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Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two populations of splenic B cells were recently identified as precursors of follicular B2 cells. Transitional B cells of type 1 (T1) represent the recent immigrants from the bone marrow. These cells are the precursors for transitional B cells of type 2 (T2), which are actively dividing cells and are found exclusively in the primary follicles of the spleen (3). Only 5–10% of the newly generated immature B cells are selected into the pool of long-lived mature B2 cells (4, 5). The precursors of MZ B cells are considered to be the newly formed T1 and T2 (6) or mature B cells (3), although the nature of this process has not been fully characterized.
The program of B cell development and differentiation is largely controlled at the level of transcription initiation. This pathway is characterized by activation or down-regulation of stage and lineage-specific genes that are governed by regulatory DNA elements and combinatorial interactions of ubiquitous and cell type-specific transcription factors and cofactors. In this study, we focused on the potential role of helix-loop-helix (HLH) proteins in controlling the differentiation of immature B cells. Transcription factors with a basic HLH (bHLH) motif have been shown to be crucial for various cell differentiation processes during development of multicellular organisms (7). The HLH domain primarily mediates homo- or heterodimerization, which is essential for DNA binding and transcription regulation. Nearly all HLH proteins possess a region of highly basic residues adjacent to the HLH domain, which facilitates binding to DNA. The E proteins are members of the highly conserved bHLH family. These proteins include the mammalian E2A, E2-2, and Hela E-box-binding proteins and the Drosophila gene product, Daughterless. E proteins were shown to be crucial for lymphocyte development. B lymphopoiesis in E2A-deficient mice is arrested at an early stage, which precedes the onset of Ig gene rearrangement (8, 9). Transcriptional activity of the E proteins can be regulated by the expression of another class of HLH proteins, the Id family. These proteins lack a DNA-binding region, and instead function solely by dimerization with other transcriptional regulators, principally those of the bHLH family, and inhibit their functions in a dominant-negative manner (10, 11, 12). Id proteins play key roles in the regulation of lineage commitment, cell fate decisions, and in the timing of differentiation during lymphopoiesis (13, 14, 15, 16, 17, 18).
In the present study, we show that Id2 is a regulator of immature B cell differentiation in the spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6, Ii-deficient (19), Id2-/+, and Id2-/- (18) mice were used at 6–8 wk of age. Id2-/+ and Id2-/- were screened as previously described (18). All animal procedures were approved by the Animal Research Committee at The Weizmann Institute (Rehovot, Israel).
Cells and separation of B cells
Spleen cells were obtained from C57BL/6, Ii-/- and Id2-/+, or Id2-/- at 6–8 wk of age as previously described (20). Control IgD- cells were separated from the IgD+ population as was previously described (21). Briefly, the IgD- population was separated with anti-CD45R (B220) magnetic using the MACS system. Ii-/- B cells were purified by the CD45R beads. To separate the IgD-, CD21 positive and negative populations, IgD- cells were divided according to their CD21 expression using the MACS system. The IgD-CD21- cells were incubated with anti-CD45R magnetic beads and reseparated. The purity of each cell population was determined by flow cytometry using appropriate mAbs.
RNA isolation and reverse transcription
Total RNA was isolated from cells using the Tri Reagent kit (Molecular Research Center, Cincinnati, OH). Reverse transcription was conducted using Superscript II RT (Life Technologies, Grand Island, NY). The primers used included: Id1, 5'-GGTGAGGTCCGAGTCAGAGTATT-3' and 5'-CCATCTGGTCCCTCAGTGC-3'; Id2, 5'-CAGCCATTTCACCAGGAGAACA-3' and 5'-CAGCATTCAGTAGGCTCGTGTCA-3'; Id3, 5'-GGAGCCCGAGAGAAGGACTG-3' and 5'-GAGTTCATAATCAGGGCAGCAGA-3'; E2A, 5'-CATCCATGTCCTGCGAAGCCAC-3' and 5'-TTCTTGTCCTCTTCGGCGTCGG-3'; hypoxanthine guanine phosphoribosyltransferase, 5'-GAGGGTAGGCTGGCCTATGGCT-3' and 5'-GTTGGA TACAGGCCAGACTTTGTTG-3'.
Detection of Id2 and E2A by Western blotting
Cells were lysed as described previously (20). Lysates were separated by 12% (w/v) SDS-PAGE. Alternatively, Id2 was immunoprecipitated using the anti-Id2 Ab (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) as was previously described (20). The proteins were transferred onto nitrocellulose, and probed with the anti-Id2 Ab (C-20; Santa Cruz Biotechnology) or with anti-E2A (YAE; Santa Cruz Biotechnology) followed by HRP-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA).
Immunofluorescence and flow cytometry
Staining was performed on freshly isolated splenocytes as previously described (22). The following Abs were used in experiments: RA3-6B2 anti-CD45R/B220 was obtained from Southern Biotechnology Associates (Birmingham, AL). Anti-IgD, R6-60.2 anti- IgM, 7G6 anti-CD21, S7 anti-CD43, 493 anti-mouse early B lineage mAb, and B3B4 anti-CD23 were obtained from BD PharMingen (San Diego, CA).
EMSA
Nuclear extracts were prepared as described (23) from Ii-/-, control, Id2-/+, or Id2-/- B cells. A double-stranded oligonucleotide, containing the Oct or E2A site was 32P-labeled with Klenow fragment of DNA polymerase I for use as a probe. The oligonucleotide’s sequence was described previously (23). The Abs were preincubated with the extracts for 15 min at 4°C. The Abs used were: anti-E2A (YAE; Santa Cruz Biotechnology) and anti-CD4 (clone GK1.5; Southern Biotechnology Associates).
Proliferation of B cells
Purified B cells were cultured in 96-well plates at 2 x 10 5 cells/well in RPMI medium supplemented with 10% FCS, 2 mM glutamate, 100 U/ml penicillin, 100 µg/ml streptomycin, and several concentrations of LPS (Sigma-Aldrich, St. Louis, MO) or anti-IgM (Jackson ImmunoResearch Laboratories). DNA synthesis was assayed by pulsing the cultures with 1 µCi of [3H]thymidine (International Chemical and Nuclear, Irvine, CA) for the last 18 h of a 2-day culture, after which the cells were harvested and counted. Assays were performed in triplicate.
5-bromo-2-deoxyuridine (BrdU) labeling of cells
Mice were fed with drinking water containing 1 mg/ml BrdU (Sigma-Aldrich) for 3 days. Spleen cells were then isolated and fixed with 70% ethanol. To determine the precentage of BrdU+ (proliferating) cells, cells were washed twice with PBS and resuspended in 0.5 ml 2N HCl/Triton X-100 and left for 20 min at room temperature. Cells were collected by centrifugation, washed, and stained with FITC-labeled anti-BrdU (BD Biosciences, Mountain View, CA) and anti-B220 (Southern Biotechnology Associates), anti-CD23, or anti-493 (BD PharMingen), and were analyzed by FACS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). To follow Id2 protein
levels, we first analyzed the specificity of the Ab by identifying the
Id2 band by Western blot analysis comparing steady state levels of the
protein in immature B cells derived from Ii-/-
immature B cells and Id2 heterozygote (Id2-/+)
and homozygote (Id2-/-) B cells. As can be seen
in Fig. 1B, high levels of Id2 were detected in the purified
Ii-/- immature B cells. In total B cell
population derived from Id2 heterozygote mice, Id2 levels were
dramatically lower, while this protein was completely absent in the
Id2-/- B cells. In addition, Western blot
analysis of Id2 expression in immature (IgD-)
from control (Fig. 1C) or Ii-/-
(Fig. 1D) and mature (IgD+) B cells
revealed reduced expression of Id2 protein in the
IgD+ cells (Fig. 1, C and
D). Thus, the HLH protein, Id2, is specifically
down-regulated during the differentiation from immature to mature B2
cells.
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A, in the absence of
Id2, B cells expressed higher levels of IgD and CD23, reflecting a more
mature phenotype. Dot plot analysis of the spleen population revealed a
shift of the mature cells to a population expressing dramatically
higher levels of IgD and CD23 and a significant down-regulation of the
IgD-IgM+ and
CD23-IgM+ immature
populations (Fig. 2B). Table I
summarizes the percentage of the various B cell populations in the
periphery of the Id2-/+ and
Id2-/- mice. To determine whether the shift in
the splenic population results from a defect in the differentiation of
B cells in the bone marrow, early B cells in the bone marrow were
analyzed. As can be seen in Fig. 2C, similar levels of the
early B cell populations were detected in the bone marrow, suggesting a
normal differentiation in this compartment. The only major difference
was in the mature CD23+ B population, which
was elevated in the Id2-deficient population. These mature cells
recycle from the periphery to the bone marrow and provide further
indication that only the peripheral differentiation of the cells is
shifted in the Id2-deficient mice.
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A). Moreover, analysis of the
more mature B cells, the 493 negative population, revealed a reduced
proportion of CD21high cells, the MZ B cells.
Thus, in the absence of Id2, there was a dramatic shift in the B2
population toward a mature B cell phenotype, while differentiation of
MZ B cells was inhibited.
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B, Id2 message was
detected mostly in the
IgD-CD21- immature B
cells. However, in cells differentiated to mature B2
(IgD+) or MZ
(IgD-CD21+) populations,
Id2 message was down-regulated. Thus, peripheral B cells express Id2
almost exclusively at the T1 stage. Transition from immature B cells in
the spleen to either follicular or MZ B cells is accompanied by a
dramatic down-regulation of Id2 mRNA expression.
To show that the half-life of the B2 cell populations from
Id2-/+ or Id2-/- mice
was similar, mice were fed with BrdU for 3 days (Fig. 4A). Similar proportions of
BrdU positive cells were detected in the Id2-/+
and Id2-/- populations, suggesting a similar
generation and rate of replacement of B cells in the presence or
absence of Id2 expression. Moreover, analysis of the immature
(493+) and mature (CD23+) B
cells revealed similar BrdU positive populations in the two mice. Thus,
in the absence of Id2, the various populations have similar half-life.
To further characterize the Id2-/- B cells, we
analyzed their functionality in response to LPS or IgM stimulation. LPS
is a B cell mitogen, which can activate both immature and mature B
cells, whereas only mature B cells respond to anti-IgM stimulation.
As can be seen in Fig. 4B, Id2-/+ and
Id2-/- B cells proliferated similarly in
response to LPS stimulation, while Id2-/- B
cells had a somewhat stronger response to anti-IgM stimulation at
the lower concentrations tested (Fig. 4C), suggesting the
existence of a larger population of mature cells in mice that do not
express Id2.
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A, this probe specifically
competed with the binding activity of a specific band in the mature
population, showing that this band represents a specific E2A binding
site. Moreover, incubation of this extract with anti-E2A Abs
resulted in a supershift of this complex (Fig. 5A). In
addition, we found that the E2A binding activity in the immature
population was dramatically reduced, whereas Oct-1 binding was
unchanged (Fig. 5B). The observed DNA binding activity is
E2A-specific, as incubation of these extracts with Abs against this
protein resulted in a supershifted complex, whereas Abs against CD4 did
not induce any complex formation. In addition,
Id2-/- B cells exhibited a profound elevation
in the DNA-binding activity to the E2 box site (Fig. 5C),
indicating that in the absence of Id2, E2A-binding activity to the DNA
is dramatically elevated.
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D, E2A was coimmunoprecipitated together with Id2 in
greater amounts from immature B cells and to a lesser extent from the
total control population, indicating that the heterodimer complex
exists at higher levels in immature B cells. Thus, high expression
levels of Id2 down-regulates the transcription activity of E2A, which
results in reduced transcription activity of the genes required for
maturation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we focused on the potential role of HLH proteins in the differentiation of immature B cells in the spleen. Our studies show that immature B cells express Id1, Id2, and Id3. However, Id2 message and protein are specifically down-regulated during the transition to a mature B2 population. This suggests that Id2 may play a role in the differentiation of immature B cells in the spleen.
To directly demonstrate the function of Id2 in this process, we
analyzed maturation of B cells in mice lacking Id2. Our results reveal
normal early B cell populations in bone marrow derived from
Id2-/- mice, suggesting that Id2 does not
regulate the differentiation to pro-, pre-, and immature B cells.
However, these Id2-/- mice exhibit a different
peripheral B cell population distribution. Although control spleen
consists of a small proportion of IgD negative immature B cells,
Id2-/- spleens lose their immature population
and instead accumulate more mature B cells. B cells derived from
Id2-deficient mice express higher levels of IgD and CD23, which are
mature cell markers. Thus, Id2 controls the peripheral differentiation
of immature B cells in the spleen. The Id2 homozygote and heterozygote
B cell mature and immature populations have a similar lifespan,
suggesting that the accumulation of the more mature B2 cells in the
Id2-/- mice does not result from their
transformation into longer-lived cells. In addition, B cells derived
from the Id2-/+ and
Id2-/- mice proliferated similarly in response
to LPS. However, these Id2-/- cells responded
more efficiently to anti-IgM stimulation, which can activate only
mature B cells, in agreement with the ability of these cells to respond
more strongly to SRBC (18). Therefore, in the absence of
Id2, B cells differentiate to a more mature population that does not
normally exist in the control mice or represents only a small
proportion of the cells. The existence of a more mature phenotype
suggests that maturation of splenic B cells is down-regulated by Id2.
In the absence of this control, the steady state of the cells shifts,
the immature B population disappears, and the cells undergo
uncontrolled differentiation, resulting in greater numbers of mature
cells (Fig. 6).
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). Studies from both Drosophila and mammals support the view that the primary mechanisms through which ID proteins function is by antagonism of bHLH transcriptional regulators. The mammalian ID proteins preferentially target the ubiquitously expressed E proteins, which belong to the group of class A bHLH proteins (10, 11, 12). Although all ID proteins interact avidly with each of the E proteins, biochemical data indicate that individual ID proteins have distinct preferences for specific E protein targets (26, 27). It was shown that bHLH proteins encoded by the E2A gene and the early B cell development factor are required for the initiation of B lymphopoiesis (25), and that PAX-5 is crucial for the commitment of the cells to the B lineage (28, 29). Our results indicate that Id2 and E2A form heterodimeric complexes that reduce the binding activity of E2A in cells of the immature stage. In the mature stage, Id2 expression is down-regulated, and this enables an up-regulation of the binding activity of E2A and transcription of essential genes required for differentiation of these mature B2 cells. Id2 controls differentiation by inhibiting E2A-dependent transcription of essential genes required for maturation and may be the heart of a mechanism regulating differentiation of immature B cells in the spleen by antagonizing E2A activity. It remains necessary to identify the genes regulated by E2A that are essential for this control.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Idit Shachar, Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel. E-mail address: Idit.Shachar{at}weizmann.ac.il
3 Abbreviations used in this paper: MZ, marginal zone; HLH, helix-loop-helix; bHLH, basic HLH; BrdU, 5-bromo-2-deoxyuridine; T1, transitional B cell type 1; T2, transitional B cell type 2.
Received for publication November 28, 2001. Accepted for publication March 25, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Nat. Immunol. 2:242.[Medline]
but not of 
T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors. EMBO J. 18:2793.[Medline]
negatively regulates homing of immature B cells. J. Exp. Med. 192:1381.
B dependent transcription program. J. Biol. Chem. 276:27203.This article has been cited by other articles:
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