in B cells in this study. In addition, myelopoiesis was unaffected, and in fact RelA-/- FL-injected Rag-1-/- mice showed elevated numbers of granulocytes both in blood and tissues (see below). These results contrast with previous studies using CD45.1 recipient mice, which showed that RelA-/- lymphocyte generation is relatively normal (23, 25, 29). Thus, the genetic background of the recipient mouse also plays a crucial role in hemopoietic reconstitution.



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  		FIGURE 1. Impaired RelA-/- B lymphocyte generation in Rag-1-/- mice. RelA+/+ and RelA-/- FL cells were transplanted into Rag-1-/- mice. Seven weeks after transplantation, recipient mice were sacrificed. Spleen and bone marrow cells were stained with anti-B220 and anti-IgM Abs before FACS analysis. B220+IgM- and B220+IgM+ populations are shown in boxes along with the respective percentages.

 
Absence of TNFR1 rescues RelA-/- B lymphocyte generation in Rag-1-/- mice

The development of RelA-/- lymphocytes (e.g., in CD45.1 mice) suggests that there is no cell-autonomous function for RelA in lymphocyte generation. Thus, one possibility is that RelA-/- B lymphocytes generated in Rag-1-/- mice fail to survive. Because RelA-/- hepatocyte loss and embryonic lethality are TNF-{alpha} dependent (14, 15), we determined whether loss of RelA-/- B lymphocytes was also dependent on a TNF-{alpha}-induced mechanism. To test this possibility, we injected RelA-/-TNFR1-/- FL cells into irradiated Rag-1-/- mice (previous studies have indicated that TNFR1 mediates most TNF-{alpha}-induced responses, including embryonic lethality in RelA-/- mice) (15). Interestingly, greatly enhanced numbers of B cells were generated after injection of RelA-/-TNFR1-/- FL cells into Rag-1-/- mice, with no difference in the splenic B220+ population compared with RelA+/+TNFR1-/- FL-transplanted mice (Fig. 2). In the BM, the B220+ population was also considerably increased, although still less than in RelA+/+TNFR1-/- FL-transplanted mice (Fig. 2). Thus, loss of RelA-/- B lymphocytes occurs in large part by a TNFR1-dependent mechanism. Between 6 and 8 wk, Rag-1-/- mice transplanted with RelA-/-, but not RelA+/+ FL cells also showed severe infiltration of neutrophils into tissues in the apparent absence of infection (data not shown). Significantly, Rag-1-/- mice in which RelA-/-TNFR1-/- FL cells had been injected also showed this phenotype, suggesting that B lymphocyte loss and neutrophil infiltration occur by different mechanisms, with only the former being TNFR1 dependent.



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  		FIGURE 2. Absence of TNFR1 rescues RelA-/- B lymphocyte generation in Rag-1-/- mice. RelA+/+TNFR1-/- and RelA-/-TNFR1-/- FL cells were transplanted into Rag-1-/- mice. Seven weeks after transplantation, recipient mice were sacrificed. Spleen and bone marrow cells were stained with anti-B220 and anti-IgM Abs before FACS analysis. B220+IgM- and B220+IgM+ populations are shown in boxes along with the respective percentages.

 
In vitro development of RelA-/- B cells is normal despite significantly reduced {kappa}B site-binding activity

Our results suggest that the disappearance of RelA-/- B cells may be due to TNF-{alpha}-induced loss of developing B cells (Figs. 1 and 2). To test this possibility, we generated RelA-/- B lymphocytes from FL hemopoietic precursors in vitro. FL cells from RelA+/+ and RelA-/- embryos were obtained at day 13 of development and cultured in the presence of IL-7 for 8 days, after which nonadherent cells were analyzed by FACS. On day 8 of culture, >90% of the cells stained positive for B220 (Fig. 3A). The same results were obtained using CD19 as a marker for the B cells (data not shown). Significantly, double staining with anti-IgM HC showed less than 5% cells to be B220+IgM+ in both RelA+/+ and RelA-/- cell populations (Fig. 3A). Both RelA+/+ and RelA-/- showed very little cell surface expression of pre-BCR, but high intracellular expression. These results therefore indicate that the vast majority of cells are at the early pre-B cell stage. Significantly, these results also show normal in vitro development of RelA-/- B cells.



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  		FIGURE 3. In vitro development of RelA-/- B cells is normal despite significantly reduced {kappa}B site-binding activity. A, FL-derived RelA+/+ and RelA-/- B cells were stained with anti-B220, anti-IgM, and anti-pre-BCR Abs. The percentage of different populations of cells is indicated. For pre-BCR, both cell surface and intracellular staining is shown. B, EMSA analysis of nuclear extracts from FL-derived RelA+/+ and RelA-/- pre-B cells was performed after incubation of cells for 2 h with or without TNF-{alpha} (10 ng/ml). The mouse H-2 binding site was used. As control, the constitutive Oct-1 binding site was used.

 
We then analyzed {kappa}B site-binding activity in RelA+/+ and RelA-/- cells, and in particular, the effect of TNF-{alpha} treatment on {kappa}B site-binding activity. FL-derived B cells incubated overnight without IL-7 were left untreated or treated with TNF-{alpha} for 2 h. Significantly, nuclear extracts from untreated RelA-/- B cells showed considerably decreased constitutive {kappa}B site-binding activity compared with RelA+/+ B cells (6.4-fold) (Fig. 3B). After treatment with TNF-{alpha} for 2 h, a moderate increase in DNA-binding activity was observed in RelA+/+ (1.4-fold), but not in RelA-/- B cells (Fig. 3B). Our results also demonstrate equivalent Octamer 1 site binding, indicating that nuclear extracts from RelA-/- B lymphocytes are not degraded. These results indicate that RelA is a major component of {kappa}B site-binding activity present in FL-derived pre-B cells. Notably, these results also suggest that decreased {kappa}B site-binding activity in RelA-/- B cells has no apparent effect on pre-B cell development.

Sensitivity of developing RelA-/- B cells to TNF-{alpha} killing in vitro

The results shown above indicate that loss of RelA-/- B cells occurs in a TNFR1-dependent manner. We next determined whether this was due to direct killing of RelA-/- B cells by TNF-{alpha}. To this end, B cells were removed from plates on day 8 and cultured without IL-7 for 16 h. On day 9, mouse TNF-{alpha} was added to both RelA+/+ and RelA-/- B cells for 48 h. Cells were then stained with propidium iodide (PI), and FACS analysis was performed to determine the percentage of cell death. Our results revealed significant differences between RelA+/+ and RelA-/- B cells in susceptibility to TNF-{alpha} killing (Fig. 4A). RelA-/- B cells showed 58% cell death after TNF-{alpha} treatment compared with 17% cell death in untreated cells. RelA+/+ cells treated with TNF-{alpha}, in contrast, showed only 19% killing, while untreated cells showed 12% cell death (Fig. 4A). Thus, RelA-/- B cells show 3-fold more cell death after TNF-{alpha} treatment compared with RelA+/+ B cells. We also analyzed the surface expression of TNFR1 on RelA+/+ and RelA-/- B cells (Fig. 4B). The surface expression of TNFR1 in developing B cells was low, but detectable. Nevertheless, RelA+/+ and RelA-/- B cells were found to have similar levels of TNFR1 cell surface expression. The mean fluorescence intensity values for TNFR1 were 20.11 in RelA+/+ and 21.30 in RelA-/- (the mean values in unstained samples were 12.63 in RelA+/+ and 12.71 in RelA-/-). We have found that the kinetics of cell death of TNF-{alpha}-treated RelA-/- cells are rather slow, compared with mouse embryonic fibroblasts (30). Very little cell death was detected until 24 h, which typically progressed to 50–75% by 48 h in different experiments (Fig. 4 and data not shown). Wild-type B cells also reproducibly show some cytotoxicity, but which was significantly less than in RelA-/- B cells. In contrast, untreated RelA+/+ and RelA-/- cells did not reproducibly show differences in amount of spontaneous cell death (12 and 17%, in the results shown in this study). Therefore, these results demonstrate that in the absence of RelA, TNF-{alpha} can induce cell death of developing B cells. The disappearance of developing RelA-/- B cells in Rag-1-/- mice is thus most likely the result of direct killing by TNF-{alpha}.



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  		FIGURE 4. A, Sensitivity of developing RelA-/- B cells to TNF-{alpha} killing in vitro. In vitro developed B cell precursors from RelA+/+ and RelA-/- embryos were kept untreated or treated with TNF-{alpha} (10 ng/ml) for 48 h. The cells were then stained with PI, and FACS analysis was performed to determine the percentage of cell death. B, Surface expression of TNFR1 in in vitro developed B cells. FL-derived RelA+/+ and RelA-/- B cells were stained with purified TNFR1 Abs. Filled histograms show unstained cells, while open histograms show TNFR1 Ab-stained cells.

 
Reduced levels of cFLIP and Bcl-2 in RelA-/- B cells

The TNF-{alpha} sensitivity and greatly reduced {kappa}B site-binding activity in RelA-/- B cells suggest that NF-{kappa}B-regulated gene expression, including expression of antiapoptotic genes, may be compromised in these cells. To test this possibility, we first determined expression levels of I-{kappa}B{alpha}, a well-characterized NF-{kappa}B target gene (31, 32), in RelA-/- B cells. Northern blot analysis showed that the constitutive levels of I-{kappa}B{alpha} mRNA were significantly reduced in RelA-/- B cells (Fig. 5A). Induction of I-{kappa}B{alpha} expression by TNF-{alpha} was also significantly reduced in RelA-/- cells (Fig. 5A). These results indicate that overall reduction in {kappa}B site-binding activity in RelA-/- B cells significantly impairs expression of the NF-{kappa}B target gene, I-{kappa}B{alpha}.



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  		FIGURE 5. Reduced levels of cFLIP and Bcl-2 in RelA-/- B cells. A, Northern blotting using I-{kappa}B{alpha} and {beta}-actin-specific probes. Total RNA (10 µg) was used per sample. B, RPA was performed using 40 µg of total RNA from RelA+/+ and RelA-/- in in vitro developed B cells. The cells were untreated or treated with TNF-{alpha} (10 ng/ml) for 2 or 6 h. Mobilities corresponding to Bcl-xL, Bcl-2, cFLIP, and {beta}-actin are indicated.

 
We next determined mRNA expression of three putative NF-{kappa}B target genes (33, 34, 35), which are also thought to be important in regulating lymphocyte survival: Bcl-2, Bcl-xL, and cFLIP (long) (cFLIP(L)). Notably, previous studies have shown that cFLIP(L) can prevent both Fas and TNFR1-induced cell death by blocking activation of caspase 8 (36, 37). Strikingly, constitutive expression of Bcl-2 and cFLIP(L) was significantly reduced in RelA-/- cells, while, in contrast, Bcl-xL levels were not significantly affected (Fig. 5B). Shorter exposure showed that Bcl-xL expression was also slightly reduced in untreated RelA-/- B cells, which was increased to levels similar to RelA+/+ B cells after TNF-{alpha} treatment (data not shown). In contrast, no significant increase in mRNA levels of Bcl-2 and cFLIP was observed after TNF-{alpha} treatment in either cell type. These results thus indicate that the absence of RelA considerably reduces constitutive Bcl-2 and cFLIP expression levels, consistent with reduced constitutive {kappa}B site-binding activity in RelA-/- B cells. Thus, susceptibility of RelA-/- B cells to TNF-{alpha} may be due to reduced expression of these antiapoptotic genes.

Retrovirus-mediated expression of Bcl-2 or cFLIP inhibits TNF-{alpha} killing of RelA-/- B cells

To further study the potential role of Bcl-2 and cFLIP in regulating TNF-{alpha}-induced cell death, we infected RelA-/- B cells with retroviruses expressing these genes. For these studies, we used the MIG retroviral vector (27). B cells from two different RelA-/- embryos were obtained, as described above. On days 6 and 7 of culture, the developing B cells were infected with MIG, MIG-Bcl-2, or MIG-cFLIP retroviral supernatants. The efficiency of infection of B cells (i.e., GFP+ cells) is shown in Fig. 6A. In comparison with MIG-infected RelA-/- B cells, Bcl-2 and cFLIP expression significantly reduced TNF-{alpha}-specific killing of RelA-/- B cells (Fig. 6B). These results therefore suggest that susceptibility of RelA-/- B cells to TNF-{alpha}-induced cell death may be due to reduced expression of Bcl-2 and cFLIP.



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  		FIGURE 6. Retrovirus-mediated expression of Bcl-2 or cFLIP inhibits TNF-{alpha} killing of RelA-/- B cells. A, FL-derived B cells from two different RelA-/- embryos were obtained as above. On days 6 and 7 of culture, they were infected with MIG, Bcl-2-MIG, and cFLIP-MIG retroviral supernatants. The percentage of infection of B cell from embryos 1 and 2 was determined by FACS from GFP fluorescence on day 9. The cells were also stained with B220 as marker for B cells. B, After infection, TNF-{alpha} was added at a final concentration of 10 ng/ml for 48 h. Following this incubation, the B cells were removed from the wells and stained with PI, and FACS analysis was performed to determine TNF-{alpha}-specific apoptosis of the infected cells (see Materials and Methods). The percentage of TNF-{alpha}-specific cell death is shown in MIG-, Bcl-2-MIG-, and cFLIP-MIG-infected cells.

 
The findings presented in this work have revealed a key role for the RelA subunit of NF-{kappa}B in regulating B cell survival. To study the function of RelA in lymphocyte development, we adoptively transferred RelA-/- FL cells into lethally irradiated mature lymphocyte-deficient Rag-1-/- mice. Our results indicate greatly impaired generation of RelA-/- B lymphocytes in Rag-1-/- mice. Significantly, in the absence of TNFR1 signaling, the disappearance of B cells was largely rescued. Our in vitro results show that in RelA-/- developing B cells, both constitutive and TNF-{alpha}-induced NF-{kappa}B activity is dramatically reduced. Significantly, RelA-/-, but not RelA+/+ B cells, underwent cell death in the presence of TNF-{alpha}, demonstrating that TNF-{alpha} is directly cytotoxic to RelA-/- B cells. Our results further suggest that susceptibility to TNF-{alpha} is due to reduced constitutive expression of Bcl-2 and cFLIP in RelA-/- cells. Interestingly, while TNF-{alpha} treatment moderately increased NF-{kappa}B activity in RelA+/+ cells, expression of Bcl-2 or cFLIP was not affected by TNF-{alpha}. The reason for this is presently unclear, but may suggest a requirement for additional transcriptional factors for enhancement of expression of these genes, which may not be induced by TNF-{alpha}. Together, these results suggest that a key function of RelA-containing NF-{kappa}B complexes in developing B cells is to prevent TNF-{alpha} killing by enhancing expression of antiapoptotic genes.

Interestingly, previous studies have shown that, unlike cRel, RelA is not required for mature B cell proliferation (29). Furthermore, cRel-containing complexes are predominant in mature B cells, most likely due to inefficient removal of cRel from nuclei (38, 39). Our results indicate that RelA-containing complexes are predominant in pre-B cells (Fig. 3). In addition, we have also found that pre-B cells lacking cRel (and p50) are not susceptible to TNF-{alpha} killing (data not shown). Together, these results suggest distinct functions for the RelA and cRel in B cells at different developmental stages: RelA inhibits TNF-{alpha} cytotoxicity in developing B cells, but is not essential for mature B cell proliferation, while for cRel, the converse is true.

In contrast to Bcl-2 and cFLIP, we have found that significant expression of Bcl-xL still occurs in RelA-/- B cells. Successful rearrangement of the Ig HC leading to pre-BCR expression is essential for in vivo pre-B cell survival. Significantly, induction of Bcl-xL expression by the pre-BCR may be one of the key mechanisms for pre-B cell survival (4). Our results indicate that RelA is not essential for Bcl-xL expression in pre-B cells. In addition, there is no cell-intrinsic requirement for RelA or p50 + RelA (23, 29)in B cell development. Together, these findings suggest that the key function of RelA-containing complexes during B cell development may be to prevent TNF-{alpha} killing, but not to regulate survival pathways that play an integral role in B cell development, such as pre-BCR-induced Bcl-xL expression.

The source of cytotoxic TNF-{alpha} that kills developing B cells is presently unclear. However, it is likely that stromal cells in the bone marrow, especially macrophages, may be an important source. These cells are present in close proximity to developing B cells within the BM microenvironment and are known to be important producers of TNF-{alpha}. Our results also indicate an important role for the recipient mouse background in determining the outcome of hemopoietic reconstitution. In contrast to Rag-1-/--injected mice, significant numbers of RelA-/- B lymphocytes were generated after transfer of RelA-/- FL cells into CD45.1 congenic mice (~2-fold less than in RelA+/+ FL-injected mice; data not shown) (23, 25, 29). These results suggest that RelA-/- lymphocytes are generated and survive after transfer of RelA-/- FL cells into lymphocyte-containing mice (CD45.1 mice), but not in mice devoid of lymphocytes (Rag-1-/- mice). Significantly, we have found that even after receiving a lethal dose of radiation, CD45.1 mice retain a small, but significant number of radioresistant recipient CD45.1 T cells (~15% of total T cells; data not shown) (25). Thus, it is possible that radioresistant WT T cells may enhance RelA-/- lymphocyte survival by inhibiting TNF-{alpha} generation and/or cytotoxic mechanisms.

In conclusion, our findings have revealed a key role for the RelA subunit of NF-{kappa}B in regulating B cell survival. Although RelA is apparently not required for B cell development, it plays a crucial role in preventing TNF-{alpha} killing of B cells. Our results further suggest that RelA may inhibit TNF-{alpha} killing by regulating expression of the key antiapoptotic genes, Bcl-2 and cFLIP. Together, these findings help define a key mechanism that plays a crucial role in developing B cell survival.


   Acknowledgments
 
We thank Dr. Christopher Roman (Down State University, New York, NY) for very helpful discussions. We also thank Dr. Luk Van Parijs (MIT) for providing the MIG vectors, Fang Li for technical assistance, and members of the lab for discussions.


   Footnotes
 
1 This work was supported by National Institutes of Health R01 CA074892 to A.A.B. Back

2 Address correspondence and reprint requests to Dr. Amer A. Beg, 1110 Fairchild Center, Department of Biological Sciences, 1212 Amsterdam Avenue, Columbia University, New York, NY 10027. E-mail address: aab41{at}columbia.edu Back

3 Abbreviations used in this paper: HC, H chain; BCR, B cell receptor; BM, bone marrow; cFLIP, cellular FLICE-inhibitory protein; cFLIP(L), cFLIP (long); FL, fetal liver; GFP, green fluorescence protein; IKK, I-{kappa}B kinase; MIG, mouse stem cell virus 2.2-internal ribosomal entry site-GFP 3M; PI, propidium iodide; Rag, recombination-activating gene; RPA, RNase protection assay. Back

Received for publication February 7, 2003. Accepted for publication August 6, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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