Early growth response genes regulate B cell development, proliferation and immune response

Abstract

SUMMARY

Egr-1 (Early growth response gene-1) is an immediate early gene encoding a zinc finger motif containing transcription factor. Upon cross-linking of B cell receptor (BCR), mature B cells undergo proliferation with an increase in Egr-1. Immature B lymphoma cells that express Egr-1 constitutively are growth inhibited when Egr-1 is downregulated by negative signals from BCR or by antisense oligonucelotides. To test the hypothesis that Egr-1 is important for B-cell development, we examined B cells from primary and secondary lymphoid organs in Egr-1-/- mice. Marginal zone B cell development was arrested in these mice while the B cells in all other compartments were increased. To test the hypothesis that Egr-1 function may be partially compensated by other Egr family members, we developed transgenic mice expressing a dominant negative form of Egr-1, which lacks the transactivation domain but retains the DNA binding domain, in a B cell specific manner. There was a decrease in B lymphopioesis in the bone marrow accompanied by a reduction in splenic immature and mature B cells as well as marginal zone B cells in the transgenic mice. Moreover, transgenic mice respond poorly to BCR cross-linking in vitro and T-independent and T-dependent antigens in vivo.

INTRODUCTION

In response to infection, B and T cells undergo rapid proliferation and subsequent differentiation leading to the generation of effector cells that successfully combat the infection. Many cell types including lymphocytes express specific class of genes called immediate early genes in response to stimulation via antigen receptor, growth factor and cytokines (1). Immediate early genes are rapidly induced (within minutes) and their induction does not require new protein synthesis. Genes included within this category are c-fos and Egr-1. The Egr-1 gene encoded protein Egr-1, a nuclear zinc finger containing transcription factor, binds to the consensus DNA motif GCGG/TGGGCG and modulates the transcriptional activity of several target genes including IL-2, CD44, ICAM-1 and TNF-alpha in B lymphocytes (1). Target genes for EGR-1 identified in other cell types include c-Myc, Cyclin D2, p19 and Cyclin G2 (2-4).

Egr-1 has both anti-apoptotic and pro-apoptotic roles in different cell types depending on the nature of the stimulus. Egr-1 can up- or down-regulate transcription of p300 and CBP based on the nature of its post-translational modification (5). Acetylation of Egr-1 by CBP and p300 upon serum stimulation increases its stability and promotes cell survival, while phosphorylation of Egr-1 following UV radiation represses p300/CBP transcription and favors cell death (5). Egr-1 has been shown to be important for prostate tumorigenesis, as knockout mice have delayed tumorigenesis when crossed to TRAMP mice, which spontaneously develop prostate invasive neoplasia (PIN) (6).

Egr-1 is expressed ubiquitously by many cell types whereas Egr-2 and Egr-3 are restricted in their expression (7). Even though Egr family members share redundancy in their functions as evident by 90% homology in their DNA binding region, they do have unique functions as revealed by specific phenotypes in knockout mice. Egr-1^-/- mice are characterized by female infertility, Egr2^-/- mice have hindbrain abnormalities, Egr-3^-/- mice have motor neuron disorders, and Egr-4^-/- mice have male infertility (8). Mice homozygous for Egr-2 deficiency die during the first two weeks after birth due to brain abnormalities (9).

The importance of EGR-1 in T cell biology has been studied quite extensively. Egr-1 is expressed in thymocytes and peripheral T cells, and its expression is rapidly induced upon TCR engagement in an ERK-dependent manner (10). Egr-1-deficient mice have defects in positive selection beyond the β-selection checkpoint, resulting in a reduced percentage of CD4 and CD8 single positive mature cells in the thymus (11). On TCR transgenic backgrounds, Egr-1-deficient mice express reduced numbers of naive T cells. Egr-1 overexpression in thymus under lck promoter allowed positive selection of thymocytes, possibly by lowering the threshold of avidity required for positive selection in the thymus (12). Although Egr-1-deficient animals have a low percentage of mature thymocytes, the absolute number of mature thymocytes is only slightly reduced due to an increase in thymus size. Recently, Frederick and colleagues demonstrated that Egr-1 is required for survival of mature thymocytes and newly emigrated thymocytes (13). All four family members are induced upon T cell receptor ligation. Overexpression of Egr-2 and Egr-3 is associated with an increase in the E3 ubiquitin ligase Cbl-b and inhibition of T cell activation. Also, T cells from Egr-3^-/- mice display lower Cbl-b and are resistant to in vivo peptide-induced tolerance (14). These data support the idea that Egr-2 and Egr-3 are involved in promoting TCR-induced negative signaling. The role of Egr-1 in macrophage differentiation has also been studied in detail. Using a variety of differentiation-inducible myeloid cell lines, Krishnaraju and colleagues showed that the ectopic Egr-1 expression in normal hematopoietic progenitors stimulates development along the macrophage lineage at the expense of development along the granulocyte or erythroid lineages, regardless of the cytokine used (15-17). These observations are in contrast to the phenotype observed in Egr-1^-/- mice where differentiation along the macrophage lineage remains normal (18).

In B lymphocytes, cross-linking of the antigen receptor results in egr-1 expression through activation of the p21 ras/MAPK pathway (1, 19). Detailed analysis of the Egr-1 promoter showed that two most distal serum response elements mediate Egr-1 gene induction (20). Egr-1 binding sites are found within the promoter regions of the genes that encode ICAM-1 and CD44, two cell adhesion molecules important for cell trafficking (20-22). ICAM-1 and CD44 are upregulated in B cells upon receptor cross-linking. Though the Egr-1 promoter has been studied extensively, its role in B cell development and function is not well understood. The role of Egr-1 in pre-B cell development has been demonstrated previously and Egr-1 was shown to be important for BP-1 expression in the bone marrow pre-B cells (23). Differential expression of Egr-1 in anergic and naïve B cells has also been studied previously with differing conclusions, with an increase in Egr-1 in anergic B cells in one of the studies and no such increase in another (24, 25). It was shown that Egr-1 gene is methylated and not induced upon BCR ligation of an immature B cell line WEHI-231 (26). This suggests that inactivation of Egr-1 gene might be important for tolerance induction in B cells. In our laboratory, we find that when mature B cells are cross-linked, they undergo proliferation with an increase in Egr-1 expression whereas neonatal B cells, which are immature, become unresponsive with only a modest increase in Egr-1 (19). This differential expression pattern of Egr-1 during different stages of B cell development could represent an important comparison during induction of tolerance versus clonal expansion in B cells. Accordingly, microarray analysis of naïve and tolerant B cells showed a difference in Egr-1 expression subsequent to BCR cross-linking, followed by a rapid decrease in Egr-1 (27). Moreover, BKS-2 B lymphoma cells, that have an immature B cell phenotype and express Egr-1 constitutively, undergo growth arrest when treated with antisense oligos specific for Egr-1 (28). Microarray studies demonstrate a 100-fold decrease in Egr-1 mRNA when surface BCR is deleted in immature B cells derived from the bone marrow of Cre-LoxP conditional transgenic mice in which Cre activation leads to heavy chain deletion (29). These data show that Egr-1 is important for B cell survival and that Egr-1 induction downstream of BCR might be important for B cell development. Despite these studies, the in vivo role of Egr-1 during B cell-development and functional responses remains to be elucidated.

Although Egr-1^-/- mice have been generated, no significant effect of Egr-1 deficiency on B cell development has been reported (30). This could be due to the redundancy of Egr-1 function as this family has three other members with similar transcriptional activation properties (31, 32). Previous studies of Egr-1^-/- mice did not examine the effect of Egr-1 deficiency on specific stages of B-cell development. Hence, we performed a detailed analysis of B cell development in Egr-1^-/- mice. In addition, we generated transgenic mice in which the function of all Egr-1 family members is inhibited by a dominant negative Egr-1 construct in a B-cell specific manner. We show that Egr family members are important for B lymphopoiesis and proliferation, and that follicular B cells lacking this transcription factor are defective in proliferative response to antigen receptor stimulation. Egr-1 in particular, is essential for marginal zone B cell development. We also demonstrate that Egr family members are critical for immune responses in vivo.

MATERIALS AND METHODS

Mice

Egr-1^-/- mice were generated and backcrossed to BL/6 as described earlier and were a kind gift from Dr. Jeffrey Milbrandt (30). Dominant-negative Egr-1 (ΔEgr-1) construct was generated as described previously (20, 33). A FLAG tag was added to this construct at the C-terminus and was then cloned into a plasmid containing the immunoglobulin heavy chain promoter and μ enhancer (obtained from Dr. N. Muthusamy, Ohio State University, Columbus) so that the transgene will be expressed only in B cells (34). Dominant-negative Egr-1 (DN-Egr-1) transgenic mice were generated by pronuclear microinjection by the University of Kentucky Transgenic Facility. We generated four founders in the (C3HxBL/6)F1 background. One line has been backcrossed for 9 generations onto C57BL/6 background.

Reagents and cell lines

Antibodies to Egr-1 (C-19), c-Myc and cyclin D2 were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA) and antibodies to Egr-2 were purchased from Covance Research Products (Berkeley, CA). Monoclonal anti-β-actin Ab was obtained from Sigma (St. Louis, MO). The immature B lymphoma cell line BKS-2 was isolated and maintained in vivo as a splenic tumor in our laboratory (35). Female CBA/N (Xid) mice were obtained from the Jackson Lab (Bar Harbor, ME). Mice were housed in microisolator cages in our American Association for Laboratory Animal Accreditation and Certification-approved rodent facility. BKS-2 B lymphoma cells obtained from the spleens of CBA/N mice were depleted of T cells with a cocktail of anti-T cell antibodies and complement as described (35). Normal splenic B cells were prepared according to procedures described previously (36). The characteristics of the monoclonal rat anti-mouse μ chain Ab, AK11, were described previously (37, 38).

Cell preparation and staining

Suspensions of BM cells were flushed from tibias and femurs and splenocytes were prepared through crushing of spleens in Hank’s Balanced Salt Solution (HBSS). Cells were washed and then incubated with optimal dilutions of the indicated antibodies in polystyrene round-bottom tubes in a final volume of 100 μl. After 30 min on ice, cells were washed twice with FACS buffer and then, when appropriate, cells were incubated for 20 min on ice before two final washes with fluorochrome-conjugated streptavidin to reveal staining by biotinylated antibodies.

Flow cytometric analyses

PE-Cy5 (Cy) anti-CD45R/B220 (RA3-6B2), PE anti-BP-1, FITC anti-CD43, FITC anti-IgM, PE anti-IgM, PE anti-CD23, FITC anti-CD21, FITC anti-CD5, Biotin-anti-CD24/HSA (30F1) and anti-sIgD antibodies were obtained from BD Pharmingen (San Jose, CA) and allophycocyanin-AA4.1 (AA4.1.1) was obtained from eBiosciences (San Diego, CA). Analyses were conducted on a dual laser flow cytometer (FACSCaliber; BD Immunocytometry Systems, San Jose, CA) or a MoFlo cell sorter (DakoCytomation, Fort Collins, CO). All flow cytometry data were analyzed by CELL QUEST software.

Retroviral production and transduction of B lymphoma cells

Egr-2 and Wilm’s tumor Egr-1 (WT-Egr) constructs cloned into the retroviral vector LZRSpBMN-linker-internal ribosomal entry site (IRES)-enhanced green fluorescence protein (eGFP; LZRS) encompassing an IRES were described previously (32). Retroviral vectors were transiently transfected into Phoenix packaging cells using the Lipofectamine transfection system (InVitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Transfection efficiency was assessed by determining the percentage of Phoenix packaging cells expressing eGFP by FACS analysis. Virus-containing supernatants were harvested from transfected Phoenix cells and pretreated with 5 μg/ml of Polybrene (Sigma, St. Louis, MO). BKS-2 B lymphoma cells were washed in serum-free opti-MEM and single-cell suspensions were incubated at a concentration of 1× 10⁶ cells/ml/well of a 6 well plate for 2 h at 30°C in 2 ml of viral supernatants and spin infection of the plates was performed. At the end of the 2-h infection period, virus supernatant was discarded and fresh IF-12 medium was added to the cells, which were cultured for 48 hours in a 5% CO₂-humidified incubator. After 2 days, cells were sorted for GFP using a Mo-Flo cell sorter and sorted cells were plated and proliferation measured 48 hours later as described below.

Proliferation Assay

T-depleted B cells from the Egr-1^-/- mice, DN-Egr transgenic mice and appropriate littermate controls were incubated at 2 × 10⁵ cells/well in triplicate in 96-well flat-bottom plates in medium consisting of IMDM and Ham’s F12 supplemented with 10 mM glutamine, 10 mM HEPES, 0.5 mg/ml gentamicin, and 5 × 10^-5 2-ME. Stimuli added included F(ab’)₂ goat anti-IgM (μ-chain specific; ICN Pharmaceuticals, Cappel, ICN Pharmaceuticals, Aurora, OH), LPS (Sigma, St. Louis, MO) or anti-CD40 (clone 1C10). After 44 h, cultures were pulsed with 1 μCi of [³H]thymidine and harvested 4 h later for scintillation counting. BKS-2 cells were cultured in IF-12 medium [1:1 mixture of Iscove’s Modified Dulbecco’s Medium (IMDM) and Ham’s F12] + 10% Fetal Calf Serum (FCS) (Atlanta Biologicals, Norcross, GA). To measure proliferation, 2 × 10⁴ cells were cultured in 200 μL of medium. The cells were harvested onto filter mats using a cell harvester (Packard, Meriden, CT). The levels of radionucleotide incorporation were measured with a Matrix 96 ß-radiation counter (Packard, Downers Grove, IL). Results are presented as arithmetic mean of triplicate cultures ± SE and statistical significance of different treatments is evaluated by the Student’s t-test. Percentage of control response was defined as (cpm in the treated group/cpm in the untreated group) × 100.

Real-time PCR

Total RNA was isolated from B cells with the Tri-reagent (Sigma, St. Louis, MO). 2 μg of total RNA was subsequently used to make cDNA using the Superscript II reverse transcriptase (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s protocol. RT-PCR was performed on an ABI Prism 7000 machine using Taqman based Egr-1, Egr-2 and Egr-3 specific primers and probe (Applied Biosystems, Foster City, CA). The GAPDH specific primers and probe were used for loading control (Applied Biosystems, Foster City, CA).

Western blotting

T-depleted B cells from the Egr-1-knockout, DN-Egr transgenic and littermates were rested for 3 hours in SIP (Serum, Insulin, Progesterone) free IF-12 medium and then were stimulated with anti-IgM (25 μg/ml) or PMA (10 mg/ml) for 1 hour. Cell lysates were prepared in 1× SDS sample buffer or 1% Triton-X 100 as described earlier and were subjected to SDS-PAGE and Western blot analysis (36). Western blots were analyzed by probing the membrane using various primary antibodies (Egr-1 and Egr-2) followed by HRP-conjugated secondary antibodies (Santa Cruz). The blots were developed with Pico Chemiluminescence substrate (Pierce Biotechnology, Rockford, IL) and exposed to Kodak X-O mat films which were scanned with a flat-bed scanner (UMAX Technologies, Hsinchu, Taiwan). Alternatively, the blots were scanned by a Kodak Image Station 2000RT (Eastman Kodak, New Haven, CT). For reprobing, membranes were stripped using a solution containing 62.5 mM Tris-HCl, 2% SDS, and 100 mM ß-mercaptoethanol at 65°C for 20 min. The relative integrated OD of the protein bands was estimated using Scion Image software (Scion Corporation) or Kodak Image Station software.

In vivo immunizations and PFC assays

Mice (both DN-Egr transgenic and littermate controls) were immunized intraperitoneally with 10 μg of TNP-Ficoll or 10% v/v of SRBC in 300 μl of 1× PBS. The number of IgM anti-TNP-secreting cells was determined 5 days after immunization using a glass slide version of the technique of localized hemolysis in a gel as described (39). Briefly, a 1-ml packed cell volume of SRBC (Colorado Serum, Denver, CO) was coupled with 2,4,6-trinitrobenzene sulfonic acid (Eastman Kodak, Rochester, NY) following published protocols. The splenocytes were washed with HBSS and then mixed with 50 μl of 13.5% (v/v) TNP-coupled SRBC, 200 μl of 2× basal Eagle’s medium (Life Technologies), and 200 μl of 1.6% agarose (FMC Bioproducts, Rockland, ME) and poured onto a glass microscope slide (Goldseal, Portsmouth, NH). The slides were incubated for 1 h at 37°C, and the plaques were developed during an additional 1-h incubation at 37°C with guinea pig complement (Pel-Freez Biologics, Rogers, AR). The plaques representing antibody forming cells (AFC) were viewed under a low power microscope. Ag-specific AFC were calculated by taking the number of plaques for the antigen immunized and subtracting the number of plaques obtained in the vehicle immunized mice. Results are provided as the arithmetic mean ± SE, and the statistical significance of different treatments is evaluated by Student’s t test.

ELISA

Serum Ig levels were measured by mouse immunoglobulin isotyping kit (BD Biosciences, Carlsbad, CA).

RESULTS

Increase in Immature B cells in the bone marrow and spleen of Egr-1^-/- mice

The number of B220⁺ cells in the bone marrow was increased in Egr-1^-/- mice (Table 1). Analysis of B cell development according to the Hardy protocol showed a significant increase in the number of pre-pro B cells, pro-B cells, pre-B cells (B220⁺/CD43⁺/IgM^-) and immature B cells, transitional and mature re-circulating B cells (B220⁺/CD43^-/IgM⁺) in the bone marrow of mice lacking Egr-1 (Figure 1A and Table 1) (40). Further subfractionation of pro-B cells using the BP-1 and HSA markers into A (Pre-proB), B (Pro-B), C (Pre-BI), and C’(Pre-BII) populations revealed a decrease in Fraction C’ in Egr-1^-/- mice compared to wildtype mice (Figure 1B). Typical FACS profiles are shown in various panels of Figure 1 and summary data based on several mice is shown in Table 1. The reduction in Fraction C’ is likely due to a decrease in high BP-1 expression, a marker for this fraction, which in turn is likely due to a direct role of Egr-1 in enhancing BP-1 expression (23). Decreased BP-1 expression does not appear to affect B cell maturation as we observed an increase in Fractions D and E, which are thought to be derived from Fraction C’ (Figure 1C). There appeared to be a decrease in Fraction F (recirculating follicular B cells) in the Egr-1^-/- mice. Currently, it is not clear whether this is due to homing and/or retention defect in the bone marrow. A homing defect is consistent with the finding that Egr-1 regulates ICAM-1 and CD44, adhesion molecules involved in trafficking (20, 21). Thus, in the Egr-1^-/- mice, there is an overall increase in Pre-Pro B (Fraction A), Pro B (Fraction B), Pre-BI (Fraction C), and immature (Fraction E) B cells in the bone marrow suggesting that Egr-1 regulates B lymphopoiesis at a very early stage possibly the Common Lymphoid Progenitors (CLPs).

Table 1.

Immature B cell accumulation in the bone marrow and spleen of Egr-1^-/- mice and a decrease in marginal zone but not follicular B cells in the spleen of Egr-1^-/- mice

	Bone marrow			Spleen
	B220+ (Pre-pro, Pro, Pre, Immature, transitional and mature recirculating ) B cells	B220+ IgM- (Pre-pro, Pro and Pre) B cells	B220+ IgM+ (Immature, transitional and mature recirculating) B cells	B220+ AA4+ Transitional B cells	B220+ AA4- Follicular and marginal zone B cells	CD23- HSA+ CD21+ Marginal zone B cells
× 106 cells/organ
Wild type (+/+)	1.8± 0.4 (15%±2%)	0.16 ± 0.2 (10%±3%)	1.64 ± 0.6 (5%±2%)	5.1 ± 0.8 (4%±2%)	25± 5 (28%±4%)	4 ± 0.3 (16%±2%)
Egr-1-/-	3.0± 0.3 (34%±4%)	0.72 ± 0.2 (29%±2%)	2.28 ± 0.3 (9%±2%)	12.5 ± 0.3 (14%±2%)	26± 3 (30%±3%)	1 ± 0.1 (4%±1%)
p value	< 0.05	< 0.05	< 0.05	< 0.05	NS	< 0.05

Absolute numbers of the indicated B-cell populations are obtained by multiplying the frequencies of cells by the number of cells harvested from two tibia and two femurs or spleen from each mouse. Various B cell precursor members were estimated by FACS as shown in Fig.1. Values represent mean ± SE of B cell numbers from 6 mice each in all cases except for MZ B cells where 4 mice each of Egr-1^+/+ and Egr-1^-/- were used. Numbers in parentheses represent % of cells with indicated phenotype in each tissue. *Values of P were calculated for absolute numbers using Student’s t test using data from littermate mice as the control population.

Figure 1. Altered B cell subsets in the bone marrow and spleen of Egr-1^-/- mice.

Flow cytometric analysis of bone marrow cells obtained from Egr-1^-/- and littermate controls stained with Cychrome anti-B220 and PE anti-IgM. B220+ IgM- cells represent Pre-pro B, pro-B and Pre-B cells. B220+ IgM+ cells represent immature B, transitional and recirculating mature B cells (panel A). Bone marrow cells were stained with Cy anti-B220, FITC anti-CD43, PE anti-BP-1 and APC anti-HSA for Fractions A to C’ (panel B). B220+ CD43+ HSA- BP-1- represent Fraction A, B220+ CD43+ HSAhi BP-1lo represent Fraction B, B220+ CD43+ HSAint BP-1int represent Fraction C, B220+ CD43+ HSAhi BP-1hi represent Fraction C’. Bone marrow cells were stained for Cy anti-B220, FITC anti-CD43, PE anti-IgM and APC anti-IgD for Fractions D to F (panel C). B220+ CD43- IgM- IgD- represent Fraction D, B220+ CD43- IgM+ IgD- represent Fraction E and B220+ CD43- IgM+ IgD+ represent Fraction F. Percentages indicate relative values of each subset compared to other subsets in the same histogram. (D) Resolution of immature transitional B cell subsets and the follicular B cells in the spleen of 10 week old Egr-1^-/- and littermate controls by staining splenocytes with FITC anti-IgM, Cy-anti-B220, allophycocyanin-anti-AA4.1.1, and PE-anti-CD23 and analyzed on a FACS Calibur. B220+ AA4+ cells represent immature transitional B, B220+ AA4- cells represent mature follicular B. 100,000 events were analyzed and data are representative of the average of six mice in each group. (E) Flow cytometric analysis of marginal zone B cells in the spleen of Egr-1-/- and the littermate control by staining with FITC anti-CD21, Cy-anti-B220, allophycocyanin-anti-HSA, and PE-anti-CD23. Marginal zone B cells are HSA+ CD21+ gated on CD23^- splenocytes. (F) Peritoneal B cell subsets were identified by staining with Cy-anti-B220 and PE-anti-CD23 and analyzed by flow cytometry. Peritoneal B cells represent B220+ CD23- cells. Data are representative of three independent experiments.

The increased output of immature B cells in the bone marrow was reflected in the spleen of Egr-1^-/- mice, which showed a three fold increase in the frequency and two fold increases in the absolute numbers of B220⁺/AA4.1⁺ immature transitional B cells (Figure 1D and Table 1). Further analysis of B220⁺/AA4.1⁺ cells for IgM and CD23 expression revealed an increase in all three transitional B cell populations (IgM⁺/CD23^- for T1, IgM⁺/CD23⁺ for T2 and IgM^-/CD23⁺ for T3 cells) (data not shown). Interestingly, the mature follicular B cell population as identified by B220⁺/AA4.1^- markers remained unaffected despite an increase in transitional B cells. Marginal zone (MZ) B cells are thought to be the main cells in T independent immune responses. We found a significant decrease (three to four fold) in MZ B cell population (B220⁺/CD23^-/HSA⁺/ CD21⁺ fraction) in the spleen of Egr-1^-/- mice (Figure 1E and Table 1). Alternatively, we looked at the MZ B cell population based on CD21 and CD23 staining and found that CD21⁺ CD23^- MZ B cells were reduced in the Egr-1^-/- mice compared to the wildtype mice. There was a significant decrease in B-1 cells in the peritoneum of the knockout mice based on their B220⁺/CD23^- phenotype (Figure 1F).

Hyperproliferation of B cells from mice which lack Egr-1

Since Egr-1 is rapidly induced upon antigen receptor stimulation and Egr-1 was shown to promote cell proliferation in other cell types (41, 42), we determined if Egr-1 is critical for BCR induced B cell proliferation. B cells from both Egr-1^-/- mice and littermate control mice were stimulated with anti-IgM for 48 hours and proliferation was measured by thymidine incorporation. There was a two fold increase in the proliferative response of Egr-1^-/- splenic B cells to anti-IgM at three different doses (5, 10 and 25 μg/ml) (Figure 2A). The increased BCR response in Egr-1^-/- mice might be due to compensation by other family members like Egr-2 (which was also shown to be upregulated during BCR cross-linking in normal B cells) and Egr-3 (27, 43). The other possibility is that Egr-1 is a negative regulator of B cell activation and the response is higher upon deletion. The former hypothesis is supported by our observations that Egr-2 is expressed by the Egr-1^-/- B cells and a lower mobility band appears in anti-IgM or PMA activated cells in the Egr-1^-/- but not the littermate wild type mice (Figure 2B). This slow moving band appeared to be due to phosphorylation, since it became undetectable when Egr-1^-/- B cells were stimulated with anti-IgM in the presence of ERK (PD98059) or JNK (SP600125) inhibitor. Unlike the Egr-1 knockouts, Egr-2 is not modified in the littermate controls. In addition, the Egr-2 and Egr-3 genes were also upregulated in Egr-1^-/- B cells and wild type B cells upon BCR cross-linking (Figure 2B-D).

Figure 2. Proliferative response of Egr-1^-/- B cells and the critical role for Egr-2 in B cell proliferation.

(A) T-depleted B cells from wild type and the Egr-1^-/- mice were cultured for 48 hours with medium alone or with indicated concentrations of anti-IgM or anti-CD40 and proliferation measured as described in Materials and Methods. (B) B cells from wild type and Egr-1^-/- mice were stimulated with anti-IgM or PMA for 1 hour and cell lysates were prepared (top panel). Bottom panel is the same as the top panel except for the addition of PD98059 (ERK inhibitor) and SP600125 (JNK inhibitor) in the presence of BCR cross-linking. Cell lysates were analyzed by immunoblotting with an antibody to Egr-2 which were then stripped and probed for β-actin. Results are representative of two experiments. (C and D), T-depleted Egr-1-/- and wildtype B cells were stimulated for 1 h with 25 μg/ml anti-IgM or PMA (10 ng/ml) and RNA was extracted. The cDNA was transcribed from the extracted RNA. The levels of cDNA for Egr-2 (panel C) and Egr-3 (panel D) were determined by Real time PCR. Results represent duplicate determinations of cDNA from two different mice in each group. (E) Sorted BKS-2 B lymphoma cells, infected with control (LZRS) or Egr-2 expressing retrovirus in the presence or absence of indicated concentration of anti-IgM (AK11), were cultured for 48 hours and proliferation measured as described in Materials and Methods. Data are representative of three independent experiments.

To test the compensatory role of Egr-2, we utilized the BKS-2 B lymphoma model in which lymphoma growth is inhibited by BCR cross-linking with an accompanying decrease in Egr-1 (19, 28). Ectopic expression of Egr-2 using a retroviral vector rescued immature BKS-2 B lymphoma cells from BCR induced growth inhibition (Figure 2E). On the other hand, the CD40 response remained comparable between the knockout and the littermates suggesting that Egr-1 may not function downstream of CD40 signaling pathways.

Generation of DN-Egr transgenic mice

To understand further the role of Egr-1 in B cells, we generated transgenic mice expressing a dominant negative form of EGR-1 in a B-cell restricted manner using V_H promoter and the Eμ enhancer. The DN-Egr construct contained the DNA binding domain of Egr-1 but lacked the N-terminal transactivation domain (22). When cotransfected into fibroblasts with an Egr-1-dependent reporter, this mutant was shown to inhibit the transcriptional activity of both endogenous Egr-1, as well as exogenously expressed wild-type Egr-1 protein (22). In addition, this construct was shown to block the activity of all four Egr family members (32). The founders, #50, #36, #18 were generated in (C3HxBL/6)F1 mice. The founders and the transgene positive offspring were backcrossed to C57BL/6 mice for nine generations. The presence of the transgene in the founders was confirmed by Southern blot and in the offspring of subsequent generations by tail DNA PCR (Figure 3A, top panel). Expression of the transgene in splenic B cells was verified using RT-PCR (Figure 3A, middle panel) and by Western Blot (Figure 3A, bottom panel). Line 50 was used for all the studies except when indicated specifically.

Figure 3. Genotypic and Phenotypic characterization of dominant negative Egr-1 transgenic mice.

(A) PCR of tail DNA samples of the transgenic and the littermate controls of four lines (top panel) and the RT-PCR of the RNA samples obtained from B220⁺ B cells of indicated mice (middle panel). Western blot analysis of DN-Egr expression based on the expected molecular weight of ∼ 25 kDa of the truncated protein (bottom panel). (B) Resolution of immature transitional B cell subsets and the follicular B cells in the spleen of 10 week old dominant negative Egr transgenic mice and littermate controls by staining splenocytes with FITC anti-IgM, Cy-anti-B220, allophycocyanin-anti-AA4.1.1, and PE-anti-CD23 and analyzed on a FACS Calibur. B220+ AA4+ cells represent immature transitional B cells; B220+ AA4- cells represent mature follicular B cells and marginal zone B cells. Subfractionation of B220+ AA4+ immature B cells based on IgM and CD23 expression reveals IgM+ CD23- transitional T1 cells and IgM+ CD23+ transitional T2 cells. Subfractionation of B220+ AA4- mature B cells based on IgM and CD23 expression reveals IgM+ CD23- marginal zone B cells and IgM+ CD23+ follicular B cells.100,000 events were analyzed and data are representative of the average of 8 mice in each group. (C) Flow cytometric analysis of Marginal zone B cells in the spleen of transgenic and the littermate control by staining with FITC anti-CD21, Cy-anti-B220, allophycocyanin-anti-HSA, and PE-anti-CD23. Data are representative of four independent experiments. (D) Flow cytometric analysis of Peritoneal B cells in the peritoneum of transgenic and the littermate control by staining with FITC anti-B220 and PE-anti-CD23. The cells were gated on B220 (hence there are no cells in the upper and bottom left quadrants) and the B220 gated B220+ CD23+ cells represent the B-2 B cells whereas B220+ CD23- cells represent the B-1 B cells. Profiles for two individual mice are shown.

Reduced B lymphopoiesis in the bone marrow and reduced mature B cells in the spleen of dominant negative Egr transgenic mice

We observed a significant decrease in the number of pre-B cells and immature B cells in the bone marrow (B220⁺/CD43⁺/IgM^- and B220⁺/CD43^-/IgM⁺) of transgenic mice expressing the dominant negative Egr-1 (Table 2). Thus, in the DN-Egr transgenic mice, there was an overall decrease in Pre-Pro B (Fraction A), Pro B (Fraction B), Pre-BI (Fraction C), Pre-BII (Fraction C’), and immature (Fraction E) B cells in the bone marrow suggesting that Egr transcription factors regulate B lymphopoiesis at a very early stage possibly the CLPs. The decrease in the output of immature B cells in the bone marrow was reflected in the spleen B cells of transgenic mice which showed a two fold reduction (Table 3). There was a three fold decrease in the frequency and two to three fold decrease in absolute numbers of B220⁺/AA4.1⁺ immature transitional B cells and the B220⁺/AA4.1^- mature follicular B cell population (Figure 3B, Table 2, and Table 3). Further analysis of B220⁺/AA4.1⁺ cells for IgM and CD23 expression revealed a decrease in all three transitional B cell populations (IgM⁺/CD23^- for T1, IgM⁺/CD23⁺ for T2 and IgM^-/CD23⁺ for T3 cells) (data not shown). In addition, analysis of B220⁺/AA4.1^- cells for IgM and CD23 expression revealed a decrease in follicular B cell populations (IgM⁺/CD23⁺). We found a significant decrease (three fold) in the MZ B cell population in the spleen of transgenic mice compared to wildtype controls (B220⁺/CD23^-/HSA⁺/CD21⁺ fraction) (Figure 3C and Table 2). There was a significant decrease in B-1 cells in the peritoneum of the transgenic mice based on their B220⁺/CD23^- phenotype (Figure 3D).

Table 2.

Decrease in B-lymphopoiesis in DN-Egr mice

	B220+ Bone marrow (Pre-pro, Pro-, Pre- Immature, transitional and mature recirculating) B cells	B220+ AA4+ Transitional B cells (spleen)	B220+ AA4- Follicular and marginal zone B cells (Spleen)	CD23- HSA+ CD21+ Marginal zone B cells (Spleen)
		106 cells/ organ
Wild type (+/+)	2.0± 0.4 (30%±2%)	9.1 ± 1.0 (7%±2%)	26± 5 (38%±6%)	3 ± 0.5 (16%±2%)
DN-Egr transgenic	0.9± 0.3 (14%±4%)	4.5 ± 0.6 (3%±2%)	14± 3 (12%±5%)	1.2 ± 0.3 (4%±1%)
p value	< 0.05	< 0.05	< 0.05	< 0.05

Mean ± SE of absolute numbers in millions of the indicated populations were calculated by multiplying the frequencies of cells by the number of cells harvested from two tibia and two femurs and spleen from each mouse (n=8). *Values of P were calculated for absolute numbers using Student’s t test using data from littermate mice as the control population.

Table 3.

Reduction in absolute number of splenocytes and B cells in the spleen of DN-Egr transgenic mice

	Total splenocytes (× 106)		B220+ B cells (× 106)
Line # 50	Mean	n	Mean	n
Wild type	88 ± 10	10	40 ± 7	10
Transgenic	56 ± 6	10	21 ± 6	10
Line # 36
Wild type	62 ± 8	12	24 ± 5	12
Transgenic	37 ± 8	12	13 ± 2	12

Mean ± SE of absolute numbers of the indicated populations were calculated by multiplying the frequencies of cells by the number of cells harvested from the spleen of each mouse. p < 0.05; Values of P were calculated for absolute numbers using Student’s t test using data from littermate mice as the control population.

Defective proliferative response of B cells that lack Egr-1 activity

Since Egr-1-/- mice showed an enhancement of B cell proliferation in response to BCR cross-linking, presumably due to compensation by Egr-2 and Egr-3, we asked whether inhibition of all family members affected B cell proliferation. B cells from both transgenic and non-transgenic littermate control mice were stimulated with anti-IgM for 48 hours and the proliferation was measured by thymidine incorporation. There was a decrease in the proliferative response of splenic B cells to anti-IgM at all doses (Figure 4A). This defective response was evident in all the three founder mice (Figure 4B). Similar results were obtained with a fourth founder line (data not shown). On the other hand, this defect was not observed when B cells were treated with LPS or CD40 ligation, suggesting that Egr family members were downstream of BCR signaling but not TLR4 or CD40 pathways (Figure 4C). The reduced proliferative response of splenic B cells from the transgenic mice can be attributed to failure of the follicular B cells to respond to antigen receptor stimulation or due to an increase in the proportion of immature B cells in the periphery. To clarify this issue, we sorted the follicular B cell population (mature B cells) from both transgenic and littermate controls based on their CD21 and HSA expression and measured their proliferative response to BCR cross-linking. The HSA^lo CD21⁺ fraction from the transgenic mice proliferated less efficiently compared to the littermate at two different doses of anti-IgM (Figure 4D). Interestingly, cyclin D2 upregulation was defective at 25 μg/ml but not at 50 μg/ml of anti-IgM suggesting that higher doses of anti-IgM might overcome the defective cyclin D2 upregulation (Figure 4E). Moreover, transgenic B cells failed to upregulate c-Myc in response to two different doses of anti-IgM. On the other hand, CD40 ligation induced c-Myc upregulation remained comparable to wildtype, suggesting that Egr is downstream of B-cell receptor, and is critically dependent on c-Myc to enter cell cycle (Figure 4F). Nevertheless, our data provide mechanistic basis for Egr-1 induced cell proliferation in B cells as evident by its role in regulating c-Myc levels and to some extent cyclin D2 levels. These data suggest that Egr family members are critical for BCR induced proliferation of normal mature B cells. Also, cell adhesion molecule CD44 failed to upregulate, when B cells from DN-Egr transgenic mice were stimulated with anti-IgM compared to littermate controls (Figure 4G). There is a 5 fold decrease in CD44 expression in DN-Egr B cells compared to littermate controls. This result is consistent with the previous studies demonstrating CD44 as a target gene of Egr-1 in B cells (20).

Figure 4. Defective proliferation of B cells from DN-Egr transgenic mice.

(A) T-depleted B cells from littermate and the DN-Egr (Line 50) transgenic mice were cultured for 48 hours with medium alone or with indicated concentrations of anti-IgM and proliferation measured as described in Materials and Methods. (B) T-depleted B cells from littermate and the DN-Egr-1 transgenic mice of three different founder lines were cultured for 48 hours with medium alone or with indicated concentrations of anti-IgM and proliferation measured as described in Materials and Methods. n indicates the number of mice tested. (C) T-depleted B cells from littermate and the Line#50 DN-Egr transgenic mice were cultured for 48 hours with indicated concentrations of LPS or anti-CD40 and proliferation measured as described in Materials and Methods. (D) Sorted follicular B cells (B220⁺ HSA^lo CD21⁺) from littermate and the DN-Egr-1 transgenic mice were cultured for 48 hours with medium alone or with indicated concentrations of anti-IgM and proliferation measured as described in Materials and Methods. The differences between the transgenic and wild type mice stimulated with anti-IgM (at all doses) were statistically significant (P<0.05). Data are representative of five independent experiments. (E and F) B220 bead purified B cells were cultured in vitro for 6 hours in the presence or absence of indicated stimuli and lysates were probed for c-Myc and cyclin D2 protein levels by Western blotting. Blots were stripped and probed for beta-actin for protein loading. (G) T-depleted B cells from littermate and the Line#50 DN-Egr transgenics (n = 2) and the littermate mice (n = 2) were cultured for 48 hours with or without anti-IgM and cells were analyzed for CD44 expression by flow cytometry. The average MFI of CD44 expression in anti-IgM stimulated cells from two mice is 112 ± 4 for DN-Egr B cells and 480 ± 20 for littermate contols. Data is plotted as histogram comparing untreated and treated groups in littermates and transgenics. The p value for CD44 expression is < 0.05 comparing littermate and the DN-Egr transgenics.

Normal T-cell responses in DN-Egr transgenic mice

To rule out the possible effects of the transgene in T-cell lineage, we analyzed the thymocytes and probed peripheral T-cell responses. We did not observe any defect in the thymocyte population (both percentages and the absolute numbers) of the transgenic mice (Table 4). T-cell proliferation (Figure 5A), IL-2 production (Figure 5B) and CD40 ligand expression in response to anti-CD3 stimulation remained comparable between wildtype and transgenics suggesting that the effects of the transgene are restricted to B cells. Nevertheless, the effect of transgene in cell types other than B cells cannot be completely ruled out.

Table 4.

Normal thymocyte populations in the thymus of DN-Egr transgenic mice

	CD4+CD8+ thymocytes		CD4+ thymocytes		CD8+ thymocytes
	% gated	n	% gated	n	% gated	n
Transgene positive	87 ± 3	4	10 ± 4	4	2 ± 1	4
Transgene negative	89 ± 2	4	7 ± 1	4	2 ± 1	4

Mean percentages of the indicated populations from the thymus of each mouse.

Figure 5. T-cell responses are normal in DN-Egr transgenic mice.

B220 microbead depleted splenic T cells were activated in culture for three days with anti-CD3 coated plates. Proliferation was measured by thymidine incorporation as described in Materials and Methods (A). IL-2 was measured in the culture supernatants by ELISA (B) and CD40 ligand expression by FACS analysis (C).

Egr family members are critical for immune responses in vivo

Since Egr-1 was important for BCR-induced B cell proliferation, we next immunized the transgenic mice and tested the in vivo B cell responses. The basal immunoglobulin levels of DN-Egr transgenic mice remained comparable to the wildtype except for a modest increase in IgG1 (Figure 6A). There was a significant reduction in antibody formation by the B cells from the transgenic mice compared to littermates in response to both T-independent type 2 antigen TNP-Ficoll (Figure 6C) and T-dependent antigen SRBC (Figure 6D) but not T-independent type 1 antigen TNP-LPS (Figure 6B). Overall, these studies suggest that Egr family members are critical for immune responses in vivo.

Figure 6. Egr family members are critical for both T-independent type 2 and T-dependent immune responses in vivo.

(A) Basal immunoglobulin levels (pre-immune) in the serum of transgenic mice and the littermates as measured by ELISA. Results are representative of serum from three mice in each group. (B) For in vitro immunization, cultures were set up in 1 ml IMDM + Ham’s F12 (+10% fetal bovine serum) in 48-well plates (Costar, Corning, NY) with TNP-LPS (1 and 2 μg/ml), splenocytes (1×10⁶ per culture), for 4 days in 5% CO₂ and at 37°C. The number of IgM anti-TNP-secreting cells was determined using a glass-slide version of the Ab-forming cell (AFC) assay as described earlier (59). (C & D) Mice were immunized with 10 μg of TNP-Ficoll or 10% v/v SRBC i/p. 5 days after immunization, the anti-TNP response for TNP-Ficoll and the anti-SRBC response for SRBC were detected by a PFC assay. * indicates p<0.01. This experiment is representative of 3 mice in each group with duplicate slides for PFC assays for each mouse. Results are representative of three experiments for the TNP-Ficoll response and two experiments for the SRBC response.

DISCUSSION

In the present study, the importance of Egr-1 in B cell development and proliferation was studied using knockout mice in which Egr-1 gene is deleted and using transgenic mice that express a dominant negative form of Egr-1 in a B cell restricted manner. Egr-1^-/- mice exhibited an increase in B lymphopoiesis in the bone marrow leading to an increase of B cells (mostly immature) in the bone marrow, spleen and the peritoneum. However, follicular (FO) mature B cells were not increased in these knockouts. Egr-1^-/- B cells exhibited an increased proliferation response to BCR cross-linking, but responded normally to CD40 ligation. In contrast, B cell lymphopoiesis was reduced in dominant negative Egr transgenic mice resulting in a reduction in B cell numbers in the bone marrow and spleen (both immature and mature). In addition, B cells were defective in their proliferative response to BCR cross-linking but not for CD40 ligation or TLR4 stimulation.

This is the first report demonstrating the importance of the transcription factor Egr-1 in B cell development and functional response. The molecular circuitry involving B cell lineage commitment is fairly well characterized, with PU.1 playing a key role in the decision between myeloid and B-cell lineage. In this context, the role of the Egr family of immediate early gene transcription factors assumes greater importance since Egr-1 has been shown to be downstream of PU.1 (50). Immediate early genes are crucial for cellular responses including the immune cells since they are rapidly induced upon receptor ligation which primes the cells for subsequent late events that regulate cell survival, proliferation and differentiation (1). The Egr-1 gene is induced by growth factors and cytokines in many cell types (1). The role of Egr-1 in B cell development and proliferation is not known. Egr-1 was shown to be important for BP-1 expression, a marker for Pre-B cells in the bone marrow using transgenic mice that express Egr-1 in a B-cell specific manner (23). We and others showed that Egr-1 is rapidly induced in B cells upon BCR ligation.

Cytokine receptors Flt3 and IL-7, transcription factors PU.1, Ikaros, E2A, Bcl11a, EBF, and Pax-5 are crucial for the development of B cell lineage precursor cells (44-47). Expression of the receptor tyrosine kinase Flt3 within a subset of multipotent progenitors is one of the earliest events in B-cell development. PU.1 and Ikaros are required for expression of Flt3. Flt3 signaling in coordination with PU.1 induces IL-7 receptor. IL-7R signaling induces E2A, which in turn regulates EBF gene in coordination with PU.1. EBF co-operates with E2A and activates the early B lineage gene expression determining the B cell fate. In addition to commitment towards the B cell fate, EBF induces the expression of Pax-5. Pax-5 shuts down alternate lineage specifications and promotes commitment to the B cell fate (44-46). In the periphery, Bcl-6, another key transcription factor, is important for the maintenance of germinal center B cells (48).

The phenotype observed in both the knockout and the transgenic mice is contrasting and at the same time highlights the importance of the relative roles of Egr family members in B cell biology. As noted earlier, there are four family members viz., Egr-1, Egr-2, Egr-3 and Egr-4. The increase in B lymphopoiesis in Egr-1^-/- could be due to a negative role of Egr-1 in B cell development or due to a compensatory increase in other family members that further enhance B lymphopoiesis. The later possibility is supported by the finding that B lymphopoiesis is similarly decreased both in the bone marrow and spleen of DN-Egr mice. The fact that Egr-1^-/- B cells hyperproliferate and have increased expression of Egr-2 and Egr-3 provide further support to the second model. The possibility of different role for various Egr family members cannot be ruled out.

Egr-1, however, has a nonredundant role in the development of marginal zone (MZ) B cells, since this population is significantly decreased in both Egr-1^-/- and the DN-Egr transgenic mice. This is consistent with the notion that BCR signaling is required for marginal zone B cell development (49). Similarly, Egr-1 appears to be uniquely required for the high rate of expression of BP-1, which is decreased in Egr-1^-/- mice. Presently, it is not precisely clear at what stage of B cell commitment does Egr-1 have a regulatory role. It is possible that Egr-1 might be affecting the developmental stages as early as the commitment of CLPs towards the B cell lineage. This hypothesis is supported by our preliminary observations that DN-Egr transgenic mice have a two fold increase in B220⁺ Sca-1^lo early lineage cells, which are defined as CLPs in the bone marrow (50). Moreover, the model put forth by Singh and coworkers suggests that low levels of PU.1 (transcription factor required for both B and myeloid lineage commitment) in B cells activates Egr-1/2, thus establishing a connection between PU.1 and Egr-1 in lineage stability (51). On the other hand, high PU.1 activates Egr-1 very strongly and promotes macrophage differentiation. These data further support our observations that Egr family members regulate B cell commitment at a very early stage of their development in the bone marrow.

The BCR signal strength model put forward by Pillai and coworkers proposes that if the BCR of the B cell reacts fairly well (intermediate affinity) to an antigen, then the B cells are induced to differentiate into follicular B cells. But if the BCR of the B cell reacts weakly (weak affinity) to a cognate self antigen, it is prone to receive signals that drive the B cell to become a marginal zone B cell. The development of B-1 B cells is dependent upon strong signals from the BCR (strong affinity) (49). Although there is strong support to this hypothesis, there are some observations that are not fully consistent with this hypothesis. One such observation comes from Rajewsky and coworkers where they showed that expression of the Epstein-Barr virus LMP2A gene specifically in B cells driven by a weak promoter, leads to the generation of both MZ and FO B cells whereas a strong promoter gives rise to B-1 B cells (52). Our data suggest that Egr-1 affects the development of both marginal zone and B-1 B cell development but not the FO B cells in the Egr-1^-/- mice since there is a decrease in both MZ and B-1 B cell populations in the spleen and peritoneum respectively. Presently, it is not clear why a deficiency in Egr-1 affects the development of two B cell subsets (MZ and B-1) but not the development of FO B cells. One possibility is that the other Egr-1 family members can replace Egr-1 function for FO B cell development but not for MZ or B-1 B cell development. This hypothesis is supported by the finding that all three subsets are reduced in DN-Egr transgenic mice (Tables 2 & 3).

We think Egr-1 has an intrinsic role in B cells, because BCR signal strength is known to affect the development of B-cell subsets, as shown elegantly by Casola et al (52) and several other studies that use BCR signal deficient mutant mice (53). The fact that the dominant negative Egr-1 mice that express the DN-Egr protein in a B cell specific manner exhibit similar MZ B cell defect further strengthens this argument. One such factor regulated by Egr-1 could be Notch2, which has been shown to be important for MZ B cell development (54). Delta like 1 (DL1), one of the Notch ligands is also important for MZ B cells and is originally reported to be expressed by B cells (55) but a more recent paper by Moriyama et al reports that DL1 is expressed by macrophages and dendritic cells but not B cells (56). It is conceivable that Egr-1 regulates DL1 expression in macrophages or some other stromal cell molecule that affects MZ B cell development. The possibility that Egr-1 may affect stromal cells rather than B cells cannot be completely ruled out at this time.

Egr family members are not only important for B lymphocyte development but also for B cell clonal expansion initiated by B cell triggering. Just as in overall B lymphopoiesis, Egr-1 function can be substituted by other family members in the BCR induced proliferation responses as shown by their increase in Egr-1^-/- B cells, and the ability of ectopically expressed Egr-2 to overcome BCR induced growth inhibition of immature B cell lymphoma cells. When the functions of all Egr family members are suppressed by DN-Egr, then the follicular B cells are hyporesponsive. The requirement for Egr family members is unique for BCR pathway, since B cell proliferation induced by TLR4 or CD40 signaling is not affected either in Egr-1^-/- or in DN-Egr mice. This observation is consistent with the findings that The BCR signaling defect observed in DN-Egr mice is important for in vivo B-cell clonal expansion as antibody responses to both TI-2 and TD antigens are decreased.

Upon BCR ligation, there is a significant increase in Egr-1 message as well as protein levels in Egr-1^+/+ B cells. In contrast to Egr-1, resting B cells express a high basal level of Egr-2 protein which is being modified (most likely phosphorylated) upon BCR ligation in Egr-1^-/- but not wildtype B cells. This is the first report of Egr-2 being phosphorylated, a property it shares with Egr-1. This modification is likely to be important because others have shown that phosphorylation versus acetylation potentially modulates the activity of Egr-1 in terms of survival and apoptosis in prostate cancer cells (5). In addition, we find that Egr-2 and Egr-3 message levels are elevated upon BCR cross-linking in Egr-1^-/- B cells. Since Egr family members share 90% homology in their DNA binding region, we propose that in Egr-1^-/- mice, Egr-2 and possibly Egr-3 assume a critical compensatory role. Whether such a compensation involves regulation of some of the Egr-1 target genes remains to be explored. At present, target genes that are critically modulated by Egr-2 are not known except that Egr-2 regulates FasL expression in T cells (57, 58). This hypothesis is strengthened by our observations that over-expression of Egr-2 partially overcomes BCR induced growth inhibition in an immature B cell lymphoma model.

We conclude that Egr family members are positive modulators of B lymphopoiesis, marginal zone B cell development, BCR induced B cell proliferation, T-independent type 2 and T-dependent immune responses (Figure 6.1). Our observations demonstrate that Egr-1 is critical for MZ B cell development and its associated TI-2 immune responses. This phenotype is very significant since there are only few instances where deficiency of a transcription factor leads to a defect in MZ B cell development and TI-2 responses (49). Currently we are looking at some of the target genes that could be potentially modified by Egr-2 in lymphoma cells over-expressing this transcription factor. Moreover, mice deleted for Egr-2 and or Egr-3 could reveal some interesting roles for Egr-2 and Egr-3 in B cell growth response. Further studies on Egr family members will provide new insights into novel players in B cell development.