|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
IMMUNOBIOLOGY
From the Departments of Medicine and Immunology,
University of Washington, Seattle.
Decisions about cell survival or death are central components of
adaptive immunity and occur at several levels in immune system development and function. The Bcl-2 family of homologous proteins plays
an important role in these decisions in lymphoid cells. Bcl-2, Bcl-xL,
and A1 are differentially expressed during B- and T-cell development,
and they have shared and distinct roles in regulating cell death. We
sought to gain insight into the role of A1 in immune system development
and function. A murine A1-a transgene was expressed under
the control of the Eµ enhancer, and mice with A1 overexpression in B-
and T-cell lineages were derived. Thymocytes and early B cells in
Eµ-A1 mice showed extended survival. B-lineage development was
altered, with expansion of the pro-B cell subset at the expense of
pre-B cells, suggesting an impairment of the pro- to pre-B-cell
transition. This early B-cell phenotype resembled Eµ-Bcl-xL mice but
did not preferentially rescue cells with completed V(D)J rearrangements
of the immunoglobulin heavy chain. In contrast to Eµ-Bcl-2
transgenes, A1 expression in pro-B cells did not rescue pre-B-cell
development in SCID mice. These studies indicate that A1 protects
lymphocytes from apoptosis in vitro but that it has lineage- and
stage-specific effects on lymphoid development. Comparison with the
effects of Bcl-2 and Bcl-xL expressed under similar control elements
supports the model that antiapoptotic Bcl-2 homologs interact
differentially with intracellular pathways affecting development and
apoptosis in lymphoid cells.
(Blood. 2002;99:3350-3359) Regulation of cell survival is a critical process
in the development and function of multicellular
organisms.1,2 The family of proteins sharing homology with
Bcl-2 plays key roles in regulating cell fate and includes members that
antagonize and others that promote cell death.3,4
Available studies suggest a role for the antiapoptotic proteins Bcl-2,
Bcl-xL, and A1 in the development, function, or both of the immune
system. In mice, Bcl-2 plays a critical role in the survival of mature
T and B cells.5-7 Variations in its expression may enhance
specific immunity or may limit the extent of antigen
responses.4,8 Bcl-xL is developmentally regulated in the
thymus and is required for the survival of immature thymocytes,
suggesting a potential role in the generation of central
tolerance.9-11 In the bone marrow, Bcl-xL is expressed in
pre-B cells and is required for the survival of early B-lineage
cells.10,12 Bcl-xL is down-regulated at later stages of
B-cell development, but it is induced in activated lymphocytes and may
play a role in the selection of beneficial clones.12-14 Less is known regarding A1, which is inducible as an early-response gene to a variety of stimuli in myeloid, lymphoid, and endothelial cells.15-20 Like Bcl-2 and Bcl-xL, A1 is developmentally
regulated in the immune system and is induced on cellular activation,
suggesting a role in development and immune responses.18
A1 is unusual in that it is encoded by 4 distinct genes in mice, termed
A1-a through A1-d.21 Aside
from A1-c, which bears a frameshift insertion, A1-a, A1-b, and A1-d are 97%
identical, and in neutrophils all 3 isoforms are expressed. Increased
apoptosis of neutrophils has been demonstrated in mice with targeted
inactivation of the A1-a isoform, but this subtle phenotype
likely reflects functional compensation by the other genes.
Although Bcl-2, Bcl-xL, and A1 all have the property of enhancing cell
survival, it remains unclear whether the individual functions of these
proteins represent the same fundamental activity targeted to particular
developmental and physiologic circumstances. Alternatively, these
homologs could have important individual activities within the cell,
such as regulating different categories of death signals or other types
of cellular stimuli. To study the functional properties of A1 in
lymphoid cells in vivo, we have constructed transgenic mice that
overexpress A1-a under the control of the immunoglobulin
intronic enhancer Eµ. This control element has been well
characterized in transgenic systems. Moreover, numerous studies have
been performed using Bcl-2 and Bcl-xL overexpressed under Eµ control.
By comparing the similarities and differences in the phenotypes of
Eµ-Bcl-2, Eµ-Bcl-xL, and Eµ-A1 mice, we sought to gain insight
into the distinct cellular functions of these homologs.
Eµ-A1 transgenic mice
To assess transgene expression, total RNA was harvested from thymus,
spleen, bone marrow, and kidney using Tri-reagent (Sigma, St Louis,
MO), according to the manufacturer's instructions. RNA (1.5 µg per
sample) was used in a ribonuclease protection assay (Multi-probe RPA;
BD PharMingen, San Diego, CA) as previously described.17,23 The probe used in this assay protected
nucleotides 140 to 503 in the A1-a cDNA. Within the range of
this A1-a probe, there were 6 mismatches for the
A1-b mRNA subtype, 9 mismatches for A1-c, and 4 mismatches for A1-d. As a result, only the A1-a subtype mRNA was expected to yield the longest (364 base pair [bp])
protected fragment. Quantitation of protected bands was performed using
densitometric analysis of autoradiograms (National Institutes of Health
Image software, version 1.62).
Polymerase chain reaction assays
Analysis of lymphoid development
Cell survival assays Thymocyte survival in vitro was assessed after disrupting thymic lobes over nylon mesh and plating cells at a density of 1 × 106/mL in RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, nonessential amino acids, and antibiotics. Cells were cultured in 24-well plates at 37% in a humidified 6% CO2 incubator in medium or in dexamethasone (0.2 or 1 µM) or were exposed to -irradiation (2.5 or 10 Gy) and
were studied for the ensuing 4 days. Cells were counted using a
hemacytometer, and viability was determined by trypan blue exclusion.
Bone marrow survival was studied as described.28
Increased A1 mRNA expression in lymphoid tissues of Eµ-A1 transgenic mice To study the in vivo effects of A1 overexpression on immune system development, we inserted cDNA representing the A1-a isoform into an expression system based on the IgH intronic enhancer Eµ and the H-2Kb promoter22 (Figure 1). Steady-state levels of A1-a mRNA were studied using RNase protection assays in 3 independent transgenic lines. We found that A1-a mRNA levels were substantially increased in thymus, bone marrow, and spleen but not in kidney in 3 independent transgenic lines. Expression of transgenic mRNA was subsequently confirmed using PCR to detect A1 transcripts containing sequences from the heterologous control element (see below and Figure 7). The observed expression pattern was consistent with previous studies using this transgene regulatory system.22Effects of A1 overexpression on immune system development Four independent lines of Eµ-A1 transgenic mice were analyzed for effects on primary and secondary lymphoid tissues using flow cytometry. In all lines, no discernible effects on thymic cellularity or subsets defined by CD4 and CD8 expression were observed (data not shown). B lymphopoiesis was analyzed using 3-color flow cytometry with markers for IgM, B220, and CD43. In all 4 lines, an increase in the percentage of B220+IgM CD43+
pro-B cells was observed (Figure 2,
Table 1). This effect was most
pronounced in line 8, which was selected for more detailed studies.
Analysis of absolute cell numbers contained in 2 femurs from line 8 or
control mice indicated that the proportional increase in the ratio of
pro- to pre-B cells was the result of an increase in pro-B-cell
numbers and a decrease in pre-B-cell numbers. B-cell developmental
subsets were further analyzed using 4-color flow cytometry according to
the scheme of Li et al25 and Hardy et al29
(Figures 2, 3). This analysis showed that
the expanded population of
B220+IgMCD43+ pro-B cells was
accounted for primarily by an increase in fraction B (heat-stable
antigen [HSA+] BP1), a population that
precedes delivery of the pre-B-cell receptor signal (Figure 3). Taken
together, these data indicate that the Eµ-A1 transgene led to an
expansion of pro-B cells, and they suggest that this was in part
caused by impaired progression from the pro-B-cell to the
pre-B-cell stage.
The pro-B-cell stage is characterized by ordered D to J, followed by V
to DJ rearrangement of the IgH locus. Successful in-frame IgH
rearrangement leads to expression of the µH protein and signals the
transition to the pre-B-cell stage.30 We assessed IgH DJ and V(D)J rearrangement in the expanded pro-B-cell population in
Eµ-A1 transgenic mice. B220+ bone marrow cells
representing pro- and pre-B-cell subsets were purified by cell
sorting (Figure 4). Genomic DNA from
equivalent cell numbers was extracted and subjected to PCR as
previously described24 using a primer downstream of
JH3, paired with a degenerate primer complementary to 5'
DH segments or to VH segments. Amplification of
polyclonal B-lineage populations gives a 3-part ladder representing
rearrangements to JH1, JH2, and
JH3. The CD14 gene was amplified from each sample as a
control. As expected, the V(D)J signal was lower in pro-B cells than
in pre-B cells, indicating that only a fraction of IgH alleles in the
former population had completed rearrangement. There were no
significant differences in DJ or V(D)J rearrangements when pro-B cells
from wild-type and Eµ-A1 transgenic mice were compared, suggesting
that rearrangement of the IgH locus was not blocked by the transgene
and that pro-B-cell expansion did not represent selective accumulation
of cells with completed V(D)J rearrangements of the IgH locus.
Peripheral lymphoid populations were analyzed in line 8 mice (Table
2). We noted a modest but statistically
significant decrease in splenic B cells in transgene-positive mice.
Similar analyses performed on the other Eµ-A1 lines showed no
definable difference in peripheral B cell number compared with
littermate controls (data not shown). Serum immunoglobulin levels were
also analyzed in 6- to 8-week-old line 8 mice and showed a
correspondingly modest decrease in mean levels of serum IgG1 and IgG2a
but not of IgM (Table 3).
Increased survival of Eµ-A1 transgenic thymocytes and B-cell precursors in vitro Enforced expression of A1 in cell lines leads to enhanced survival in the absence of growth signals or in response to apoptotic stimuli.31 Similar effects are noted when other antiapoptotic Bcl-2 homologs are overexpressed in lymphoid cells in vivo.32-34 To verify the expression of A1 protein in Eµ-A1 transgenic lymphoid cells, we studied the survival of thymocytes and bone marrow B-lineage cells. Compared with nontransgenic controls, thymocytes from transgenic animals survived better ex vivo in tissue culture medium and were more tolerant toward death-inducing stimuli, including-irradiation (2.5 Gy) and 0.2 µM dexamethasone
(Figure 5A). Similar protective effects
were observed with higher doses of -irradiation (10 Gy) and
dexamethasone (1 µM) over the same culture period (data not shown).
Survival of bone marrow B-lineage cells was studied by in vitro
culture, followed by flow cytometric analysis of surviving cells
defined by light-scatter characteristics (Figure 5B). In 2 independent
experiments, after 11 and 12 days, respectively, substantially more
cells survived in the lymphoid gate in transgenic mice. When these
events were analyzed, essentially all cells were B220+. In
wild-type mice, rare surviving B-lineage cells were almost exclusively
IgM+, whereas in Eµ-A1 mice, surface IgM
B-precursor cells and IgM+ B cells survived.
Short-term in vitro survival assays were also performed on lymph node
and spleen cells in which no significant differences were found between
wild-type and Eµ-A1 transgenic mice (data not shown). These data
demonstrate that Eµ-A1 transgenic thymocytes and bone marrow
B-lineage cells exhibit a survival advantage in vitro, and they suggest
that the transgene leads to overexpression of functional A1
protein.
Failure of the Eµ-A1 transgene to rescue V(D)J recombination and pre-B cells in SCID mice In SCID mice, mutation of the Prkdc gene, encoding the DNA-dependent protein kinase catalytic subunit, leads to impairment of V(D)J recombination and failure of T- and B-cell development.30 The defect is leaky, and T and B cells accumulate in these mice as they age. Eµ-Bcl-2 transgenes expressed in the SCID background lead to rescue of pre-B-cell development, a phenotype that is dependent on functional IgH rearrangement.35,36 To determine whether the expression of A1 in SCID B-lineage cells had a similar effect on developmental progression, we introduced the Eµ-A1 transgene into the SCID background. The number of B220+ cells recovered from both femurs in Eµ-A1 SCID mice was comparable to that in SCID littermates (Table 4). Surprisingly, we found no effect on the phenotype of early B-lineage cells in the bone marrow of transgenic SCID mice (Figure 6A). We next assayed bone marrow cells from SCID and Eµ-A1 SCID mice for DJ and V(D)J rearrangements (Figure 6B). Faint DJ signals were seen more strongly in Eµ-A1 SCID than in SCID bone marrow, suggesting that there might have been a small difference in the number of cells undergoing the first step of IgH rearrangement in the presence of the Eµ-A1 transgene. However, no V(D)J rearrangements were detected in either Eµ-A1 SCID or SCID mice. These findings indicate that A1 overexpression does not lead to successful V(D)J rearrangement in SCID pro-B cells or to rescue of pre-B cells as seen in Eµ-Bcl-2 SCID mice. Bcl-2 expression in either the SCID or the Rag-2/
background leads to the accumulation of B220+ cells in the
spleen.35,36 SCID mice expressing the A1 transgene had no
such accumulation of B-lineage cells (Table
4).
To understand potential reasons for the developmental delay of pro-B
cells in Eµ-A1 transgenic mice, pro-B cells arrested at this stage
by the SCID defect were examined. Cells at this developmental stage
express Rag-1 and Rag-2 endonuclease genes, transcribe the unrearranged
IgH locus, and express pre-B-cell receptor signaling components. After
successful IgH rearrangement and expression of Cµ protein,
pre-B-cell receptor signals lead to proliferation and phenotypic
changes that characterize the pre-B-cell compartment. We used
semiquantitative RT-PCR to characterize the levels of mRNA for Rag-1,
Rag-2, Cµ sterile transcripts, and
Irradiation of newborn SCID mice leads to the partial rescue of
thymocyte development, with an increase in thymic cellularity and
appearance of CD4+CD8+ immature thymocytes that
bear functional T-cell receptor (TCR)-
Eµ-A1 mice do not develop lymphoid tumors Overexpression of Bcl-2 in B cells is associated with the development of tumors as mice age.32,40 Similarly, overexpression of Mcl-1, another cytoprotective member of the Bcl-2 family, also leads to B-cell tumors.41 In multiple lines of Eµ-A1 transgenic mice followed up for as long as 18 months, no B-cell tumors were observed. One Eµ-A1 SCID mouse had thymic lymphoma at age 4 months. These observations suggest that the oncogenic potential for A1 overexpression in lymphoid cells may be less than that for Bcl-2 or Mcl-1.
The immune system uses unique forms of targeted genomic instability to randomize parts of the primary sequences of immunoglobulin and TCR genes to create a nearly unlimited repertoire of antigenic specificities. This process generates many out-of-frame or otherwise nonfunctional or deleterious alleles, and selective processes ensure the survival of a primary repertoire of cells with functional and self-tolerant antigen receptors. Further diversification of immunoglobulin genes occurs in B cells during immune responses, requiring additional selection events to ensure survival of the small minority of cells with improved affinity for antigen. These processes illustrate a fundamental aspect of adaptive immunity: survival or death decisions are required on a cell-by-cell basis at several points in immune system development and function. Cell fate decisions in immunity represent a complex integration of signals delivered by antigen receptors, coreceptors, cytokines, and cell death surface receptor proteins such as Fas. Among the intracellular consequences of several of these signals is the modulation of Bcl-2 family proteins, which may promote or antagonize the activation of downstream apoptotic pathways.8,42 Among the antiapoptotic Bcl-2 homologs, little is known regarding the
function of A1 in vivo. Analysis of gene-deficient mice is complicated
by the apparent redundancy engendered by the presence in mice of 4 nearly identical A1 genes, 3 of which are expressed.43 Although these animals can be shown to have increased neutrophil and
macrophage apoptosis,21,44 lack of an apparent phenotype in the lymphoid lineage is difficult to interpret because of gene redundancy. An A1-a transgene under the control of the
Eµ/H2K expression cassette led to broad overexpression of
A1-a mRNA in the T- and B-cell lineages (Figure 1),
consistent with previous results using these control
elements.22 We were unable to detect A1 protein in vivo
using antibodies available commercially or antibodies generated in our
laboratory that interacted specifically with A1 protein in vitro or
using a reagent previously shown16 to detect A1 in
macrophages with highly inducible A1 expression (P.I.C., D.M.W.,
unpublished observations, January 2001; May 2001). However, we
did find evidence of extended in vitro survival of bone marrow
B-lineage cells and thymocytes derived from transgenic mice (Figure 5).
For thymocytes, these studies indicated that A1 antagonized apoptosis
induced not only by ex vivo culture but also by treatment with death
agonist dexamethasone and DNA damage induced by Despite evidence for A1 overexpression in the thymus of Eµ-A1 transgenic mice, we saw no discernible effect on thymic cellularity or composition. Maturation of immature CD4+CD8+ double-positive T-cell precursors to the CD4+ or CD8+ single-positive stage is largely determined by positive and negative survival signals, which govern the selection of cells bearing major histocompatibility complex-reactive, self-tolerant TCR specificities.30,45 Bcl-xL and A1 are both expressed at high levels at the double-positive stage and are down-regulated with maturation, whereas Bcl-2 expression exhibits the reverse pattern.9,19,46 The limited thymic phenotype of Eµ-A1 mice is similar to that reported for Eµ-Bcl-2 and Eµ-Bcl-xL mice.9,33,34,47-49 Partial inhibition of negative-selection processes and rescue of cells otherwise dying for lack of positive selection has been reported in Eµ-Bcl-2 and Eµ-Bcl-xL mice, suggesting that the modulation of antiapoptotic Bcl-2 homologs may play a role in thymic maturation.33,47,50,51 The antiapoptotic effect of these proteins appears to balance the activities of proapoptotic Bcl-2 homologs.4,8 For example, mice lacking the proapoptotic homolog Bim display an accumulation of thymocytes in early life and a defect in peripheral lymphoid homeostasis.52 In the thymus, CD4+CD8+ pre-T cells undergo massive apoptosis in Bcl-xL-deficient mice, implicating Bcl-xL in thymic selection processes.10,11 This result also indicates that A1 expression at physiologic levels is not functionally redundant with Bcl-xL at this stage. Despite transgene expression in T- and B-cell lineages, including significant increases in A1-a mRNA in peripheral lymphoid tissue, we saw no accumulation of T cells in the periphery of Eµ-A1 transgenic mice. Although we cannot rule out poor A1 protein expression in this cellular subset, these results suggest that enforced A1 expression is insufficient to overcome normal homeostatic control of peripheral T-cell populations, similar to findings in Eµ-Bcl-2 mice with T-lineage expression.33,48 In contrast, Eµ-Bcl-xL mice expressing the transgene in the T-cell lineage exhibited 3- to 5-fold increases in lymph node T-cell numbers.9 The major phenotype we observed in Eµ-A1 transgenic mice was the
expansion of B-cell progenitors at the pro-B-cell stage. This finding
was present in multiple independent mouse lines, indicating that it was
a function of the transgene per se, independent of variability
conferred by insertional effects (Table 1). The central differentiation
event within the pro-B-cell compartment is the V(D)J rearrangement of
the IgH locus, which, if successful and in-frame, leads to synthesis of
the Igµ protein. Expression of Igµ in the context of the
pre-B-cell receptor complex delivers an intracellular signal that
leads to cellular proliferation and maturation to the pre-B-cell
stage.30 Differentiation events within the pro-B-cell
compartment correlate with distinct cellular subfractions defined by
the expression of HSA and BP-1.25,29 Within
fraction A (HSA Mice with defective pre-B-cell receptor signals Fang et al28 found that pro-B cells were expanded in Eµ-Bcl-xL transgenic mice, similar to the phenotype observed in Eµ-A1 mice. The expanded population of pro-B cells in these mice had increased levels of IgH V(D)J rearrangements and contained an increased frequency of IgH alleles with nonproductive V(D)J rearrangements, leading to the suggestion that pro-B-cell accumulation reflected the survival of cells that had failed to achieve an in-frame IgH rearrangement and that would normally undergo apoptosis. As noted above, the pro-B-cell accumulation in Eµ-A1 mice did not reflect a bias toward cells with completed V(D)J joining of IgH alleles (Figure 4), suggesting that this explanation does not apply. A reduction in the efficiency of IgH gene assembly at the pro-B-cell stage could explain the phenotype of Eµ-A1 transgenic mice. We did not observe a significant effect of the transgene on the expression of Rag-1 or Rag-2 mRNA or on germline transcription of the IgH locus. However, these data do not exclude subtle effects or differences at the level of Rag protein expression. A final question is whether the apparent block in pro- to pre-B-cell development could simply reflect differences in expression of the A1 transgene in these compartments. Although this is not the expected expression pattern based on the Eµ control element, the lack of reagents capable of detecting A1 protein in vivo precludes direct testing of this possibility. However, this scenario would not readily explain the absolute reduction in pre-B cells observed in transgenic mice. To further investigate the properties of A1 overexpression in pro-B
cells, we studied the effects of this transgene in the SCID background.
The SCID defect impairs rejoining of coding DNA ends in V(D)J
recombination, leading to a developmental block at the pro-B stage
that is similar to the developmental block in Rag-1 Exposure of newborn SCID mice to low levels of A1 is normally expressed at low levels in early B cells and is up-regulated in long-lived peripheral B cells, where it may enhance B-cell memory.18 In contrast, Bcl-2 is expressed in pro-B and mature B cells and is down-regulated at the pre-B-cell and immature B-cell stages, whereas Bcl-xL is expressed in pre-B cells and is down-regulated at the mature B stage.12,60,61 A1 and Bcl-xL are up-regulated in response to CD40 signals, by which they appear to contribute to cell survival in the context of B-cell receptor signaling.23,62-68 These data suggest that the physiologic role of A1 is more likely to be important in later stages of B-cell function than at the pro-B-cell stage. It is probable that Eµ-A1 mice express the transgene in peripheral B cells based on the known properties of Eµ-dependent transgenes and on the observed increases in splenic A1-a mRNA. Unexpectedly, we observed a decrease in peripheral B cells in Eµ-A1 transgenic mice, in contrast to B-cell accumulation reported in Eµ-Bcl-2 and Eµ-Bcl-xL transgenic mice.12,28,32,48 This observation corresponded with a modest decrease in serum immunoglobulin levels of switched subclasses; however, serum immunoglobulin levels in mice continued to increase up to 6 months of age, and it remains unknown whether the Eµ-A1 transgene would affect steady state immunoglobulin levels. These observations may reflect the early developmental effects of the transgene in the B-cell lineage rather than indicating a negative effect of A1 overexpression on peripheral B cells. Bcl-2, Bcl-xL, and A1 share the property of antagonizing cell death. Genetic experiments show that overexpression of Bcl-xL in the T-cell lineage can rescue the Bcl-2 null phenotype in mature T cells,49 illustrating the functional overlap in these proteins in vivo. Because the regulation of these proteins differs, the extent to which they may have distinct individual functions remains unclear. Transgenic mice made under the control of similar expression elements permit comparison of the effects of Bcl-2 homologs on the development and function of the immune system. Our studies indicate that the phenotype of Eµ-A1 mice most resembles that of Eµ-Bcl-xL mice in that the pro-B-cell compartment is strongly affected. However, differences in the IgH rearrangement status of the accumulated pro-B cells suggest that the underlying causes of this phenotype may be different in the 2 strains. In contrast, Bcl-2 appears to have a less prominent effect on the pro-B-cell compartment. Despite these observations, Bcl-2 can rescue the pro- to pre-B-cell transition in SCID mice, whereas A1 does not. Inasmuch as neither the complete spectrum of cellular effects nor the relative activity of antiapoptotic Bcl-2 family members is known, it cannot be ruled out that differences in expression levels among the different transgenic mouse lines reported are responsible for these phenotypic contrasts. However, given the consistency of these phenotypic differences across numerous individual mouse lines, these studies lend further support to the notion that antiapoptotic Bcl-2 homologs interact differentially with intracellular pathways affecting cell fate in lymphoid cells. It is unclear whether all the available data can be accounted for by differences in cell-survival effects of these proteins. Further consideration of the potential roles for Bcl-2 homologs in other cellular pathways are, therefore, warranted in future studies.
Submitted August 8, 2001; accepted December 31, 2001.
Supported by National Institutes of Health grants PHS03174 (J.M.H.), AI41051 (D.M.W.), and CA88075 (D.M.W.) and by an American Lung Association- Washington Affiliate Research Grant (P.I.C.). P.I.C. is the recipient of an American Heart Association Clinician Scientist Award. C.Y.L. is the recipient of a Chang-Gung Memorial Hospital Sabbatical Scholarship Award.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Dennis M. Willerford, Division of Hematology, University of Washington School of Medicine, Box 357710, 1959 NE Pacific St, Seattle, WA 98195; e-mail: dwiller{at}u.washington.edu.
1. Vaux DL, Korsmeyer SJ. Cell death in development. Cell. 1999;96:245-254[CrossRef][Medline] [Order article via Infotrieve]. 2. Jacobson MD, Weil M, Raff MC. Programmed cell death in animal development. Cell. 1997;88:347-354[CrossRef][Medline] [Order article via Infotrieve].
3.
Adams JM, Cory S.
The Bcl-2 protein family: arbiters of cell survival.
Science.
1998;281:1322-1326
4.
Yang E, Korsmeyer SJ.
Molecular thanatopsis: a discourse on the BCL2 family and cell death.
Blood.
1996;88:386-401
5.
Nakayama K, Negishi I, Kuida K, Sawa H, Loh DY.
Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.
Proc Natl Acad Sci U S A.
1994;91:3700-3704
6.
Nakayama K, Negishi I, Kuida K, et al.
Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.
Science.
1993;261:1584-1588 7. Veis DJ, Sorenson CM, Shutter JR, Korsmeyer SJ. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell. 1993;75:229-240[CrossRef][Medline] [Order article via Infotrieve]. 8. Strasser A, O'Connor L, Dixit VM. Apoptosis signaling. Annu Rev Biochem. 2000;69:217-245[CrossRef][Medline] [Order article via Infotrieve].
9.
Grillot DA, Merino R, Nunez G.
Bcl-XL displays restricted distribution during T cell development and inhibits multiple forms of apoptosis but not clonal deletion in transgenic mice.
J Exp Med.
1995;182:1973-1983
10.
Motoyama N, Wang F, Roth KA, et al.
Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.
Science.
1995;267:1506-1510
11.
Ma A, Pena JC, Chang B, et al.
Bclx regulates the survival of double-positive thymocytes.
Proc Natl Acad Sci U S A.
1995;92:4763-4767
12.
Grillot DA, Merino R, Pena JC, et al.
Bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice.
J Exp Med.
1996;183:381-391 13. Yang XF, Weber GF, Cantor H. A novel Bcl-x isoform connected to the T cell receptor regulates apoptosis in T cells. Immunity. 1997;7:629-639[CrossRef][Medline] [Order article via Infotrieve]. 14. Mueller DL, Seiffert S, Fang W, Behrens TW. Differential regulation of bcl-2 and bcl-x by CD3, CD28, and the IL-2 receptor in cloned CD4+ helper T cells: a model for the long-term survival of memory cells. J Immunol. 1996;156:1764-1771[Abstract]. 15. Lin EY, Orlofsky A, Berger MS, Prystowsky MB. Characterization of A1, a novel hemopoietic-specific early-response gene with sequence similarity to bcl-2. J Immunol. 1993;151:1979-1988[Abstract].
16.
Orlofsky A, Somogyi RD, Weiss LM, Prystowsky MB.
The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils.
J Immunol.
1999;163:412-419 17. Chuang PI, Yee E, Karsan A, Winn RK, Harlan JM. A1 is a constitutive and inducible Bcl-2 homologue in mature human neutrophils. Biochem Biophys Res Commun. 1998;249:361-365[CrossRef][Medline] [Order article via Infotrieve].
18.
Tomayko MM, Cancro MP.
Long-lived B cells are distinguished by elevated expression of A1.
J Immunol.
1998;160:107-111
19.
Tomayko MM, Punt JA, Bolcavage JM, Levy SL, Allman DM, Cancro MP.
Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.
Int Immunol.
1999;11:1753-1761
20.
Karsan A, Yee E, Kaushansky K, Harlan JM.
Cloning of human Bcl-2 homologue: inflammatory cytokines induce human A1 in cultured endothelial cells.
Blood.
1996;87:3089-3096
21.
Hatakeyama S, Hamasaki A, Negishi I, Loh DY, Sendo F, Nakayama K.
Multiple gene duplication and expression of mouse bcl-2-related genes, A1.
Int Immunol.
1998;10:631-637 22. Pircher H, Mak TW, Lang R, et al. T cell tolerance to Mlsa encoded antigens in T cell receptor V beta 8.1 chain transgenic mice. EMBO J. 1989;8:719-727[Medline] [Order article via Infotrieve]. 23. Craxton A, Chuang PI, Shu G, Harlan JM, Clark EA. The CD40-inducible Bcl-2 family member A1 protects B cells from antigen receptor-mediated apoptosis. Cell Immunol. 2000;200:56-62[CrossRef][Medline] [Order article via Infotrieve]. 24. Schlissel MS, Baltimore D. Activation of immunoglobulin kappa gene rearrangement correlates with induction of germline kappa gene transcription. Cell. 1989;58:1001-1007[CrossRef][Medline] [Order article via Infotrieve].
25.
Li YS, Hayakawa K, Hardy RR.
The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver.
J Exp Med.
1993;178:951-960
26.
Leung DT, Morefield S, Willerford DM.
Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo.
J Immunol.
2000;164:3527-3534 27. Willerford DM, Chen J, Ferry JA, Davidson L, Ma A, Alt FW. Interleukin-2 receptor alpha chain regulates the size and content of the peripheral lymphoid compartment. Immunity. 1995;3:521-530[CrossRef][Medline] [Order article via Infotrieve]. 28. Fang W, Mueller DL, Pennell CA, et al. Frequent aberrant immunoglobulin gene rearrangements in pro-B cells revealed by a bcl-xL transgene. Immunity. 1996;4:291-299[CrossRef][Medline] [Order article via Infotrieve].
29.
Hardy RR, Carmack CE, Shinton SA, Kemp JD, Hayakawa K.
Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.
J Exp Med.
1991;173:1213-1225 30. Willerford DM, Swat W, Alt FW. Developmental regulation of V(D)J recombination and lymphocyte differentiation. Curr Opin Genet Dev. 1996;6:603-609[CrossRef][Medline] [Order article via Infotrieve].
31.
Lin EY, Orlofsky A, Wang HG, Reed JC, Prystowsky MB.
A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation.
Blood.
1996;87:983-992 32. McDonnell TJ, Deane N, Platt FM, et al. Bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell. 1989;57:79-88[CrossRef][Medline] [Order article via Infotrieve]. 33. Strasser A, Harris AW, Cory S. Bcl-2 transgene inhibits T cell death and perturbs thymic self-censorship. Cell. 1991;67:889-899[CrossRef][Medline] [Order article via Infotrieve]. 34. Sentman CL, Shutter JR, Hockenbery D, Kanagawa O, Korsmeyer SJ. Bcl-2 inhibits multiple forms of apoptosis but not negative selection in thymocytes. Cell. 1991;67:879-888[CrossRef][Medline] [Order article via Infotrieve]. 35. Strasser A, Harris AW, Corcoran LM, Cory S. Bcl-2 expression promotes B- but not T-lymphoid development in SCID mice. Nature. 1994;368:457-460[CrossRef][Medline] [Order article via Infotrieve]. 36. Young F, Mizoguchi E, Bhan AK, Alt FW. Constitutive Bcl-2 expression during immunoglobulin heavy chain-promoted B cell differentiation expands novel precursor B cells. Immunity. 1997;6:23-33[CrossRef][Medline] [Order article via Infotrieve]. 37. O'Connor L, Huang DC, O'Reilly LA, Strasser A. Apoptosis and cell division. Curr Opin Cell Biol. 2000;12:257-263[CrossRef][Medline] [Order article via Infotrieve].
38.
O'Reilly LA, Harris AW, Tarlinton DM, Corcoran LM, Strasser A.
Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo.
J Immunol.
1997;159:2301-2311
39.
Danska JS, Pflumio F, Williams CJ, Huner O, Dick JE, Guidos CJ.
Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents.
Science.
1994;266:450-455 40. Strasser A, Harris AW, Cory S. E mu-bcl-2 transgene facilitates spontaneous transformation of early pre-B and immunoglobulin-secreting cells but not T cells. Oncogene. 1993;8:1-9[Medline] [Order article via Infotrieve].
41.
Zhou P, Levy NB, Xie H, et al.
MCL1 transgenic mice exhibit a high incidence of B-cell lymphoma manifested as a spectrum of histologic subtypes.
Blood.
2001;97:3902-3909 42. Chao DT, Korsmeyer SJ. BCL-2 family: regulators of cell death. Annu Rev Immunol. 1998;16:395-419[CrossRef][Medline] [Order article via Infotrieve].
43.
Hamasaki A, Sendo F, Nakayama K, et al.
Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene.
J Exp Med.
1998;188:1985-1992
44.
Kausalya S, Somogyi R, Orlofsky A, Prystowsky MB.
Requirement of A1-a for bacillus Calmette-Guerin-mediated protection of macrophages against nitric oxide-induced apoptosis.
J Immunol.
2001;166:4721-4727 45. Rothenberg EV. The development of functionally responsive T cells. Adv Immunol. 1992;51:85-214[Medline] [Order article via Infotrieve]. 46. Gratiot-Deans J, Ding L, Turka LA, Nunez G. Bcl-2 proto-oncogene expression during human T cell development: evidence for biphasic regulation. J Immunol. 1993;151:83-91[Abstract].
47.
Strasser A, Harris AW, von Boehmer H, Cory S.
Positive and negative selection of T cells in T-cell receptor transgenic mice expressing a bcl-2 transgene.
Proc Natl Acad Sci U S A.
1994;91:1376-1380
48.
Katsumata M, Siegel RM, Louie DC, et al.
Differential effects of Bcl-2 on T and B cells in transgenic mice.
Proc Natl Acad Sci U S A.
1992;89:11376-11380
49.
Chao DT, Linette GP, Boise LH, White LS, Thompson CB, Korsmeyer SJ.
Bcl-XL and Bcl-2 repress a common pathway of cell death.
J Exp Med.
1995;182:821-828
50.
Tao W, Teh SJ, Melhado I, Jirik F, Korsmeyer SJ, Teh HS.
The T cell receptor repertoire of CD4
51.
Chao DT, Korsmeyer SJ.
BCL-XL-regulated apoptosis in T cell development.
Int Immunol.
1997;9:1375-1384
52.
Bouillet P, Metcalf D, Huang DC, et al.
Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity.
Science.
1999;286:1735-1738 53. Ehlich A, Schaal S, Gu H, Kitamura D, Muller W, Rajewsky K. Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development. Cell. 1993;72:695-704[CrossRef][Medline] [Order article via Infotrieve]. 54. Cheng AM, Rowley B, Pao W, Hayday A, Bolen JB, Pawson T. Syk tyrosine kinase required for mouse viability and B-cell development. Nature. 1995;378:303-306[CrossRef][Medline] [Order article via Infotrieve]. 55. Huang DC, O'Reilly LA, Strasser A, Cory S. The anti-apoptosis function of Bcl-2 can be genetically separated from its inhibitory effect on cell cycle entry. EMBO J. 1997;16:4628-4638[CrossRef][Medline] [Order article via Infotrieve]. 56. D'Sa-Eipper C, Chinnadurai G. Functional dissection of Bfl-1, a Bcl-2 homolog: anti-apoptosis, oncogene-cooperation and cell proliferation activities. Oncogene. 1998;16:3105-3114[CrossRef][Medline] [Order article via Infotrieve]. 57. Roth DB, Menetski JP, Nakajima PB, Bosma MJ, Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992;70:983-991[CrossRef][Medline] [Order article via Infotrieve].
58.
Tarlinton DM, Corcoran LM, Strasser A.
Continued differentiation during B lymphopoiesis requires signals in addition to cell survival.
Int Immunol.
1997;9:1481-1494
59.
Wang C, Bogue MA, Levitt JM, Roth DB.
Irradiation-mediated rescue of T cell-specific V(D)J recombination and thymocyte differentiation in severe combined immunodeficient mice by bone marrow cells.
J Exp Med.
1999;190:1257-1262 60. Merino R, Ding L, Veis DJ, Korsmeyer SJ, Nunez G. Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes. EMBO J. 1994;13:683-691[Medline] [Order article via Infotrieve]. 61. Choi MS, Holmann M, Atkins CJ, Klaus GG. Expression of blc-x during mouse B cell differentiation and following activation by various stimuli. Eur J Immunol. 1996;26:676-682[Medline] [Order article via Infotrieve].
62.
Lee HH, Dadgostar H, Cheng Q, Shu J, Cheng G.
NF- 63. Kuss AW, Knodel M, Berberich-Siebelt F, Lindemann D, Schimpl A, Berberich I. A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death. Eur J Immunol. 1999;29:3077-3088[CrossRef][Medline] [Order article via Infotrieve].
64.
Fang W, Nath KA, Mackey MF, Noelle RJ, Mueller DL, Behrens TW.
CD40 inhibits B cell apoptosis by upregulating bcl-xL expression and blocking oxidant accumulation.
Am J Physiol.
1997;272:C950-C956 65. Ishida T, Kobayashi N, Tojo T, Ishida S, Yamamoto T, Inoue J. CD40 signaling-mediated induction of Bcl-XL, Cdk4, and Cdk6: implication of their cooperation in selective B cell growth. J Immunol. 1995;155:5527-5535[Abstract]. 66. Merino R, Grillot DA, Simonian PL, et al. Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells: dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction. J Immunol. 1995;155:3830-3838[Abstract]. 67. Choi MS, Boise LH, Gottschalk AR, Quintans J, Thompson CB, Klaus GG. The role of bcl-XL in CD40-mediated rescue from anti-mu-induced apoptosis in WEHI-231 B lymphoma cells. Eur J Immunol. 1995;25:1352-1357[Medline] [Order article via Infotrieve].
68.
Grumont RJ, Rourke IJ, Gerondakis S.
Rel-dependent induction of A1 transcription is required to protect B cells from antigen receptor ligation-induced apoptosis.
Genes Dev.
1999;13:400-411
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. J. Herold, J. Zeitz, C. Pelzer, C. Kraus, A. Peters, G. Wohlleben, and I. Berberich The Stability and Anti-apoptotic Function of A1 Are Controlled by Its C Terminus J. Biol. Chem., May 12, 2006; 281(19): 13663 - 13671. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Shahaf, K. Johnson, and R. Mehr B cell development in aging mice: lessons from mathematical modeling Int. Immunol., January 1, 2006; 18(1): 31 - 39. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Mahadevan, C. Spier, K. Della Croce, S. Miller, B. George, C. Riley, S. Warner, T. M. Grogan, and T. P. Miller Transcript profiling in peripheral T-cell lymphoma, not otherwise specified, and diffuse large B-cell lymphoma identifies distinct tumor profile signatures Mol. Cancer Ther., December 1, 2005; 4(12): 1867 - 1879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Xiang, O. Windl, G. Wunsch, M. Dugas, A. Kohlmann, N. Dierkes, I. M. Westner, and H. A. Kretzschmar Identification of Differentially Expressed Genes in Scrapie-Infected Mouse Brains by Using Global Gene Expression Technology J. Virol., October 15, 2004; 78(20): 11051 - 11060. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. G. Sandler, M. M. Mentink-Kane, A. W. Cheever, and T. A. Wynn Global Gene Expression Profiles During Acute Pathogen-Induced Pulmonary Inflammation Reveal Divergent Roles for Th1 and Th2 Responses in Tissue Repair J. Immunol., October 1, 2003; 171(7): 3655 - 3667. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-actin, 
5, RAG-1, and
RAG-2, as described by Li et al.
for example, through
mutation of 
B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes.
Proc Natl Acad Sci U S A.
1999;96:9136-9141