Early Growth Response Regulate B Cell Development, Proliferation, and Immune Response

This information is current as Murali Gururajan, Alan Simmons, Trivikram Dasu, Brett T. of September 29, 2021. Spear, Christopher Calulot, Darrell A. Robertson, David L. Wiest, John G. Monroe and Subbarao Bondada J Immunol 2008; 181:4590-4602; ; doi: 10.4049/jimmunol.181.7.4590

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Early Growth Response Genes Regulate B Cell Development, Proliferation, and Immune Response1

Murali Gururajan,2*†‡ Alan Simmons,*‡ Trivikram Dasu,3*‡ Brett T. Spear,*†§ Christopher Calulot,* Darrell A. Robertson,*‡ David L. Wiest,¶ John G. Monroe,ሻ and Subbarao Bondada4*†‡§

Egr-1 (early growth response -1) is an immediate early gene encoding a zinc finger motif-containing . Upon cross-linking of BCR, mature B cells undergo proliferation with an increase in Egr-1 message. Immature B lymphoma cells that express Egr-1 message and protein constitutively are growth inhibited when Egr-1 is down-regulated by negative signals from BCR or by antisense oligonucleotides. 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, whereas 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 Downloaded from members, we developed transgenic mice expressing a dominant negative form of Egr-1, which lacks the trans activation domain but retains the DNA-binding domain, in a B cell-specific manner. There was a decrease in B lymphopoiesis 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 Ags in vivo. The Journal of Immunology, 2008, 181: 4590–4602. http://www.jimmunol.org/ n response to infection, B and T cells undergo rapid prolif- lational modification (5). Acetylation of Egr-1 by CBP and p300 eration and subsequent differentiation leading to the genera- upon serum stimulation increases its stability and promotes cell I tion of effector cells that successfully combat the infection. survival, whereas phosphorylation of Egr-1 after UV radiation re- Many cell types including lymphocytes express a specific class of presses p300/CBP transcription and favors cell death (5). Egr-1 has genes called immediate early genes in response to stimulation via been shown to be important for prostate tumorigenesis, because Ag receptor, growth factor, and cytokines (1). Immediate early genes knockout mice have delayed tumorigenesis when crossed to trans- are rapidly induced (within minutes), and their induction does not genic adenocarcinoma mouse prostate model mice, which sponta- require new protein synthesis. Genes included within this category are neously develop prostate invasive neoplasia (6). c-fos and Egr-1. The Egr-1 gene-encoded protein Egr-1, a nuclear Egr-1 is expressed ubiquitously by many cell types, whereas Egr-2 by guest on September 29, 2021 zinc finger-containing transcription factor, binds to the consensus and Egr-3 are restricted in their expression (7). Even though Egr DNA motif GCGGTGGGCG and modulates the transcriptional ac- family members share redundancy in their functions as evidenced tivity of several target genes including IL-2, CD44, ICAM-1, and by 90% homology in their DNA-binding region, they do have TNF-␣ in B lymphocytes (1). Target genes for EGR-1 identified in unique functions as revealed by specific phenotypes in knockout other cell types include c-, cyclin D2, p19, and cyclin G2 (2–4). mice. Egr-1Ϫ/Ϫ mice are characterized by female infertility, Egr-1 has both antiapoptotic and proapoptotic roles in different Egr2Ϫ/Ϫ mice have hindbrain abnormalities, Egr-3Ϫ/Ϫ mice have cell types depending on the nature of the stimulus. Egr-1 can up- motor disorders, and Egr-4Ϫ/Ϫ mice have male infertility or down-regulate transcription of p300 and cAMP-responsive el- (8). Mice homozygous for Egr-2 deficiency die during the first 2 ement-binding protein (CBP)5 based on the nature of its posttrans- wk after birth due to abnormalities (9). The importance of EGR-1 in T cell biology has been studied quite extensively. Egr-1 is expressed in thymocytes and peripheral Departments of *Microbiology, Immunology, and ‡Molecular , Sanders Brown Center on Aging, †Graduate Center for Toxicology, and §Markey Cancer T cells, and its expression is rapidly induced upon TCR engage- Center, University of Kentucky, Lexington, KY 40536; ¶Basic Science Division, Fox ment in an ERK-dependent manner (10). Egr-1-deficient mice ʈ Chase Cancer Center, Philadelphia, PA 19111; and Pathology and Laboratory Med- have defects in positive selection beyond the ␤ selection check- icine, University of Pennsylvania, Philadelphia, PA 19104 point, resulting in a reduced percentage of CD4 and CD8 single- Received for publication December 4, 2007. Accepted for publication July 30, 2008. positive mature cells in the thymus (11). On TCR-transgenic back- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance grounds, Egr-1-deficient mice express reduced numbers of naive T with 18 U.S.C. Section 1734 solely to indicate this fact. cells. Egr-1 overexpression in thymus under lck allowed 1 This work was supported by National Institutes of Health Grants AI 21490, AG positive selection of thymocytes, possibly by lowering the thresh- 05731, and CA 92372 (to S.B.). old of avidity required for positive selection in the thymus (12). 2 Current address: Department of Pathology, Emory University, 1462 Clifton Road Although Egr-1-deficient animals have a low percentage of mature N.E., DSB 405, Atlanta, GA 30322. thymocytes, the absolute number of mature thymocytes is only 3 Current address: Clinical Immunology Laboratory, Rosalind Franklin University of slightly reduced due to an increase in thymus size. Recently, Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064. 4 Address correspondence and reprint requests to Dr. Subbarao Bondada, Room 303 Combs Cancer Building, Markey Cancer Research Center, University of Kentucky, lymphoid progenitors; EBF, early B cell transcription factor; DL1, delta-like 1; PFC, Lexington, KY 40536-0096. E-mail address: [email protected] plaque-forming cell. 5 Abbreviations used in this paper: CBP, cAMP-responsive element-binding protein; DN, dominant negative; MZ, marginal zone; TNP, trinitrophenyl; CLP, common Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 www.jimmunol.org The Journal of Immunology 4591 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. Altered B cell subsets in the bone marrow and spleen of Egr-1Ϫ/Ϫ mice. Flow cytometric analysis of bone marrow cells obtained from Egr-1Ϫ/Ϫ and littermate controls stained with CyChrome anti-B220 and PE anti-IgM. B220ϩIgMϪ cells represent pre-pro B, pro-B, and pre-B cells. B220ϩIgMϩ cells represent immature B, transitional, and recirculating mature B cells (A). Bone marrow cells were stained with PE-Cy anti-B220, 4592 B CELL INTRINSIC ROLE FOR Egr

Schnell et al demonstrated that Egr-1 is required for survival of marrow of Cre-LoxP conditional transgenic mice in which Cre mature thymocytes and newly emigrated thymocytes (13). All four activation leads to H chain deletion (29). These data show that family members are induced upon TCR ligation. Overexpression Egr-1 is important for B cell survival and that Egr-1 induction of Egr-2 and Egr-3 is associated with an increase in the E3 ubiq- downstream of BCR might be important for B cell development. uitin ligase Cbl-b and inhibition of T cell activation. Also, T cells Despite these studies, the in vivo role of Egr-1 during B cell de- from Egr-3Ϫ/Ϫ mice display lower Cbl-b and are resistant to in velopment and functional responses remains to be elucidated. vivo peptide-induced tolerance (14). These data support the idea Although Egr-1Ϫ/Ϫ mice have been generated, no significant that Egr-2 and Egr-3 are involved in promoting TCR-induced neg- effect of Egr-1 deficiency on B cell development has been reported ative signaling. The role of Egr-1 in macrophage differentiation (30). This could be due to the redundancy of Egr-1 function given has also been studied in detail. Using a variety of differentiation- that this family has three other members with similar transcrip- inducible myeloid cell lines, Krishnaraju et al. (15–17) showed tional activation properties (31, 32). Previous studies of Egr-1Ϫ/Ϫ that the ectopic Egr-1 expression in normal hemopoietic progeni- mice did not examine the effect of Egr-1 deficiency on specific tors stimulates development along the macrophage lineage at the stages of B cell development. Hence, we performed a detailed expense of development along the granulocyte or erythroid lin- analysis of B cell development in Egr-1Ϫ/Ϫ mice. In addition, we eages, regardless of the cytokine used. These observations are in generated transgenic mice in which the function of all Egr-1 family Ϫ Ϫ contrast to the phenotype observed in Egr-1 / mice where dif- members is inhibited by a dominant-negative (DN) Egr-1 con- ferentiation along the macrophage lineage remains normal (18). struct in a B cell-specific manner. We show that Egr family mem- In B lymphocytes, cross-linking of the Ag receptor results in bers are important for B lymphopoiesis and proliferation, and that

ras Downloaded from egr-1 expression through activation of the p21 /MAPK pathway follicular B cells lacking this transcription factor are defective in (1, 19). Detailed analysis of the Egr-1 promoter showed that the proliferative response to Ag receptor stimulation. Egr-1, in partic- two most distal serum response elements mediate Egr-1 gene in- ular, is essential for marginal zone (MZ) B cell development. We duction (20). Egr-1-binding sites are found within the promoter also demonstrate that Egr family members are critical for immune regions of the genes that encode ICAM-1 and CD44, two cell responses in vivo. adhesion molecules important for cell trafficking (20–22).

ICAM-1 and CD44 are up-regulated in B cells upon receptor http://www.jimmunol.org/ cross-linking. Although the Egr-1 promoter has been studied ex- Materials and Methods tensively, its role in B cell development and function is not well Mice understood. The role of Egr-1 in pre-B cell development has been Ϫ/Ϫ demonstrated previously, and Egr-1 was shown to be important for Egr-1 mice were generated and backcrossed to C57BL/6 (B6) mice as BP-1 expression in the bone marrow pre-B cells (23). Differential described earlier and were a gift from Dr. Jeffrey Milbrandt (30). Domi- nant-negative Egr-1 (⌬Egr-1) construct was generated as described previ- expression of Egr-1 in anergic and naive B cells has also been ously (20, 33). A FLAG tag was added to this construct at the C terminus studied previously with differing conclusions, with an increase in and was then cloned into a plasmid containing the Ig H chain promoter and Egr-1 in anergic B cells in one of the studies and no such increase ␮ enhancer (obtained from Dr. N. Muthusamy, Ohio State University, Co- in another (24, 25). It was shown that the Egr-1 gene is methylated lumbus, OH) so that the transgene would be expressed only in B cells (34). by guest on September 29, 2021 Egr-1 and not induced upon BCR ligation of an immature B cell line DN- -transgenic mice were generated by pronuclear microinjection by the University of Kentucky Transgenic Facility (Lexington, KY). We gen- WEHI-231 (26). This suggests that inactivation of the Egr-1 gene ϫ erated four founders in the (C3H B6)F1 background. One line has been might be important for tolerance induction in B cells. In our lab- backcrossed for nine generations onto a B6 background. oratory, 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 Reagents and cell lines only a modest increase in Egr-1 (19). This differential expression Abs to Egr-1 (C-19), c-Myc, and cyclin D2 were obtained from Santa Cruz pattern of Egr-1 during different stages of B cell development may Biotechnologies, and Abs to Egr-2 were purchased from Covance Research be related to induction of tolerance vs clonal expansion in B cells. Products. Anti-␤-actin mAb was obtained from Sigma-Aldrich. The im- mature B lymphoma cell line BKS-2 was isolated and maintained in vivo Accordingly, microarray analysis of naive and tolerant B cells as a splenic tumor in our laboratory (35). Female CBA/N (Xid) mice were showed a difference in Egr-1 expression subsequent to BCR cross- obtained from The Jackson Laboratory. Mice were housed in microisolator linking, followed by a rapid decrease in Egr-1 (27). Moreover, cages in our American Association for Laboratory Animal Accreditation BKS-2 B lymphoma cells, which have an immature B cell pheno- and Certification-approved rodent facility. BKS-2 B lymphoma cells ob- type and express Egr-1 constitutively, undergo growth arrest when tained from the spleens of CBA/N mice were depleted of T cells with a mixture of anti-T cell Abs and complement as described (35). Normal treated with antisense oligos specific for Egr-1 (28). Microarray splenic B cells were prepared according to procedures described previously studies demonstrate a 100-fold decrease in Egr-1 mRNA when (36). The characteristics of the monoclonal rat anti-mouse ␮ chain Ab, surface BCR is deleted in immature B cells derived from the bone AK11, were described previously (37, 38).

FITC anti-CD43, PE anti-BP-1, and allophycocyanin anti-HSA for fractions A–CЈ (B). B220ϩCD43ϩHSAϪBP-1Ϫ represents fraction A, B220ϩCD43ϩHSAhighBP-1low represents fraction B, B220ϩCD43ϩHSAintermediateBP-1intermediate represents fraction C, and B220ϩCD43ϩHSAhighBP-1high represents fraction CЈ. Bone marrow cells were stained for PE-Cy anti-B220, FITC anti-CD43, PE anti-IgM, and allophycocyanin anti-IgD for fractions D–F (C). B220ϩCD43ϪIgMϪIgDϪ represents fraction D, B220ϩCD43ϪIgMϩIgDϪ represents fraction E, and B220ϩCD43ϪIgMϩIgDϩ represents fraction F. Percentages indicate relative values of each subset compared with other subsets in the same histogram. D, Resolution of immature transitional B cell subsets and the follicular B cells in the spleen of 10-wk-old Egr-1Ϫ/Ϫ and littermate controls by staining splenocytes with FITC anti-IgM, PE-Cy-anti-B220, allophycocyanin-anti-AA4.1.1, and PE-anti-CD23 and analyzed on a FACSCalibur. B220ϩAA4ϩ cells represent immature transitional B, B220ϩAA4Ϫ cells represent mature follicular B. There were 100,000 events analyzed; 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-1Ϫ/Ϫ and the littermate control by staining with FITC anti-CD21, PE-Cy-anti-B220, allophycocyanin- anti-HSA, and PE-anti-CD23. MZ B cells are HSAϩCD21ϩ gated on CD23Ϫ splenocytes. F, Peritoneal B cell subsets were identified by staining with PE-Cy-anti-B220 and PE-anti-CD23 and analyzed by flow cytometry. Peritoneal B-1 cells represent B220ϩCD23Ϫ cells. Data are representative of three independent experiments. The Journal of Immunology 4593

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

Bone Marrow (ϫ106 cells/organ) Spleen (ϫ106 cells/organ)

B220ϩ (pre-pro, pro, pre, B220ϩAA4Ϫ immature, transitional, B220ϩIgMϩ (immature, B220ϩAA4ϩ follicular and CD23ϪHSAϩCD21ϩ and mature recirculating) B220ϩIgMϪ (pre-pro, transitional, and mature transitional marginal zone marginal zone B cells pro, and pre) B cells recirculating) B cells B cells B cells B cells

Wild type (ϩ/ϩ) 1.8 Ϯ 0.4 (15 Ϯ 2) 0.16 Ϯ 0.2 (10 Ϯ 3) 1.64 Ϯ 0.6 (5 Ϯ 2) 5.1 Ϯ 0.8 (4 Ϯ 2) 25 Ϯ 5 (28 Ϯ 4) 4 Ϯ 0.3 (16 Ϯ 2) Egr-1Ϫ/Ϫ 3.0 Ϯ 0.3 (34 Ϯ 4) 0.72 Ϯ 0.2 (29 Ϯ 2) 2.28 Ϯ 0.3 (9 Ϯ 2) 12.5 Ϯ 0.3 (14 Ϯ 2) 26 Ϯ 3 (30 Ϯ 3) 1 Ϯ 0.1 (4 Ϯ 1) p Ͻ0.05 Ͻ0.05 Ͻ0.05 Ͻ0.05 NS Ͻ0.05

a Absolute numbers of the indicated B cell populations are obtained by multiplying the frequencies of cells by the number of cells harvested from two tibias and two femurs or the spleen from each mouse. Various B cell precursor members were estimated by FACS as shown in Fig.1. Values represent mean Ϯ SE of B cell numbers from six mice each in all cases except for MZ B cells where four mice each of Egr-1ϩ/ϩ and Egr-1Ϫ/Ϫ were used. Numbers in parentheses represent percent of cells with indicated phenotype in each tissue. p values were calculated for absolute numbers with Student’s t test, using data from littermate mice as the control population.

Cell preparation and staining is evaluated by Student’s t test. Percentage of control response was defined ϫ as (cpm in the treated group/cpm in the untreated group) 100. Downloaded from Suspensions of BM cells were flushed from tibias and femurs and spleno- cytes were prepared through crushing of spleens in HBSS. Cells were Real-time PCR washed and then incubated with optimal dilutions of the indicated Abs in polystyrene round-bottom tubes in a final volume of 100 ␮l. After 30 min Total RNA was isolated from B cells with the Tri-reagent (Sigma-Aldrich), on ice, cells were washed twice with FACS buffer and, when appropriate, and 2 ␮g of total RNA was subsequently used to make cDNA using the cells were incubated for 20 min on ice before two final washes with flu- Superscript II reverse transcriptase (Invitrogen) according to the manufac- orochrome-conjugated streptavidin to reveal staining by biotinylated Abs. turer’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 http://www.jimmunol.org/ Flow cytometric analyses (Applied Biosystems). The GAPDH-specific primers and probe were used PE-Cy5 anti-CD45R/B220 (RA3-6B2), PE anti-BP-1, FITC anti-CD43, for loading control (Applied Biosystems). FITC anti-IgM, PE anti-IgM, PE anti-CD23, FITC anti-CD21, FITC anti- CD5, biotin-anti-CD24/HSA (30F1), and anti-sIgD Abs were obtained Western blotting from BD Pharmingen, and allophycocyanin-AA4.1 (AA4.1.1) was ob- T-depleted B cells from the Egr-1 knockout, DN Egr-transgenic and lit- tained from eBiosciences. Analyses were conducted on a dual-laser flow termates were rested for3hinserum, insulin, progesterone-free IF-12 cytometer (FACSCalibur; BD Immunocytometry Systems) or a MoFlo cell medium and then were stimulated with anti-IgM (25 ␮g/ml) or PMA (10 sorter (DakoCytomation). All flow cytometry data were analyzed with ng/ml) for 1 h. Cell lysates were prepared in 1ϫ SDS sample buffer or 1% CellQuest software. Triton X-100 as described earlier and were subjected to SDS-PAGE and by guest on September 29, 2021 Retroviral production and transduction of B lymphoma cells Western blot analysis (36). Western blots were analyzed by probing the membrane using various primary Abs (Egr-1 and Egr-2) followed by HRP- Egr-2 and Wilms’ tumor Egr-1 (WT-Egr) constructs cloned into the ret- conjugated secondary Abs (Santa Cruz). The blots were developed with roviral vector LZRSpBMN-linker-internal ribosomal entry site-enhanced Pico Chemiluminescence substrate (Pierce Biotechnology) and exposed to GFP (LZRS) encompassing an internal ribosomal entry site were described Kodak X-Omat films which were scanned with a flat-bed scanner (UMAX previously (32). Retroviral vectors were transiently transfected into Phoe- Technologies). Alternatively, the blots were scanned on a Kodak Image nix packaging cells using the Lipofectamine transfection system (Invitro- Station 2000RT (Eastman Kodak). For reprobing, membranes were gen) according to the manufacturer’s protocol. Transfection efficiency was stripped using a solution containing 62.5 mM Tris-HCl, 2% SDS, and 100 assessed by determining the percentage of Phoenix packaging cells ex- mM 2-ME at 65°C for 20 min. The relative integrated OD of the protein pressing enhanced GFP by FACS. Virus-containing supernatants were bands was estimated using Scion Image software (Scion Corp.) or Kodak harvested from transfected Phoenix cells and pretreated with 5 ␮g/ml po- Image Station software. lybrene (Sigma-Aldrich). BKS-2 B lymphoma cells were washed in serum- free opti-MEM, single-cell suspensions were incubated at a concentration In vivo immunizations and plaque-forming cell (PFC) assays of 1 ϫ 106 cells/ml/well of a six-well plate for2hat30°C in 2 ml of viral supernatants, and the plates were spin infected. At the end of the 2-h in- Mice (both DN-Egr-transgenic and littermate controls) were immunized ␮ fection period, virus supernatant was discarded, and fresh IF-12 medium i.p. with 10 g of trinitrophenyl (TNP)-Ficoll or 10% v/v of SRBC in 300 ␮lof1ϫ PBS. The number of IgM anti-TNP-secreting cells was deter- was added to the cells, which were cultured for 48 h in a 5% CO2 humid- ified incubator. After 2 days, cells were sorted for GFP using a MoFlo cell mined 5 days after immunization using a glass slide version of the tech- sorter, and sorted cells were plated and proliferation measured 48 h later as nique of localized hemolysis in a gel as described (39). Briefly, a 1-ml described in the next section. packed cell volume of SRBC (Colorado Serum) was coupled with 2,4,6- trinitrobenzenesulfonic acid (Eastman Kodak) following published proto- Proliferation assay cols. The splenocytes were washed with HBSS and then mixed with 50 ␮l of 13.5% (v/v) TNP-coupled SRBC, 200 ␮lof2ϫ basal Eagle’s medium Ϫ Ϫ T-depleted B cells from the Egr-1 / mice, DN-Egr transgenic mice, and (Life Technologies), and 200 ␮l of 1.6% agarose (FMC Bioproducts) and appropriate littermate controls were incubated at 2 ϫ 105 cells/well in poured onto a glass microscope slide (Goldseal). The slides were incubated triplicate in 96-well flat-bottom plates in medium consisting of IMDM and for1hat37°C, and the plaques were developed during an additional 1-h Ham’s F-12 medium supplemented with 10 mM glutamine, 10 mM incubation at 37°C with guinea pig complement (Pel-Freez Biologics). The Ϫ HEPES, 0.5 mg/ml gentamicin, and 5 ϫ 10 5 2-ME. Stimuli added in- plaques representing Ab-forming cells were viewed under a low power Ј ␮ cluded F(ab )2 goat anti-IgM ( -chain specific; ICN Pharmaceuticals), LPS microscope. Ag-specific Ab-forming cells were calculated by taking the (Sigma-Aldrich), or anti-CD40 (clone 1C10). After 44 h, cultures were number of plaques for the Ag-mmunized mice and subtracting the number pulsed with 1 ␮Ci of [3H]thymidine and harvested 4 h later for scintillation of plaques obtained in the vehicle-immunized mice. Results are provided as counting. BKS-2 cells were cultured in IF-12 medium (1:1 mixture of the arithmetic mean Ϯ SE, and the statistical significance of different treat- IMDM and Ham’s F-12 (ϩ10% FCS); Atlanta Biologicals). To measure ments is evaluated by Student’s t test. proliferation, 2 ϫ 104 cells were cultured in 200 ␮l of medium. The cells were harvested onto filter mats using a cell harvester (Packard). The levels ELISA of radionucleotide incorporation were measured with a Matrix 96 beta radiation counter (Packard). Results are presented as the arithmetic mean of Serum Ig levels were measured by mouse Ig isotyping kit (BD triplicate cultures Ϯ SE and statistical significance of different treatments Biosciences). 4594 B CELL INTRINSIC ROLE FOR Egr Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. Proliferative response of Egr-1Ϫ/Ϫ B cells and the critical role for Egr-2 in B cell proliferation. A, T-depleted B cells from wild-type and Egr-1Ϫ/Ϫ mice were cultured for 48 h 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-1Ϫ/Ϫ mice were stimulated with anti-IgM or PMA for 1 h, and cell lysates were prepared (top). 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 Ab to Egr-2, which were then stripped and probed for ␤-actin. Results are representative of two experiments. C and D, T-depleted Egr-1Ϫ/Ϫ and wild-type B cells were stimulated for 1 h with 25 ␮g/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 (C) and Egr-3 (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 in the presence or absence of indicated concentration of anti-IgM (AK11), were cultured for 48 h, and proliferation measured as described in Materials and Methods. Data are representative of three independent experiments.

Results shown in various panels of Fig. 1, and summary data based on Increase in immature B cells in the bone marrow and spleen of several mice are shown in Table I. The reduction in fraction CЈ is Egr-1Ϫ/Ϫ mice likely due to a decrease in high BP-1 expression, a marker for this ϩ fraction, which in turn is likely due to a direct role of Egr-1 in The number of B220 cells in the bone marrow was increased in Egr-1Ϫ/Ϫ mice (Table I). Analysis of B cell development accord- enhancing BP-1 expression (23). Decreased BP-1 expression does ing to the Hardy protocol showed a significant increase in the not appear to affect B cell maturation given that we observed an number of pre-pro B cells, pro-B cells, pre-B cells increase in fractions D and E, which are thought to be derived from ϩ Ϫ Ϫ Ј (B220 CD43 IgM ) and immature B cells, transitional and ma- fraction C (Fig. 1C). There appeared to be a decrease in fraction Ϫ/Ϫ ture recirculating B cells (B220ϩCD43ϪIgMϩ) in the bone mar- F (recirculating follicular B cells) in the Egr-1 mice. Currently, row of mice lacking Egr-1 (Fig. 1A and Table I; Ref. 40). Further it is not clear whether this is due to homing and/or retention defect subfractionation of pro-B cells using the BP-1 and HSA markers in the bone marrow. A homing defect is consistent with the finding into A (pre-pro-B), B (pro-B), C (pre-BI), and CЈ (pre-BII) pop- that Egr-1 regulates ICAM-1 and CD44, adhesion molecules in- ulations revealed a decrease in fraction CЈ in Egr-1Ϫ/Ϫ mice com- volved in trafficking (20, 21). Thus, in the Egr-1Ϫ/Ϫ mice, there is pared with wild-type mice (Fig. 1B). Typical FACS profiles are an overall increase in pre-pro-B (fraction A), pro-B (fraction B), The Journal of Immunology 4595 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 3. Genotypic and phenotypic characterization of DN-Egr-1-transgenic mice. A, PCR of tail DNA samples of the transgenic and the littermate controls of four lines (top) and the RT-PCR of the RNA samples obtained from B220ϩ B cells of indicated mice (middle). Western blot analysis of DN-Egr expression based on the expected molecular mass of ϳ25 kDa of the truncated protein (bottom). B, Resolution of immature transitional B cell subsets and the follicular B cells in the spleen of 10-wk-old DN-Egr-transgenic mice and littermate controls by staining splenocytes with FITC anti-IgM, PE-Cy-anti-B220, allophycocyanin-anti-AA4.1.1, and PE-anti-CD23 and analyzed on a FACSCalibur. B220ϩAA4ϩ cells represent immature transitional B cells; B220ϩAA4Ϫ cells represent mature follicular B cells and MZ B cells. Subfractionation of B220ϩAA4ϩ immature B cells based on IgM and CD23 expression reveals IgMϩCD23Ϫ transitional T1 cells and IgMϩCD23ϩ transitional T2 cells. Subfractionation of B220ϩAA4Ϫ mature B cells based on IgM and CD23 expression reveals IgMϩCD23Ϫ MZ B cells and IgMϩCD23ϩ follicular B cells. There were 100,000 events analyzed; data are representative of the average of eight mice in each group. C, Flow cytometric analysis of MZ B cells in the spleen of transgenic and the littermate controls by staining with FITC anti-CD21, PE-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 B220ϩCD23ϩ cells represent the B-2 B cells, whereas B220ϩCD23Ϫ cells represent the B-1 B cells. Profiles for two individual mice are shown. pre-BI (fraction C), and immature (fraction E) B cells in the bone and CD23 expression revealed an increase in all three transitional marrow, suggesting that Egr-1 regulates B lymphopoiesis at a very B cell populations (IgMϩCD23Ϫ for T1, IgMϩCD23ϩ for T2, and early stage possibly the common lymphoid progenitors (CLP). IgMϪCD23ϩ for T3 cells; data not shown). The mature follicular The increased output of immature B cells in the bone marrow B cell population as identified by B220ϩAA4.1Ϫ markers was reflected in the spleen of Egr-1Ϫ/Ϫ mice, which showed a remained unaffected despite an increase in transitional B cells. MZ 3-fold increase in the frequency and 2-fold increases in the abso- B cells are thought to be the main cells in T-independent immune lute numbers of B220ϩAA4.1ϩ immature transitional B cells (Fig. responses. We found a significant decrease (3- to 4-fold) in the MZ 1D and Table I). Further analysis of B220ϩAA4.1ϩ cells for IgM B cell population (B220ϩCD23Ϫ/HSAϩCD21ϩ fraction) in the 4596 B CELL INTRINSIC ROLE FOR Egr

Table II. Decrease in B-lymphopoiesis in DN-Egr micea

B220ϩ Bone Marrow (Pre-pro, B220ϩAA4Ϫ Follicular Pro-, Pre-, Immature, Transitional B220ϩAA4ϩ Transitional and Marginal Zone CD23ϪHSAϩ CD21ϩ Marginal and Mature Recirculating) B Cells B Cells (Spleen) B Cells (Spleen) Zone B Cells (Spleen) (ϫ106 cells/organ) (ϫ106 cells/organ) (ϫ106 cells/organ) (ϫ106 cells/organ)

Wild type (ϩ/ϩ) 2.0 Ϯ 0.4 (30 Ϯ 2) 9.1 Ϯ 1.0 (7 Ϯ 2) 26 Ϯ 5 (38 Ϯ 6) 3 Ϯ 0.5 (16 Ϯ 2) DN-Egr transgenic 0.9 Ϯ 0.3 (14 Ϯ 4) 4.5 Ϯ 0.6 (3 Ϯ 2) 14 Ϯ 3 (12 Ϯ 5) 1.2 Ϯ 0.3 (4 Ϯ 1) P Ͻ0.05 Ͻ0.05 Ͻ0.05 Ͻ0.05

a Mean Ϯ 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 ϭ 8). p values were calculated for absolute numbers with Student’s t test, using data from littermate mice as the control population. Numbers in parentheses represent the percentage of cells in the given tissue. spleen of Egr-1Ϫ/Ϫ mice (Fig. 1E and Table I). Alternatively, we protein (22). In addition, this construct was shown to block the looked at the MZ B cell population based on CD21 and CD23 activity of all four Egr family members (32). The founders, lines ϩ Ϫ ϫ staining and found that CD21 CD23 MZ B cells were reduced in 50, 36, and 18 were generated in (C3H B6)F1 mice. The the Egr-1Ϫ/Ϫ mice compared with the wild-type mice. There was founders and the transgene positive offspring were backcrossed to a significant decrease in B-1 cells in the peritoneum of the knock- B6 mice for nine generations. The presence of the transgene in the ϩ Ϫ out mice based on their B220 CD23 phenotype (Fig. 1F). founders was confirmed by Southern blot and in the offspring of Downloaded from subsequent generations by tail DNA PCR (Fig. 3A, top). Expres- Hyperproliferation of B cells from mice that lack Egr-1 sion of the transgene in splenic B cells was verified using RT-PCR Because Egr-1 is rapidly induced upon Ag receptor stimulation, (Fig. 3A, middle) and by Western blot (Fig. 3A, bottom). Line 50 and Egr-1 was shown to promote cell proliferation in other cell was used for all the studies except when indicated specifically. types (41, 42), we determined whether Egr-1 is critical for BCR- Ϫ/Ϫ Reduced B lymphopoiesis in the bone marrow and reduced induced B cell proliferation. B cells from both Egr-1 mice and http://www.jimmunol.org/ littermate control mice were stimulated with anti-IgM for 48 h, and mature B cells in the spleen of dominant negative proliferation was measured by thymidine incorporation. There was Egr-transgenic mice a 2-fold increase in the proliferative response of Egr-1Ϫ/Ϫ splenic We observed a significant decrease in the number of pre-B cells B cells to anti-IgM at three different doses (5, 10 and 25 ␮g/ml; and immature B cells in the bone marrow (B220ϩCD43ϩBP- Fig. 2A). The increased BCR response in Egr-1Ϫ/Ϫ mice might be 1ϩIgMϪ and B220ϩCD43ϪIgMϩ, respectively) of transgenic due to compensation by other family members like Egr-2 (which mice expressing the DN Egr-1 (Table II). Thus, in the DN Egr- was also shown to be up-regulated during BCR cross-linking in transgenic mice, there was an overall decrease in pre-pro-B (frac- normal B cells) and Egr-3 (27, 43). The other possibility is that tion A), pro-B (fraction B), pre-BI (fraction C), pre-BII (fraction Egr-1 is a negative regulator of B cell activation and that the re- CЈ), and immature (fraction E) B cells in the bone marrow sug- by guest on September 29, 2021 sponse is higher upon deletion. The former hypothesis is supported gesting that Egr transcription factors regulate B lymphopoiesis at by our observations that Egr-2 is expressed by the Egr-1Ϫ/Ϫ B a very early stage, possibly the CLPs. The decrease in the output cells and that a lower mobility band appears in anti-IgM- or PMA- of immature B cells in the bone marrow was reflected in the spleen activated cells in the Egr-1Ϫ/Ϫ but not the littermate wild-type B cells of transgenic mice that showed a 2-fold reduction (Table mice (Fig. 2B). This slow moving band appeared to be due to III). There was a 2-fold decrease in the frequency in the absolute phosphorylation, because it became undetectable when Egr-1Ϫ/Ϫ numbers of B220ϩAA4.1ϩ immature transitional B cells and B cells were stimulated with anti-IgM in the presence of ERK the B220ϩAA4.1Ϫ mature follicular B cell population (Fig. 3B, (PD98059) or JNK (SP600125) inhibitor. Unlike the Egr-1 knock- Table II, and Table III). Further analysis of B220ϩAA4.1ϩ cells outs, Egr-2 is not modified in the littermate controls. In addition, for IgM and CD23 expression revealed a decrease in all three the Egr-2 and Egr-3 genes were also up-regulated in Egr-1Ϫ/Ϫ B transitional B cell populations (IgMϩCD23Ϫ for T1, IgMϩCD23ϩ cells and wild-type B cells upon BCR cross-linking (Fig. 2, B–D). for T2 and IgMϪCD23ϩ for T3 cells; data not shown). In addition, To test the compensatory role of Egr-2, we used the BKS-2 B analysis of B220ϩAA4.1Ϫ cells for IgM and CD23 expression lymphoma model in which lymphoma growth is inhibited by BCR revealed a decrease in follicular B cell populations (IgMϩCD23ϩ). cross-linking with an accompanying decrease in Egr-1 (19, 28). Ectopic expression of Egr-2 using a retroviral vector rescued im- mature BKS-2 B lymphoma cells from BCR induced growth in- Table III. Reduction in absolute number of splenocytes and B cells in hibition (Fig. 2E). On the other hand, the CD40 response remained the spleen of DN-Egr-transgenic micea comparable between the knockout and the littermates, suggesting that Egr-1 may not function downstream of CD40 signaling Total Splenocytes B220ϩ B Cells ϫ 6 ϫ 6 pathways. ( 10 ) ( 10 ) Mean n Mean n Generation of DN Egr-transgenic mice Line 50 To understand further the role of Egr-1 in B cells, we generated Wild type 88 Ϯ 10 10 40 Ϯ 710 transgenic mice expressing a DN form of EGR-1 in a B cell-re- Transgenic 56 Ϯ 61021Ϯ 610 stricted manner using V promoter and the E␮ enhancer. The DN- Line 36 H Ϯ Ϯ Egr construct contained the DNA-binding domain of Egr-1 but Wild type 62 81224512 Transgenic 37 Ϯ 81213Ϯ 212 lacked the N-terminal trans activation domain (22). When cotrans- fected into fibroblasts with an Egr-1-dependent reporter, this mu- a Mean Ϯ 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 tant was shown to inhibit the transcriptional activity of both en- of each mouse. p Ͻ 0.05; p values were calculated for absolute numbers with Stu- dogenous Egr-1 and exogenously expressed wild-type Egr-1 dent’s t test, using data from littermate mice as the control population. The Journal of Immunology 4597 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

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 h 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 h with medium alone or with indicated concentrations of anti-IgM and proliferation measured as described in Materials and Methods. C, T-depleted B cells from littermates and the line 50 DN-Egr-transgenic mice were cultured for 48 h with indicated concentrations of LPS or anti-CD40 and proliferation measured as described in Materials and Methods. D, Sorted follicular B cells (B220ϩHSAlowCD21ϩ) from littermate and the DN-Egr-1-transgenic mice were cultured for 48 h 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 Ͻ 0.05). Data are representative of five 4598 B CELL INTRINSIC ROLE FOR Egr

Table IV. Normal thymocyte populations in the thymus of DN-Egr transgenic micea

CD4ϩCD8ϩ CD4ϩ CD8ϩ Thymocytes Thymocytes Thymocytes

% gated n % gated n % gated n

Transgene positive 87 Ϯ 3410Ϯ 44 2Ϯ 14 Transgene negative 89 Ϯ 24 7Ϯ 14 2Ϯ 14

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

We found a significant decrease (3-fold) in the MZ B cell popu- lation in the spleen of transgenic mice compared with wild-type controls (B220ϩCD23ϪHSAϩCD21ϩ fraction; Fig. 3C and Table II). There was a significant decrease in B-1 cells in the peritoneum of the transgenic mice based on their B220ϩCD23Ϫ phenotype (Fig. 3D).

Defective proliferative response of B cells that lack Egr-1 activity Downloaded from Because Egr-1Ϫ/Ϫ mice showed an enhancement of B cell prolif- eration in response to BCR cross-linking, presumably due to com- pensation by Egr-2 and Egr-3, we asked whether inhibition of all family members affected B cell proliferation. B cells from both transgenic and nontransgenic littermate control mice were stimu- lated with anti-IgM for 48 h and the proliferation was measured by http://www.jimmunol.org/ thymidine incorporation. There was a decrease in the proliferative response of splenic B cells to anti-IgM at all doses (Fig. 4A). This defective response was evident in all the three founder mice (Fig. 4B). Similar results were obtained with a fourth founder line (data not shown). Alternatively, 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 (Fig. 4C). The reduced proliferative response of by guest on September 29, 2021 splenic B cells from the transgenic mice can be attributed to failure of the follicular B cells to respond to Ag 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 HSAlowCD21ϩ fraction from the transgenic mice proliferated less efficiently than did the littermate at two different doses of anti-IgM (Fig. 4D). Interestingly, cyclin D2 up-regulation was defective at 25 ␮g/ml but not at 50 ␮g/ml anti-IgM, suggesting that higher doses of anti-IgM might overcome the defective cyclin D2 up- FIGURE 5. T cell responses are normal in DN-Egr-transgenic mice. regulation (Fig. 4E). Moreover, transgenic B cells failed to up- B220 microbead-depleted splenic T cells were activated in culture for 3 regulate c-Myc in response to two different doses of anti-IgM. days in anti-CD3-coated plates. Proliferation was measured by thymidine Alternatively, CD40 ligation-induced c-Myc up-regulation re- incorporation as described in Materials and Methods (A). IL-2 was mea- mained comparable with wild-type, suggesting that Egr is down- sured in the culture supernatants by ELISA (B) and CD40L expression by FACS (C). stream of B cell receptor and is critically dependent on c-Myc to enter the cell cycle (Fig. 4F). Nevertheless, our data provide a mechanistic basis for Egr-1-induced cell proliferation in B cells as evidenced by its role in regulating c-Myc levels and to some extent B cells from DN Egr-transgenic mice were stimulated with anti- cyclin D2 levels. These data suggest that Egr family members are IgM compared with littermate controls (Fig. 4G). There is a critical for BCR-induced proliferation of normal mature B cells. 5-fold decrease in CD44 expression in DN-Egr B cells com- Also, cell adhesion molecule CD44 failed to up-regulate, when pared with littermate controls. This result is consistent with the independent experiments. E and F, B220 bead-purified B cells were cultured in vitro for6hinthepresence 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 ␤-actin for protein loading. G, T-depleted B cells from littermates and the line 50 DN-Egr transgenics (n ϭ 2) and the littermate mice (n ϭ 2) were cultured for 48 h 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 Ϯ 4 for DN-Egr B cells and 480 Ϯ 20 for littermate contols. Data are plotted as a histogram comparing untreated and treated groups in littermates and transgenics. The p value for CD44 expression is Ͻ0.05 comparing littermate and the DN-Egr transgenics. The Journal of Immunology 4599

previous studies demonstrating CD44 as a target gene of Egr-1 in B cells (20).

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 pop- ulation (both percentages and the absolute numbers) of the trans- genic mice (Table IV). T cell proliferation (Fig. 5A), IL-2 produc- tion (Fig. 5B), and CD40L expression in response to anti-CD3 stimulation remained comparable between wild-type and trans- genic mice, suggesting that the effects of the transgene are re- stricted to B cells. Nevertheless, the effect of transgene in cell types other than B cells cannot be completely ruled out.

Egr family members are critical for immune responses in vivo Because Egr-1 was important for BCR-induced B cell prolifera- tion, we next immunized the transgenic mice and tested the in vivo B cell responses. The basal Ig levels of DN-Egr-transgenic mice Downloaded from remained comparable with those of the wild type except for a modest increase in IgG1 (Fig. 6A). There was a significant reduc- tion in Ab formation by the B cells from the transgenic mice com- pared with littermates in response to both T-independent type 2 Ag TNP-Ficoll (Fig. 6C) and T-dependent Ag SRBC (Fig. 6D) but not

T-independent type 1 Ag TNP-LPS (Fig. 6B). Overall, these stud- http://www.jimmunol.org/ ies suggest that Egr family members are critical for immune re- sponses in vivo.

Discussion In this 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 DN form of Egr-1 in a B cell-restricted manner. Egr-1Ϫ/Ϫ mice exhibited an by guest on September 29, 2021 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 mature B cells were not increased in these knockouts. Egr-1Ϫ/Ϫ B cells exhibited an in- creased proliferation response to BCR cross-linking, but responded normally to CD40 ligation. In contrast, B cell lymphopoiesis was reduced in DN-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 tran- scription factor Egr-1 in B cell development and functional re- sponse. The molecular circuitry involving B cell lineage commit- ment 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 given that Egr-1 is downstream

of IMDM ϩ Ham’s F-12 (ϩ10% FBS) in 48-well plates (Costar) with TNP-LPS (1 and 2 ␮g/ml) and splenocytes (1 ϫ 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 assay as described earlier (44). C and D, Mice were immunized with 10 ␮gof TNP-Ficoll or 10% v/v SRBC i.p. 5 days after immunization; the anti-TNP FIGURE 6. Egr family members are critical for both T-independent response for TNP-Ficoll and the anti-SRBC response for SRBC were de- p Ͻ 0.01. This experiment is representative of ,ء .type 2 and T-dependent immune responses in vivo. A, Basal Ig levels tected by a PFC assay (preimmune) in the serum of transgenic mice and the littermates as mea- three mice in each group with duplicate slides for PFC assays for each sured by ELISA. Results are representative of serum from three mice in mouse. Results are representative of three experiments for the TNP-Ficoll each group. B, For in vitro immunization, cultures were set up in 1 ml response and two experiments for the SRBC response. 4600 B CELL INTRINSIC ROLE FOR Egr of PU.1 (45, 46). Immediate early genes are crucial for cellular affinity) to an Ag, then the B cells are induced to differentiate into responses including the immune cells because they are rapidly in- follicular B cells; but if the BCR of the B cell reacts weakly (weak duced upon receptor ligation which primes the cells for subsequent affinity) to a cognate self Ag, it is prone to receive signals that late events that regulate cell survival, proliferation, and differen- drive the B cell to become a MZ B cell. The development of B-1 tiation (1). The Egr-1 gene is induced by growth factors and cy- B cells is dependent on strong signals from the BCR (strong af- tokines in many cell types (1). The role of Egr-1 in B cell devel- finity) (51). Although there is strong support to this hypothesis, opment and proliferation is not known. Egr-1 was shown to be there are some observations that are not fully consistent with this important for BP-1 expression, a marker for pre-B cells in the bone hypothesis. One such observation comes from Rajewsky and co- marrow using transgenic mice that express Egr-1 in a B cell-spe- workers (53) who showed that expression of the EBV LMP2A cific manner (23). We and others showed that Egr-1 is rapidly gene specifically in B cells driven by a weak promoter leads to the induced in B cells upon BCR ligation. generation of both MZ and follicular B cells whereas a strong Cytokine receptors Flt3 and IL-7 and transcription factors PU.1, promoter gives rise to B-1 B cells. Our data suggest that Egr-1 Ikaros, E2A, Bcl11a, early B cell transcription factor (EBF), and affects the development of both MZ and B-1 B cell development Pax-5 are crucial for the development of B cell lineage precursor but not the follicular B cells in the Egr-1Ϫ/Ϫ mice, given that there cells (46–49). Expression of the receptor tyrosine kinase Flt3 is a decrease in both MZ and B-1 B cell populations in the spleen within a subset of multipotent progenitors is one of the earliest and peritoneum, respectively. It is not now clear why a deficiency events in B cell development. PU.1 and Ikaros are required for in Egr-1 affects the development of two B cell subsets (MZ and expression of Flt3. Flt3 signaling in coordination with PU.1 in- B-1) but not the development of follicular B cells. One possibility duces IL-7R. IL-7R signaling induces E2A, which in turn regulates is that the other Egr-1 family members can replace Egr-1 function Downloaded from EBF gene in coordination with PU.1. EBF cooperates with E2A for follicular B cell development but not for MZ or B-1 B cell and activates the early B lineage determining the development. This hypothesis is supported by the finding that all B cell fate. In addition to commitment toward the B cell fate, EBF three subsets are reduced in DN-Egr-transgenic mice (Tables II induces the expression of Pax-5. Pax-5 shuts down alternate lin- and III). eage specifications and promotes commitment to the B cell fate We think that Egr-1 has an intrinsic role in B cells, because BCR

(46–48). In the periphery, Bcl-6, another key transcription factor, signal strength affects the development of B cell subsets, as shown http://www.jimmunol.org/ is important for the maintenance of germinal center B cells (50). by Casola et al. (53) and in several other studies that use BCR The phenotype observed in both the knockout and the transgenic signal-deficient mutant mice (54). The fact that the DN-Egr-1 mice mice is contrasting and at the same time highlights the importance that express the DN-Egr protein in a B cell-specific manner exhibit of the relative roles of Egr family members in B cell biology. As a similar MZ B cell defect further strengthens this argument. One noted earlier, there are four family members, namely, Egr-1, Egr-2, such factor regulated by Egr-1 could be Notch2, which has been Egr-3, and Egr-4. The increase in B lymphopoiesis in Egr-1Ϫ/Ϫ shown to be important for MZ B cell development (55). Delta-like could be due to a negative role of Egr-1 in B cell development or 1 (DL1), one of the Notch ligands is also important for MZ B cells due to a compensatory increase in other family members that fur- and is originally reported to be expressed by B cells (56) but a ther enhance B lymphopoiesis. The later possibility is supported by more recent paper by Moriyama et al. (57) reports that DL1 is by guest on September 29, 2021 the finding that B lymphopoiesis is similarly decreased both in the expressed by macrophages and dendritic cells but not B cells. It is bone marrow and spleen of DN-Egr mice. The fact that Egr-1Ϫ/Ϫ conceivable that Egr-1 regulates DL1 expression in macrophages B cells hyperproliferate and have increased expression of Egr-2 or some other stromal cell molecule that affects MZ B cell devel- and Egr-3 provides further support to the second model. The pos- opment. The possibility that Egr-1 may affect stromal cells rather sibility of different role for various Egr family members cannot be than B cells cannot be completely ruled out at this time. ruled out. Egr family members are important not only for B lymphocyte Egr-1, however, has a nonredundant role in the development of development but also for B cell clonal expansion initiated by B cell MZ B cells, because this population is significantly decreased in triggering. Just as in overall B lymphopoiesis, Egr-1 function can both Egr-1Ϫ/Ϫ and the DN-Egr-transgenic mice. This is consistent be substituted by other family members in the BCR-induced pro- with the notion that BCR signaling is required for MZ B cell de- liferation responses as shown by their increase in Egr-1Ϫ/Ϫ B cells, velopment (51). Similarly, Egr-1 appears to be uniquely required and the ability of ectopically expressed Egr-2 to overcome BCR- for the high rate of expression of BP-1, which is decreased in induced growth inhibition of immature B cell lymphoma cells. Egr-1Ϫ/Ϫ mice. Presently, it is not precisely clear at what stage of When the functions of all Egr family members are suppressed by B cell commitment does Egr-1 have a regulatory role. It is possible DN-Egr, then the follicular B cells are hyporesponsive. The re- that Egr-1 might be affecting the developmental stages as early as quirement for Egr family members is unique for BCR pathway, the commitment of CLPs toward the B cell lineage. This hypoth- because B cell proliferation induced by TLR4 or CD40 signaling esis is supported by our preliminary observations that DN-Egr- is not affected either in Egr-1Ϫ/Ϫ or in DN-Egr mice. This obser- transgenic mice have a 2-fold increase in B220ϩSca-1low early vation is consistent with the findings that the BCR signaling defect lineage cells, which are defined as CLPs in the bone marrow (45). observed in DN-Egr mice is important for in vivo B cell clonal Moreover, the model put forth by Singh and coworkers suggests expansion as Ab responses to both TI-2 and TD Ags are decreased. that low levels of PU.1 (transcription factor required for both B Upon BCR ligation, there is a significant increase in Egr-1 mes- and myeloid lineage commitment) in B cells activates Egr-1/2, sage as well as protein levels in Egr-1ϩ/ϩ B cells. In contrast to thus establishing a connection between PU.1 and Egr-1 in lineage Egr-1, resting B cells express a high basal level of Egr-2 protein stability (52). Alternatively, high PU.1 activates Egr-1 very which is being modified (most likely phosphorylated) upon BCR strongly and promotes macrophage differentiation. These data fur- ligation in Egr-1Ϫ/Ϫ but not wild-type B cells. This is the first ther support our observations that Egr family members regulate B report of Egr-2 being phosphorylated, a property it shares with cell commitment at a very early stage of their development in the Egr-1. This modification is likely to be important because others bone marrow. have shown that phosphorylation vs acetylation potentially mod- The BCR signal strength model put forward by Pillai et al. pro- ulates the activity of Egr-1 in terms of survival and apoptosis in poses that if the BCR of the B cell reacts fairly well (intermediate prostate cancer cells (5). In addition, we find that Egr-2 and Egr-3 The Journal of Immunology 4601

Ϫ Ϫ message levels are elevated upon BCR cross-linking in Egr-1 / 15. Krishnaraju, K., B. Hoffman, and D. A. Liebermann. 2001. Early growth response B cells. Because Egr family members share 90% homology in their gene 1 stimulates development of hematopoietic progenitor cells along the mac- rophage lineage at the expense of the granulocyte and erythroid lineages. Blood Ϫ/Ϫ DNA-binding region, we propose that in Egr-1 mice, Egr-2 97: 1298–1305. and possibly Egr-3 assume a critical compensatory role. Whether 16. Krishnaraju, K., B. Hoffman, and D. A. Liebermann. 1998. The zinc finger tran- scription factor Egr-1 activates macrophage differentiation in M1 myeloblastic such a compensation involves regulation of some of the Egr-1 leukemia cells. Blood 92: 1957–1966. target genes remains to be explored. At present, target genes that 17. Krishnaraju, K., H. Q. Nguyen, D. A. Liebermann, and B. Hoffman. 1995. The are critically modulated by Egr-2 are not known except that Egr-2 zinc finger transcription factor Egr-1 potentiates macrophage differentiation of hematopoietic cells. Mol. Cell. Biol. 15: 5499–5507. regulates Fas ligand expression in T cells (58, 59). This hypothesis 18. Lee, S. L., Y. Wang, and J. Milbrandt. 1996. Unimpaired macrophage differen- is strengthened by our observations that overexpression of Egr-2 tiation and activation in mice lacking the zinc finger transplantation factor partially overcomes BCR-induced growth inhibition in an imma- NGFI-A (EGR1). Mol. Cell. Biol. 16: 4566–4572. 19. Ke, J., M. Gururajan, A. Kumar, A. Simmons, L. Turcios, R. L. Chelvarajan, ture B cell lymphoma model. D. M. Cohen, D. L. Wiest, J. G. Monroe, and S. Bondada. 2006. The role of We conclude that Egr family members are positive modulators MAPKs in B cell receptor-induced down-regulation of Egr-1 in immature B of B lymphopoiesis, MZ B cell development, BCR-induced B cell lymphoma cells. J. Biol. Chem. 281: 39806–39818. 20. Maltzman, J. S., J. A. Carman, and J. G. Monroe. 1996. Role of EGR-1 in proliferation, and T-independent type 2 and T-dependent immune regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. responses (Fig. 6). Our observations demonstrate that Egr-1 is crit- Cell. Biol. 16: 2283–2294. ical for MZ B cell development and its associated TI-2 immune 21. Maltzman, J. S., J. A. Carmen, and J. G. Monroe. 1996. Transcriptional regula- tion of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lym- responses. This phenotype is very significant because there are phocytes: role for transcription factor EGR1. J. Exp. Med. 183: 1747–1759. only few instances in which deficiency of a transcription factor 22. Carman, J. A., and J. G. Monroe. 1995. The EGR1 protein contains a discrete leads to a defect in MZ B cell development and TI-2 responses transcriptional regulatory domain whose deletion results in a truncated protein Downloaded from that blocks EGR1-induced transcription. DNA Cell Biol. 14: 581–589. (51). Currently, we are examining some of the target genes that 23. Dinkel, A., K. Warnatz, B. Ledermann, A. Rolink, P. F. Zipfel, K. Burki, and could be potentially modified by Egr-2 in lymphoma cells over- H. Eibel. 1998. The transcription factor early growth response 1 (Egr-1) advances expressing this transcription factor. Moreover, mice deleted for differentiation of pre-B and immature B cells. J. Exp. Med. 188: 2215–2224. 24. Glynne, R., S. Akkaraju, J. I. Healy, J. Rayner, C. C. Goodnow, and D. H. Mack. Egr-2 and or Egr-3 could reveal some interesting roles for Egr-2 2000. How self-tolerance and the immunosuppressive drug FK506 prevent B-cell and Egr-3 in B cell growth response. Further studies on Egr family mitogenesis. Nature 403: 672–676. 25. Merrell, K. T., R. J. Benschop, S. B. Gauld, K. Aviszus, D. Decote-Ricardo, members will provide new insights into novel players in B cell http://www.jimmunol.org/ L. J. Wysocki, and J. C. Cambier. 2006. Identification of anergic B cells within development. a wild-type repertoire. Immunity 25: 953–962. 26. Seyfert, V. L., S. B. McMahon, W. D. Glenn, A. J. Yellen, V. P. Sukhatme, X. M. Cao, and J. G. Monroe. 1990. Methylation of an immediate-early inducible Acknowledgments gene as a mechanism for B cell tolerance induction. Science 250: 797–800. We thank Dr. E. C. Snow for critical reading of the manuscript. We also 27. Glynne, R., G. Ghandour, J. Rayner, D. H. Mack, and C. C. Goodnow. 2000. thank Jennifer Strange and Greg Bauman for help with flow cytometry. B-lymphocyte quiescence, tolerance and activation as viewed by global gene expression profiling on microarrays. Immunol. Rev. 176: 216–246. 28. Muthukkumar, S., S. S. Han, V. M. Rangnekar, and S. Bondada. 1997. Role of Disclosures Egr-1 gene expression in B cell receptor-induced apoptosis in an immature B cell The authors have no financial conflict of interest. lymphoma. J. Biol. Chem. 272: 27987–27993.

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