Activation of NF-{kappa}B promotes the transition of large, CD43+ pre-B cells to small, CD43 pre-B cells

Eijiro Jimi1,2,*, Roderick J. Phillips1,2,5,*, Mercedes Rincon1,6, Reinhard Voll1,2,7, Hajime Karasuyama3, Richard Flavell1,4 and Sankar Ghosh1,2

1 Section of Immunobiology, 2 Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520, USA
3 Department of Immune Regulation, Tokyo Medical and Dental University, Graduate School of Medicine, Tokyo 113-8519, Japan
4 Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520, USA
5 Present address: Department of Physiology, UCLA, Los Angeles, CA 90401, USA
6 Present address: Department of Medicine, University of Vermont, Burlington, VT 05405, USA
7 Present address: Institute for Clinical Immunology, Friedrich-Alexander University, Erlangen, Germany

Correspondence to: S. Ghosh; E-mail: sankar.ghosh{at}yale.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The regulation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) during B-cell development was examined using cells isolated from the bone marrow of transgenic mice expressing a {kappa}B luciferase reporter gene. The results indicate that the highest level of NF-{kappa}B activity is present in cells expressing the pre-B-cell receptor. Furthermore, cross-linking of Igß on CD43+ pre-B cells is able to activate NF-{kappa}B in recombination-activating gene 1-deficient mice, preceding their further differentiation into CD43 pre-B cells. Expression of a dominant negative form of I{kappa}B{alpha} using a transgenic approach or by retroviral infection leads to a reduction in the number of CD43+ pre-B cells. These data therefore indicate that activation of NF-{kappa}B in CD43+ pre-B cells, as a result of signaling by the pre-B-cell receptor, facilitates the continued development of large, CD43+ pre-B cells into small CD43 pre-B cells.

Keywords: NF-kappa B, B-cells, lymphocytes, development, apoptosis


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lymphocyte development is dependent upon and closely regulated by the expression of the antigen receptor genes [reviewed in (1, 2)]. The great diversity of the antigen receptor repertoire is generated through a somatic DNA rearrangement process; however, only a certain percentage of gene rearrangement events lead to the expression of a functional antigen receptor (3). Lymphocytes that fail to undergo productive gene rearrangement are arrested during development and undergo apoptosis, whereas lymphocytes expressing functional antigen receptors undergo further selection and differentiation before exiting the bone marrow or the thymus (1, 2).

The antigen receptors are composed of two subunits, and genes encoding these subunits are rearranged and expressed sequentially. In B-lymphocyte development, rearrangement of the µ heavy chain occurs first, whereas in {alpha}/ß T-lymphocyte development the TCR-ß chain is the first one to be rearranged (1, 2). Pre-B cells that have successfully completed the first round of gene rearrangement, express the Igµ heavy-chain protein that assembles with the {lambda}5 and V pre-B surrogate light chains to form a pre-B-cell receptor (2). Analogously, in pre-T cells that have undergone successful ß-chain rearrangement, the ß protein associates with the pre-T{alpha} protein, thus forming a pre-TCR (1). The assembly of a pre-B or pre-T receptor serves as the first checkpoint in lymphocyte development and is critical for further proliferation and differentiation. In mutant mice that lack the recombinase proteins recombination-activating gene (RAG) 1 or 2 or µ heavy chain, {lambda}5, TCR-ß or pre-T{alpha}, cannot form the pre-BCR or pre-TCR and lymphocyte development is significantly arrested prior to the rearrangement of the {kappa}/{lambda} light chains or the TCR {alpha} chain [reviewed in (1, 2, 4)]. Therefore, successful µ heavy-chain or TCR-ß-chain rearrangement is essential for cells to proceed to the next developmental stage.

The signals originating from the pre-BCR or pre-TCR that cause proliferation and differentiation of developing B or T lymphocytes, respectively, remain to be fully characterized. Requirement of the signaling chains Ig{alpha}/ß and CD3 in the expression and functioning of pre-B and pre-T receptors suggests that the signaling from these receptors are likely to be similar to those emanating from the mature antigen receptors (4). Such a conclusion is further bolstered by the observation that mice lacking signaling molecules such as lck or SLP-65 exhibit a block in lymphocyte differentiation at the stage of µ or ß-chain selection (4). Recent studies have focused on identifying the transcription factors that are activated by these receptors. In thymocytes, signaling from the pre-TCR leads to the activation of both nuclear factor-{kappa}B (NF-{kappa}B) and NFAT, as well as increased Ca2+ mobilization (5, 6). Using a variety of approaches we have recently demonstrated that active NF-{kappa}B in thymocytes provides a survival signal, but not a proliferative signal, which helps promote thymocyte development (5). Because of the similarity between B- and T-cell development, and the overall regulation of pre-B and pre-T-cell receptors, we felt it was important to assess the possible regulation of NF-{kappa}B by the pre-B-cell receptor in developing pre-B cells.

Developing B cells can be separated into distinct stages using a panel of cell surface markers (79). Although recently, there has been some controversy regarding the characterization of the early stages in the differentiation pathway, the scheme developed by Hardy and colleagues is generally used to distinguish stages of B-cell development (10, 11). According to this scheme, fractions A–C are characterized by the expression of B220, CD43 and the differential expression of BP-1 and HSA. Fraction A (BP-1HSA) contains cells that appear committed to the B lineage; these cells are termed pre–pro-B cells (9). Fraction B (early pro-B cells) and fraction C (late pro-B cells) both express HSA and both undergo rearrangement at the IgH locus; however, only late pro-B cells express BP-1. In the next stage of B-cell development (fraction D), CD43 is down-regulated and the cells express intracellular Igµ; the cells in this fraction are termed pre-B cells. Although some rearrangement at the {kappa} locus is observed in fractions B and C, the majority of V{kappa}J{kappa} recombination occurs in fraction D (12, 13). Once productive rearrangements have occurred at the heavy- and light-chain loci, IgM is expressed on the cell surface; this is fraction E. It is thought that B cells leave the bone marrow at this point and enter the spleen, where they undergo a final maturation process enabling them to express IgD as well as IgM; this is fraction F. Surface-positive IgD and IgM cells are then able to enter the recirculating pool of naive, competent B lymphocytes.

To determine how NF-{kappa}B is regulated during B-cell development, we have studied the activity of this transcription factor in B lineage cells freshly isolated from murine bone marrow. Using a combination of electrophoretic mobility shift assay (EMSA) and transgenic mice that express the luciferase gene under the control of two {kappa}B elements (5), we show that the activity of NF-{kappa}B in developing B cells is highest in large, CD43+ pre-B cells. We show that cross-linking of Ig accessory chain, Igß, on CD43+ pre-B cells from RAG1-deficient mice activates NF-{kappa}B, preceding their further differentiation into CD43 pre-B cells, suggesting that signaling from the pre-B-cell receptor is responsible for activation of NF-{kappa}B in CD43+ pre-B cells. Finally, we find that forced inhibition of NF-{kappa}B activity in pro-B cells by the transgenic or retroviral expression of an I{kappa}B{alpha} super-repressor leads to a selective loss of CD43+ pre-B cells. Therefore, our studies show that similar to the role of NF-{kappa}B in thymocytes (5), NF-{kappa}B in CD43+ pre-B cells appears to provide a critical signal that enables large CD43+ pre-B cells to develop into small CD43 pre-B cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of transgenic mice
The I{kappa}B{alpha}-SS-PEST-dominant negative (DN) construct has been described previously (5). In this study, the construct was driven by a B-cell-specific promoter (mb-1) and enhancer (Igµ) elements (14) and also contains an influenza epitope tag hemagglutinin (HA) which is recognized by the HA antibody. The 3' region contains the human growth hormone 3'-untranslated region and poly (A) to improve the efficiency of transcription and translation. The plasmid was micro-injected into fertilized C57BL/6 x SJL F2 eggs and transgenic mice were generated as described previously (5). Four founders were obtained with different levels of expression: 6, 35, 57 and 76 (Fig. 5).



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Fig. 5. Generation of transgenic mice expressing a constitutively inhibiting I{kappa}B{alpha} in B-cell lineage. (A) The I{kappa}B{alpha}-SS-PEST-DN construct contains mutations in the PEST domain to improve the stability of the protein (5). The construct is driven by B-cell-specific promoter and enhancer elements and also contains an HA tag which is recognized by the flu antibody. The 3' region contains the human growth hormone 3'-untranslated region and poly (A) to improve transcription and translation efficiencies. (B) Micro-injection of the construct into mice produced four founders 6, 35, 57 and 76. Western analysis of these founders plus appropriate littermate controls was performed using an anti-I{kappa}B{alpha} polyclonal antibody. The I{kappa}B{alpha}-DN protein is observed as the upper band in each case. (C) The I{kappa}B{alpha}-DN protein blocks B-cell development at the pro-B-cell stage. Mice expressing the I{kappa}B{alpha}-SS-PEST construct and appropriate littermate controls were sacrificed and cells from the B lineage were isolated. These bone marrow-derived cells were then stained as described in Fig. 1. Data are presented from founder 76, although comparable data were obtained for the other founders. (D) The I{kappa}B{alpha}-DN transgenic mice have a dose-dependent reduction in pro-B-cell stage. These bone marrow-derived cells were then stained as described in Fig. 1. The results from the transgenic mice are the average from multiple mice. The relative amount of I{kappa}B{alpha}-DN protein expressed was measured by densitometry.

 
The generation and characterization of the {kappa}B luciferase transgenic mice has been described previously (5).

Reagents
The p65 (RelA), NF-{kappa}B1 and c-Rel antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); each was an affinity-purified rabbit polyclonal antibody raised against an appropriate peptide. The remaining antibodies were obtained from Pharmingen (San Diego, CA, USA), except for Quantum Red670 directly conjugated anti-B220 mAb, which was purchased from Sigma (St Louis, MO, USA). SCA-1 (anti-Ly6A) was a gift from A. Bothwell (Yale University), and FITC–BP-1 was a gift from Richard Hardy (Fox Chase Cancer Center, Philadelphia, PA, USA). The micro-BCA protein assay kit was supplied by Pierce (Rockford, IL, USA), while the Luciferase Assay System was purchased from Promega (Madison, WI, USA).

Cell preparation, staining and sorting
Bone marrow was isolated from the femur and tibia of either NF-{kappa}B-luciferase transgenic mice or non-transgenic littermates and pre-incubated with Fc BlockTM (anti-CD16 and anti-CD32; 5 µg ml–1) for 5 min on ice. The cells were then stained with a combination of anti-CD43, anti-B220, F(ab')2 anti-IgM, anti-HSA, anti-SCA-1 (Ly6A) and anti-BP-1 mAbs for 30 min on ice in the dark, and washed twice in sorting medium (PBS plus 5% FCS). The stained bone marrow cells were sorted on a Becton Dickinson FACStarPLUS cell sorter, where the average purity of each fraction was 99%. Cells were collected into Bruffs medium plus 20% FCS and kept on ice until assayed.

Preparation of nuclear extracts and EMSA
Cells were pelleted by centrifugation, washed in PBS and re-suspended in buffer A [10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT) and 0.5 mM phenylmethylsulphonylfluoride (PMSF)]. Following NP-40 detergent lysis and centrifugation, the supernatant (the cytoplasmic fraction) was collected and spun again in an ultracentrifuge to remove cellular debris. The pellet (the nuclear fraction) was re-suspended in buffer C (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT and 1 mM PMSF) and incubated for 15 min at 4°C. Subsequently, the nuclear extract was centrifuged and the supernatant was collected. Both cytoplasmic and nuclear extracts were finally frozen (in 10% glycerol) and stored at –70°C. After determination of protein concentration, using the micro-BCA protein assay reagent kit, 5 µg of nuclear extracts was taken and made up to a final volume of 10 µl with water. Subsequently, a cocktail containing the 32P-labeled {kappa}B probe (from the Ig {kappa} gene intronic enhancer), dIdC (competitor DNA), GTP, BSA and the lipage-binding buffer were prepared. Next, 10 µl of the cocktail was added to each sample and the mixture was incubated at room temperature for 15 min. Finally, the samples were electrophoresed under standard conditions, dried and the gels were exposed to film for ~24 h.

For supershift analysis, 1 µl of the appropriate rel antibody was added to each sample, and these were then incubated for 2 h at 4°C; in these experiments, 4 µg of total protein was assayed for each sample. Subsequently, 10 µl of the cocktail was added, and the gel shift was performed as described above, except that the samples were electrophoresed at 4°C, rather than at room temperature.

Immunoblotting analysis
Immunoblotting was performed on 25 µg of cytoplasmic protein. Following SDSP, the proteins were electrophoretically transferred to a PVDF membrane at 100 V for 1 h at 4°C. The membranes were then incubated with an I{kappa}B{alpha} antibody at a 1 : 1000 dilution in 5% dry milk solution plus 0.01% azide overnight at 4°C. Subsequently, the blots were washed in TTBS and then incubated with HRP-conjugated donkey anti-rabbit secondary antibody (Pierce) for 30 min at room temperature. The immunoreactive proteins were finally visualized using enhanced chemiluminescence (Amersham).

Luciferase assay
Luciferase activity was determined on cell lysates derived from different B lineage populations using the Luciferase Assay System (Promega) and following the manufacturer's instructions. Briefly, after lysing the cells, the supernatant was recovered and mixed with the luciferase substrate and activity was measured in a luminometer. All results were normalized and presented as luciferase activity (relative luciferase units) per 106 cells.

In vivo injection of anti-Igß antibody
RAG1-deficient mice (8 weeks old) were injected intravenously with 200 µg of anti-Igß (HM79) mAb (purified from hybridoma supernatant). After 12 or 24 h of treatment, B220+CD43+ cells were isolated from the bone marrow and then examined for NF-{kappa}B and Oct-1 DNA-binding activity. In addition, for some mice, bone marrow cells were collected 7 days following treatment and analyzed by flow cytometry.

Generation of recombinant retroviruses
A murine stem cell virus (MSCV) vector containing an internal ribosome entry site (IRES) and green fluorescent protein (GFP) cDNA were kind gifts of Kenneth M. Murphy (Washington University, St Louis) (15). The 1.0-kb fragment of the DN form of human I{kappa}B{alpha} was generated by PCR. The I{kappa}B{alpha}DN fragment was cloned into BglII-digested MSCV 2.2-IRES GFP vector to generate MSCV-I{kappa}B{alpha}DN.

Retrovirus was produced by transfecting the ecotropic Phoenix E packaging cell line with MSCV-GFP or MSCV-I{kappa}B{alpha}DN plasmid. The Phoenix packaging cell line was obtained from American Type Tissue Collection. Virus titer was determined by infecting NIH3T3 cells and analyzing GFP+ cells by FACS after 2 days of infection. Immunoblot analysis was used to determine the level of expression of I{kappa}B{alpha}DN protein in infected cells using a rabbit anti-I{kappa}B{alpha} antibody.

Bone marrow reconstitution experiments
Bone marrow cells were prepared from the tibia and femur of 8-week-old female B6 mice, 5 days after they had received 5 mg per mouse of 5-fluorouracil (Sigma) via intra-peritoneal injection. Bone marrow cells were cultured at a concentration of 4 x 106 cells ml–1 in DMEM with 10% FBS, 10 ng ml–1 mouse recombinant IL-3 and IL-6, 100 ng ml–1 mouse recombinant stem cell factor, 100 U ml–1 streptomycin, 100 U ml–1 penicillin and 2 mM L-glutamine (GIBCO). After overnight stimulation, non-adherent bone marrow cells were spin infected with control MSCV-GFP virus or MSCV-I{kappa}B{alpha}DN virus in the medium as described above, plus 4 µg ml–1 polybrene (Sigma). A second spin infection was performed after 16 h. After 48 h incubation, bone marrow cells were washed to remove free virus and were suspended in PBS for injection. A total of 5 x 105 cells were injected into irradiated (700 rads) RAG1-deficient females (8 weeks old) via the tail vein. Six weeks after bone marrow reconstitution, recipient mice were sacrificed and the B cells from the bone marrow were analyzed.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NF-{kappa}B transcriptional activity is only present in B lineage cells in the bone marrow
Bone marrow was isolated and sorted into B-cell and non-B-cell fractions, using B220 as a pan B-cell marker. These sorted cell fractions were then assayed for NF-{kappa}B by EMSA. To determine whether the DNA-binding activity seen in B-cell fractions correlated with NF-{kappa}B transcriptional activity, we measured {kappa}B luciferase activity in these fractions using the {kappa}B luciferase reporter mice (5). We observed that constitutive NF-{kappa}B activity was found exclusively in the B-cell fraction, with no detectable activity in the non-B-cell population (Fig. 1A, left panel). As a control, we tested the binding activity of constitutive Oct-1 transcription factor, which as expected did not exhibit the same difference between these fractions (Fig. 1A, right panel). Next, we isolated bone marrow-derived B lineage cells and separated them into IgM+ and IgM fractions. We observed slightly increased levels of {kappa}B-binding complexes (Fig. 1B), and significantly greater NF-{kappa}B transcriptional activity (Fig. 1C), in the IgM fraction than in the IgM+ population. These results therefore indicate that NF-{kappa}B activity correlates with B lineage cells in the bone marrow and can be detected in immature pre-B cells.



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Fig. 1. Constitutive NF-{kappa}B transcriptional activity is restricted to B lineage cells in the bone marrow. (A) Bone marrow was isolated from the femur and tibia of wild-type mice and stained with a combination of FITC–anti-IgM and Red670–anti-B220. Five micrograms of nuclear extract was then prepared and analyzed for NF-{kappa}B and Oct-1 DNA-binding activity by to EMSA. (B) Nuclear extract of B220+IgM+ B cells and the B220+IgM pro- and pre-B cells were analyzed for NF-{kappa}B and Oct-1 DNA-binding activity by EMSA. (C) Cells from NF-{kappa}B luciferase reporter mice were isolated as described above and then assayed for luciferase activity. Data are presented as relative luciferase units per 106 cells.

 
NF-{kappa}B activity varies across different stages of development of B cells in the bone marrow
To identify the exact stage in B-cell differentiation where NF-{kappa}B becomes active, we separated B lineage cells isolated from the bone marrow into fractions corresponding to different stages of development using the protocol devised by Hardy and colleagues, as described earlier (7). A panel of cell surface markers was used to fractionate bone marrow B cells into pre–pro- (fraction A), early pro- (fraction B), late pro- (fraction C), pre- (fraction D), immature (fraction E) and mature B cells (fraction F; Fig. 2A). Cells from the different fractions were assayed for luciferase activity (Fig. 2B). The majority of NF-{kappa}B transcriptional activity was observed in fraction B/C (at the pro-B-cell stage of B-cell development; Fig. 2B), whereas significantly less activity was found in the pre-B- and mature B-cell stages (fractions D–F). Low level of luciferase activity was detectable in fraction A (the pre–pro-B-cell population), which is the first identifiable compartment committed to the B-cell lineage. We verified the luciferase data from the reporter transgene by EMSA (Fig. 2C) finding once again that fraction B/C contained the majority of the NF-{kappa}B activity (Fig. 2C). Surprisingly, although fractions D/E/F contained significant levels of {kappa}B-binding complexes, they exhibited very low levels of NF-{kappa}B transcriptional activity as revealed by analysis of the luciferase reporter mice. The significance of this discordance between DNA-binding and transcriptional activity in this B-cell fraction remains to be explored. In summary, our results show that only fraction B/C cells within the bone marrow express both high levels of NF-{kappa}B DNA-binding and transcriptional activity.



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Fig. 2. All B lineage cells isolated from the bone marrow express constitutively activate NF-{kappa}B. (A) Bone marrow-derived B lineage cells were separated into discrete fractions, using the following combinations of antibodies and dyes. Red670–anti-B220, PE–anti-CD43, biotinylated HSA (which was revealed by streptavidin Texas Red) and FITC–anti-BP-1. (B) According to the above staining procedures, cells from NF-{kappa}B luciferase reporter mice were sorted into four populations: non-B cells, fraction A, fraction B/C and fraction D/E/F. These fractions were then analyzed for NF-{kappa}B transcriptional activity using the luciferase assay. Data are presented as relative luciferase units per 106 cells. (C) Five micrograms of nuclear extract from each fraction was analyzed for NF-{kappa}B and Oct-1 DNA-binding activity by EMSA. (D) Cells from fractions A and B/C were again isolated from wild-type mice. This time 4 µg of the nuclear extract was incubated with nothing (lanes 1 and 6), anti-NF-{kappa}B1 antibody (lanes 2 and 7), anti-RelA antibody (lanes 3 and 8), anti-c-Rel antibody (lanes 4 and 9) or a combination of the anti-RelA and anti-c-Rel antibodies (lanes 5 and 10) for 2 h at 4°C. Subsequently, the samples were assayed by EMSA.

 
To determine which rel proteins were present in the NF-{kappa}B DNA-binding complexes in developing B cells, we performed supershift analysis on fraction A (B220+CD43+HSA), the combined fraction B/C (B220+CD43+HSA+) and fraction D/F (B220+CD43) (Fig. 2D). NF-{kappa}B complexes from all three populations were completely supershifted with the anti-p50 antibody. However, neither anti-p65 nor anti-c-Rel antibodies alone were capable of totally inhibiting the interaction between the rel proteins and DNA, suggesting that complexes composed of p50–p65 and p50-c-Rel heterodimers were present in the nucleus of these cells.

The peak of NF-{kappa}B transcriptional activity is in the large, CD43+ pro-B cells (fraction C')
As shown in Fig. 2(B and C), the combined fraction B/C contains the highest levels of NF-{kappa}B transcriptional activity in developing B cells in the bone marrow. Further fractionation revealed that both fractions B and C by themselves contained significant amounts of NF-{kappa}B activity (Fig. 3A). In addition, fraction C itself can be sub-divided into two distinct populations, C and C', based on the higher level of expression of HSA in fraction C'. Functionally, fractions C and C' represent critical steps in B lineage development, since fraction C represents the small CD43+ pro-B cells, which are yet to express the pre-B-cell receptor (7, 8, 12, 13). By contrast, fraction C' contains cells expressing the pre-B-cell receptor and hence destined to survive (large, CD43+ pre-B cells). We therefore wanted to examine which population contained the majority of NF-{kappa}B activity. As shown in Fig. 3(A), fraction C' (the large, CD43+ pre-B cells) contained the highest amount of NF-{kappa}B (approximately double the level in fraction C) among the B lineage fractions assayed (Fig. 3A, right panel). It is therefore likely that similar to thymocytes, NF-{kappa}B provides a stimulus (possibly an anti-apoptotic survival signal) that facilitates the continued development of CD43+ pre-B cells into small, CD43 pre-B cells.



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Fig. 3. The peak of NF-{kappa}B activity is found in the large, CD43+ pro-B cells. (A) B lineage cells from NF-{kappa}B luciferase reporter mice were stained with Red670–anti-B220, PE–anti-CD43, biotinylated HSA (which was revealed by streptavidin Texas Red) and FITC–anti-BP-1. Fraction C and C' cells were then separated as follows: fraction C cells are B220+CD43+BP-1+HSA+, whereas fraction C' cells are B220+CD43+BP-1+HSA++. B lineage cells collected in this way were then analyzed for luciferase activity; the results are presented in relative luciferase units per 106 cells. (B, C) The NF-{kappa}B luciferase reporter mice (luc+/–) was crossed with either the µMT knockout (µMT–/–) or the {lambda}5 knockout ({lambda}5–/–) to generate the following chimeras: luc+/–/µMT–/– (B) and luc+/–/{lambda}5–/–(C). Pro-B cells from both these chimeras were isolated and analyzed for NF-{kappa}B luciferase activity. Data are presented as relative luciferase units per 106 cells. (D) Decrease DNA-binding activity in nuclear extracts from {lambda}5–/– mice. Pro-B cells from {lambda}5–/– mice were isolated and analyzed for NF-{kappa}B and Oct-1 DNA-binding activity by EMSA.

 
Since large, CD43+ pre-B cells are the only known population in B lineage development to express the pre-B-cell receptor (7, 8, 12, 13), it is possible that activation of NF-{kappa}B depends on the expression of this receptor. To more closely examine the link between the pre-B-cell receptor and NF-{kappa}B activation, we crossed the luc+/– mouse with the µMT–/– and the {lambda}5–/– mice (12, 18). Both these mutations lead to a block in B-cell development at the large, CD43+ pre-B-cell stage, since they fail to assemble the pre-B-cell receptor. CD43+ B lineage cells isolated from both the µMT–/–/luc+/– mouse and the {lambda}5–/–/luc+/– mouse showed significant reductions in constitutive NF-{kappa}B transcriptional activity in comparison with wild-type cells (Fig. 3B and C). As shown in Fig. 3(D), the NF-{kappa}B DNA-binding activity was also dramatically decreased in nuclear extracts of CD43+ pre-B cells isolated from {lambda}5–/– mice.

Activation of the pre-BCR leads to activation of NF-{kappa}B
Pre-BCR complex consists of Igµ chain in association with Ig{alpha}/Igß heterodimer on the cell surface. Nagata et al. have shown that cross-linking of Igß using anti-Igß mAb (CD79b) activates several serine/threonine or tyrosine kinases in vitro and can partly overcome the block in early B-cell development in RAG2-deficient mice (19). These results suggest that anti-Igß mAb treatment provides signals that normally originate from the pre-BCR complex. We therefore first examined whether cross-linking with anti-Igß mAb directly activates NF-{kappa}B in the pro-B-cell line, 63-12 derived from RAG2-deficient fetal liver cells, which expresses Igß (20). NF-{kappa}B was activated within 1 h and then decreased to basal levels 4 h after cross-linking (Fig. 4A, upper panel). As a control, Oct-1 DNA-binding activity was not significantly changed (Fig. 4A, lower panel). This indicates that cross-linking of Igß directly activates NF-{kappa}B in pro-B cells.



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Fig. 4. Activation of NF-{kappa}B in pro-B cells in RAG1-deficient mice by the treatment of anti-Igß mAb (HM79). (A) Pro-B-cell line, 63-12 derived from RAG2-deficient fetal liver, cells were incubated with plate-bound anti-Igß mAb (HM79; 20 µg ml–1) for indicated periods. NF-{kappa}B and Oct-1 DNA-binding activity in the nuclear extracts was determined by EMSA. The amount of Oct-1 complex was determined by quantitation of the scanned autoradiograph using NIH Image 1.63, and the relative levels of NF-{kappa}B complexes were adjusted by normalizing the signals to the Oct-1 complexes. The normalized values of the intensity of the NF-{kappa}B complexes are indicated in arbitrary units. (B) RAG1-deficeint mice (8–10 weeks old) were injected intravenously with 200 µg of anti-Igß mAb. After 12 or 24 h of treatment, B220+CD43+ cells were isolated and then examined for NF-{kappa}B and Oct-1 DNA-binding activity. The normalized values of the NF-{kappa}B complexes are indicated as described above. (C) In vivo treatment with anti-Igß mAb (HM79) induced pre-B-cell differentiation in RAG1-deficient mice. RAG1-deficeint mice (8–10 weeks old) were injected intravenously with 200 µg of anti-Igß mAb (HM79). On day 7 post-injection, bone marrow cells were stained with Red670–anti-B220, PE–anti-CD43 and FITC–anti-BP-1. Cells present in the lymphocyte gate defined by size scatted were analyzed by three-color flow cytometry. Data shown are representative of three repeated analyses. (D) The average number of cells in the indicated fraction from bone marrow cells of RAG1-deficient mice with or without the treatment of anti-Igß mAb is shown.

 
We next injected the anti-Igß mAb into RAG1-deficient mice and then isolated B220+CD43+ cells after 0, 12 and 24 h, and examined NF-{kappa}B DNA-binding activity. The B220 and CD43 profiles did not change after 24 h of anti-Igß mAb treatment (data not shown). However, anti-Igß mAb clearly activated NF-{kappa}B in CD43+ pro-B cells in the injected mice within 24 h (Fig. 4B, upper panel). The anti-Igß mAb treatment had no effect on the constitutive transcription factor Oct-1 (Fig. 4B, lower panel). Seven days after antibody treatment, the total cell number was not significantly altered, however B220+CD43 pre-B-cell numbers were significantly increased (Fig. 4C and D). In addition the expression of BP-1 (Fig. 4C) and CD25 (data not shown) were up-regulated. These changes have been used to characterize events associated with the transition from pro-B cells into pre-B cells (7) and therefore these results demonstrate that activation of NF-{kappa}B by the treatment of anti-Igß mAb strongly correlates with pre-B-cell differentiation in RAG1-deficient mice.

Inhibition of NF-{kappa}B in developing B cells leads to a reduction in the number of CD43pro-B cells
To test whether NF-{kappa}B promotes the survival of large, CD43+ pro-B cells, we blocked the expression of constitutive NF-{kappa}B activity using a DN version of I{kappa}B{alpha} (5). This super-repressor form of I{kappa}B{alpha} was cloned into a vector under the control of the Eµ-enhancer and the mb-1 promoter to restrict the expression of this DN protein to the B lineage (14).

Using a similar approach to that described for the luciferase construct (5), we generated four transgenic lines (6, 35, 57 and 76) expressing the I{kappa}B{alpha}-SS-PEST-DN protein at different levels in murine B cells. The molecular weight of the DN form is slightly higher than that of endogenous I{kappa}B{alpha} because it is tagged with the HA-epitope. Therefore, we were able to identify both forms of the inhibitor, using antibodies specific for I{kappa}B{alpha} (Fig. 5B). Unfortunately, the levels of expression of our transgene were actually lower than those of the endogenous I{kappa}B{alpha} (compare upper and lower bands, Fig. 5B). However, the level of expression was sufficient to significantly interfere with B-cell development (Fig. 5C). I{kappa}B{alpha}-SS-PEST founder 76 proved to be the most effective since it reduced the numbers of pro-B cells by >40% in comparison with the appropriate littermate control. Figure 5(D) shows that the other three expressing founders also had reduced numbers of pro-B cells, and the degree of reduction was in keeping with the original level of expression of the transgene (Fig. 5B). The reduction in pro-B-cell numbers also led to reductions in the number of cells in the CD43 fraction by ~20% (Fig. 5C and data not shown). These data therefore indicate that inhibition of NF-{kappa}B in developing B cells leads to a selective loss of pro-B cells, suggesting that NF-{kappa}B activation by the pre-BCR provides a survival signal that enables large, CD43+ pro-B cells to differentiate into small, CD43 pre-B cells. Alternatively, the reduction in cell numbers in all pro-B-cell fractions could be due to a previously unrecognized role of NF-{kappa}B activation in earlier stages of B-cell development.

To overcome the difficulty of being unable to express the I{kappa}B{alpha}-SS-PEST in sufficiently high levels using a transgenic approach, we employed a retroviral approach to introduce I{kappa}B{alpha}DN into hematopoietic cells derived from B6 mice. We cloned the I{kappa}B{alpha}DN cDNA into a MSCV-derived vector (15) (gift of K. Murphy, Washington University School of Medicine), encoding a bi-cistronic mRNA that expresses both I{kappa}B{alpha}DN and GFP (Fig. 6A). Transfection of Phoenix E packaging cells with MSCV-I{kappa}B{alpha}DN vector produced high titer of virus-expressing I{kappa}B{alpha}DN protein, as demonstrated by immunoblotting of MSCV-I{kappa}B{alpha}DN-infected NIH3T3 cells (data not shown).



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Fig. 6. Over-expression of DN form of I{kappa}B{alpha} protein prevents B-cell development during pro-B to pre-B-cell stage. (A) A diagram of the DN form of the I{kappa}B{alpha} cloned into the MSCV retroviral vector. The 5' long terminal repeat-mediated transcription generates a bi-cistronic mRNA, with the GFP reading frame accessed by IRES. (B) Representative data from a single mouse are shown. Similar results were obtained from multiple experiments. Bone marrow cells from wild-type B6 mice were infected with either MSCV-GFP or MSCV-I{kappa}B{alpha}DN and then injected via the tail vein into irradiated (700 rads) RAG1-deficient females (8 weeks old). Six weeks after bone marrow reconstitution, recipient mice were sacrificed. The percentages of GFP+ and B220+/CD43 cells from total bone marrow were determined by immunostaining and flow cytometry. GFP+ or GFP populations were then analyzed for B220+/CD43 cells.

 
To examine the in vivo consequences of expressing high levels of I{kappa}B{alpha}DN in developing B cells, we performed bone marrow engraftment of irradiated RAG1-deficient mice (21). Bone marrow cells isolated from B6 wild-type mice were infected with control MSCV-GFP or MSCV-I{kappa}B{alpha}DN retroviruses and then injected into lethally irradiated RAG1-deficient mice (21). After 6 weeks, reconstituted mice were sacrificed, and bone marrow cells were prepared for FACS analysis of B cells. Representative data are shown in Fig. 6(B). Approximately 10% of cells from the bone marrow expressed GFP, indicating that these cells were derived from retrovirus-infected bone marrow precursors. To examine the consequences of inhibition of NF-{kappa}B by I{kappa}B{alpha}DN protein in the infected cells, B-cell fractions in GFP+ cells and GFP bone marrow cells were analyzed. In the wild-type B6 mice, ~15% of GFP-positive cells are B220+/CD43 pre-B cells. However, <1% of GFP/I{kappa}B{alpha}DN-positive cells are pre-B cells. B-cell development in GFP cells in I{kappa}B{alpha}DN-infected mice were comparable with control vector-infected mice. Therefore, higher level of over-expression of I{kappa}B{alpha}DN protein by the retroviral approach leads to a more severe phenotype where B-cell development is almost completely blocked at a stage that appears to precede the pro-B to pre-B-cell transition.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this paper we have presented a detailed analysis of the pattern of expression of NF-{kappa}B activity in different stages of B-cell development (Fig. 7). We found that the peak of NF-{kappa}B transcriptional activity corresponded to large, CD43+ pro-B cells. Our analysis showed that similar to the role of pre-TCR-activated NF-{kappa}B in thymocytes, NF-{kappa}B in developing B cells appears to provide a signal that is necessary for progression of the large, CD43+ pro-B cells to the small, CD43 pre-B cells (5, 6). Therefore, these results reinforce the similarity in the processes that occur during development of lymphocytes leading to the selection of cells with productively rearranged antigen receptor genes (4).



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Fig. 7. Summary of NF-{kappa}B DNA-binding and transcriptional activity in bone marrow-derived B lineage cells. Shown are steps in the B-cell development sequence defined using mAbs. The arrows indicated the defect of B-cell development observed in gene knockout mice. The expression levels of Bcl-2 are based on studies reported in Merino et al. (24). ND: not done.

 
The pre-BCR plays a crucial role in B-cell development by allowing maturing B lymphocytes to ‘sense’ the generation of a functional Ig heavy chain (22, 23). It is believed that signaling from the pre-B-cell receptor provides both a survival advantage to the cells successful in rearranging the heavy chain and also gives a growth or proliferation advantage that allows the pre-BCR-expressing cells to multiply rapidly (23). It has been shown previously that whereas early pro-B cells, particularly CD43+ cells, express high levels of Bcl-2, cells at later stages of development show a dramatic drop in the expression of Bcl-2 (24) (Fig. 7). Therefore, it appears that Bcl-2 and NF-{kappa}B promote survival of B cells at distinct stages of development. The replacement of Bcl-2, which is expressed in the entire cohort of developing early pro-B cells, with NF-{kappa}B, which is only expressed in cells that have successfully rearranged the heavy chain and are poised to develop further, would also allow the previously reported inhibitory effect of Bcl-2 on proliferation to be relieved (25, 26). It has been reported that subsequently, in fraction D and later, the survival function of NF-{kappa}B is most likely replaced by Bcl-xL (27). An identical pattern of regulation of Bcl-2 and NF-{kappa}B is also seen in developing thymocytes (5).

In contrast to our previous study in thymocytes, where we used an lck promoter to drive thymocyte-specific expression of the transgenes, the transgenic expression system used in this study, mb-1 promoter with the µ-enhancer, resulted in significantly lower levels of transgene expression. The reduced level of transgene expression prevented us from expressing sufficient amounts of the I{kappa}B{alpha}-SS-PEST construct, and despite the derivation of numerous founders, we failed to obtain any mice where the expression of the I{kappa}B{alpha}-SS-PEST was greater than the endogenous I{kappa}B{alpha} protein. Because I{kappa}B{alpha}-SS-PEST inhibits NF-{kappa}B through competitive displacement of wild-type I{kappa}B{alpha} for the mutant form, the B cells from the transgenics showed only partial inhibition of NF-{kappa}B activity. Therefore, it was not surprising that the effect on the number of CD43 cells was relatively modest.

To overcome the limitation of transgenic expression we cloned the I{kappa}B{alpha}-SS-PEST in a retroviral vector that contained a IRES-driven GFP cassette. We transduced bone marrow cells with this retrovirus and reconstituted lethally irradiated RAG1 knockout mice. Although the degree of reconstitution was modest, it was clear that expression of the I{kappa}B{alpha}DN almost completely blocked development of B cells past the stage where the pre-BCR is expressed. In fact, development might actually be blocked at an even earlier stage due to early onset of expression of the I{kappa}B{alpha}DN protein. Characterization of thymocytes from these mice also revealed a similarly dramatic block at stages preceding ß-selection (data not shown). The results with the retroviral constructs suggest that the consequence of completely inhibiting NF-{kappa}B activity in developing B lymphocytes may be more severe than originally suspected. In fact, the reduction of all pro-B-cell fractions in the I{kappa}B{alpha}DN transgenic mice as well as the retroviral expression experiments suggests that NF-{kappa}B may play a crucial role in an early stage of B-cell development, and this role may not be analogous to the pre-TCR-induced NF-{kappa}B-dependent survival signal observed in developing thymocytes. Recent generation of conditionally targeted alleles of NEMO and IKK2 (28, 29), however, raises the possibility that more careful testing of the requirement of NF-{kappa}B in early B-cell development can be carried out, provided early B-cell-specific cre-recombinase expression can be achieved.

The overall arrangement and function of the pre-BCR and the pre-TCR are very similar and therefore it would not be surprising if they functioned in a similar manner. The Ig{alpha}/ß heterodimer associates with Igµ in the plasma membrane and is required for signaling by the pre-BCR. In T cells, productive TCR-ß gene rearrangement results in the formation of the pre-TCR signaling complex comprised of a TCR-ß chain, a pre-T{alpha} surrogate a chain and a multisubunit of CD3 signaling complex (1). Targeted gene disruption of any of the components of pre-BCR or pre-TCR leads to a developmental block at pro-B to pre-B cells and DNIII thymocytes, respectively (1, 2). However, the cross-linking of the CD3 complex in DN thymocytes using anti-CD3{varepsilon} antibody can strongly activate NF-{kappa}B in stage III thymocytes and promote differentiation into the double-positive stage (3032). Recently, a similar technique has been used to manipulate early B-cell development by using anti-Igß mAb to cross-link the Ig{alpha}/ß heterodimer (19). Using this technique we find that anti-Igß mAb strongly induces NF-{kappa}B in CD43+ pro-B cells and can release the developmental block at pro-B-cell stage in RAG1-deficient mice. Our results demonstrate for the first time that pre-BCR signaling can activate NF-{kappa}B in pro-B cells.

In summary, using a combination of EMSA and transgenic mice we have been able to comprehensively analyze NF-{kappa}B transcriptional activity throughout B lineage development in the bone marrow. Our results indicate that the role of NF-{kappa}B in early B-cell development is to act as a critical factor that help cells expressing the pre-BCR to proceed to subsequent stages of development. Such a role for NF-{kappa}B is similar to our previous findings in thymocytes and therefore further establishes the similarity in the role of the pre-BCR and pre-TCR. Identifying the genes that are regulated by NF-{kappa}B at these stages of lymphocyte development remains a challenge for the future.


    Acknowledgements
 
We would like to thank Crystal Bussey and Iris Douglas for technical help. This work was supported by a grant from NIH (R37-AI33443).


    Abbreviations
 
DN   dominant negative
DTT   dithiothreitol
EMSA   electrophoretic mobility shift assay
GFP   green fluorescent protein
HA   hemagglutinin
IRES   internal ribosome entry site
MSCV   murine stem cell virus
NF-{kappa}B   nuclear factor-{kappa}B
PMSF   phenylmethylsulphonylfluoride
RAG   recombination-activating gene

    Notes
 
* These authors contributed equally to the study. Back

Transmitting editor: W. Leonard

Received 20 August 2004, accepted 24 March 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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