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Research Unit on Autoimmune Diseases, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, México City, Distrito Federal, México;
Department of Molecular Virology, Center for Research in Infectious Diseases, Instituto Nacional de Salud Pública, Cuernavaca, México;
Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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EP-CD154). Precursors beyond pro-B
cells were absent in adult bone marrow but were increased in the fetal
liver. Newborn EP-CD154 mice had largely increased numbers of
peripheral B cells, which were CD154+, and that 36 h
after birth expressed high surface levels of CD23 and MHC class II,
resembling activated mature B cells. Nevertheless, EP-CD154 mice
were hypogammaglobulinemic, indicating that the expanded population of
apparently activated B cells was nonfunctional. Further analysis
revealed that soon after birth, EP-CD154 mice-derived B cells became
CD5+/Fas+, after which progressively decreased
in the periphery in a CD154-CD40-dependent manner. These results
indicate that CD40 ligation during B cell ontogeny induces negative
selection characterized by either hyporesponsiveness or an arrest in
maturation depending on the time of analysis and the anatomic site
studied. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During ontogeny, B cell precursors sequentially express several genes and receptors, some of which are involved in their maturation. The CD40 receptor is expressed during B cell ontogeny but its possible function in early B cell precursors is unknown. In mice, CD40 expression was first detected at the pre-B cell stage (15), differing from humans, where CD40 is first seen on pro-B cells (4).
The majority of information on the role of CD40 in B cell function relates to its positive effects on proliferation and differentiation. However, depending on the stage of differentiation and/or functional state, CD40 can also negatively influence B cells (16, 17). Thus, CD40 plus BCR signaling on immature B cells leads to tolerance and Fas-FasL dependent exclusion from germinal centers (16). Moreover, under certain circumstances, CD40 ligation stops terminal B cell differentiation in mice (17) and can also inhibit Ig secretion by human tonsilar IgD- memory B cells (18).
In normal individuals, autoreactive B cells are inactivated in different ways leading to tolerance. After BCR signaling, mainly in the periphery, immature IgM+ B cells follow one of three fates: receptor editing (19, 20), clonal deletion (21), or clonal anergy (22). Although CD40 appears to participate in B cell clonal anergy (23, 24), its possible role in other forms of B cell tolerance has not been clarified.
However, abnormal CD154 expression can induce a pathological state. Thus, constitutive CD154 expression in the skin results in T cell-mediated local autoimmune disease, through activation of Langerhans dendritic cells, in addition to autoantibody-mediated systemic connective tissue disease (25). The latter could be due to a direct effect of CD154 on B cells, leading to B cell hyperresponsiveness to BCR signaling, which in the case of self-reactive B cells would result in autoantibody switch recombination and secretion (7, 14). Most pathogenic autoantibodies bear isotypes which are dependent of CD40-CD154 interactions such as IgG. It was reported that peripheral blood B cells of systemic lupus erythematosus patients (27, 28, 29), as well as mice models of systemic lupus erythematosus (30) constitutively express CD154. However, its possible role in the pathogenesis of autoantibody production was not examined. Although normal B cells have also been found to express CD154 (31, 32), its meaning is unknown.
CD40 ligation could also influence B cell fate during positive and/or negative selection, as suggested by the increase in autoreactive B cells in human CD154 deficiency (33). Thus, while CD154-CD40 is necessary for terminal B cell differentiation and pathogenic autoimmunity, its absence alters shaping of the B cell repertoire.
To gain insight on the effects of augmented CD40 ligation on B cells, and to examine a possible role of CD154-CD40 in B cell ontogeny in vivo, we generated transgenic mice (tgM) with selective expression of CD154 by B cells. Although these mice have an early expansion of B cells, adult mice show a profound reduction of the peripheral B cell compartment due to both peripheral loss and failure of B cell maturation during early stages of bone marrow (BM) (2) lymphopoiesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CD154 cDNA (kindly provided by Dr. M. Berton, University of
Arizona, Tuscon, AZ (34) was inserted in a plasmid
containing the K14 
-globin cassette (kindly provided by Dr. E.
Fuchs, University of Michigan, Ann Arbor, MI), where the K14 promoter
was substituted by a 1.6-kb fragment of the plasmid pKenVpr, a
gift of Dr. U. Storb (University of Chicago, Chicago, IL; Fig. 1
a), which contains a
cassette with the V intronic enhancer, linked to the V promoter.
To excise the entire enhancer/promoter cassette, a second
HindIII site of pKenVpr located between the enhancer
and promoter was eliminated by partial HindIII
digestion, followed by isolation of the linearized plasmid, which was
then filled-in with Klenow DNA polymerase and re-ligated with T4 DNA
ligase (Life Technologies, Rockville, MD). The enhancer/promoter
cassette was subcloned by blunt ligation of a filled-in
HindIII-XbaI fragment into a filled-in
AvaI site of the CD154/K14 plasmid, where the 0.8-kb
human globin intron was left downstream of the promoter as a
splicing donor, followed by CD154, and the 3' untranslated region of
the human K14 gene, respectively. We refer to this construct as
EP-CD154.
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tgM expressing CD154 on B cells (EP-CD154 mice) were
generated by microinjection of (C57BL/6xC3H)F1 zygotes with
a HindIII-EcoRI 3.6 kb fragment of
EP-CD154 containing the Ig regulatory elements, CD154 cDNA, plus
K14-splicing and poly(A) signals. The potentially transgenic progeny
was screened by Southern blot of 10 µg XbaI-digested
tail genomic DNA with a [32P]dCTP-labeled 0.8-kb human
-globin probe. Further screening was conducted in genomic tail DNA
by PCR amplification of a CD154 cDNA 0.8 kb fragment with the primers:
5'-GGA ATT CTG CAG ATC ATG ATA GAA ACA-3' and 5'-GGG CCC TCT AGA ACA
GCG CAC TGT TCA-3' in 1x PCR buffer, 1.5 mM MgCl2,
at 95°C, 55°C, and 72°C for 30 s each, for 35 cycles.
CD40 KO in 129/v background mice (kindly provided by Dr. R. Geha,
Harvard Medical School, Boston, MA; Ref. 35) were
maintained in 129/v background and were crossed with EP-CD154 mice.
Screening of CD40-/- progeny was conducted with
appropriate primers by PCR as described (35) and
homozigocity for null CD40 alleles was determined by flow cytometry
analysis of peripheral blood lymphocytes with
anti-CD40-PE-conjugated mAb (BD PharMingen, San Diego, CA).
C57BL/6 MD4 anti-hen egg white lysosome (HEL) Ig transgenics
(22), were maintained in C57BL/6 background and identified
by flow cytometry analysis of peripheral blood lymphocytes with
anti-B220-FITC-conjugated mAb and biotinylated HEL, followed by
streptavidin-PE (Jackson ImmunoResearch Laboratories, West Grove, PA).
Due the virtual absence of peripheral B cells, genotyping of
EP-CD154xMD4 double tgM was achieved by PCR as described in
http://www.jax.org/resources.
Because most EP-CD154 had growth retardation, reduced lifespan (an
average of 6 mo), and lower breeding capacity than normal mice, they
were maintained in a 129/v CD40-/- background,
which improved their health status. F1 crosses of
EP-CD154/CD40-/- with C57BL/6 mice were used
for most experiments. All mice strains were maintained in the
conventional facility at the Instituto Nacional de Salud Pública
(Cuernavaca, Mexico). All mice experiments were performed in compliance
with international and institutional guidelines, and the protocol was
approved by the Ethics Committee of the Coordinación de
Investigación Médica, Instituto Mexicano del Seguro
Social, Mexico.
Semiquantitative southern RT-PCR
Total RNA was obtained from tissue homogenates in TRIzol reagent
(Life Technologies) according to manufacturer instructions with a
second re-extraction to ensure elimination of genomic DNA. A total of 5
µg RNA was reverse-transcribed with Superscript II RT (Life
Technologies) and 1/25 reaction volume was amplified by PCR with the
oligonucleotide pairs: CD154 Gp39F (363–385): 5'-GGA
TCC TCA AAT TGC AGC ACA CG-3' and CD154 Isoser
891–617(891–617): 5'-CTC TCA GAT CCA ATG CTG GGC TTC AGC-3' described by
Armitage et al. (36) for CD154 expression and, as an
internal control with -actin primers 5'-CCA AGG CCA ACC GCG AGA AGA
TGA C-3' and 5'-AGG GTA CAT GGT GGT GCC GCC AGA C-3'. The CD154 primers
amplify a 255 bp fragment spanning exons 4 and 5, which in wild-type
(WT) mice allows the distinction between genomic DNA from cDNA. After a
15 cycle amplification, samples were run in 2% agarose gels and
transferred onto nylon membranes (HyBond N; Amersham Pharmacia,
Buckinghamshire, U.K.) in 20 x SCC, then hybridized at 60°C
with alkaline phoshatase-labeled CD154 and -actin cDNA probes,
detected with a chemilumescent kit according to manufacturer
instructions (AlkPhos; Amersham Pharmacia) and exposed for 1 h to
Hyperfilm ECL (Amersham Pharmacia).
Lymphoid cell preparations
Spleen cells were obtained in FACS buffer (PBS, pH 7.4, 2% FBS, 5 mM EDTA, 1% rabbit serum, and 0.1% NaN3) by dissociation of spleens between frosted glass slides. BM cells were obtained by injecting 0.75 ml of FACS buffer in one end of the femur, and collecting the cells at the other end. Fetal liver cells were obtained from timed matings of the different mice combinations, as described for spleen cells. Contaminating RBC in all cell suspensions were eliminated with 0.16 M NH4Cl buffer, and then washed twice in FACS buffer. The total cell number in each suspension was estimated by counting in a hemocytometer.
Cell staining and flow cytometry analysis
A total of 1 x 106 cells were reacted in FACS buffer for 20 min on ice with combinations of the following conjugated mAb: anti-B220/CyChrome or FITC, anti-CD43 (S7)-PE, anti-BP-1-FITC (Ly-64), anti-CD23-PE, anti-FAS (Jo2)/biotin, anti-CD24-biotin (30-F1), anti-IgM (LoMe9)-FITC, anti-IgD-biotin, anti-CD40-FITC, anti-CD154 (MR1)-biotin, anti-CD5-PE, and anti-Thy 1.2-biotin (BD PharMingen). In experiments with MD4 tgM, specific B cells were labeled with biotinylated HEL. Streptavidin-FITC, streptavidin-PE or streptavidin-PerCP (BD PharMingen) were used as a second label for biotin-conjugated reagents. Samples were run on a dual laser FACSCalibur and analyzed with CellQuest software (BD Immunocytometry Systems, Mountain View, CA).
Serum Igs
These were determined by an ELISA method with biotinylated rabbit anti-mouse isotype-specific Abs followed by HRP-labeled streptavidin in polystyrene plates (Nunc, Roskilde, Denmark) coated with polyvalent goat anti-mouse Ig. Plates were read in an automated ELISA reader (Labsystems, Helsinki, Finland) at 492 nm. All ELISA reagents were purchased from Zymed Laboratories (San Francisco, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EP-CD154 mice
Six of 10 EP-CD154 transgenic founders were runted, and died
before 4 wk of age, which precluded further analysis. The remaining
four founders were backcrossed to C57BL/6 mice for further analysis.
Semiquantitative RT-PCR showed maximal transgene expression in the
spleen, followed by lymph nodes (LN), thymus, lung, and adult liver
(Fig. 1
c).
Decreased number of peripheral B cells in adult EP-CD154
mice
Preliminary flow cytometric analysis aiming at defining transgene
expression in 8-wk-old bEP-CD154 mice revealed a marked decrease of
B220+ cells in peripheral blood, LN, spleen, BM, and
peritoneal cavity (not shown). The microscopic architecture of LN and
spleen was abnormal with increased white pulp, poorly defined
follicles, and absent germinal centers (Fig. 2). As these findings were consistent in
all surviving transgenic lines, the results presented hereafter derive
from analysis of one representative line, 28.
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EP-CD154 mice had about half
the total cell number as nontransgenic littermates, B cells were
decreased by at least 15-fold (Fig. 3a). This was more evident in
LN, where B220+ cells were less than 3% (data
not shown). The few remaining B220+, but no other
cells, in the spleens of EP-CD154 mice expressed low density CD154
(Fig. 1b). Additional findings included slightly increased T
cell numbers and greatly increased
Mac-1+/Gr-1+ cells in
spleens and LN of 10- to 12-wk-old EP-CD154 mice (data not shown).
The B cell defect in EP-CD154 mice was due to CD154-CD40
ligand-receptor interaction, as B cell numbers in adult
EP-CD154-transgenic-CD40-/- mice
(35) were normal (Fig. 2b).
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EP-CD154 mice
Ten to 12-wk-old EP-CD154 mice had few peripheral B cells,
which in older mice were only vestiges. Therefore, we examined the
EP-CD154 B cell compartment from newborn to adult animals in greater
detail. Newborn EP-CD154 spleens, had twice total B cell numbers as
control mice (Fig. 3c), indicating that during fetal
life, B cells were generated and expanded in EP-CD154 mice. At two
weeks, EP-CD154 mice had enlarged spleens (Fig. 2a)
and the number of peripheral B cells was even larger, until 4 wk, when
both relative and absolute B cell counts progressively declined until
almost absent (Fig. 3c). These data suggest that the B
cell defect in EP-CD154 mice, is due to a peripheral loss that
occurs after birth.
Deficient B cell maturation in the BM but not in the fetal liver of
EP-CD154 mice
During fetal life in mammals, B cell lymphopoiesis occurs in the
liver, although after birth it takes place in BM
(37). Fetal EP-CD154 mice appeared to have a
spared B cell lymphopoiesis as indicated by the B cell expansion from
birth to 2 wk of age. However, the inability to replenish the B cell
compartment after this time suggested that in addition to the
peripheral B cell loss, EP-CD154 BM was unable of generating new B
cells. Hence, B220+ cells in BM or in newborn livers of
EP-CD154 and nontransgenic littermates, were examined by flow
cytometry (Fig. 3d). As seen, while B220+
cell numbers in newborn EP-CD154 mice livers were increased when
compared with nontransgenic littermates, from 2 to 32 wk of age (the
earliest and latest ages examined), total B220+ cells in BM
of EP-CD154 mice were markedly decreased. Thus, in EP-CD154 mice,
B cell lymphopoiesis is enhanced in the fetal liver, but it is
profoundly impaired in the BM. Of note, both
IgM+/B220+ and
IgM-/B220+ cells were increased in the livers
of newborn EP-CD154 mice (see below).
B cell maturation in the BM of EP-CD154 mice halts during or
after the pro-B cell stage
B cell ontogeny in the liver and BM, in mice, are slightly
different (37). However, in both sites, the earliest
identifiable B cell precursor is B220low/CD43+
(pro-B), whereas pre-B cells (the next stage of differentiation) are
B220+/CD43- (38). Therefore, we
conducted a detailed two-color FACS analysis of B cell precursor
profile in newborn liver and adult BM of EP-CD154 mice to define at
which stage was B cell ontogeny impaired in EP-CD154 mice, and to
establish possible differences between adult BM and newborn liver.
As shown in Fig. 4a, at all
ages studied, the BM of EP-CD154 mice had an almost complete absence
of B220low/CD43- pre-B
cells, but not of
B220low/CD43+ pro-B cells.
In contrast, and as shown above, B220+ cells were
increased in the livers of newborn EP-CD154 mice. Indeed, all B cell
precursors, regardless of their stage of maturation, were increased in
the livers of newborn EP-CD154 mice (Figs. 3b and
4b). Similar findings were obtained in 18-day fetal livers
(data not shown).
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EP-CD154 liver B cells (which in normal
newborn mice are immature B cells) were higher than in controls (data
not shown), resembling the phenotype of mature B cells. Control
32-wk-old BM had a reduced number of B cell precursors, which is
age-related (37); yet, EP-CD154 BM had one-tenth the
number of pre-B cells. Finally, young EP-CD154 mice had a
considerable number of
B220high/CD43- cells in
the BM, which have been considered by some (38) as
recirculating B cells.
Taken together, these results indicate that transition from pro-B to
pre-B takes place in neonatal EP-CD154 livers and their B220 levels
resemble recirculating B cells (38), suggesting an
accelerated rate of maturation, whereas in EP-CD154 BM, B cell
development stops during pro-B to pre-B cell transition. The fact that
IgM-/B220+ cells,
representing all B cell precursor stages before immature B cells, are
also increased in the fetal liver, indicates that B cell expansion in
fetal and newborn EP-CD154 mice is due to a signal delivered in the
fetal liver at either pro-B or pre-B cell stages. As the only
difference between EP-CD154 and normal mice is the transgene
(CD154), it seems reasonable to conclude that CD40 signaling at early
stages of B cell ontogeny in the fetal liver or in the BM has distinct
effects. Hence, CD40 ligation in the BM prevents B cell precursor
maturation beyond the pro-B cell stage, whereas in the fetal liver, the
same signal induces B cell expansion followed by early maturation and
apparent elimination in the periphery.
Arrest of B cell differentiation in the BM and expansion in the
fetal liver of EP-CD154 mice occurs at a late pro-B cell stage
Expression by B220+/CD43+ cells of the
heat-stable Ag (CD24) and Ly51 (BP-1) subdivide pro-B cells into three
maturational stages (38). The earliest (fraction A) is
CD24-/BP-1-, fraction B is
CD24+/BP-1-, and fraction C is
CD24+/BP-1+. To examine at which pro-B cell
stage B cell ontogeny was arrested in EP-CD154, BM cells were
examined by four-color FACS analysis for B220, CD43, CD24, and BP-1
expression. Fig. 5a shows
that A and B fractions (CD24-/BP-1- and
CD24+/BP-1-, respectively) were present in the
BM of 6- to 8-wk-old mice, whereas late pro-B cells (fraction C) were
absent. Further analysis of B220+ cells with CD25 and
c-Kit, as defined by Melchers and Rolink (Ref. 37 ; Fig. 5b) confirmed the marked decrease of B cell precursors
after the small pre-B1 stage (CD25+/c-Kit-;
see Ref. 37). This indicates that B cell precursor
development in BM of EP-CD154 mice halts after the first heavy chain
gene rearrangement (D–J).
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EP-CD154 liver cells with
C-Kit and B220 revealed an increase of
B220+/c-Kit- B cells that
include all stages of B cell differentiation beginning with small
pre-B1 cells, further suggesting that the signal responsible for B cell
expansion in the liver of EP-CD154 mice is the same as the signal
that prevents B cell maturation in the BM, most likely CD154-CD40.
B cell maturation in BM and in the fetal liver of EP-CD154 mice
is disturbed at the time of CD154-CD40 expression
As it was clear that changes in B cell ontogeny in newborn liver
and adult BM bEP-CD154 were occurring at a discrete stage during
late pro-B cell differentiation, we examined the expression of CD154 in
EP-CD154 mice and CD40 in normal B6 mice by three-color FACS
analysis with B220 and CD43 plus CD40 or CD154. This was important
because it was reported that, in mouse, CD40 is first expressed at the
pre-B cell stage, whereas in the human, CD40 is first seen on pro-B
cells. Fig. 6a shows that
all B cell precursors in normal mice, including
CD43+/B220+ cells (pro-B) were
CD40+. Similarly, B220+ cells in the liver and BM of
EP-CD154 mice were CD154+ (Fig. 6b). This
indicates that the arrest of pro-B cell differentiation in
EP-CD154 BM, as well as B cell expansion in the
liver, are coincident with CD40 and CD154 expression. The earliest
stage where CD154 was expressed in EP-CD154 B cell
precursors was in pro-B cells (data not shown).
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EP-CD154 mice have an activated phenotype
The finding that B220+ cells in young
EP-CD154 mice were increased after the expression CD40 and its
ligand, suggested that CD154-CD40 B-B cell interaction in the fetal
liver was inducing expansion of B cell precursors, which could
no longer be found in the periphery beyond four wk of age. Therefore,
it was important to examine the functional and phenotypic consequences
of CD40 signaling during fetal B cell ontogeny in newborn and young
adult EP-CD154 in peripheral B cells. Initial analysis of the cell
cycle in 7-wk-old EP-CD154 mice showed that among the few B cells
remaining in their spleens, the proportion of cells in S,
G2, and M phases of the cell cycle was higher than in
normal controls (data not shown). Fig. 7, a and b, show that, while at birth,
splenic EP-CD154 B220+ cells had the same phenotype as
controls. As early as 36 h later, EP-CD154 B cells expressed
continuously increasing levels of CD23 and MHC class II. Moreover,
their cell size was increased as demonstrated by a higher forward
scatter (data not shown). These results indicate that perinatal
engagement of CD40 on EP-CD154 B cells or their precursors, most
likely in the fetal liver, induce early maturation and expression of
activation markers. Thus, homotypic pro-B or pre-B cell CD40-CD154
interaction in fetal EP-CD154 mice liver appears to induce
functional changes on their progeny reminiscent of B cell activation,
followed by peripheral loss.
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EP-CD154 mice are not functional
Anergic B cells, before their elimination, express activation
markers and are excluded from entering the spleen follicles, after
which they are eliminated (16). The activated phenotype of
the increased B cells found in young EP-CD154 mice suggested that
these cells could be undergoing clonal inactivation before being
apparently eliminated in the spleen. Therefore, we indirectly examined
the functional status of EP-CD154 mice B cells in vivo by measuring
serum Ig levels. Two-week-old EP-CD154 mice had slightly increased
IgM, but normal or decreased levels of all other Ig isotypes (Fig. 8). These were even lower when corrected
for the total B cell number at each stage (data not shown), and
considering that, at least part of the Ig detected was
maternally-derived. Thus, despite the high numbers of activated B cells
present, young EP-CD154 mice do not have increased serum Igs,
indicating that B cells in young EP-CD154 mice are not functional,
probably anergic.
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EP-CD154 B cells acquire expression of Fas and CD5,
after which they can no longer be detected
Peripheral elimination of anergic B cells is mediated by T cells
in a CD95 (Fas)-CD40L-BCR-dependent manner (16). Moreover,
other studies have indicated that B-2 type B cells can express CD5
(39), which has been associated to tolerance induction
(40). Therefore, we examined the expression of CD95 and
CD5 by EP-CD154 B cells. As seen in Fig. 7, c and
d, in parallel with their acquisition of activation markers,
EP-CD154 B cells became
Fas+/CD5+, remaining
positive as they progressively decreased. Thus, the expanded B cell
population in EP-CD154 mice express surface molecules, which are
involved in deletion or tolerance induction and resemble clonally
anergic B cells.
CD5+ B cells in EP-CD154 are B-2 type B cells
Many authors have long considered CD5 as a marker for the B-1 type
B cell subset, which uses different sets of H and L chain V region
segments (41). Transgenic expression of rearranged Ig
genes that use V regions belonging to either B-1 or B-2 type B cells
leads to development of only the B cell subset corresponding to the V
region in the rearranged gene (42). To determine whether
the CD5+ B cell population present in EP-CD154
mice represented B-1 or B-2 type B cells, EP-CD154 mice were crossed
to mice carrying rearranged transgenic B-2 type Ig H and L chain genes
specific for HEL (22). This experiment was important for
two reasons: first, to define if expression of a rearranged Ag receptor
before CD40 ligation could rescue B cell precursors; second, because
these mice should only develop B-2 type B cells, and therefore, if CD5
were only a B-1 cell marker, no CD5+ cells would
be found in double tgM for anti-HEL-EP and CD154. Fig. 9 shows that HEL-binding B cells in
anti-HEL-EP-CD154 double tgM were CD5+
with no remarkable differences to EP-CD154 mice. These results
indicate that CD5+ B cells in both EP-CD154
and anti-HEL-EP-CD154 double tgM are B-2 type B cells and are,
therefore, in agreement with observations, that CD5 expression by B
cells is not restricted to the B-1 subset (39). Moreover,
no B-1 type Ab specificities were detected in EP-CD154 sera (data
not shown). Although, Ig transgenes did not rescue
anti-HEL-EP-CD154 double tgM B cells from deletion, this was
slightly delayed (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The essential role of CD40 in mature B cell differentiation and isotype switching is beyond controversy. In addition, CD40 ligation rescues B cells from apoptosis in germinal centers during affinity maturation (43, 44, 45, 46). However, the later is not so straightforward, as CD40 can also facilitate elimination of tolerant B cells in a T cell- and Ag-dependent manner (16, 17, 18). Ligation of CD40 plus Fas on the surface B cells rendered tolerant by BCR-signaling when immature leads to their elimination (16), indicating that timing of BCR ligation is critical for the outcome of CD40-signaling. Moreover, CD40 ligation can inhibit proliferation and Ig secretion by normal human B cells (18), human or mouse B cell lymphomas (47), and hybridomas (48). Thus, CD40-signaling can mediate either B cell survival or death in different situations.
Studies on the role of CD40 during early B cell ontogeny are scarce and
in some cases conflicting. In one study (49), CD40
ligation on human fetal BM pro-B cells
(CD34+/CD10+) inhibited
IL-7-induced proliferation in vitro, whereas in another, it induced
surface Ig negative human B cell precursors to proliferate, acquire
expression of CD23, and to become CD10-/surface
Ig-, in response to IL-3 and other cytokines
(50). Although it was reported that CD40 expression both
at the cell surface and mRNA in mice begins in pre-B cells, which are
refractory to in vitro CD40-stimulation (51), in the
current studies, CD40 expression was evident from the pro-B cell stage
both in normal and in EP-CD154 tgM and was functional in vivo, as
demonstrated by the CD154-CD40-dependent B cell depletion in
EP-CD154. It is unlikely that this merely reflects an increased
susceptibility of pro-B cells to signal-induced cell death, as newborn
EP-CD154 B220+ cells in liver and peripheral
lymphoid tissues were expanded. Thus, the profound B cell deficiency in
adult EP-CD154 mice was due to a halt on B cell ontogeny during
pro-B to pre-B cell transition in the BM as well as to peripheral loss
of fetal liver-derived B cells which resembled the phenotype found in
anergic B cells.
CD40-mediated apoptosis has been described for transformed nonlymphoid
cells (52), but the mechanism involved is unclear since
the cytoplasmic tail of CD40 lacks the TNFR family death domains. We
found that B cell precursors in adult BM, but not in the liver of
newborn EP-CD154 mice, expressed Fas (not shown). However, in the
latter, peripheral B cells expressed maturation and activation markers,
and became Fas+ 36 h after birth. Although
we did not show the involvement of Fas in the loss of B cells and their
precursors in EP-CD154 mice, taken together the data suggests
that Fas is involved in the apparent B cell deletion in this model, and
could explain why this loss is delayed in liver-derived, but immediate
in BM-derived B cells. As in mature B cells, CD40 stimulation of early
B cell precursors could induce Fas expression, resulting in increased
susceptibility to apoptosis (53, 54). Overall, what this
implies is not readily apparent, but suggests that B cell development
in the BM and in the liver, is governed by different signals.
The possibility that EP-CD154 B cells did not undergo CD40 ligation
before leaving the liver and the CD154-CD40 signal was instead
delivered in the periphery is unlikely for at least two reasons. First,
liver B cells were expanded in EP-CD154, and the only possible
explanation for this was an increased CD154-CD40 interaction. The
second explanation requires the assumption that only B cell precursors
receiving two signals were the targets of elimination/expansion, and if
the same stimulus led to both events. The first signal is mediated
through the BCR, and the second through CD40. If this is true,
EP-CD154 pre-B cells would receive the dual signaling event.
Although the signaling events following CD40 ligation in mature B cells
are synergistic to BCR signaling, leading to a fully activated state;
in immature B cells, BCR signaling leads to anergy or cell death
(reviewed in Ref. 39).
What could the current findings mean in normal mice? The pre-BCR is a
constitutively signaling molecule (37) that plays a role
in positive selection of pre-B cells expressing a functional H chain,
apparently without additional signals. In contrast, the BCR on immature
B cells signals only after Ag-binding (which is predominantly the case
for self-reactive B cells). This takes place in the periphery, in the
presence of CD154 expressing cells, which do not normally appear to be
present in the BM or liver. Hence, in the periphery, only autoreactive,
immature B cells would concomitantly signal through the BCR plus CD40.
Such dual signaling would delete immature B cells, whereas CD40-only
signaling allows immature B cells to continue their development into
mature µ
B cells and further (55, 56). This
hypothesis is in agreement with the finding that CD154-defective
individuals have an expanded B cell repertoire and persistence of many
autoreactive B cells (33). As there is no information in
the literature concerning CD154 expression in the normal BM and in the
present study, normal mouse BM did not contain CD154 mRNA (Fig. 1), we
propose that this mechanism takes place mainly in the periphery. In
EP-CD154 mice, B cell precursors undergo homotypic
CD154-CD40 interaction at the time their pre-B cell receptor starts
signaling. Like EP-CD154 mice, all cells with a
functional pre-BCR receive both signals. We interpret this as
that they are suffering the same fate as immature autoreactive B cells
in the periphery of normal mice. The nature of the second signal could
not be only restricted to CD40, as expression of other surface
molecules, including other TNF-TNFR family members (57)
can produce a similar effect. This could also have implications in
pathologic states of immune stimulation, where activated T cells could
enter the BM, thus providing CD40 signaling, which could impair B
lymphopoiesis.
The failure of a functional BCR to rescue B cells in
anti-HEL-EP-CD154 double tgM is not clear, as these mice should
not have suffer B cell loss in the absence of their specific Ag or Ig
crosslinking, unless low affinity BCR-Ag interactions were taking place
in the BM, according to our hypothesis that BCR signaling plus
costimulatory signals eliminate B cell precursors. The delayed B cell
deletion seen in EP-CD154 mice suggest that additional mechanisms
are involved.
One last relevant observation of the current studies was the finding
that most B cells in EP-CD154 were CD5+, and
that a transgenic B-2 type BCR did not prevent the expression of CD5.
According to the hypothesis that B-1 or B-2 type phenotypes are
dictated by the BCR V region usage, this indicates that these B cells
are indeed B-2 and not B-1 type cells. CD5 expression by B cells is not
restricted to the B-1 subset, as activated B-2 type B cells can express
CD5 (39), which could be a marker of self-reactive B cells
undergoing tolerance induction, as this phenotype resembles
clonally inactivated B cells (16, 40), and is compatible
with the hypogammaglobulinemia seen in young EP-CD154 mice. Thus,
CD40 ligation on late pro-B or, perhaps, early pre-B cells prevents
their maturation, which is immediate in the BM but delayed in the
liver. Fetal liver-derived B cells in EP-CD154 mice move
into in the periphery, after they further expand until 4 wk of age,
after which they can no longer be found. Such expansion and apparent
elimination is likely to take place in the spleen. The cellular
mechanism responsible for this event was not defined, but it has been
suggested that T cells are responsible for the elimination of clonally
anergic B cells (16), which could be similar to our
findings if the expanded B cell population in EP-CD154
indeed represents clonally anergic B cells. The roles of Fas and CD5
expression on B cell fate and function in EP-CD154 mice,
respectively, deserve further studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to: Dr. José Moreno, Research Unit on Autoimmune Diseases, Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social, Apartado Postal 73-032, 03020 Mexico, D. F. Mexico. E-mail address: jmoreno49{at}prodigy.net.mx
3 Abbreviations used in this paper: BCR, B cell Ag receptor; BM, bone marrow; HEL, hen egg lysosome; LN, lymph node; tgM, transgenic mice; WT, wild type.
Received for publication October 9, 2001. Accepted for publication November 29, 2001.
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References |
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1 and 
Ig gene expression in murine B cells. IL-4 and the CD40 ligand-CD40 interaction provide distinct but synergistic signals. J. Immunol. 155:5637.[Abstract]
-transgenic mice. J. Immunol. 167:2671.
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), or littermate control
mice (
). d, B lymphopoiesis in 
) mice at two, four, and 12 wk. Lines
(