The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wikström, I.
Right arrow Articles by Holmberg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wikström, I.
Right arrow Articles by Holmberg, D.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Stem Cells
The Journal of Immunology, 2006, 177: 6723-6729.
Copyright © 2006 by The American Association of Immunologists, Inc.

E2-2 Regulates the Expansion of Pro-B Cells and Follicular versus Marginal Zone Decisions1

Ingela Wikström2,*, Johan Forssell2,*, Mario Goncalves3,*, Francesco Colucci{dagger} and Dan Holmberg4,*

* Department of Medical Biosciences, Umeå University, Umeå, Sweden; and {dagger} Unité des Cytokines et Développement Lymphoïde, Institut Nationale de la Santé et Recherche Médicale Equipe 101, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The E-proteins E2A, HeLa E-box binding protein, and E2-2 constitute a class of basic helix-loop-helix transcription factors that differentially affect B cell development. E2A is by far the most investigated and appears to operate at several levels during B cell ontogeny. Less is known concerning the role of the other E-proteins. To address the role of E2-2, we have performed transfers of fetal liver (FL) cells into irradiated Rag-deficient mice. Although the transfer of E2-2-deficient cells alone can reconstitute all B cell subpopulations, albeit with a moderate reduction in cellularity, E2-2-deficient cells have a disadvantage when transferred together with wild-type cells. Cultivation of E2-2–/– day 14.5 FL cells on stromal cells and IL-7 revealed a reduced frequency of responding B cell progenitors despite normal IL-7R{alpha} surface expression. Real-time PCR analysis revealed that E2-2 mRNA expression is high at the pro-B cell stage and drops sharply at the pre-B cell stage, consistent with a role for E2-2 in pro-B cells. In contrast, E2A mRNA was most abundant in pre-B cells. Analysis of the peripheral repertoire revealed that mice reconstituted with E2-2–/– FL cells had an increased proportion of marginal zone (MZ) B cells. Interestingly, E2-2 mRNA was elevated ~2-fold (p < 0.01) in follicular compared with MZ B cells. Although E2A mRNA showed a similar tendency, the difference was not significant. Collectively, our findings indicate that E2-2 is required for optimal expansion of pro-B cells, and also influences the follicular vs MZ decision.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
B cell differentiation from hemopoietic progenitors depends on stromal cell-derived factors, rearrangements of IgH and IgL chains, and appropriate expression of B lineage-associated genes (1). A complex network of transcription factors that ensures proper expression of B lineage-specific genes, while repressing alternative gene expression programs, regulates B lineage commitment. Key roles in this process have been assigned to E2A, early B cell factor (EBF),5 and Pax-5 (2). E2A is a basic helix-loop-helix transcription factor that, together with HeLa E-box binding protein (HEB) and E2-2, constitutes the E-protein family (3, 4, 5). E-proteins are broadly expressed and can, depending on cell-type, form heterodimers with other tissue-specific helix-loop-helix proteins. However, in lymphocytes, E-proteins appear to operate primarily as hetero- or homodimers with other E-proteins (6). The activity of E-proteins are counteracted by the Id proteins, which can efficiently heterodimerize with E-proteins and inhibit their binding to DNA in a dominant-negative manner (7, 8). There are four vertebrate Id proteins, Id1–4 (7, 8, 9, 10), of which only Id2 and Id3 are abundantly expressed in B and T lymphocytes (1, 11).

Targeted mutations have revealed critical roles for the E-proteins during both T- and, in particular, B-lymphocyte development (6). Thus, in the absence of E2A, B lymphopoiesis is completely inhibited (12, 13). A similar phenotype is seen in mice transgenic for Id1, an inhibitor of E-protein activity (14). B cell development in the absence of E2A can be partially restored by EBF expression, indicating that EBF is a critical target of E2A (15). E2A plays an additional role during peripheral B lymphopoiesis through its apparent involvement in receptor editing and regulation of marginal zone (MZ) vs follicular B cell fates (11). Moreover, E2A is induced upon B cell activation and is required for Ig class switching (16).

Although most of the work conducted so far has been focused toward directly addressing the function of E2A, it is clear that HEB and E2-2 are also involved in the regulation of lymphocyte development. Direct analysis of HEB–/– and E2-2–/– mice is hampered by their postnatal lethality, and data on these E-proteins are scarce, but analysis of day 18.5 fetal liver (FL) cells from HEB–/– and E2-2–/– embryos revealed a roughly 2-fold reduction in pro-B cells (17). Furthermore, mice transheterozygous for E2A and HEB or E2-2 have a notable reduction of B lymphopoiesis compared with E2A heterozygous mice (17). Moreover, knock-in of the HEB gene into the E2A locus through gene targeting could functionally rescue B cell development caused by deletion of E2A (18). Thus, several lines of evidence suggest considerable redundancy among the different E-proteins, and such observations have contributed to the view that overall timing and dosage of E-protein activity may be the key determinant in B cell progression rather than the activity of a specific E-protein (19). This would in turn be determined primarily by the amount of available E-protein and the E:Id protein ratio. Therefore, it is reasonable to assume that at least some of the specificity among the different E-proteins should be reflected by their respective expression pattern at a given E-protein-regulated selection point.

Despite the fact that HEB and E2-2 clearly influence lymphocyte development, their roles have remained poorly characterized. Previous studies in our laboratory have detected a profound disadvantage of E2-2-/lox alleles in the spleen and B cells from c-kitCre-mosaic mice, but not in several other organs (20). To further address the role of E2-2 in B cell development, we performed a series of transfer experiments to investigate the ability of E2-2-deficient B cell progenitors to mature in Rag-deficient mice under different conditions. This approach was combined with real-time PCR analysis of E protein mRNA expression in sorted wild-type B cell populations to gain an estimation of specific E-protein fluctuations in different populations, ultimately aiming to discriminate between E2-2-specific and global E-protein-regulated events. Our findings point to a role for E2-2 operating at the pro-B cell stage, where high mRNA expression was observed, to facilitate B cell commitment and to expand early B lineage cells. In addition, we provide evidence that E2-2-deficient cells modulate the follicular vs MZ decision, and that this correlates with modulation of E2-2 mRNA levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mouse strains and screening

E2-2+/– mice were previously generated in our laboratory (20). E2A+/– and HEB+/– mice were provided by Dr. Y. Zhuang (Duke University Medical Center, Durham, NC). Each of these strains was backcrossed to C57BL6/J mice. Heterozygous animals were crossed to get homozygous knockout mice, and each litter was genotyped by PCR using the following primers: E2-2: 5'-TGT GTA TGT GCA TAT TTC CTT TAT ATT CC-3', 5'-AGA TAC CAT GCG CAG AAA TCT TT-3', and 5'-ATG TAA CTG GTA GTT CAG GAC AAC AAA-3'; E2A, 5'-ATG TGT GGT GGC CCA CAC TTG T-3' and 5'-CGC AAC CTG AAC CCC AAA GC-3'; HEB, 5'-GAC ATC AAG GTC TCA TCT AGG-3' and 5'-TCT CAC TTG CTG TTC TAG ACT-3'; E2A/HEB, 5'-TCG CAG CGC ATC GCC TTC TA-3'.

Animals were maintained in the animal facilities of Umeå University, under pathogen-free conditions, according to institutional animal care and use guidelines. The local animal ethical committee approved all animal procedures. The Rag2–/–{gamma}c–/– mice were kept at the animal facilities of Institut Pasteur.

Flow cytometry cell sorting

Spleens and bone marrow were removed, and cell suspensions were prepared in HBSS. Spleen suspensions were depleted of erythrocytes with Gey’s lysis buffer. Cells were then stained with anti-B220 biotin, anti-CD43 FITC, and anti-IgM Cy5 for defining pro-B, pre-B, and total B cells—with anti-IgM Cy5, anti-CD21 FITC, and anti-CD23 PE to identify MZ and follicular B cells, and with anti-Ly5.1 PE, anti-Ly5.2 biotin, to define Ly5.1 and Ly5.2 donor cells. All Abs were purchased from BD Pharmingen. Briefly, cells were washed in FACS medium (PBS containing 3% FCS and 0.01% sodium azide) and stained for 20 min at 4°C. The cells stained with biotinylated Abs were then washed, and a streptavidin-CyC conjugate was added for 20 min at 4°C. Cell sorting was performed on a FACSVantage DiVa cytometer (BD Biosciences), and cells were collected in PBSA with 3% FCS. All sorted cell populations were >97% pure.

RNA and cDNA preparation

Cells were washed with 1x PBS and stored at –86°C. RNA was prepared from these cells using RNeasy Mini Kit (Qiagen; VWR International), according to the manufacturer’s instructions, and dissolved in 40 µl of RNase-free water followed by treatment with DNase I (Ambion, Applied Biosystems). RNA concentrations were measured using a spectrophotometer, and cDNA was prepared from 300 ng of total RNA using the Reverse Transcription Reagents (TaqMan) purchased from Applied Biosystems.

Real-time PCR analysis

Primers and probes used in this study to measure the RNA expression for E2A were as follows: forward, 5'-TTC TCC TCC CGC TTG ATC TC-3'; reverse, 5'-AGT TCC CTC CCT GAC CTC TCA-3'; probe, 5'-(VIC)-CCT GCC CTC CCG AGT CCA CTA TAG GA-(TAMRA)-3'; and endogenous control genes (acidic ribosomal phosphoprotein (36B4): forward, 5'-CCCTGAAGTGCTCGACATCA-3'; reverse, 5'-TGCGGACACCCTCCAGAA-3'; probe, 5'-(VIC)-AGAGCAGGCCCTGCACTCTCGC-(TAMRA)-3') were designed using Primer Express (Applied Biosystems) and purchased from Applied Biosystems.

HEB, E2-2, and Id1–4 assays were ordered as "assay on demand" from Applied Biosystems: HEB, Mm00441699_m1; E2-2, Mm00443198_m1; Id1, Mm00775963_g1; Id2, Mm00711781_m1; Id3, Mm00492575_m1; Id4, Mm00499701_m1.

Relative expression of the transcripts was measured in the ABI Prism 7900HT Sequence detection system (Applied Biosystems), and E2A relative RNA levels were determined using the "standard curve method" as described in the Applied Biosystems users bulletin (21); standard curves were constructed for all cDNAs. Relative RNA quantification of HEB, E2-2, and Id1–4 was performed using the "Comparative Ct (comparative threshold cycle) method" as described in the Applied Biosystems users bulletin (21). Briefly, the amount of target transcript normalized to the endogenous control gene 36B4 and relative to a calibrator sample (total RNA prepared from naive B6 thymus is given by the following formula: 2–{Delta}{Delta}Ct, where {Delta}{Delta}Ct = (Cttarget – Ctendogenous control) sample in study – (Cttarget – Ctendogenous control) calibrator, and the value of the calibrator sample was set as 1.

FL transfers

FLs were dissected out from E14-E15 E2-2-deficient embryos and their littermates. Cell suspensions were prepared in HBSS with 3% FCS. The cells were shipped to Institut Pasteur and injected into irradiated Ly5.1Rag2–/–{gamma}c–/– mice, 2–4 x 106 cells/mouse. After 8–9 wk, the mice were sacrificed and the cell compartments were analyzed by flow cytometry. For chimeras, FLs dissected as described above were mixed at a 1:1 ratio with gestation day matched wild-type Ly5.1 congenic FL cells. Mixes of ~30 x 106 cells were injected into irradiated Rag–/– mice, and these mice were subsequently sacrificed for flow cytometric analysis 8–9 wk later.

Limiting dilution and FL cultures

FL cells were dissected out from E14-E15 E2-2-deficient embryos and their littermates. To estimate the frequency, FL cells from 5 to 6 pooled embryos of each genotype were plated and serially diluted in 96-well plates (starting at 40,000 cells/well) containing a semiconfluent layer of irradiated S-17 cells, in Iscoves medium supplemented with 10% FCS and 2.5% IL-7-conditioned medium (22). All wells (16–24 wells/cell concentration) were scored for lymphoid growth after 8–9 days, and the frequency of responding cells was calculated as described previously (23). FL cultures that were >95% B220+ were generated by pooling responding wells from each genotype and reculturing with IL-7 on freshly irradiated stroma every 3–4 days. After reculturing 5–6 times on stroma and IL-7, pro-B cells were seeded at 105 cells/well in 24-well plates, and viable cell counts were determined by trypan blue exclusion after 3 days. For some experiments, Ly5.2 E2-2+/+, E2-2+/–, and E2-2–/– cells were mixed with Ly5.1 congenic wild-type cells in a 1:1 ratio, and the mixes were recultured at 105 cells/well. After 3 days, cells were counted and the Ly5.2:Ly5.1 ratio was determined by FACS analysis.

For Ig secretion analyses, pro-B cells were washed twice with medium devoid of IL-7 and recultured on irradiated S-17 in Iscoves medium devoid of IL-7, but containing 10 µg/ml LPS. After 6 days, supernatant was collected, filtered, and analyzed with ELISA. Plates were coated overnight at 4°C with 5 µg/ml capturing Ab (unlabeled goat anti-mouse IgM and IgG; Southern Biotechnology Associates). Plates were washed and blocked for 2 h with BSA, washed again, and the filtered cell supernatant was serially diluted and incubated for 1 h at room temperature. Following six washes in PBS, AP-conjugated goat anti-mouse IgM or IgG (Southern Biotechnology Associates) was added at 1:1000 and incubated for 45 min. Plates were developed with p-nitrophenyl tablets (Sigma-Aldrich) dissolved in 100 mM glycin containing 10 mM MgCL2 and 10 mM ZnCL2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
E2-2 is predominantly expressed at the pro-B cell stage

The redundant nature of E-proteins makes predictions concerning their specific roles during B cell development a delicate task. However, one should be able to make some predictions about their individual roles by determining their expression profiles during different developmental stages. Using such reasoning as a basis, we analyzed the mRNA expression of the various E proteins in FACS-sorted populations by a real-time quantitative PCR approach. Interestingly, E2-2 displayed a pronounced expression at the pro-B cell stage, with a sharp drop in pre-B cells, and low expression in splenic B cells (Fig. 1A). In contrast, the E2A level was found to be relatively low in pro-B cells, followed by a profound increase at the pre-B cell stage, and was down-regulated again in splenic B cells (Fig. 1B). HEB was very scarcely expressed in any of these populations (Fig. 1C). These results indicate that HEB has only a minor role during B cell development, whereas E2A would be predicted to function at several stages, most notably at the pre-B cell stage. The restricted expression pattern of E2-2 suggests a specific role at the pro-B cell stage, which only partially overlaps with E2A.


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 1. Predominant expression of E2-2 at the pro-B cell stage. Cells at specific stages of B cell development were sorted and distinguished as follows; pro-B cells (B220+CD43+), pre-B cells (B220+CD43–), and total splenic (spl) B cells (B220+). Expression of E2-2 (A), E2A (B), and HEB (C) mRNA in the respective populations were measured using TaqMan real-time PCR and normalized to an endogenous control (36B4) and a calibrator set to 1. SEM is calculated from three independent experiments using B6 mice.

 
E2-2 deficiency causes an impaired expansion of B lineage cells

To more precisely identify the stages at which B lineage cells lacking E2-2 are compromised, we transferred E14–15 FL cells from E2-2+/+, E2-2+/–, and E2-2–/– embryos into sublethally irradiated Rag2{gamma}c-deficient mice. Following 8–9 wk of reconstitution, the number of pro-B cells (B220+CD43+) and pre-B cells (B220+CD43–) was determined in the bone marrow of the reconstituted mice. E2-2–/– FL cells had a 40–50% reduction in cellularity at both the pro-B and pre-B cell stages (Fig. 2A). We also analyzed the peripheral B cell populations in the reconstituted mice. E2-2–/– B cells showed reconstitution with a roughly 30% reduction in peripheral B cell numbers, compared with 40–50% in the bone marrow (Fig. 2B). We could not detect any significant difference between E2-2+/– and E2-2–/– FL cells in the capacity to compensate by expanding IgM+ cells (Fig. 2B).


Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 2. Impaired expansion of E2-2-deficient B lineage cells. A, Irradiated Rag2–/–c{gamma}–/– mice reconstituted with E2-2+/+ (n = 3), E2-2+/– (n = 3), or E2-2–/– (n = 5) FL cells were sacrificed after 8 wk, and the average number of Ly5.2+ cells was determined at the pro-B (B220+CD43+) and pre-B stages (B220+CD43–) in the bone marrow (BM). B, Average number of IgM+ cells in the spleen.

 
A higher proportion of MZ B cells in the periphery of E2-2–/–-reconstituted mice correlates with the differential expression pattern of E2-2 in follicular vs MZ B cells

To determine whether the absence of E2-2 is translated into any alterations in the composition of the peripheral repertoire, we also analyzed the number of follicular and MZ B cells. The follicular B cell numbers were decreased by ~30%, whereas MZ B cells were formed in normal numbers in E2-2–/–-reconstituted mice, thus promoting a higher proportion of B cells with a MZ phenotype (Fig. 3A). Interestingly, analysis of E-protein expression in sorted populations revealed higher E2-2 levels in follicular compared with MZ B cells (Fig. 3B). As observed in pro-B cells, E2A showed an expression pattern overlapping with E2-2s and with a similar tendency for higher expression in follicular B cells (Fig. 3B). Notably, however, whereas the difference in E2-2 expression was statistically significant using Student’s t test (p = 0.004), this was not the case for E2A (p = 0.112).


Figure 3
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 3. A relative enrichment of MZ B cells in E2-2–/–-reconstituted mice. A, Spleens were removed from mice reconstituted with FL from E2-2+/+ (n = 3), E2-2+/– (n = 3), or E2-2–/– (n = 5) mice after 8–9 wk. The cells were stained for MZ B cells (IgM+CD21highCD23low) and follicular B cells (IgM+CD21+CD23+). B, Average numbers of follicular B cells and MZ B cells. C and D, Follicular B cells and MZ B cells were sorted from 6- to 8-wk-old B6 mice, and the expression of E2-2 and E2A (C) and Id2 and Id3 (D) were measured using TaqMan real-time PCR. Average values are from the analysis of three separate cell sortings. Values of p were calculated using Student’s t test.

 
Id proteins down-modulate the activity of E-proteins, but do not appear to alter expression levels (24). To get a more complete picture of how E-protein activity is modulated in follicular vs MZ B cells, we analyzed Id mRNA expression in sorted cell populations. Expression studies of Id1–4 revealed that only Id2 and Id3 were expressed in the sorted cell populations (Fig. 3C and data not shown). Id3 levels were higher in follicular B cells than in MZ B cells, whereas the opposite expression pattern was seen for Id2.

The impaired expansion of E2-2–/– cells is more pronounced in a competitive environment

To analyze the ability of E2-2–/– FL cells to compete with wild-type cells, we transferred day 14.5 FL cells from E2-2+/+, E2-2+/–, and E2-2–/– embryos (all Ly5.2) into sublethally irradiated Rag-deficient mice as 1:1 mixes with Ly5.1 congeneic wild-type FL cells (FL chimeras). Following 8–9 wk of reconstitution, the number of Ly5.2+ pro-B (B220+CD43+), pre-B (B220+CD43–), and B220+IgM+ B cells was determined in the bone marrow of the chimeras. These analyses revealed that E2-2–/– FL cells have a dramatic disadvantage to reconstitute B cell compartments when developing with competing wild-type cells (Fig. 4A). There seemed, however, to be no exacerbation of the phenotype as cells progressed from pro-B to IgM+ cells, because the ratio of Ly5.2:Ly5.1 cells was unaltered at these different stages (Fig. 4A). We also analyzed the peripheral B cell populations in the chimeric mice. E2-2–/– cells displayed a disadvantage to develop into B220+IgM+ cells when competing with wild-type cells, albeit less pronounced compared with observations in the bone marrow (i.e., a 3-fold reduction compared with 5- to 6-fold in the bone marrow; Fig. 4B).


Figure 4
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 4. A competitive disadvantage for E2-2-deficient cells to reconstitute B cell populations in the bone marrow of irradiated Rag2–/– mice. A, Ly5.2+ FL cells of E2-2+/+ (n = 3) or E2-2–/– (n = 3) origin were mixed at a 1:1 ratio with Ly5.1+ wild-type FL cells and injected into irradiated Rag2–/– mice. Following 8 wk of reconstitution, the number of Ly5.2+ cells in the bone marrow was determined at the pro-B (B220+CD43+), pre-B (B220+CD43–), and IgM+ B cell stages, represented as the average cell number and SEM. B, Average number of Ly5.2+IgM+ in the spleen.

 
Reduced frequency of IL-7-responsive cells in E2-2–/– FL cultures

The fact that the profound disadvantage of E2-2-deficient cells to reconstitute the B cell compartment manifests at an early stage in the mixed wild-type/E2-2 chimeras, combined with the observed reduction in cellularity when E2-2-deficient cells were transferred alone, points to a role for E2-2 in expanding early B cell progenitors. We therefore cultivated FL cells from E2-2–/–, E2-2+/–, and E2-2+/+ day 14 or 15 embryos on an irradiated layer of semiconfluent S-17 stromal cells in the presence of IL-7. After growing FL cells in bulk cultures on S-17 and IL-7 for 6–9 days, wild-type cultures consisted mainly of small round cells that were 50–90% B220+ cells, whereas E2-2–/– cultures contained only 13–24% B220+ cells with an abundance of irregular cells. Although we observed a 5- to 10-fold reduction of B220+ cells in E2-2–/– cultures compared with wild-type cultures, intermediate numbers were observed in E2-2+/– cultures, supporting the notion that E2-2 was required in a dose-dependent fashion for optimal cell expansion (data not shown). These data suggest that E2-2 plays an important role in expanding early B cell progenitors. In line with this result, FACS analysis revealed that the proportion of B lineage cells marked with B220 and CD43 at day 15.5 of gestation, dropped down from 1.57%, of the total FL cell population in wild-type animals, to 1.29 and 1.20%, for E2-2+/– and E2-2–/–, respectively (Fig. 5). At day 17.5, the corresponding numbers were 4.22% in wild-type, 3.37% in E2-2+/–, and 2.65% in E2-2–/– mice (Fig. 5). This is comparable to results reported by Zhuang et al. (17) in day 18.5 embryos.


Figure 5
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 5. A moderate reduction of pro-B cell numbers in E2-2-deficient FLs. FL cells from E2-2-deficient E15.5 (A) and E17.5 (B) embryos and littermate controls were stained with B220 and CD43, and the percentage of pro-B cells (B220dullCD43+) were determined in E2-2–/–, E2-2+/–, and E2-2+/+ mice (n = 2 for each genotype).

 
Although this difference explains some of the reduced E2-2–/– cell numbers, it cannot account for the 4- to 5-fold dominance of E2-2+/+ cells over E2-2–/– cells when transferred at a 1:1 ratio mix in the chimeras. We therefore analyzed the ability of unsorted bulk FL cells to give rise to B cell growth when serially diluted on S-17 stromal cells and supplemented with IL-7. Wells were scored positive for B lymphoid growth when colonies of small, round cells appeared within 10 days of culture initiation. Colonies defined by those morphological criteria were shown to express B220 and secrete Igs (IgM and IgG) when stimulated for 6 days with LPS in the absence of IL-7 (data not shown). To estimate the frequency of IL-7-responding cells, FL cells from 5 to 6 pooled embryos of each genotype were plated and serially diluted. Cell culture plates were scored for lymphoid growth after 8–9 days, and the frequency of responding cells was determined (23). As illustrated in Fig. 6 and Table I, 1 of 8 of the B220+/CD43+ pro-B cells were found to respond in wild-type mice, whereas this frequency was reduced to 1 of 29 and 1 of 62 in E2-2+/– and E2-2–/– embryos, respectively.


Figure 6
View larger version (9K):
[in this window]
[in a new window]

  		FIGURE 6. Reduced IL-7 responsiveness in vitro of unsorted of E2-2-deficient FL cells. FL cells from E2-2–/–, E2-2+/–, and E2-2+/+ were plated onto irradiated S-17 stromal cells and serially diluted in 96-well plates with IL-7-supplemented medium. B lymphoid growth was evaluated after 8–9 days, and the frequency of responding cells was determined as the cell concentration where 37% of the wells were nonresponding.

 

View this table:
[in this window]
[in a new window]

  		Table I. B lymphoid potential of fetal liver cells

 
Normal IL-7R{alpha} expression in E2-2–/– FL cells and pro-B cell lines

One possible explanation for the impaired IL-7 responsiveness could be the reduced expression of the IL-7R. Low but similar levels of IL-7R{alpha} were detected among B220+ FL cells from the different genotypes (Fig. 7A), indicating that reduced expression of the IL-7R was not causing the reduced responsiveness. Likewise, there were no differences seen in IL-7R{alpha} expression when comparing pro-B cell lines established from the different genotypes (Fig. 7B).


Figure 7
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 7. E2-2-deficient FL cells express normal levels of IL-7R{alpha}. A, The expression of IL-7R{alpha} was determined by FACS analysis of freshly isolated FL cells. FL cells stained with secondary reagent only (thick line) are overlaid with IL-7R{alpha}-stained E2-2–/– FL cells (thin line) and E2-2+/+ FL cells (dotted line). B, IL-7R{alpha} expression on IL-7-dependent E2-2+/+ and E2-2–/– pro-B cells. The shaded area is from staining with secondary reagent only. The continuous, non-IL-7-dependent but IL-7R{alpha}+, pre-B cell line 38B9 was used as a positive control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Based on the marked expression of E2-2 mRNA at the pro-B cell stage, E2-2 could be predicted to play a role at this stage of B cell development. This is functionally supported by the impaired expansion of pro-B cells observed in E2-2–/–-reconstituted mice and the defective IL-7 responsiveness of E2-2–/– FL B cell progenitors in vitro. Because E2A is also significantly expressed at the pro-B cell stage, and has been suggested to be similarly important for optimal IL-7 responsiveness in vitro (15), it is difficult to evaluate the relative contribution of these two E-proteins at this stage of B cell development.

The sharp down-regulation of E2-2 to low levels at the pre-B cell stage may indicate that E2-2 plays no major role in the selective events that occur in the bone marrow at the pro- to pre-B cell stages or after IgM appears at the cell surface. Accordingly, mice reconstituted with E2-2–/– FL cells were able to generate all populations of B cells, albeit with a 40–50% reduction in cellularity. In contrast to E2-2, E2A mRNA expression increased significantly at the pre-B cell stage and was down-regulated again in mature B cells. This resembles data by Herblot et al. (25), where E2A up-regulation in pre-B cells and subsequent down-regulation in mature B cells was also seen at the protein level. A similar expression pattern was observed in E2A-GFP-fused knock-in mice, with the exception that E2A-GFP levels did not increase further in pre-B cells (24). Together, our mRNA expression data would predict roles for E2A, but not E2-2, beyond the pro-B cell stage in the bone marrow. Indeed, E2A has been suggested to be involved in receptor editing, based on impaired abilities for secondary rearrangements following expression of an autoreactive BCR in E2A+/– mice (11). In contrast, the results from the expression analysis of HEB performed in the present study indicate a minor role, if any, for this E protein in B cell development.

Mice reconstituted with E2-2–/– FL cells displayed a decrease in follicular B cells, whereas MZ B cells numbers were normal. E2A has previously been suggested to regulate MZ B vs follicular B decisions based on similar observations in E2A heterozygous mice (11). Conversely, increasing the available E-protein levels through deletion of Id proteins results in a decreased proportion of MZ B cells in both Id2 and Id3-deficient mice (11, 26). These results fit well the significantly higher E2-2 mRNA expression that we observed in follicular compared with MZ B cells and the similar (albeit weaker) tendency for E2A. The opposite expression patterns of Id2 and Id3 makes predictions concerning their functional role in this event more difficult. Nevertheless, all these studies converge on the observation that decreasing the available E-protein levels, either by decreasing the E2A levels (11) or E2-2 levels (present study) results in a decrease in the follicular B cell compartment, whereas increasing the available E-protein activity by removing Id2 (26) or Id3 (11) favors follicular B cells over MZ B cells.

The fact that E2-2 and E2A appear to share roles both in early B cell development as well as in the selection of peripheral B cell subsets is consistent with the overlapping mRNA expression of these proteins detected at those stages, and could indicate that E2-2 and E2A co-operate tightly during B cell development, analogous to interactions described between HEB and E2A during T cell development (27, 28). In this context, it is interesting to note that mRNA expression of the stem cell leukemia (SCL) gene, an apparent negative regulator of E-proteins, was essentially overlapping with E2-2 expression, being high in pro-B cells and subsequently down-regulated to low/undetectable levels (25). Moreover, transgenic overexpression of SCL results in a phenotype similar to our E2-2–/–-reconstituted Rag mice, with a reduction in the number of pro-B cells that is essentially maintained up to mature B cells (25). Although this was interpreted by Herblot et al. (25) as a partial effect on E2A by virtue of SCL’s claimed effect on DNA binding of E2A homodimers, the DNA binding result on which this conclusion was based was somewhat unclear, partly because E2A DNA binding in pro-B cells appeared very low in both wild-type and SCL transgenics. In addition, it is puzzling that there were no apparent further effects of SCL overexpression past the pro-B cell stage, despite the fact that both E2A expression and particularly E2A DNA-binding activity was considerably higher at the pre-B cell stage compared with pro-B cells (25). It would, however, be fully compatible with an effect on E2-2, given the similar phenotype observed by SCL overexpression and E2-2 deficiency and the fact that E2-2 expression is down-regulated beyond the pro-B cell stage.

Collectively, the findings described in this study support the notion of a threshold model for E-protein activity (18), where a minimum E-protein threshold must be exceeded for B lineage entry and efficient pro-B cell expansion, but cells are further selected on the basis of total E-protein expression. Such a model would explain why the loss of E2-2 moderately reduces B cell numbers, whereas the situation changes dramatically if E2-2–/– progenitors are forced to compete with wild-type cells, causing a profound reduction in E2-2–/– B cells.

The impaired IL-7-responsiveness of E2-2–/– pro-B cell lines and FL cells is not a consequence of decreased expression of IL-7R{alpha}. It is previously shown that E2A proteins are required for IL-7-dependent proliferation, in part due to a role in optimal expression of N-myc (15), a transcription factor involved in cell proliferation and a known target for IL-7R signaling in B lymphocyte precursors (29). It is plausible, therefore, that this is the case also for E2-2-deficient cells.

Although the E-proteins appear to have roles throughout B cell development, the most striking B cell dependence on E-protein expression appears to be at B lineage entry. At the pro-B cell stage, E2-2 has an important role to promote B lineage entry that can be partially, but not fully, compensated for by the other E proteins. The partial overlap in expression between E2-2 and E2A, combined with the low expression of HEB, strongly suggests that it is mainly E2A that provides this redundancy. Nevertheless, the fact that HEB expression, driven by the endogenous E2A promoter, can rescue B cell development in E2A null mice (18), implies that the expression pattern to a large extent determines the different functions of E proteins.

These results may seem difficult to unite with the fact that E2-2 is totally unable to compensate for E2A deficiency to promote B lineage entry, despite high expression in pro-B cells. However, based on the E-protein expression profile, there is a fundamental difference facing developing E2-2–/– as opposed to E2A–/– cells that is likely to be relevant for the distinct outcome of the mutations. Because both E2-2 and E2A are expressed at the CD43+B220+ cell stage, deletion of either of them would be expected to impair B lineage entry. This is observed for both mutations, only to a more extreme extent for E2A. In case of E2-2 deletion, the formation of pro-B cells is reduced, but cells that can traverse this early block and progress to late pro-B cells will face a significant up-regulation of E2A, allowing B cell commitment and B cell developmental progression. In contrast, E2A-deficient progenitors would, even if B lineage entry occurred by some rare CD43+B220+ cells, instead face a dramatic drop in total E-protein levels (i.e., E2-2). Thus, E2A-deficient progenitors may face both an early block for B lineage entry, similar to E2-2–/– progenitors, but also an additional block caused by lack of the normal up-regulation of E-protein activity (in wild-type cells executed exclusively by E2A) at the pro-B to pre-B cell transition. This up-regulation of E2A proteins, concomitant with a dramatic drop in E2-2 levels, may be critical to provide the E-protein activity required for initiation of Ig-gene rearrangements and, more importantly, to drive the expression of B lineage-associated genes, promote developmental progression and B lineage commitment. This would be consistent with the report that E2A-deficient FL cells cannot reconstitute B cells when transferred to lethally irradiated mice, but rather remain pluripotent (30), similar to Pax-5–/– FL cells (31, 32).

Although differential expression alone, in our view, may provide important insight to the different functions of E2-2 and E2A, it cannot be excluded that E2A could have some features that make it unique in relation to E2-2. For example, the ability of E47 to form DNA-binding dimers might serve a critical function for B cell commitment, and although one of the alternatively spliced variants of E2A, E12, is able to form dimers, they bind DNA poorly owing to an inhibitory domain not active in E47 dimers or E12/E47 heterodimers (33). Whether E2-2 has a similarly acting inhibitory domain is to our knowledge unknown, as well as whether E2-2 can be subjected to the posttranslational modifications claimed to be required for E47 homodimerization (34). However, because HEB can apparently replace E2A (18), such limitations seem less likely to cause the total paucity of B cells in E2A null mice. Therefore, the idea that the functional diversification of E-proteins is dictated to a significant extent by their expression profile and threshold-regulated events appears an appealing hypothesis. Further studies on HEB, and in particular E2-2, are clearly needed for a comprehensive view on the role of E-proteins during lymphocyte development.


   Acknowledgments
 
We thank Dr. Leif Carlsson for the S-17 stromal cell line and Åsa Larefalk for cell sorting expertise.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Cancer Foundation. M.G. was supported by funds from Fundacao para a Ciencia e a Tecnologia, Portugal. I.W., J.F., and D.H. were supported by funds from the Swedish Cancer Foundation. Back

2 I.W. and J.F. contributed equally to this paper. Back

3 Mario Goncalves was deceased February 2006. Back

4 Address correspondence and reprint requests to Dr. Dan Holmberg, Department of Medical Biosciences, Division of Medical and Clinical Genetics, Umeå University, S-901 85 Umeå, Sweden. E-mail address: dan.holmberg{at}medbio.umu.se Back

5 Abbreviations used in this paper: EBF, early B cell factor; HEB, HeLa E-box binding protein; MZ, marginal zone; FL, fetal liver; Ct, comparative threshold cycle; SCL, stem cell leukemia. Back

Received for publication April 26, 2006. Accepted for publication August 15, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kee, B. L., C. J. Paige. 1995. Murine B cell development: commitment and progression from multipotential progenitors to mature B lymphocytes. Int. Rev. Cytol. 157: 129-179. [Medline]
  2. Busslinger, M.. 2004. Transcriptional control of early B cell development. Annu. Rev. Immunol. 22: 55-79. [Medline]
  3. Henthorn, P., M. Kiledjian, T. Kadesch. 1990. Two distinct transcription factors that bind the immunoglobulin enhancer microE5/{kappa}2 motif. Science 247: 467-470. [Abstract/Free Full Text]
  4. Hu, J. S., E. N. Olson, R. E. Kingston. 1992. HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors. Mol. Cell Biol. 12: 1031-1042. [Abstract/Free Full Text]
  5. Corneliussen, B., A. Thornell, B. Hallberg, T. Grundstrom. 1991. Helix-loop-helix transcriptional activators bind to a sequence in glucocorticoid response elements of retrovirus enhancers. J. Virol. 65: 6084-6093. [Abstract/Free Full Text]
  6. Engel, I., C. Murre. 2001. The function of E- and Id proteins in lymphocyte development. Nat. Rev. Immunol. 1: 193-199. [Medline]
  7. Benezra, R., R. L. Davis, D. Lockshon, D. L. Turner, H. Weintraub. 1990. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61: 49-59. [Medline]
  8. Sun, X. H., N. G. Copeland, N. A. Jenkins, D. Baltimore. 1991. Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins. Mol. Cell. Biol. 11: 5603-5611. [Abstract/Free Full Text]
  9. Christy, B. A., L. K. Sanders, L. F. Lau, N. G. Copeland, N. A. Jenkins, D. Nathans. 1991. An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc. Natl. Acad. Sci. USA 88: 1815-1819. [Abstract/Free Full Text]
  10. Riechmann, V., I. van Cruchten, F. Sablitzky. 1994. The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3. Nucleic Acids Res. 22: 749-755. [Abstract/Free Full Text]
  11. Quong, M. W., A. Martensson, A. W. Langerak, R. R. Rivera, D. Nemazee, C. Murre. 2004. Receptor editing and marginal zone B cell development are regulated by the helix-loop-helix protein, E2A. J. Exp. Med. 199: 1101-1112. [Abstract/Free Full Text]
  12. Bain, G., E. C. Maandag, D. J. Izon, D. Amsen, A. M. Kruisbeek, B. C. Weintraub, I. Krop, M. S. Schlissel, A. J. Feeney, M. van Roon, et al 1994. E2A proteins are required for proper B cell development and initiation of immunoglobulin gene rearrangements. Cell 79: 885-892. [Medline]
  13. Zhuang, Y., P. Soriano, H. Weintraub. 1994. The helix-loop-helix gene E2A is required for B cell formation. Cell 79: 875-884. [Medline]
  14. Sun, X. H.. 1994. Constitutive expression of the Id1 gene impairs mouse B cell development. Cell 79: 893-900. [Medline]
  15. Seet, C. S., R. L. Brumbaugh, B. L. Kee. 2004. Early B cell factor promotes B lymphopoiesis with reduced interleukin 7 responsiveness in the absence of E2A. J. Exp. Med. 199: 1689-1700. [Abstract/Free Full Text]
  16. Quong, M. W., D. P. Harris, S. L. Swain, C. Murre. 1999. E2A activity is induced during B-cell activation to promote immunoglobulin class switch recombination. EMBO J. 18: 6307-6318. [Medline]
  17. Zhuang, Y., P. Cheng, H. Weintraub. 1996. B-lymphocyte development is regulated by the combined dosage of three basic helix-loop-helix genes, E2A, E2-2, and HEB. Mol. Cell. Biol. 16: 2898-2905. [Abstract]
  18. Zhuang, Y., R. J. Barndt, L. Pan, R. Kelley, M. Dai. 1998. Functional replacement of the mouse E2A gene with a human HEB cDNA. Mol. Cell. Biol. 18: 3340-3349. [Abstract/Free Full Text]
  19. Lazorchak, A., M. E. Jones, Y. Zhuang. 2005. New insights into E-protein function in lymphocyte development. Trends Immunol. 26: 334-338. [Medline]
  20. Bergqvist, I., M. Eriksson, J. Saarikettu, B. Eriksson, B. Corneliussen, T. Grundstrom, D. Holmberg. 2000. The basic helix-loop-helix transcription factor E2-2 is involved in T lymphocyte development. Eur. J. Immunol. 30: 2857-2863. [Medline]
  21. Pfaffl, M. W., G. W. Horgan, L. Dempfle. 2002. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30: e36[Abstract/Free Full Text]
  22. Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, F. Melchers. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10: 327-336. [Medline]
  23. Cumano, A., C. J. Paige. 1992. Enrichment and characterization of uncommitted B-cell precursors from fetal liver at day 12 of gestation. EMBO J. 11: 593-601. [Medline]
  24. Zhuang, Y., A. Jackson, L. Pan, K. Shen, M. Dai. 2004. Regulation of E2A gene expression in B-lymphocyte development. Mol. Immunol. 40: 1165-1177. [Medline]
  25. Herblot, S., P. D. Aplan, T. Hoang. 2002. Gradient of E2A activity in B-cell development. Mol. Cell. Biol. 22: 886-900. [Abstract/Free Full Text]
  26. Becker-Herman, S., F. Lantner, I. Shachar. 2002. Id2 negatively regulates B cell differentiation in the spleen. J. Immunol. 168: 5507-5513. [Abstract/Free Full Text]
  27. Bain, G., I. Engel, E. C. Robanus Maandag, H. P. te Riele, J. R. Voland, L. L. Sharp, J. Chun, B. Huey, D. Pinkel, C. Murre. 1997. E2A deficiency leads to abnormalities in {alpha}β T-cell development and to rapid development of T-cell lymphomas. Mol. Cell. Biol. 17: 4782-4791. [Abstract]
  28. Sawada, S., D. R. Littman. 1993. A heterodimer of HEB and an E12-related protein interacts with the CD4 enhancer and regulates its activity in T-cell lines. Mol. Cell. Biol. 13: 5620-5628. [Abstract/Free Full Text]
  29. Morrow, M. A., G. Lee, S. Gillis, G. D. Yancopoulos, F. W. Alt. 1992. Interleukin-7 induces N-myc and c-myc expression in normal precursor B lymphocytes. Genes Dev. 6: 61-70. [Abstract/Free Full Text]
  30. Ikawa, T., H. Kawamoto, L. Y. Wright, C. Murre. 2004. Long-term cultured E2A-deficient hematopoietic progenitor cells are pluripotent. Immunity 20: 349-360. [Medline]
  31. Rolink, A. G., S. L. Nutt, F. Melchers, M. Busslinger. 1999. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401: 603-606. [Medline]
  32. Nutt, S. L., B. Heavey, A. G. Rolink, M. Busslinger. 1999. Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401: 556-562. [Medline]
  33. Sun, X. H., D. Baltimore. 1991. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell 64: 459-470. [Medline]
  34. Sloan, S. R., C. P. Shen, R. McCarrick-Walmsley, T. Kadesch. 1996. Phosphorylation of E47 as a potential determinant of B-cell-specific activity. Mol. Cell. Biol. 16: 6900-6908. [Abstract]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
C. Pridans, M. L. Holmes, M. Polli, J. M. Wettenhall, A. Dakic, L. M. Corcoran, G. K. Smyth, and S. L. Nutt
Identification of Pax5 Target Genes in Early B Cell Differentiation
J. Immunol., February 1, 2008; 180(3): 1719 - 1728.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wikström, I.
Right arrow Articles by Holmberg, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wikström, I.
Right arrow Articles by Holmberg, D.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene
*Compound via MeSH
*Substance via MeSH
Medline Plus Health Information
*Stem Cells

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS