By
¶
§
From the * Howard Hughes Medical Institute, Boston, Massachusetts 02115; The Children's
Hospital, Boston, Massachusetts 02115; the § Center for Blood Research and the
Department of
Genetics, Harvard Medical School, Boston, Massachusetts 02115; and the ¶ Infectious Disease Unit,
Massachusetts General Hospital, Boston, Massachusetts 02114
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Abstract |
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To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras-RAG). Similar to the effects of an
immunoglobulin (Ig) µ heavy chain (HC) transgene, activated Ras caused progression of
RAG1-deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of 5,
RAG2, and germline
locus transcripts. However, these Ras-RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG-
deficient background as a result of expression of an Ig µ HC together with a Bcl-2 transgene.
Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.
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Introduction |
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Blymphocyte development proceeds through a series of
stages defined by the expression of surface markers and
by the status of Ig gene rearrangement (1). In this developmental program, upon productive rearrangement and expression of Ig µ heavy chain (HC)1 genes, B220+CD43+
pro-B cells progress to B220+CD43 pre-B cells. This
transition requires expression of the pre-B cell receptor
(pre-BCR) complex consisting of µ HC associated with
the invariant surrogate light chain proteins
5 and VpreB, most likely on the cell surface (2). Consistent with this notion, targeted germline deletion of the µ membrane exon
arrested murine B cell development at the pro-B cell stage
(3). Moreover, germline inactivation in mice of either the
recombinase-activating gene (RAG)1 or RAG2 genes,
which encode components of the V(D)J recombinase required for initiation of antigen receptor gene rearrangement, again resulted in a block in B lymphocyte development at the pro-B cell stage (4, 5). However, expression of
a rearranged µ HC transgene in the RAG-deficient background partially rescued this developmental block in the B
lineage, leading to the generation of B220+CD43
pre-B
cells and demonstrating that µ chain expression was sufficient to drive this developmental transition (6, 7).
Because the pre-BCR, like the mature BCR, has no
known intrinsic enzymatic functions, it must rely upon
associated proteins to provide a functional linkage with intracellular signaling pathways. The mature and pre-BCR-
associated Ig and Ig
contain immunoreceptor tyrosine-based activation motifs (ITAMs), which are targets for phosphorylation by tyrosine kinases (8); these proteins are required for normal B cell development (9, 10). Furthermore, the importance of an ITAM-associated tyrosine kinase activity during early B lymphopoiesis was demonstrated in mice deficient in the syk tyrosine kinase, in
which an incomplete block in development was observed
at the B220+CD43+ pro-B cell stage (11, 12). Although
several downstream signaling pathways can be induced in B
cell lines (13), the identity of the targets downstream of the
nonreceptor tyrosine kinases that are activated by the pre-BCR complex has remained unclear. In this context, the
Ras family of GTPases (14) represents an attractive candidate. In numerous vertebrate systems, Ras proteins have
been implicated in linking tyrosine kinase-mediated signal
transduction to downstream effectors (15). In the T cell
lineage, constitutive expression of activated Ras in a RAG-deficient background has been shown to drive the expansion and differentiation of double negative thymocytes to
the CD4+ CD8+ (double positive, or DP) stage (16).
Moreover, Ras-dependent signaling after cross-linking of
the mature BCR has also been observed in lymphocyte cell
lines (17, 18). We hypothesized that if activation of endogenous Ras represents a necessary event in pre-BCR signaling, then introduction of constitutively activated Ras into
RAG-deficient pro-B cells could mimic signaling by the pre-BCR and result in developmental progression.
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Materials and Methods |
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DNA Constructs.
The plasmid pEµ was constructed through ligation of a 1,042-bp fragment containing the Ig HC enhancer (Eµ) linked to a variable region promoter (19) into the SmaI site of Bluescript II SK. A BamHI/PstI fragment containing two exons of the humanEmbryonic Stem Cell Transfection and RAG2-deficient Chimera Generation.
Cotransfection of RAG1Analysis of RAG2-deficient Chimeras.
RAG2-deficient chimeras were maintained in a pathogen-free environment, and were analyzed at 4-6 wk of age. FACS® analyses of bone marrow, spleen, and lymph nodes were carried out as previously described (22). Antibodies were purchased from PharMingen and were: Cy-Chrome conjugated B220/CD45R (clone RA3-6B2); FITC-conjugated CD21/35 (CR2/CR1) (7G6), CD22.2 (Cy34.1), IgM (II/41), Ly 9.1 (30C7), B220/CD45R, and CD43 (S7); and PE-conjugated CD43 (S7), CD2 (RM2-5), CD23 (B3B4), and CD22.2. Analysis of stained samples was performed on a Becton Dickinson FACSCalibur®, and sorting of B220+ Ly 9.1+ B lineage cells was carried out on an Ortho Cytofluorograf II or Becton Dickinson FACScan®; dot plots were generated using Cell Quest software (Becton Dickinson).Western Blot Analysis.
After red blood cell lysis in ammonium chloride, single-cell splenocyte or lymph node suspensions were treated with RIPA lysis solution (0.15 mM NaCl, 0.05 mM Tris-HCl, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) at 108 cells per ml, and postnuclear supernatants were prepared following standard procedures. Proteins were resolved using SDS/10% PAGE (loading 2 × 106 cell equivalents of lysate per lane), transferred to Immobilon-P membranes (Millipore), and probed with an anti-Ha-ras monoclonal antibody (clone F235; Calbiochem), followed by a horseradish peroxidase-linked F(ab')2 sheep anti-mouse Ig (Boehringer Mannheim). For detection we used the ECL system (Amersham), and Ponceau-S staining was employed to verify equivalent protein loading.Reverse Transcription PCR Analysis.
RNA was isolated from 106 sorted B220+Ly 9.1+ cells derived from Ras-RAG1 ![]() |
Results |
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To direct expression of activated Ras to B lineage cells,
we used an expression construct containing a c-Ha-rasV12
cDNA and Ig HC regulatory sequences (Fig. 1 A). This expression construct was transfected into RAG1-deficient ES
cells (16), and the resulting ES clones were tested for their
ability to generate B lineage cells in the RAG2-deficient
blastocyst complementation assay (21). Flow cytometry
analysis of bone marrow cells from Ras-RAG chimeras revealed low numbers of IgMB220+CD43
B lineage cells
(which are absent in RAG-deficient mice); however, these
cells were also found in the spleen and lymph nodes in
numbers approaching those of normal, Ig-positive B cells
in wild-type mice (Fig. 2 and data not shown). To verify
that B220+ cells were derived from ES cells, the clonotypic
marker Ly 9.1 was used (data not shown). Furthermore,
expression of the Ha-ras protein in these chimeric mice
was confirmed by Western analysis of spleen and lymph
node cell lysates using an Ha-ras-specific monoclonal antibody (expression of endogenous Ras in lymphocytes is limited to N-ras and K-ras; reference 27) (Fig. 1 B). Therefore, activated Ras expression results in the generation of B
lineage cell populations that substantially populate the peripheral lymphoid tissues of RAG1-deficient mice.
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To further delineate the stage of maturation of B lineage
cells in Ras-RAG mice, we assayed for expression of various genes used to define stages of B cell differentiation.
RT-PCR assays demonstrated that populations of splenic
or lymph node Ras-RAG B lineage cells expressed
substantial levels of 5 and RAG2 transcripts, which are
normally transcribed in the pro-B and pre-B cells but generally are absent during later stages of development (23, 28). On the basis of semiquantitative RT-PCR analyses,
we determined that Ras-RAG cell populations expressed
5 and RAG2 at levels comparable to those in purified
wild-type pre-B cells (Fig. 3). In normal mice, productive
rearrangement and expression of µ HC genes in developing B cell progenitors leads to the transcriptional activation
and rearrangement of
light chain genes (29). To study
if signaling by activated Ras could mimic the induction of
germline transcription normally induced by expression of HC in pro-B cells, we determined the levels of germline
transcripts in Ras-RAG B lineage cells. We observed that
such transcripts were present in Ras-RAG B lineage cells
at levels similar to those in wild-type pre-B cells (Fig. 4).
These results suggest that activated Ras signaling in RAG-deficient B lineage cells promotes transcriptional activation
of the
light chain gene locus.
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Given the large number of peripheral B lineage cells in Ras-RAG mice, we further assayed for staining of more mature B cell surface markers. On the basis of these assays, we also found that the Ras-RAG B lineage cells in both the bone marrow and periphery expressed surface antigens usually associated with later stages of B cell development, such as the low affinity IgE Fc receptor CD23 (34), the BCR coreceptor CD22 (35), and complement receptor CD21/CD35 (36) (Fig. 2 and data not shown). Thus, our data suggest that expression of activated Ras results in the development of RAG-deficient B lineage cells that retain major properties of pre-B lymphocytes while also expressing cell surface markers usually found only in mature B cell stages. These Ras-RAG B lineage cells are distinct from those generated in the RAG-deficient background via expression of an Ig µ HC transgene, which only promotes differentiation to cells that show pre-B cell characteristics and remain primarily in the bone marrow (6, 7), but are similar in patterns of gene expression and tissue distribution to those observed in µ HC/Bcl-2 double transgenic, RAG-deficient mice (22, 37).
The similarity of the Ras-RAG phenotype to that promoted by µ HC plus Bcl-2 transgenes suggested to us that activated Ras may signal both differentiative and cell survival processes. During normal B cell development, the antiapoptotic gene Bcl-2 is expressed at the pro-B stage, but is downregulated in pre-B cells and later upregulated in mature B lymphocytes (23, 38). In contrast, the cell survival gene Bcl-xL displays a reciprocal pattern of expression, with high levels in pre-B cells that are downregulated in mature B cells (39). To assay for Bcl-2 and Bcl-xL expression in sorted Ras-RAG peripheral B lineage cells, we used RT-PCR analysis and determined that the expression levels of Bcl-2 and Bcl-xL in Ras-RAG B cells were more comparable with those in wild-type mature B cells with substantial levels of Bcl-2 expression (Fig. 5).
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Discussion |
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Several studies have implicated Ras as an intermediate in
signal transduction downstream of the BCR (17, 18). Our
studies of Ras-RAG mice demonstrate that activated Ras
can induce differentiation of pro-B cells in the absence of µ HC. B lineage cells that develop in Ras-RAG mice acquired characteristics shared with normal pre-B cells, including expression of RAG and 5, and the induction of germline
light chain locus transcripts. However, these
cells also expressed surface markers characteristic of more
mature stages of B lymphopoiesis. Thus, we believe it is
most likely that Ras-RAG B cells have progressed in their
differentiation beyond the pre-B cell stage, at least to the
pre-B-B cell junction. It also remains possible, given their
peripheral location, that Ras-RAG B cells may be developmentally similar to the recently described subset of germinal center B cells that reinitiate light chain gene rearrangement as a result of antigenic challenge (40).
Although such cells, unlike Ras-RAG B cells, lack CD23
expression (43), both cell types demonstrate the concurrent
expression of
5 and RAG genes with mature B cell surface markers. The accumulation of the Ras-RAG B lineage cells in the periphery could be due to several potential effects of activated Ras expression, including acquisition of surface markers necessary for transit from the bone marrow
or prolonged survival allowing exit from the marrow and
accumulation in the periphery.
Previous work has demonstrated that the introduction of a rearranged µ transgene into RAG-deficient pro-B cells induced their differentiation to pre-B cells (6, 7). Although such cells remained predominantly within the bone marrow and did not express surface markers characteristic of more mature B cells, the expression of a Bcl-2 transgene in the B lineage of µ-RAG mice resulted in the appearance of cells in the marrow and periphery with a phenotype that resembles B lineage cells found in Ras-RAG mice (22, 37). These findings suggested that survival signals provided by Bcl-2 may advance B lymphopoiesis beyond the stage achieved by µ HC alone. In this regard, we found that Ras-RAG cells expressed significantly higher levels of endogenous Bcl-2 than normal pre-B cells; in fact, the observed Bcl-2 expression levels approached those in mature B cells. At present, we do not know whether this upregulation of Bcl-2 expression in Ras-RAG cells is directly induced by activated Ras, or, alternatively, occurs as a result of developmental progression to a more mature stage. Nevertheless, the phenotypic similarities between µ HC/Bcl-2/RAG and Ras-RAG B cells suggest that introduction of activated Ras may induce and/or enable both differentiation and survival signals in RAG-deficient and, presumably, normal progenitor B lineage cells.
Our finding that B cells in Ras-RAG mice develop to a stage beyond that of B cells in µ-RAG mice indicates that signaling events triggered by constitutively activated Ras may surpass or differ from those initiated upon HC-mediated activation of endogenous Ras. In this context, in other experimental systems the effects of activated Ras on cultured cells varied depending on the level and duration of Ras expression (44). It is also possible that the expression of activated Ras in B lineage cells mimics signaling from other surface receptors, in addition to the pre-BCR, which normally trigger endogenous Ras. Numerous Ras effector pathways have been identified to date, including a well-characterized mitogen-activated protein kinase cascade and a growing number of stress-activated protein kinase cascades (45). Ras has also been shown to induce phosphatidylinositol-3 kinase (46, 47), as well as the Rho family of GTPases which regulate the actin cytoskeleton (48). It remains to be established which of these (or other) Ras-effector pathways are involved in mediating the developmental progression of pro-B cells. Selective engagement of Ras effectors using activated mutant alleles may facilitate further elucidation of these issues.
Recent data demonstrate that Ras signaling is used during several stages of B and T lymphocyte development. For example, a dominant negative Ras transgene was shown to cause an incomplete block in B cell development at the earliest known B cell precursor stage before B220+CD43+ pro-B cells (49). In T lineage cells, several studies with dominant negative alleles implicated the Ras/Raf/Mitogen-activated protein kinase pathway in the development of CD4+CD8+ (DP) thymocytes and mature T cells (50- 52). Notably, a complete reconstitution of DP thymocytes was induced by activated Ras in RAG-deficient mice; however, in these mice no developmental progression beyond the DP stage was observed, and no T cells were detected in the peripheral lymphoid organs (16). These results suggested that additional signals are required for the T cell positive selection process that normally results from signaling events accompanying ligation of the T cell receptor with self-MHC ligands of specific avidity (53). Such a requirement for additional signaling events, independent of Ras, in the development of T cells beyond the DP stage suggests that an important distinction may exist in the signals required to effect further development of precursor B versus precursor T lineage cells.
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Footnotes |
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Address correspondence to Frederick W. Alt, Howard Hughes Medical Institute, Children's Hospital, 320 Longwood Ave., Boston, MA 02115. Phone: 617-355-7290; Fax: 617-730-0432; E-mail: alt{at}rascal.med.harvard.edu
Received for publication 14 October 1998 and in revised form 27 October 1998.
We thank Juanita Campos-Torres for invaluable cell sorting assistance, and Drs. Yansong Gu, Timo Breit, and Nienke van der Stoep for helpful advice and discussions.
This work was supported in part by National Institutes of Health grants AI20047 (to F.W. Alt) and AI01532-01 (to A.C. Shaw). A.C. Shaw was a recipient of a Howard Hughes Medical Institute Postdoctoral Research Fellowship for Physicians. W. Swat is a recipient of the Arthritis Foundation Hulda Irene Duggan Investigator Award. F.W. Alt is an investigator of the Howard Hughes Medical Institute.
Abbreviations used in this paper BCR, B cell receptor; ES, embryonic stem; DP, double positive (CD4+ CD8+); HC, immunoglobulin heavy chain; RAG, recombinase-activating gene; RT, reverse transcriptase.
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