In mature B lymphocytes, the zinc finger transcription factor early growth response 1 (Egr-1) is one of the many immediate-early genes induced upon B cell antigen receptor engagement. However, its role during earlier stages of lymphopoiesis has remained unclear. By examining bone marrow B cell subsets, we found Egr-1 transcripts in pro/pre-B and immature B lymphocytes, and Egr-1 protein in pro/pre-B–I cells cultivated on stroma cells in the presence of interleukin (IL)-7. In recombinase-activating gene (RAG)-2–deficient mice overexpressing an Egr-1 transgene in the B lymphocyte lineage, pro/pre-B–I cells could differentiate past a developmental block at the B220low BP-1 stage to the stage of B220low BP-1+ pre-B–I cells, but not further to the B220low BP-1+ CD25+ stage of pre-B–II cells. Therefore, during early B lymphopoiesis progression from the B220low BP-1 IL-2R pro/pre-B–I stage to the B220low BP-1+ IL-2R+ pre-B–II stage seems to occur in at least two distinct steps, and the first step to the stage of B220low BP-1+ pre-B–I cells can be promoted by the overexpression of Egr-1 alone. Wild-type mice expressing an Egr-1 transgene had increased proportions of mature immunoglobulin (Ig)M+ B220high and decreased proportions of immature IgM+ B220low bone marrow B cells. Since transgenic and control precursor B cells show comparable proliferation patterns, overexpression of Egr-1 seems also to promote entry into the mature B cell stage. Analysis of changes in the expression pattern of potential Egr-1 target genes revealed that Egr-1 enhances the expression of the aminopeptidase BP-1/6C3 in pre-B and immature B cells and upregulates expression of the orphan nuclear receptor nur77 in IgM+ B cells.

Antigen binding to surface (s)Igs in B cells initiates a signal cascade which in the context of secondary signals leads to proliferation and differentiation of mature resting B lymphocytes into plasma or memory cells. Changes in the activity and expression of transcription factors translate activating signals into the modulated expression pattern of downstream genes. One of these transcription factors is called early growth response 1 (Egr-1;1 also known as Krox-24, NGFI-A, Tis-8, zif268, pAT225, or Z-225 [1–3]). Egr-1 is induced very rapidly in many different cell types and tissues, including fibroblasts (1), monocytes (4), lymphocytes (5, 6), kidney (7), neurons (3), and brain (8), in response to a wide range of signals (13, 5, 9). In mature B lymphocytes, transient Egr-1 expression is rapidly induced upon stimulation by B cell antigen receptor (BCR) cross-linking (5, 10), whereas signals resulting from Fc receptor cross-linking inhibit induction (11, 12). Thus, the broad spectrum of Egr-1 expression and the diverse modes of Egr-1 induction suggest that Egr-1 functions as a transcriptional regulator that links common biochemical signaling pathways to the rapid modulation of downstream gene expression.

Mature peripheral B lymphocytes originate from bone marrow precursor cells that are ordered according to their phenotype, gene expression, Ig gene rearrangement, and proliferative and developmental potential into the pro-B, pre-B, and immature B lymphocyte subsets (1317). Transcriptional regulation plays a critical role during B cell development (for a review, see reference 18) as shown by gene targeting of multiple transcription factors. Mutations in these factors that obliterate their activity were shown to arrest B lymphopoiesis at defined stages of maturation (1929).

Little is known about the expression and function of Egr-1 during early steps of B cell differentiation. Here we report that Egr-1 expression can be detected already in pre-B cells isolated from bone marrow and in fetal liver–derived pre-B cell cultures. These results suggested that Egr-1 might also have a regulatory function in early stages of B lymphopoiesis. However, mice deficient for Egr-1 fail to show defects in lymphocyte or monocyte maturation, most probably because the missing Egr-1 activity is masked by other members of the Egr transcription factor family (30, 31). To bypass the complementing activity of Egr-2, Egr-3, or Egr-4, we studied B lymphocyte differentiation in transgenic mice overexpressing Egr-1 in B cells in normal and recombinase-activating gene (RAG)-2–deficient mice. Since the RAG-2 mutation prevents rearrangement of Ig genes (32), precursor B cells are developmentally arrested in the stage of B220low BP-1 pro/pre-B–I cells (33, 34). Analyzing Egr-1 transgenic RAG-2–deficient mice, we found that pro/pre-B–I cells overcame the RAG-2−/− induced differentiation block at the stage of B220+ BP-1 pro/pre-B–I cells and differentiated into B220+ BP-1+ pre-B–I cells. Comparing B lymphocyte maturation in the bone marrow of normal transgenic and control animals, we found that Egr-1 transgenic mice had increased their fraction of mature cells. Because Egr-1–enhanced progression of developing thymocytes was also found in transgenic mice overexpressing Egr-1 in T cells (35), we propose that Egr-1 activity promotes maturation of B and T lymphocytes.

Materials And Methods

Pre-B Cell Cultures.

Fetal liver cells of day 15–18 embryos were removed and plated onto irradiated ST-2 feeder cells in Iscove's medium containing IL-7 and 10% FCS. Cells were cultured as described previously (36). Cells from transgenic lines were identified by PCR. For further analyses, nonadherent cells were collected and washed twice in ice-cold PBS. Samples from wells containing only ST-2 feeder cells were treated in parallel and served as controls.

Mice.

The detailed description of the generation of Egr-1 transgenic mice using the BALB/c embryonic stem cell line BALB/c-I will be described elsewhere. Egr-1 transgenic mice of the IA7 line were transferred to a special pathogen-free unit by implanting transgenic one-cell embryos into C57BL/6 foster animals kept under specific pathogen–free conditions. The IA7 line was then bred further by mating with wild-type BALB/c mice. RAG-2−/− mice expressing transgenic Egr-1 were obtained by backcrossing female Egr-1 transgenic IA7 mice twice with C57BL/6 RAG-2−/− males. Animals were tested by PCR for the Egr-1 transgene using genomic DNA, 5′-CTTTCGGTTTGGGGCTGGACA-3′ and 5′-CGCTGCTGGTGCTGCTGCTGCTAT-3′, as transgene-specific primer pair. The RAG-2−/− phenotype was verified by FACS® analysis of peripheral blood cells.

RNA Isolation, Northern Blot, and PCR Analysis.

RNA was extracted using the guanidinium isothiocyanate method as described (37). For Northern blotting, 10 μg of total RNA was separated in a 1% agarose gel containing 7% formaldehyde, transferred onto nylon filters, and fixed by UV cross-linking. Filters were prehybridized (50% deionized formamide, 5× SSC, 5× Denhardt's solution, 50 mM NaH2PO4, pH 7, 10 mM Na4P2O7, 0.1% SDS, 0.1 mg/ml denatured salmon sperm DNA) for 2 h at 42°C. For detection of Egr-1–specific transcripts, [α-32P]dATP-labeled probes were prepared from a 1.6-kb EcoRI-HindIII fragment from plasmid 533 (a gift from V. Sukhatme) containing the Egr-1 cDNA by the oligonucleotide priming method (38). The probe was added to the prehybridization and filters were incubated overnight, washed with 0.2× SSC, 0.1% SDS at 42°C, and exposed to X-ray films. Egr-1 expression was analyzed by PCR using cDNA reverse transcribed from total RNA with Superscript II (GIBCO BRL, Eggenstein, Germany) and the Egr-1– specific primers 5′-GCAGATCTCTGACCCGTTCGG-3′ and 5′-CCGAGCGTTTGGCTRGGGATA-3′ as described by T. Miyazaki (35). PCR was performed using Taq polymerase (MBI Fermentas, Inc., Amherst, NY) using 1/25 of the cDNA reaction as template at an annealing temperature of 54°C.

Immunoblot Analysis.

Bone marrow cells from six femurs were isolated and resuspended in FACS buffer (0.1% sodium azide, 3% FCS in PBS). B220-specific biotin-labeled antibody RA3.3A1 (39) was added and incubated for 30 min on ice. Cells were washed, magnetic streptavidin-labeled beads (Dynal, Oslo, Norway) were added, and B cells were isolated. Quality of the sorting process was verified by flow cytometric analysis. The B cells were resuspended in 30 μl lysis buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl, pH 7.0, 0.1 mM PMSF) and incubated on ice for 10 min. Cell debris was removed by centrifugation (10 min, 4°C, 22,000 g), and the extract was separated by SDS-PAGE (8%) and transferred onto nitrocellulose membrane (Hybond C extra; Amersham Pharmacia Biotech, Uppsala, Sweden). Egr-1 was detected using the antiserum C19 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 25 ng/ml followed by peroxidase-conjugated goat anti–rabbit IgG F(ab′)2 (Dianova GmbH, Hamburg, Germany) at 200 ng/ml. Expression of nur77 was analyzed using a mouse IgG anti-nur77 mAb (a gift of B. Osborne, University of Massachusetts, Amherst, MA) followed by peroxidase-conjugated goat anti–mouse IgG (Southern Biotechnology Associates, Inc., Birmingham, AL). IgM was detected by a goat anti–mouse IgM peroxidase-labeled serum (Southern Biotechnology Associates, Inc.). Signals were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech).

Flow Cytometry.

Flow cytometry was carried out as described previously (40) using the following antibodies: RS3.1-biotin specific for murine IgMa (41), 6C3-biotin for BP-1, 7D4-biotin for IL-2Rα chain, 2B8-biotin for c-kit, S7-biotin for leukosialin, AMS9.1-biotin for IgDa, RA3-6B2–PE for B220, IM7-biotin for Pgp-1, 3E2-PE for intercellular adhesion molecule 1 (ICAM-1) (all from PharMingen Europe, Hamburg, Germany), and biotinylated PB493 (42) to stain immature B lymphocytes. Cells were counterstained using PE- or APC-conjugated streptavidin (PharMingen Europe). Unspecific binding to Fc receptors was blocked by adding unlabeled mouse FcγR-specific mAb 2.4G2. Dead cells were excluded by staining with propidium iodide. Using a FACSCalibur® and CellQuest® software (Becton Dickinson, San Jose, CA), 3–5 × 104 cells were acquired according to their forward/side scatter pattern and analyzed. To analyze Egr-1 expression in bone marrow B cell subsets, 3.4 × 105 pre-B and 7 × 104 immature B cells were isolated from both femurs of a 5-wk-old BALB/c mouse by cell sorting at 4°C according to their IgMa/ PB493 staining pattern using a FACStar® cell sorter and LYSIS II® software (Becton Dickinson).

Bromodeoxyuridine Treatment and Staining.

Bone marrow cells were labeled with bromodeoxyuridine (BrdU; Sigma, Deisenhofen, Germany) starting with a single injection of 1 mg/ml i.p. BrdU and feeding mice continuously with drinking water containing 1 mg/ml BrdU for 48 h as described (43). During the labeling period, the drinking water was protected from light. Simultaneous detection of surface staining and BrdU labeling was done as described (44). After surface marker staining, cells were resuspended in 500 μl 0.15 M NaCl, 1.2 ml ice-cold 95% ethanol was added, and the cells were incubated 30 min on ice. Cells were washed and resuspended in 1 ml fixation buffer (1% paraformaldehyde and 0.01% Tween in PBS). After incubation for 30 min at room temperature, the cells were incubated for 30 min in DNase I solution (50 KU DNase I in 4.2 mM MgCl2/ 0.15 M NaCl, pH 5). Cells were washed, 10 μl anti-BrdU antibody (Becton Dickinson) was added, and the cells were then incubated for 30 min and washed.

Electrophoretic Mobility Shift Assays.

Gel shift was carried out using recombinant Egr-1 as described (45) with double-stranded radiolabeled oligonucleotides from the nur77 and BP-1 promoter regions carrying putative Egr-1 binding sites (bold): 5′-TTCCAAAGTTCCCCCTCAACCCCTC-3′ for BP-1 (position −753 to −729), 5′-GTCAGTGGCGCCCCCGCCCCTCTCCAA-3′ for nur77 (position −66 to −50), and 5′-GGATCCAGCGGGGGCGAGCGGGGGCG-3′ for Egr-1.

Results

Egr-1 Expression in Pre-B and Immature B Cell Precursors.

BCR cross-linking has previously been reported to induce Egr-1 expression in mature B cells, but not in immature B lymphocytes or in immature B cell lines (5, 46, 47). We addressed the question of whether unstimulated pre-B and immature B cells express Egr-1 by analyzing sorted B cell subsets from murine bone marrow. Transcription of the Egr-1 gene was found by PCR in both sIgM (pre-B) and sIgM+ PB493+ immature B cells (Fig. 1,A). Likewise, Egr-1 protein was detected by immunoblotting in sIgM pre-B cells isolated from fetal liver and expanded in culture on ST-2 stroma cells in the presence of IL-7 (Fig. 1 B). Both results show transcription of the Egr-1 gene and translation of Egr-1 mRNA into detectable amounts of protein as early as the pre-B cell stage before BCR surface expression.

Egr-1 Expression in Transgenic and Normal Mice.

These results suggested that Egr-1 function might also be important during early stages of B lymphopoiesis. To test this hypothesis, we generated transgenic mice expressing Egr-1 specifically in B lymphocytes using an Ig heavy chain promoter/enhancer construct. Four different founder mice showing Egr-1 germline transmission were obtained. By breeding to BALB/c mice, we established the Egr-1 transgenic lines IA7, IB10, IC4, and ID4 (to be published elsewhere). At first, we compared Egr-1 expression between transgenic and BALB/c control mice by Northern and immunoblotting (Fig. 2). Spleen cells of the line IA7 expressed 10-fold more Egr-1 mRNA than the control littermates, whereas the other lines showed Egr-1 expression levels of about two- to threefold above unstimulated spleen cells (Fig. 2,A). Since transgenic IC4 mice expressed only low levels of Egr-1 they were abandoned. Carrying out most of the experiments with mice from lines IA7, IB10, and ID4, we found only small variations between these transgenic lines. Testing Egr-1 protein expression, we found high levels in purified B220+ bone marrow B cells as well as in cultivated pre-B cells isolated from fetal liver (Fig. 2, B and C, respectively).

These results show that transgenic Egr-1 is expressed during similar stages of B cell maturation, but at far higher levels than endogenous Egr-1.

Egr-1 Expression Promotes At Least Two Different Stages of B Cell Development.

To examine whether enhanced Egr-1 expression has an effect on early stages of B cell development, we backcrossed the IA7 transgenic mice to a RAG-2–deficient background. The RAG-2 mutation prevents rearrangement of the Ig genes (32) and therefore blocks B cell maturation at the pro/pre-B–I cell stage (16, 34). These cells carry the surface markers c-kit and CD43; <5– 15% express BP-1 and <1% express the IL-2Rα chain (data not shown). Phenotypically, these pro/pre-B–I cells correspond to fraction B as classified by Hardy et al. (13). FACS® analysis of control and Egr-1 transgenic RAG-2– deficient mice revealed an unchanged expression pattern for c-kit and CD43, but a three- to fourfold increase in the fraction of BP-1+ cells, compared with control littermates (Fig. 3). Phenotypically, these BP-1+ c-kit+ CD43+ pre-B lymphocytes are defined as fraction C cells (13), and progression into this stage normally requires RAG-2 expression and Ig heavy chain gene rearrangement (16, 34), suggesting that Egr-1 might support the maturation of fraction B pre-B cells even in the absence of RAG-2 activity. The reduced cell size of transgenic BP-1+ B220+ lymphocytes as reflected by changes in forward/side scatter (Fig. 3 A, c and d) also supports the Egr-1–induced progression of B cell development. Since we did not detect cell surface markers characteristic for pre-B–II/fraction D cells, i.e., IL-2Rα and high levels of heat-stable antigen, the expression of the Egr-1 transgene seems to promote the transition of pre-B–I cells from fraction B to fraction C but not further.

These results indicated that Egr-1 expression might also change maturation of later stage bone marrow B cell subsets. According to the expression pattern of B220 and IgM, three main subsets corresponding to three consecutive stages of maturation can be distinguished: IgM B220low pre-B cells, B220low IgM+ IgD immature B cells, and finally the B220high IgM+ IgD+ mature B cells (16, 48). The proportion of mature B cells in the bone marrow increases with the age of the animal. Therefore, mice at 4 wk of age have fewer mature B cells than older animals. Comparing 23 control littermates with 23 IA7 transgenic mice between 4 and 40 wk of age, we found in general fewer immature and more mature B cells in the IA7 bone marrow than in age-matched controls (Fig. 4). These results suggest that Egr-1 also promotes the differentiation of immature B cells into mature B cells.

Normal Proliferation of B Cells in Egr-1 Transgenic Mice.

The enlarged population of BP-1+ pre-B cells in transgenic RAG-2–deficient mice and the higher percentage of mature cells in transgenic RAG-2+ animals could result from an increased proliferative potential of B cell precursors. Because dividing B lymphocytes can be traced by incorporation of the nucleotide analogue BrdU (49), we compared the proportion of BrdU+ cells in the pre-B, immature B, and mature B cell subsets between control littermates and transgenic mice after 2 d of in vivo labeling. The similar percentages of BrdU+ cells in all B cell subsets of both groups of mice suggest that the expression of transgenic Egr-1 does not change the proliferative potential of B cells (Fig. 5).

Regulation of Downstream Genes.

The increased maturation of transgenic B cells would be expected to correlate with changes in the expression of genes controlled directly or indirectly by Egr-1. Using a panel of antibodies, we screened transgenic bone marrow B cells for alterations in the expression pattern of B cell–specific surface markers by flow cytometry. Changes were not observed except for a slight increase in the percentage of BP-1+ cells and in BP-1 expression levels in all transgenic lines (Fig. 6,A). As discussed above, the proportion of BP-1+ cells might relate to the Egr-1–enhanced transition of fraction B pre-B cells into the fraction C stage, but it would not explain higher BP-1 surface levels. Therefore, we speculated that Egr-1 might regulate the transcription of the BP-1 gene and screened the BP-1 promoter for potential Egr-1 binding sites. Finding a (5′-GAGGGGGAA) sequence ∼1.6 kb upstream of the mRNA start (50) resembling an Egr-1 binding site (5′-GCGGGGGCG), we analyzed by an electrophoretic mobility shift assay (EMSA) if recombinant Egr-1 binds to an oligonucleotide containing the putative Egr-1 recognition site from the BP-1 promoter. As shown in Fig. 6 C, labeled oligonucleotides containing a cognate Egr-1 binding site (lane 1) or the binding site from the BP-1 promoter (lane 15) produced a shifted DNA–protein complex with identical electrophoretic mobility. Their intensities were reduced only by adding an excess of unlabeled oligonucleotides with an Egr-1 binding site but not by competing with an Sp-1 binding site (lanes 2–5 and 16–19, respectively). Likewise, only the addition of Egr-1– but not of Sp-1–specific antibodies retarded the migration of the complex (lanes 6 and 7, 20 and 21). Therefore, the forced expression of Egr-1 in transgenic B cells may not only help pre-B cells to proceed from fraction B into fraction C but may also enhance the expression of BP-1, which is normally upregulated during this transition.

The nur77 gene (also called NGFI-B [3] or N10 [51]) encoding an orphan nuclear receptor represents one of the transcription factors found to be induced by Egr-1 (52). Analyzing nur77 expression in purified B220+ bone marrow cells of transgenic (ID4) and control littermates by Western blotting, we found nur77 to be expressed only by transgenic but not by control B cells (Fig. 6,B). Since upregulated nur77 expression was not found in cultivated transgenic sIg pre-B cells (data not shown), the induction of the nur77 gene seems to be confined to pre-B–II or sIgM+ bone marrow cells. To analyze if Egr-1 could directly induce nur77 transcription, we tested by EMSA the binding of recombinant Egr-1 to an oligonucleotide of the nur77 promoter (position −74 to −50) containing a cognate Egr-1 binding site. As shown in Fig. 6 C, binding of recombinant Egr-1 produced a DNA–protein complex (lane 8). The specificity of Egr-1 binding was demonstrated by a competition assay using an excess of an oligonucleotide carrying an Egr-1 binding site (lanes 11 and 12) and by the decreased mobility of the complex upon the addition of an Egr-1–specific antibody (lane 13).

Since it was shown recently that nur77 activity is involved in the induction of apoptosis during negative selection of thymocytes (5356) and in the upregulation of CD95L expression (57), we looked for enhanced CD95L expression in Egr-1 transgenic mice compared with control littermates. In contrast to the results reported for nur77– expressing T cells, we did not find increased CD95L expression in Egr-1 transgenic B cells (data not shown).

In response to BCR-derived signals, Egr-1 is thought to modulate the expression pattern of downstream genes that promote further activation and differentiation of B lymphocytes. Using variants of the B cell line WEHI-231, it was shown that Egr-1 induces the expression of CD44 and of intracellular adhesion molecule 1 (ICAM-1 [58, 59]). Therefore, we tested the expression pattern of both surface markers in our transgenic mice, but did not detect differences when compared with BALB/c controls (data not shown).

Discussion

Egr-1 Accelerates B Cell Maturation.

Mature B cells respond to signals resulting from antigen receptor engagement by immediately inducing Egr-1 transcription (5), but the role of Egr-1 in earlier stages of B cell development has not been defined. The different stages and the order of B cell development are well characterized, allowing the precise typing of bone marrow B cells according to the expression of characteristic cell surface markers, the rearrangement of Ig genes, and the proliferative and differentiation potential of B cell precursors (13, 14, 16, 60, 61). By analyzing Egr-1 expression in bone marrow–derived B lymphocyte subsets and by testing Egr-1 expression in cultivated, fetal liver–derived pre-B cells, we have shown that Egr-1 is also expressed in pre-B cells lacking sIgM as well as in immature sIgM+ B cells in the absence of sIgM-induced signals. These observations suggest that Egr-1 might also have a regulatory function in pre-B cell development. By studying transgenic mice overexpressing Egr-1 from the pre-B stage on, we have found higher proportions of mature B cells and fewer immature B cells in transgenic animals than in control littermates. To identify if early stages of B lymphopoiesis are sensitive to Egr-1 activity, we arrested B cell development at the stage of pro/pre-B–I cells by backcrossing the Egr-1 transgenic line IA7 to mice deficient in RAG-2. Since the null mutation in the RAG-2 gene prevents rearrangement of Ig genes (32), B cell precursors do not receive stimulating signals required for developmental progression beyond the stage of B220low CD43+ BP-1 pro/pre-B cells (16, 62), also defined as fraction B (13). Comparing the phenotype of bone marrow pro/pre-B cells from transgenic and control mice, we found a three- to fourfold increased population of BP-1+ pre-B cells in Egr-1 transgenic mice. Since the transcription activation function of Egr-1 seems to enhance BP-1 expression in more mature B cell subsets, the increase in BP-1+ pre-B cells could also reflect the induction of BP-1 expression only and not Egr-1–induced differentiation. However, this seems to be less likely because transgenic BP-1+ cells were found to be smaller than BP-1 cells, consistent with further maturation. Therefore, these results suggest that forced expression of Egr-1 in BP-1 pro/pre-B cells induces progression into the stage of BP-1+ pre-B cells (fraction C). Since these cells failed to upregulate the IL-2Rα chain and heat-stable antigen, two markers characteristic for pre-B–II cells (fraction C′ and D [13, 16]), overexpression of Egr-1 in pro/pre-B–I cells seems to be sufficient to induce differentiation to fraction C, but not to more mature stages of B lymphopoiesis.

Progression of pro/pre-B cells developmentally arrested by a mutation in the RAG-2 gene into more mature pre-B cell stages is also induced by in vivo cross-linking of the Ig-α/Ig-β heterodimer using Ig-β–specific mAbs (63). Under those conditions, anti–Ig-β–treated pro/pre-B–I cells become smaller in size and acquire IL-2Rα expression in addition to BP-1. Since they also downregulate c-kit (CD117) and CD43, they are considered as small pre-B–II cells. In the same report, it was shown that Ig-β cross-linking stimulates tyrosine phosphorylation of several substrate proteins, including Ig-α, Syk, and Vav, and the activation of mitogen-activated protein kinase extracellular signal– regulated kinase (ERK)1. Based on these results, Nagata et al. (63) proposed that the signal cascade initiated by Ig-β activation evokes differentiation signals similar to those delivered by the pre-BCR in normal B cell development. For mature B cells it is known that BCR engagement upregulates Egr-1 transcription through a signal cascade including p21/ras and mitogen-activated protein kinase (ERK [10, 64]), and for other cell types it has been shown that ERK activation induces Egr-1 transcription (65). Since RAG-2– deficient pro/pre-B–I cells overexpressing Egr-1 do not reach the same developmental stage as anti–Ig-β–stimulated cells, it seems likely that Egr-1 activity substitutes only part of the differentiation signal originating from the pre-BCR.

Analyzing later stages of B cell development in RAG-2+/+ Egr-1 transgenic mice, we observed lower proportions of immature and increased proportions of mature bone marrow B cells compared with their wild-type littermates, whereas there was no increased proliferation of transgenic pre-B or immature B cells detectable. These findings are consistent with the current model of the development from immature to mature B cells (66, 67). Immature B cells leave the bone marrow and enter the spleen where about half of them reach the mature stage (42). Mature bone marrow B cells are thought to be part of the recirculating pool. This would suggest that Egr-1 influences this migration at one or several steps.

Egr-1 expression was also found in CD4CD8 double negative thymocytes by Miyazaki (35). Overexpression of transgenic Egr-1 in a RAG-2–deficient background allowed thymocytes to bypass the RAG-2–dependent block at the IL-2R+ Pgp-1 double negative stage and develop into immature CD8 single-positive cells, but not further to the CD4+CD8+ double-positive cell stage. In cortical CD4+CD8+ thymocytes, Egr-1 expression was reported by Shao et al. (68) to be dependent on TCR engagement, suggesting that high level expression of Egr-1 in the thymus might be a consequence of thymocyte selection. The high coincidence of Egr-1 expression in analogous B and T cell precursor subsets and the increased differentiation of pro/pre-B–I cells and thymocytes in Egr-1 transgenic mice suggest that Egr-1 activity regulates similar functions in both types of lymphocytes.

Downstream Target Genes.

Searching for potential downstream target genes responding to Egr-1, we found increased expression of the nuclear orphan receptor nur77 in bone marrow B cells from transgenic mice but not in cultivated transgenic pro/pre-B–I cells. Since we also could demonstrate binding of recombinant Egr-1 to a cognate Egr-1 binding site present in the nur77 promoter, it seems likely that Egr-1 directly induces nur77 expression in B lymphocytes before the mature B cell stage. It was reported that nur77 activity is involved in the regulation of thymocyte apoptosis (53, 54) by inducing CD95L expression (57). However, in the Egr-1 transgenic mice, we could detect neither upregulation of CD95L expression by bone marrow B cells nor an increased frequency of apoptotic cells (Warnatz, K., unpublished results). On the other hand, cellular responses other than apoptosis may be linked to nur77 function, since it is also induced upon antigen receptor ligation in B and T cells during proliferative responses (52, 69). Besides nur77 promoter, we also found enhanced expression of BP-1 in pre-B–II and in IgM+ bone marrow cells. Similar to the nur77, we could also demonstrate binding of recombinant Egr-1 to a sequence from the BP-1 promoter resembling an Egr-1 binding site. Therefore, it seems that Egr-1 activity promotes not only development to the stage of BP-1+ cells (fraction C), but also an increased surface expression of BP-1 on BP-1+ B cells. Although it is known that BP-1 acts as an aminopeptidase catalyzing the hydrolysis of acidic amino acid residues from the NH2 termini of proteins (70), its role in B lymphopoiesis remains to be clarified (71, 72).

Conclusions.

Here we provide evidence that Egr-1 supports at least two distinct steps of B cell maturation, the progression into the pre-B and into the mature B cell stage. Since Egr-1 activity is also sufficient to promote the development of double negative thymocytes into immature single-positive CD8low cells (35), as well as macrophage in vitro differentiation (73, 74), this transcription factor seems to play an important role in the differentiation of three major hematopoietic cell types.

Acknowledgments

We are grateful to Petra Fiedler for excellent technical assistance. We thank Barbara Osborne (University of Massachusetts, Amherst, MA) for generously providing nur77-specific antibodies and probes, Toru Miyazaki (Basel Institute for Immunology) for helpful discussions and for communicating unpublished results, and Harald Illges (University of Konstanz, Germany) and Mary O'Riordan (University of California, San Francisco, CA) for critically reading the manuscript.

Abbreviations used in this paper

     
  • BCR

    B cell antigen receptor

  •  
  • BrdU

    bromodeoxyuridine

  •  
  • Egr-1

    early growth response 1

  •  
  • EMSA

    electrophoretic mobility shift assay

  •  
  • ERK

    extracellular signal–regulated kinase

  •  
  • RAG

    recombinase-activating gene

References

References
1
Sukhatme
VP
,
Kartha
S
,
Toback
FG
,
Taub
R
,
Hoover
RG
,
Tsai-Morris
CH
A novel early growth response gene rapidly induced by fibroblast, epithelial cell and lymphocyte mitogens
Oncogene Res
1987
1
343
355
[PubMed]
2
Lemaire
P
,
Revelant
O
,
Bravo
R
,
Charnay
P
Two mouse genes encoding potential transcription factors with identical DNA-binding domains are activated by growth factors in cultured cells
Proc Natl Acad Sci USA
1988
85
4691
4695
[PubMed]
3
Milbrandt
J
A nerve growth factor-induced gene encodes a possible transcriptional regulatory factor
Science
1987
238
797
799
[PubMed]
4
Kharbanda
S
,
Nakamura
T
,
Stone
R
,
Hass
R
,
Bernstein
S
,
Datta
R
,
Sukhatme
VP
,
Kufe
D
Expression of the early growth response 1 and 2 zinc finger genes during induction of monocytic differentiation
J Clin Invest
1991
88
571
577
[PubMed]
5
Seyfert
VL
,
Sukhatme
VP
,
Monroe
JG
Differential expression of a zinc finger-encoding gene in response to positive versus negative signaling through receptor immunoglobulin in murine B lymphocytes
Mol Cell Biol
1989
9
2083
2088
[PubMed]
6
Zipfel
PF
,
Irving
SG
,
Kelly
K
,
Siebenlist
U
Complexity of the primary genetic response to mitogenic activation of human T cells
Mol Cell Biol
1989
9
1041
1048
[PubMed]
7
Ouellette
AJ
,
Malt
RA
,
Sukhatme
VP
,
Bonventre
JV
Expression of two “immediate early” genes, Egr-1 and c-fos, in response to renal ischemia and during compensatory renal hypertrophy in mice
J Clin Invest
1990
85
766
771
[PubMed]
8
Watson
MA
,
Milbrandt
J
Expression of the nerve growth factor-regulated NGFI-A and NGFI-B genes in the developing rat
Development (Camb)
1990
110
173
183
[PubMed]
9
Sukhatme
VP
,
Cao
XM
,
Chang
LC
,
Tsai-Morris
CH
,
Stamenkovich
D
,
Ferreira
PC
,
Cohen
DR
,
Edwards
SA
,
Shows
TB
,
Curran
T
et al
A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization
Cell
1988
53
37
43
[PubMed]
10
McMahon
SB
,
Monroe
JG
Activation of the p21ras pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 in B lymphocytes
J Exp Med
1995
181
417
422
[PubMed]
11
Klaus
SJ
,
Phillips
NE
,
Parker
DC
Effects of IL-4 and Fc gamma receptor II engagement on Egr-1 expression during stimulation of B lymphocytes by membrane immunoglobulin crosslinking
Mol Immunol
1993
30
1553
1558
[PubMed]
12
Gottschalk
AR
,
Joseph
LJ
,
Quintans
J
Fc gamma RII cross-linking inhibits anti-Ig-induced Egr-1 and Egr-2 expression in B cells
J Immunol
1994
152
2115
2122
[PubMed]
13
Hardy
RR
,
Carmack
CE
,
Shinton
SA
,
Kemp
JD
,
Hayakawa
K
Resolution and characterization of pro-B and pre–pro-B cell stages in normal mouse bone marrow
J Exp Med
1991
173
1213
1225
[PubMed]
14
Li
YS
,
Hayakawa
K
,
Hardy
RR
The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver
J Exp Med
1993
178
951
960
[PubMed]
15
Rolink
A
,
Melchers
F
B lymphopoiesis in the mouse
Adv Immunol
1993
53
123
156
[PubMed]
16
Rolink
A
,
Grawunder
U
,
Winkler
TH
,
Karasuyama
H
,
Melchers
F
IL-2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre-B cell development
Int Immunol
1994
6
1257
1264
[PubMed]
17
Osmond
DG
,
Rolink
A
,
Melchers
F
Murine B lymphopoiesis: towards a unified model
Immunol Today
1998
19
65
68
[PubMed]
18
Henderson
A
,
Calame
K
Transcriptional regulation during B cell development
Annu Rev Immunol
1998
16
163
200
[PubMed]
19
Bain
G
,
Maandag
EC
,
Izon
DJ
,
Amsen
D
,
Kruisbeek
AM
,
Weintraub
BC
,
Krop
I
,
Schlissel
MS
,
Feeney
AJ
,
van Roon
M
et al
E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements
Cell
1994
79
885
892
[PubMed]
20
Zhuang
Y
,
Soriano
P
,
Weintraub
H
The helix-loop-helix gene E2A is required for B cell formation
Cell
1994
79
875
884
[PubMed]
21
Urbanek
P
,
Wang
ZQ
,
Fetka
I
,
Wagner
EF
,
Busslinger
M
Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP
Cell
1994
79
901
912
[PubMed]
22
Lin
H
,
Grosschedl
R
Failure of B-cell differentiation in mice lacking the transcription factor EBF
Nature
1995
376
263
267
[PubMed]
23
Kaneko
H
,
Ariyasu
T
,
Inoue
R
,
Fukao
T
,
Kasahara
K
,
Teramoto
T
,
Matsui
E
,
Hayakawa
S
,
Kondo
N
Expression of Pax5 gene in human haematopoietic cells and tissues: comparison with immunodeficient donors
Clin Exp Immunol
1998
111
339
344
[PubMed]
24
Bain
G
,
Robanus
EC
,
Maandag
,
te Riele
HP
,
Feeney
AJ
,
Sheehy
A
,
Schlissel
M
,
Shinton
SA
,
Hardy
RR
,
Murre
C
Both E12 and E47 allow commitment to the B cell lineage
Immunity
1997
6
145
154
[PubMed]
25
Nutt
SL
,
Urbanek
P
,
Rolink
A
,
Busslinger
M
Essential functions of Pax5 (BSAP) in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus
Genes Dev
1997
11
476
491
[PubMed]
26
Schilham
MW
,
Oosterwegel
MA
,
Moerer
P
,
Ya
J
,
de Boer
PA
,
van de Wetering
M
,
Verbeek
S
,
Lamers
WH
,
Kruisbeek
AM
,
Cumano
A
,
Clevers
H
Defects in cardiac outflow tract formation and pro-B-lymphocyte expansion in mice lacking Sox-4
Nature
1996
380
711
714
[PubMed]
27
Kim
U
,
Qin
XF
,
Gong
S
,
Stevens
S
,
Luo
Y
,
Nussenzweig
M
,
Roeder
RG
The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes
Nature
1996
383
542
547
[PubMed]
28
Nielsen
PJ
,
Georgiev
O
,
Lorenz
B
,
Schaffner
W
B lymphocytes are impaired in mice lacking the transcriptional co-activator Bob1/OCA-B/OBF1
Eur J Immunol
1996
26
3214
3218
[PubMed]
29
Schubart
DB
,
Rolink
A
,
Kosco-Vilbois
MH
,
Botteri
F
,
Matthias
P
B-cell-specific coactivator OBF-1/ OCA-B/Bob1 required for immune response and germinal centre formation
Nature
1996
383
538
542
[PubMed]
30
Lee
SL
,
Tourtellotte
LC
,
Wesselschmidt
RL
,
Milbrandt
J
Growth and differentiation proceeds normally in cells deficient in the immediate early gene NGFI-A
J Biol Chem
1995
270
9971
9977
[PubMed]
31
Lee
SL
,
Wang
Y
,
Milbrandt
J
Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transcription factor NGFI-A (EGR1)
Mol Cell Biol
1996
16
4566
4572
[PubMed]
32
Shinkai
Y
,
Rathbun
G
,
Lam
KP
,
Oltz
EM
,
Stewart
V
,
Mendelsohn
M
,
Charron
J
,
Datta
M
,
Young
F
,
Stall
AM
et al
RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement
Cell
1992
68
855
867
[PubMed]
33
Rolink
A
,
Karasuyama
H
,
Haasner
D
,
Grawunder
U
,
Martensson
IL
,
Kudo
A
,
Melchers
F
Two pathways of B-lymphocyte development in mouse bone marrow and the roles of surrogate L chain in this development
Immunol Rev
1994
137
185
201
[PubMed]
34
Chen
J
,
Ma
A
,
Young
F
,
Alt
FW
IL-2 receptor alpha chain expression during early B lymphocyte differentiation
Int Immunol
1994
6
1265
1268
[PubMed]
35
Miyazaki
T
Two distinct steps during thymocyte maturation from CD4−CD8− to CD4+CD8+distinguished in the early growth response (Egr)-1 transgenic mice with a recombinase-activating gene–deficient background
J Exp Med
1997
186
877
885
[PubMed]
36
Rolink
A
,
Kudo
A
,
Karasuyama
H
,
Kikuchi
Y
,
Melchers
F
Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo
EMBO (Eur Mol Biol Organ) J
1991
10
327
336
[PubMed]
37
Chomczynski
P
,
Sacchi
N
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal Biochem
1987
162
156
159
[PubMed]
38
Sambrook, J., E.J. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
39
Coffman
RL
,
Weissman
IL
B220, a B cell specific member of the T200 glycoprotein family
Nature
1981
289
681
683
[PubMed]
40
Brombacher
F
,
Köhler
G
,
Eibel
H
B cell tolerance in mice transgenic for anti-CD8 immunoglobulin μ chain
J Exp Med
1991
174
1335
1346
[PubMed]
41
Schüppel
R
,
Wilke
E
,
Weiler
E
Monoclonal anti-allotype antibody towards BALB/c IgM. Analysis of specificity and site specific crossover in recombinant strain BALB-IghVa/Igh-Cb
Eur J Immunol
1987
17
553
561
42
Rolink
A
,
Andersson
J
,
Melchers
F
Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity
Eur J Immunol
1998
28
3738
3748
[PubMed]
43
Schittek
B
,
Rajewsky
K
,
Forster
I
Dividing cells in bone marrow and spleen incorporate bromodeoxyuridine with high efficiency
Eur J Immunol
1991
21
235
238
[PubMed]
44
Coligan, J.E., A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober. 1992. Current Protocols in Immunology. Vols. 1–3. John Wiley & Sons, Inc., New York.
45
Dinkel
A
,
Aicher
WK
,
Haas
C
,
Zipfel
PF
,
Peter
HH
,
Eibel
H
Transcription factor Egr-1 activity down-regulates Fas and CD23 expression in B cells
J Immunol
1997
159
2678
2684
[PubMed]
46
Seyfert
VL
,
McMahon
SB
,
Glenn
WD
,
Yellen
AJ
,
Sukhatme
VP
,
Cao
XM
,
Monroe
JG
Methylation of an immediate-early inducible gene as a mechanism for B cell tolerance induction
Science
1990
250
797
800
[PubMed]
47
Yellen
AJ
,
Glenn
W
,
Sukhatme
VP
,
Cao
XM
,
Monroe
JG
Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice
J Immunol
1991
146
1446
1454
[PubMed]
48
Rolink
A
,
Melchers
F
Generation and regeneration of cells of the B-lymphocyte lineage
Curr Opin Immunol
1993
5
207
217
[PubMed]
49
Förster
I
,
Rajewsky
K
The bulk of the peripheral B-cell pool in mice is stable and not rapidly renewed from the bone marrow
Proc Natl Acad Sci USA
1990
87
4781
4784
[PubMed]
50
Wang
J
,
Walker
H
,
Lin
Q
,
Jenkins
N
,
Copeland
NG
,
Watanabe
T
,
Burrows
PD
,
Cooper
MD
The mouse BP-1 gene: structure, chromosomal localization, and regulation of expression by type I interferons and interleukin-7
Genomics
1996
33
167
176
[PubMed]
51
Ryseck
RP
,
Macdonald-Bravo
H
,
Mattei
MG
,
Ruppert
S
,
Bravo
R
Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor
EMBO (Eur Mol Biol Organ) J
1989
8
3327
3335
[PubMed]
52
Mittelstadt
PR
,
DeFranco
AL
Induction of early response genes by cross-linking membrane Ig on B lymphocytes
J Immunol
1993
150
4822
4832
[PubMed]
53
Liu
ZG
,
Smith
SW
,
McLaughlin
KA
,
Schwartz
LM
,
Osborne
BA
Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77
Nature
1994
367
281
284
[PubMed]
54
Woronicz
JD
,
Calnan
B
,
Ngo
V
,
Winoto
A
Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas
Nature
1994
367
277
281
[PubMed]
55
Zhou
T
,
Cheng
J
,
Yang
P
,
Wang
Z
,
Liu
C
,
Su
X
,
Bluethmann
H
,
Mountz
JD
Inhibition of Nur77/ Nurr1 leads to inefficient clonal deletion of self-reactive T cells
J Exp Med
1996
183
1879
1892
[PubMed]
56
Calnan
BJ
,
Szychowski
S
,
Chan
FK
,
Cado
D
,
Winoto
A
A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection
Immunity
1995
3
273
282
[PubMed]
57
Weih
F
,
Ryseck
RP
,
Chen
L
,
Bravo
R
Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/ Fas ligand pathway
Proc Natl Acad Sci USA
1996
93
5533
5538
[PubMed]
58
Maltzman
JS
,
Carman
JA
,
Monroe
JG
Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes
Mol Cell Biol
1996
16
2283
2294
[PubMed]
59
Maltzman
JS
,
Carmen
JA
,
Monroe
JG
Transcriptional regulation of the ICAM-1 gene in antigen receptor– and phorbol ester–stimulated B lymphocytes: role for transcription factor EGR1
J Exp Med
1996
183
1747
1759
[PubMed]
60
Osmond
DG
B cell development in the bone marrow
Semin Immunol
1990
3
173
180
[PubMed]
61
Lu
L
,
Smithson
G
,
Kincade
PW
,
Osmond
DG
Two models of murine B lymphopoiesis: a correlation
Eur J Immunol
1998
28
1755
1761
[PubMed]
62
Young
F
,
Ardman
B
,
Shinkai
Y
,
Lansford
R
,
Blackwell
TK
,
Mendelsohn
M
,
Rolink
A
,
Melchers
F
,
Alt
FW
Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation
Genes Dev
1994
8
1043
1057
[PubMed]
63
Nagata
K
,
Nakamura
T
,
Kitamura
F
,
Kuramochi
S
,
Taki
S
,
Campbell
KS
,
Karasuyama
H
The Igα/ Igβ heterodimer on μ-negative proB cells is competent for transducing signals to induce early B cell differentiation
Immunity
1997
7
559
570
[PubMed]
64
McMahon
SB
,
Monroe
JG
The role of early growth response gene 1 (egr-1) in regulation of the immune response
J Leukocyte Biol
1996
60
159
166
[PubMed]
65
Cohen
DM
Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation
Proc Natl Acad Sci USA
1996
93
11242
11247
[PubMed]
66
Lortan
JE
,
Roobottom
CA
,
Oldfield
S
,
MacLennan
IC
Newly produced virgin B cells migrate to secondary lymphoid organs but their capacity to enter follicles is restricted
Eur J Immunol
1987
17
1311
1316
[PubMed]
67
Allman
DM
,
Ferguson
SE
,
Lentz
VM
,
Cancro
MP
Peripheral B cell maturation. II. Heat-stable antigenhisplenic B cells are an immature developmental intermediate in the production of long lived marrow-derived B cells
J Immunol
1993
151
4431
4444
[PubMed]
68
Shao
H
,
Kono
DH
,
Chen
LY
,
Rubin
EM
,
Kaye
J
Induction of the early growth response (Egr) family of transcription factors during thymic selection
J Exp Med
1997
185
731
744
[PubMed]
69
Garcia
I
,
Pipaon
C
,
Alemany
S
,
Perez-Castillo
A
Induction of NGFI-B gene expression during T cell activation. Role of protein phosphatases
J Immunol
1994
153
3417
3425
[PubMed]
70
Wu
Q
,
Lahti
J
,
Air
G
,
Burrows
P
,
Cooper
M
Molecular cloning of the murine BP-1/6C3 antigen: a member of the zinc-dependent metallopeptidase family
Proc Natl Acad Sci USA
1990
87
993
997
[PubMed]
71
Lin
Q
,
Taniuchi
I
,
Kitamura
D
,
Wang
J
,
Kearney
JF
,
Watanabe
T
,
Cooper
MD
T and B cell development in BP-1/6C3/aminopeptidase A-deficient mice
J Immunol
1998
160
4681
4687
[PubMed]
72
Wang
J
,
Lin
Q
,
Wu
Q
,
Cooper
MD
The enigmatic role of glutamyl aminopeptidase (BP-1/6C3 antigen) in immune system development
Immunol Rev
1998
161
71
77
[PubMed]
73
Nguyen
HQ
,
Hoffman-Liebermann
B
,
Liebermann
DA
The zinc finger transcription factor Egr-1 is essential for and restricts differentiation along the macrophage lineage
Cell
1993
72
197
209
[PubMed]
74
Krishnaraju
K
,
Nguyen
HQ
,
Liebermann
DA
,
Hoffman
B
The zinc finger transcription factor Egr-1 potentiates macrophage differentiation of hematopoietic cells
Mol Cell Biol
1995
15
5499
5507
[PubMed]

Part of this work was supported through Deutsche Forschungsgemeinschaft grants Ei235/3-1 and Ei235/4-1 (to H. Eibel).

Author notes

Address correspondence to Hermann Eibel, Clinical Research Unit for Rheumatology, Division of Rheumatology and Clinical Immunology, University Hospital Freiburg, Breisacher Str. 64, D-79106 Freiburg, Germany. Phone: 49-761-270-5294; Fax: 49-761-270-5298; E-mail: eibel@nz11.ukl.uni-freiburg.de