| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*
Integrated Department of Immunology, University of Colorado School of Medicine and National Jewish Medical and Research Center, Denver, CO 80206; and
Departments of Medicine and Pathology, Center for Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
2 is
enhanced in immature B cells and they exhibit greater capacitative
calcium entry in response to Ag. Moreover, B cell linker protein,
Bruton’s tyrosine kinase, and phospholipase C2, which are crucial
for the induction of calcium mobilization responses, are present at
3-fold higher levels in immature B cells, potentially contributing
to increased mobilization of calcium. Consistent with this possibility,
we found that the previously reported lack of
inositol-1,4,5-triphosphate production in immature B cells may be
explained by enhanced inositol-1,4,5-triphosphate breakdown. These data
demonstrate that multiple mechanisms guarantee increased Ag-induced
mobilization of calcium in immature B cells and presumably ensure
elimination of autoreactive B cells from the
repertoire. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
In both mature and immature B cells, the B cell Ag receptor
(BCR)4 is composed of
membrane (m) Ig noncovalently associated with disulfide-linked
heterodimers of immunoreceptor tyrosine-based activation motif
(ITAM)-containing CD79a and CD79b (3, 4, 5). BCR aggregation
by Ag or anti-receptor Ab mediates clustering of associated Src
family kinase molecules leading to phosphorylation of ITAM
tyrosines (6, 7). This leads to the recruitment of
additional Src family kinases as well as the tyrosine kinase Syk which,
upon recruitment, becomes tyrosyl phosphorylated and activated
(7, 8, 9). Syk phosphorylates the B cell-unique linker
protein, B cell linker protein (BLNK)/Slp-65, which is essential for
the activation of Bruton’s tyrosine kinase (Btk) and phospholipase C
(PLC) 2 (10, 11, 12). Subsequent tyrosyl phosphorylation of
CD19 leads to phosphatidylinositol 3-kinase recruitment to the membrane
and generation of phosphatidylinositol
(PtdIns)-3,4,5P3 from
PtdIns(4, 5)P2 (13, 14, 15).
In the presence of inositol 5-phosphatases, e.g., SHIP,
phosphatidylinositol 3-kinase activation should also lead to the
generation of
PtdIns(3, 4)P2. The
pleckstrin homology domain-containing signaling intermediaries
Btk and PLC translocate to the plasma membrane where they bind
PtdIns(3, 4, 5)P3 (and potentially also to
PtdIns(3, 4)P2), facilitating PLC-mediated
hydrolysis of phosphoinositides, generation of
inositol-1,4,5-triphosphate (Ins(1, 4, 5)P3), and
mobilization of calcium (16, 17, 18, 19). Comparative analysis of
signal transduction has revealed that the Src family kinase Fgr is
expressed at reduced levels in immature B cells (20, 21).
Furthermore, although BCR signaling occurs in cholesterol- and
sphingolipid-enriched membrane microdomains ("rafts") in mature B
cells, the BCR in immature B cells fails to enter lipid rafts
(22, 23). Finally, immature B cells mobilize intracellular
calcium in response to BCR aggregation by anti-receptor Abs, but
fail to produce levels of Ins(1, 4, 5)P3 detected
in mature B cells (24). These findings suggest that 1)
certain intermediary events required for Ag receptor-mediated
phosphoinositide hydrolysis are impaired in immature cells and 2)
immature B cell calcium stores are more sensitive to
Ins(1, 4, 5)P3. Neither the ability of cognate Ag
to induce distinct signal transduction responses in these cells, nor
the role of these responses in determining the unique biologic
outcome of BCR signaling in immature cells has been thoroughly
assessed.
We have previously reported that despite expression of lower levels of
Ag receptor, immature B cells exhibit increased sensitivity to Ag
compared with their mature counterparts as judged by various biologic
responses (25). This reflects, in part, increased
sensitivity of transcriptional machinery to elevation of intracellular
free calcium concentration
([Ca2+]i). Here, we
demonstrate that increased Ag sensitivity of immature B cells is also
associated with enhanced activation of BCR signaling pathways proximal
to and including calcium mobilization. As an apparent consequence,
biologically relevant responses are induced by lower Ag concentrations
in immature B cells. The data define fundamental differences between
mature and immature B cells in BCR-mediated elevation of
[Ca2+]i: specifically,
immature B cells display enhanced 1) capacitative calcium entry, 2)
Ins(1, 4, 5)P3 breakdown, and 3) phosphorylation of
CD79a, Lyn, BLNK, and PLC2 in response to Ag. Enhanced capacitative
calcium entry appears to be a function of prolonged and/or more
complete depletion of the intracellular calcium stores. Moreover, BLNK,
Btk, and PLC2 are present at 3-fold higher levels in immature B
cells, potentially aiding increased mobilization of calcium as well as
enhancing activation of parallel signaling pathways. These data suggest
that multiple mechanisms contribute to increased BCR-mediated
mobilization of calcium in immature B cells and presumably ensure
elimination of autoreactive B cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Ig-transgenic (Tg) 3-83 µ
mice expressing IgM and IgD
specific for H-2Kk (26) were used in
the experiments. Ig-Tg immature B cells were generated from IL-7-driven
bone marrow cultures as described in detail elsewhere (25, 27). Briefly, bone marrow was obtained from 3- to 4-mo-old mice;
a single-cell suspension was prepared, depleted of erythrocytes using
Gey’s solution, washed twice in IMDM, and cultured at 5 x
105 cells/ml per 10-cm petri dish (7%
CO2, 37°C) in IMDM containing 100 U/ml
penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 2 mM
L-glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, 10% FCS
(HyClone Laboratories, Logan, UT) and 50–100 U of IL-7 (derived from
culture supernatant of J558L cells transfected with murine IL-7 cDNA, a
gift from Dr. A. Rolink, Basel Institute for Immunology, Basel,
Switzerland). Typically, cells were harvested after 6–7 days in
culture, washed twice with IMDM, and used in the subsequent
experiments. Resting splenic B cells (p >
1.066) were obtained from 3- to 4-mo-old adult mice and prepared as
previously described (28). Cell viability was assessed
using trypan blue exclusion.
Reagents
Ag: An H-2Kk mimetic peptide
(CSGFGGFQHLCCGAAGA) which binds specifically to the 3-83 receptor
(28, 29) was synthesized and multimerized by coupling to
N-ethylmaleamide-activated dextran at a 100:1
peptide:dextran molar ratio. Abs directed against the following
molecules were used: CD86 (GL-1, BD PharMingen, San Diego, CA), IgM
(b-7-6; American Type Culture Collection (ATCC), Manassas, VA; IgD
(JA12.5; ATCC), and CD45R (anti-B220; RA3-3A1 and RA3-6B2; ATCC);
anti-PLC2 and Fgr were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA); affinity purified polyclonal rabbit antisera against
CD79a, Syk, BLNK, Btk, and Lyn were generated in our laboratories.
Thapsigargin and ionomycin were purchased from Calbiochem (La Jolla,
CA); Indo-1 acetoxymethyl, bis-(1,3-dibutyrylbarbituric acid)trimethine
oxonol (DiBAC4) (3), and
gramicidin from Molecular Probes (Eugene, OR).
Immunofluorescence analysis
Cells were washed once, resuspended in PBS containing 1% BSA and 0.1% sodium azide, and incubated with an optimal amount of directly fluoresceinated Ab. Cells were incubated for 30 min at 4°C and washed twice in PBS/BSA/azide. After washing, cells were analyzed by flow cytometry. Cells were gated on light scatter and dead cells were excluded using 7-amino-actinomycin D stain (Via-Probe; BD PharMingen). Histograms were constructed based on analysis of 10,000 cells.
Detection of intracellular free calcium
For measurements of [Ca2+]i, cells were loaded with Indo-1 acetoxymethyl, suspended at 106 cells/ml in IMDM, and stimulated with either Ag or anti-IgM Ab (b-7-6). Mean [Ca2+ ]i was evaluated over time using a flow cytometer (model 50H; Ortho Diagnostic Systems, Boston, MA, or LSR; BD PharMingen) with an appended data acquisition system and software. Intracellular calcium store release and extracellular calcium influx were isolated in some experiments. To exclusively detect intracellular calcium release upon stimulation, cells were resuspended in medium in which free extracellular calcium ([Ca2+]o) was buffered to 60 nM (equivalent to resting B cell [Ca2+]i) using EGTA (30, 31); calcium influx was detected by increasing the [Ca2+]o to 1.3 mM using CaCl2.
Analysis for recombinase activator gene (RAG) 2 expression
Levels of RAG-2 and G
s mRNA were determined by RT-PCR assay
as described in detail elsewhere (27, 32).
Measurement of cytoplasmic membrane potential (Em)
Determinations of Em were made by measuring the fluorescence of the negatively charged DiBAC4 (3). DiBAC4 was chosen because it primarily binds cytoplasmic membranes and is excluded from mitochondrial membranes because of its negative charge (33). Cells were incubated for 10 min in low K+ buffer (5 mM KCl, 150 mM NaCl, 20 mM HEPES, and 1.3 mM CaCl2) with DiBAC4 (final concentration, 100 nM), and fluorescence was measured immediately by flow cytometry. In some samples, the ionophore gramicidin was added simultaneously (final concentration, 10 µM) with DiBAC4 to depolarize the membrane (Em = 0). Alternatively, DiBAC4-loaded cells were spun quickly, resuspended in high K+ buffer (150 mM KCl, 5 mM NaCl, 20 mM HEPES, 1.3 mM CaCl2), and DiBAC4 fluorescence was measured. Depolarized cells display increased DiBAC4 fluorescence.
Analysis of Ins(1, 4, 5)P3 production
Cells (107) were stimulated in 400 µl of IMDM with 500 ng Ag/106 cells for various times. The stimulation was terminated by the addition of 100% ice-cold TCA and samples were put on ice for 15 min. Following centrifugation (1 min, 14,000 rpm), 2 vol of 1,1,2-trichloro-1,2,2-trifluoroethane:trioctylamine (3:1) were added to 1 vol of TCA extract. The amount of Ins(1, 4, 5)P3 in the aqueous phase was measured using a 3H-labeled radioreceptor binding inhibition assay kit (NEN, Boston, MA) according to the manufacturer’s instructions.
Analysis of phosphatidic acid (PA) production
PA production was analyzed using [32P]orthophosphate incorporation with lipid fractionation by TLC, essentially as described previously (34).
Immunoblotting
Cells were lysed in 1% Nonidet P-40 lysis buffer (150 mM NaCl,
10 mM Tris (pH 7.5), 10 mM sodium pyrophosphate, 2 mM sodium
orthovanadate, 1 mM PMSF, 10 mM NaF, 0.4 mM EDTA, 1 mM aprotinin, 1 mM
1-antitrypsin, and 1 mM leupeptin). Lysates were kept on ice for 15
min and spun at 14,000 rpm for 10 min in an Eppendorf centrifuge. In
the case of whole-cell lysate analysis, supernatants were mixed with
SDS-reducing sample buffer and boiled for 5 min; for analysis of
specific molecules, supernatants were used for immunoprecipitation of
the molecules indicated, using specific polyclonal rabbit antisera and
protein A-Sepharose beads. Proteins were separated by SDS-PAGE,
transferred to polyvinylidene difluoride membrane and visualized by
blotting using specific Abs and ECL.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
We previously demonstrated that Ag stimulation leads to mutually
exclusive biologically relevant responses, RAG-2 and CD86 expression in
immature (derived from IL-7-driven bone marrow cultures) and mature B
cells, respectively (25). Moreover, this response of
immature B cells to Ag can be mimicked simply by elevation of
[Ca2+]i, as can the
induction of CD86 expression by mature B cells. Importantly, we
demonstrated that responses of immature cells occurred following much
more modest rises in
[Ca2+]i
(25). These findings, along with the reported increased
susceptibility to tolerance induction in immature B cells (1, 2), suggested that immature B cells may respond to
lower doses of Ag. To explore this possibility, we assessed changes in
expression of CD86 in mature B cells and RAG-2 in immature B cells
following BCR stimulation with high (500 ng) or low (50 pg) doses of
Ag. In mature B cells, CD86 was induced only at a high Ag dose (Fig. 1
A). Using the same conditions
in immature B cells, we observed effective induction of RAG-2
expression at both high and low Ag doses (Fig. 1B). These
data are consistent with the increased sensitivity of immature B cells
to tolerance induction (35, 36).
|
C) despite a lower number of Ag receptors (IgM and IgD) on
the cell surface (25). Furthermore, stimulation with the
low Ag dose (50 pg) still resulted in a modest rise in
[Ca2+]i in immature B
cells, while no increase was observed in mature B cells (Fig. 1C). Similar differences were observed when anti-IgM Abs
were used to aggregate BCR (data not shown). These data demonstrate
clear differences in the BCR-induced rise in
[Ca2+]i as well as a
difference in the threshold for activation of immature and mature B
cells. Furthermore, biologic responses are induced at lower Ag
concentrations in immature B cells, suggesting that less Ag is required
to eliminate newly generated immature B cells than is necessary to
induce mature B cell activation. Increased expression and activation of signaling intermediaries in immature B cells
BCR-mediated calcium mobilization requires activation of a
signaling cascade involving sequential activation of Lyn, Syk, and Btk,
as well as recruitment and phosphorylation of BLNK and PLC2
(5). Activation of calcium influx appears to require more
sustained activation of this pathway than intracellular calcium
release. Thus, more robust activation of the pathway may enhance the
calcium mobilization response of B immature cells. To explore this
possibility, we investigated mature and immature B cells for possible
differences in the expression and activation of some of the signaling
intermediaries that intervene between BCR and calcium mobilization.
Analysis of whole-cell lysates demonstrated increased levels of
expression of BLNK, PLC2, and Btk by 3-fold in immature B cells
(Fig. 2A). Syk was slightly
overexpressed in mature B cells (40% more Syk), whereas p53/p56
lyn levels were higher in immature B cells (30 and
86%, respectively). Expression of CD79a (Ig) was found to be
similar between mature and immature B cells. Given that mature B cells
express more cell surface BCR (IgM and IgD), a significant fraction of
the CD79a in immature B cells must be cytoplasmic. Expression of the
Src family kinase member Fgr was found to be reduced in immature B
cells by >55%, consistent with previous reports (20, 21).
|
B). In agreement with the calcium
mobilization responses, we observed increased Ag-induced tyrosine
phosphorylation at all time points in immature B cells. To assess
Ag-induced tyrosine phosphorylation of specific signaling
intermediaries, immunoprecipitation and phosphotyrosine immunoblotting
were performed. Inducible CD79a and Lyn tyrosine phosphorylation was
markedly greater in immature B cells (Fig. 2C); the
differences in Lyn phosphorylation can be explained only in part by the
increased Lyn expression (Fig. 2A). CD79a phosphorylation is
the earliest phosphorylation event in BCR signaling and the observed
differences can clearly not be explained by a difference in CD79a
expression (Fig. 2A). Rather, the data indicate a more
efficient activation of Lyn and/or less activation of tyrosine
phosphatases in immature B cells. Lyn binding to phosphorylated ITAM is
greatly enhanced compared with unphosphorylated ITAM, and this binding
has been shown to result in Lyn activation (37).
The expression level of the tyrosine phosphatase SHP-1 was
roughly equal in immature and mature B cells (Fig. 2A). It
seems likely that SHP-1 activation and/or recruitment to the BCR is
less effective in immature B cells. Despite these differences in CD79a
and Lyn phosphorylation, Ag-induced Syk tyrosine phosphorylation was
comparable in immature and mature B cells, although on a
per-molecule-basis Syk phosphorylation was somewhat greater in immature
B cells. Binding of Syk to doubly phosphorylated ITAM in CD79a/CD79b
increases its kinase activity as a function of Syk autophosphorylation
and results in propagation of the signaling cascade (8, 38). Thus, while ITAM phosphorylation may be reduced in mature B
cells, increased levels of Syk expression in combination with
autophosphorylation may overcome this. Downstream from Syk, BLNK and
PLC2 phosphorylation was readily detected in mature and immature B
cells. However, consistent with the higher expression levels of these
molecules in immature cells (Fig. 2A), absolute levels of
phosphorylated BLNK and PLC2 were markedly greater in immature B
cells (Fig. 2C). Thus, while there does not appear to be a
qualitative difference between mature and immature B cells to activate
the signaling cascade leading to calcium mobilization, immature B cells
display a quantitatively greater activation of this pathway. Fundamental differences in calcium mobilization between mature and immature B cells
Data in Fig. 1 demonstrate a large difference in the amplitude of
the [Ca2+]i elevation
following BCR ligation on mature and immature B cells. Increases in
cytoplasmic calcium are generally caused by an initial release of
calcium from intracellular stores, which drives influx of calcium
across the plasma membrane by "capacitative calcium entry" or
"store-operated calcium entry" (39). We therefore
compared calcium release from intracellular stores and subsequent
calcium influx by stimulating mature and immature B cells in medium
buffered with EGTA to limit
[Ca2+]o to 60 nM;
restoration of the
[Ca2+]o to 1.3 mM allowed
subsequent measurement of calcium influx. We found that less calcium
was released from the intracellular stores of immature B cells but that
they displayed greater capacitative calcium influx compared with mature
B cells (Fig. 3A).
These data could indicate that although BCR-accessible
calcium stores are smaller in immature B cells, they are, nonetheless,
more efficiently depleted, leading to greater influx. To investigate a
potential difference in the size of intracellular calcium stores, cells
were exposed to ionomycin (Fig. 3B) or the
Ca2+-ATPase inhibitor thapsigargin that depletes
calcium stores by blocking resequestration of "leaked" calcium into
the stores (Fig. 3C). In the absence of extracellular
calcium, a greater increase in
[Ca2+]i was observed in
mature B cells following ionomycin or thapsigargin, demonstrating that
intracellular stores in immature B cells are indeed smaller. Upon
restoration of the
[Ca2+]o to 1.3 mM,
immature B cells again displayed enhanced calcium entry.
|
A). The difference in the rate of calcium
influx suggests the presence of greater numbers of calcium channels in
the cytoplasmic membrane in immature B cells. This possibility is
supported by analysis of capacitative calcium mobilization following
restoration of the
[Ca2+]o to 1.3 mM after
depletion of the internal Ca2+ stores using
ionomycin or thapsigargin (Fig. 3, B and C).
Since capacitative calcium entry depends on the degree of emptying of
the intracellular stores (39), we assessed possible
differences in the efficiency of BCR-mediated store depletion between
mature and immature B cells. To that end, cells were stimulated with Ag
in the absence of extracellular calcium, followed by ionomycin to
release any calcium remaining in the stores. Results demonstrate that,
compared with immature cells, addition of ionomycin induced a greater
increase in [Ca2+]i in
mature B cells (Fig. 3D, dashed lines), suggesting greater
reuptake of calcium in these cells. This interpretation was supported
by analysis of BCR-mediated calcium mobilization when reuptake is
blocked by thapsigargin (Fig. 3D, dotted lines). In
addition, results demonstrate that capacitative influx depends on the
degree of emptying of intracellular stores: when calcium reuptake is
prevented (thapsigargin) or delayed (ionomycin), mature B cells display
greater calcium influx than when Ag is used alone (Fig. 3D). Together, the results suggest that immature cells have smaller internal calcium stores than mature B cells and that BCR stimulation more effectively depletes these stores in immature B cells. Furthermore, the degree of store depletion limits BCR-coupled calcium influx in mature cells. In addition, immature B cells have increased capability for capacitative calcium mobilization following store depletion, possibly due to the greater number of as yet undefined calcium channels. Interestingly, a recent report demonstrated that calcium mobilization is enhanced when formation of glycosphingolipid-enriched domains (lipid rafts) is prevented, suggesting that lipid rafts may harbor molecules that negatively regulate calcium mobilization responses (40). Moreover, BCR signaling occurs outside lipid rafts in immature B cell lines (22) and it was demonstrated that, although ligated BCR colocalizes with lipid rafts in mature B cells, such an association was much less frequent in immature B cells (23). These observations suggest that enhanced calcium mobilization responses in immature B cells could be the result of inefficient localization of BCR-Ag complexes in lipid rafts.
Differences in Em do not explain differences in Ag-induced calcium influx in immature and mature B cells
An additional parameter that could contribute to the differential
seen in mature and immature B cell calcium mobilization is
Em (33). Cells actively maintain a
charge of about -80 mV across the plasma membrane with a negative
charge on the inside. Thus, a higher Em in
immature cells could result in greater Ca2+
influx responses. To investigate the possibility of a differential
contribution of Em in calcium mobilization in
mature and immature B cells, we measured Em using
a fluorescent dye (DiBAC4) and analyzed calcium
mobilization with the Em reduced to 0. It was
noted that immature B cells stain more brightly than mature cells with
DiBAC4 (Fig. 4A). Comparison of
Em in different cell types is complicated by the
fact that DiBAC4 fluorescence increases with cell
size (33). Since the immature B cells are somewhat larger
than mature B cells and thus take up more dye, size rather than
differences in Em likely explains the overall
slightly higher DiBAC4 fluorescence observed in
immature cells (Fig. 4A). Changes in
Em in each cell type, however, are relatively
easy to detect. Addition of the ionophore gramicidin causes
depolarization of cells (Em = 0), resulting in a
3.3-fold increased DiBAC4 fluorescence in both
mature and immature B cells (Fig. 4A, dotted curve).
Similarly, resuspending cells in a high potassium buffer (Fig. 4A, bold curve) depolarizes cells almost immediately
to the level achieved with gramicidin (Fig. 4A). The fact
that differences in DiBAC4 fluorescence are
maintained following depolarization support the contention that
differences in staining are a function of difference in cell size, not
Em.
|
B, lower panel) compared with cells with intact
Em (Fig. 4B, top panel).
The difference in calcium responses of immature and mature B cells was
seen in both low and high potassium buffers (Fig. 4B). We
can therefore conclude that although Em in itself
aids the influx of calcium from the extracellular space, it does not
explain the observed differences in BCR-mediated calcium influx between
mature and immature B cells. BCR-mediated Ins(1, 4, 5)P3 and PA production in mature and immature B cells
Release of calcium from the intracellular stores is a function of
Ins(1, 4, 5)P3 binding to
Ins(1, 4, 5)P3 receptors (16, 39).
Given the data presented thus far, one would expect to find increased
production of Ins(1, 4, 5)P3 in immature B cells.
However, it was previously reported that BCR ligation in immature B
cells induces below normal levels of Ins(1, 4, 5)P3
(24). Since results in that study could be compromised by
the fact that immature B cells were positively selected for mIgM from
neonatal non-Tg spleen and receptor ligation was performed using
anti-receptor Abs, we analyzed Ins(1, 4, 5)P3
levels following Ag stimulation in mature and IL-7 bone marrow-derived
immature B cells from 3-83 µ mice. Contrary to our expectations
but in accordance with previous data (24), results
demonstrate a maximal increase in Ins(1, 4, 5)P3
concentration of only 1.15-fold in immature B cells, whereas the
Ins(1, 4, 5)P3 concentration increased 2.1-fold in
mature B cells (Fig. 5A). This
was surprising given our immunoprecipitation data, which implied
greater PLC2 activation in immature B cells following BCR ligation
(Fig. 2C). However, phosphorylation of PLC does not
necessarily correlate with its enzymatic activity (41, 42)
and we therefore examined whether the reduced
Ins(1, 4, 5)P3 levels in immature cells were a
consequence of insufficient PLC activity. PLC-mediated hydrolysis
of PtdIns(4, 5)P2 results in the generation
of Ins(1, 4, 5)P3 as well as diacylglycerol (DAG);
upon phosphorylation by DAG kinase, this DAG is converted into PA
(34). To analyze PA production, cells were incubated with
[32P]orthophosphate for 1 h before Ag
stimulation and extracted lipids were analyzed by TLC as previously
described (34). Results demonstrate an equal increase in
PA at early time points and a greater increase in immature B cells
compared with mature B cells at later time points (Fig. 5B).
This can be interpreted as good induction of inositol lipid hydrolysis
in immature B cells, reflecting equivalent early PLC2 activation in
immature and mature B cells, with more prolonged PLC activation
explaining the sustained PA levels in immature B cells. This more
prolonged activation could also explain why intracellular calcium
stores are more efficiently depleted in immature B cells (Fig. 3D).
|
and PKC
(43) and it has
been demonstrated that these isozymes are similarly expressed in mature
and immature (from autoreconstituted spleen) B cells (44).
However, it has also been suggested that PKC activation is uncoupled
from BCR-induced apoptosis in immature B cells (44).
Notably, PLD can also be regulated by factors other than PKC, including
ARF and Rho proteins (43). Thus, while activation of PLD
following Ag exposure could explain the late increase in PA production
observed here in immature B cells, it is currently unknown whether PLD
activity following Ag stimulation differs between mature and immature B
cells. Studies are underway to address this issue.
The activation of PLC (measured by PA production) in combination
with the observation that Ins(1, 4, 5)P3 levels
fall below unstimulated levels in immature B cells (Fig. 5A)
suggest an increased breakdown of Ins(1, 4, 5)P3
rather than reduced Ins(1, 4, 5)P3 production. To
further address the possibility that Ins(1, 4, 5)P3
breakdown is accelerated in immature B cells, production was measured
in the presence of 20 mM LiCl, an inhibitor of
Ins(1, 4, 5)P3 breakdown pathways
(41). Indeed, in the presence of LiCl,
Ins(1, 4, 5)P3 production in immature B cells was
similar to that observed in mature B cells (Fig. 5C). These
data suggest equivalent early activation of PLC in immature and
mature B cells. The lower net concentrations of
Ins(1, 4, 5)P3 as a result of increased
Ins(1, 4, 5)P3 breakdown in immature B cells are
not likely to be limiting since the process of calcium influx seems
predominantly determined by the degree of store depletion
(45). In accordance with this assertion, we did not
observe a significant effect of the addition of LiCl on calcium
mobilization responses in either mature or immature B cells (data not
shown). Prolonged activation of PLC may ensure production of low
concentrations of Ins(1, 4, 5)P3 leading to
continued store depletion.
In addition to dephosphorylation of Ins(1, 4, 5)P3 by inositol polyphosphate 5-phosphatase, Ins(1, 4, 5)P3 levels can also be reduced by phosphorylation on the 3 position by Ins(1, 4, 5)P3 3-kinase (41). In neuronal cells, inositol 1,3,4,5-tetrakisphosphate (Ins(1, 3, 4, 5)P4) can act as an agonist of the Ins(1, 4, 5)P3 receptor (46). Furthermore, it has been estimated that at low Ins(1, 4, 5)P3 concentrations, Ins(1, 4, 5)P3 is predominantly metabolized by 3-kinase, whereas 5-phosphatase activity dominates with increasing Ins(1, 4, 5)P3 concentrations (47). Moreover, Ins(1, 3, 4, 5)P4 is degraded with slower kinetics (47) and may act to sustain calcium signals initiated by Ins(1, 4, 5)P3 (48), although this is still controversial (49) and may be cell type dependent. Interestingly, Ins(1, 3, 4, 5)P4 has been shown to interact with several intracellular proteins, including Btk (50). Clearly more work is needed to determine whether Ins(1, 3, 4, 5)P4 plays any role in the observed differences of BCR signaling in mature and immature B cells.
One possible explanation for the observed differences between immature and mature B cells is that Ig-Tg immature B cells used are propagated in IL-7. Bone marrow from non Ig-Tg animals develop to the pre-B cell stage (51) and thus Ig-Tg cells may represent pre-B cells expressing mIgM prematurely. However, mIgM+ Ig-Tg cells grown in IL-7 cultures respond to BCR ligation with the expected up-regulation of RAG-2 (this study and Refs. 25, 52), indicating initiation of receptor editing. In addition, differences in Ag sensitivity between mature and immature B cells correlates well with previously published studies using neonatal B cells (35, 36, 53). Finally, our biochemical data on the lack of Ins(1, 4, 5)P3 accumulation and reduced expression of Fgr in IL-7-cultured immature B cells are in agreement with previously published data using immature B cells from neonates or adult bone marrow (20, 21, 24). Taken together, it is our opinion that IL-7-propagated B cells function as immature B cells and are a good model to study processes of B cell tolerance in vitro.
We have demonstrated that transcriptional activation of RAG-2 in immature B cells is exquisitely sensitive to elevations in [Ca2+]i, whereas higher Ag doses are required to allow effective interaction of mature B cells with T cells by increasing CD86 expression. The increased sensitivity to Ag imposed by these mechanisms in immature B cells is likely to play a very important role in repertoire development, purging the repertoire of cells with even low affinity for self-Ags. Negative selection of immature, IgMposIgDneg, B cells in the bone marrow can be seen as an early checkpoint in B cell development critical for prevention of mature autoreactive B cell generation. A second checkpoint occurs in transitional IgMhighIgDlow B cells that emigrate from the bone marrow to the spleen (54). These cells are incapable of reactivating rag genes; however, Ag-mediated signaling leads to apoptosis (54, 55). The relatively high level of expression of mIgM in these cells may further ensure the increased sensitivity of transitional cells to negative selection (55, 56). As a result of these mechanisms, all B cells in the periphery should have negligible affinity for autoantigens. Reduced sensitivity of peripheral mature B cells to Ag has additional implications for the outcome of Ag receptor somatic mutation in germinal centers: it reduces the likelihood that B cells can acquire affinity for self as a consequence of immunogen-induced receptor somatic mutation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Lilly Research Laboratories, LCC, Indianapolis, IN 46285.
3 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated Department of Immunology, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: cambierj{at}njc.org
4 Abbreviations used in this paper: BCR, B cell Ag receptor; m, membrane; PtdIns, phosphatidylinositol; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; [Ca2+]i, intracellular free calcium concentration; RAG, recombinase activator gene; PLC, phospholipase C; DAG, diacylglycerol; PLD, phospholipase D; PKC, protein kinase C; PA, phosphatidic acid; Tg, transgenic; Em, membrane potential; ITAM, immunoreceptor tyrosine-based activation motif; Btk, Bruton’s tyrosine kinase; DiBAC4, bis-(1,3-dibutyrylbarbituric acid)trimethine oxonol; BLNK, B cell linker protein; [Ca2+]o, extracellular free Ca2+ concentration.
Received for publication April 27, 2001. Accepted for publication August 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
with distinct cytoplasmic effectors. Science 258:123.2 and Rac1-JNK in B cells. Immunity 10:117.[Medline]
-subunit gene in vivo. FEBS Lett. 338:277.[Medline]
/Ig immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.This article has been cited by other articles:
|
|
 |
|
  A. J. Gross, J. R. Lyandres, A. K. Panigrahi, E. T. L. Prak, and A. L. DeFranco Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance J. Immunol., May 1, 2009; 182(9): 5382 - 5392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. F. Andrews and D. J. Rawlings Transitional B Cells Exhibit a B Cell Receptor-Specific Nuclear Defect in Gene Transcription J. Immunol., March 1, 2009; 182(5): 2868 - 2878. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Edry, H. Azulay-Debby, and D. Melamed TOLL-like receptor ligands stimulate aberrant class switch recombination in early B cell precursors Int. Immunol., December 1, 2008; 20(12): 1575 - 1585. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Lamoureux, L. C. Watson, M. Cherrier, P. Skog, D. Nemazee, and A. J. Feeney Reduced receptor editing in lupus-prone MRL/lpr mice J. Exp. Med., November 26, 2007; 204(12): 2853 - 2864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Edry, S. B. Koralov, K. Rajewsky, and D. Melamed Spontaneous Class Switch Recombination in B Cell Lymphopoiesis Generates Aberrant Switch Junctions and Is Increased after VDJ Rearrangement J. Immunol., November 15, 2007; 179(10): 6555 - 6560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Brezski and J. G. Monroe B Cell Antigen Receptor-Induced Rac1 Activation and Rac1-Dependent Spreading Are Impaired in Transitional Immature B Cells Due to Levels of Membrane Cholesterol J. Immunol., October 1, 2007; 179(7): 4464 - 4472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. N. Teague, Y. Pan, P. A. Mudd, B. Nakken, Q. Zhang, P. Szodoray, X. Kim-Howard, P. C. Wilson, and A. D. Farris Cutting Edge: Transitional T3 B Cells Do Not Give Rise to Mature B Cells, Have Undergone Selection, and Are Reduced in Murine Lupus J. Immunol., June 15, 2007; 178(12): 7511 - 7515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu, L. J. Wysocki, and T. Manser Autoantigen-B Cell Antigen Receptor Interactions That Regulate Expression of B Cell Antigen Receptor Loci J. Immunol., April 15, 2007; 178(8): 5035 - 5047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Melamed, O. Messika, L. Glass-Marmor, and A. Miller Modulation of matrix metalloproteinase-9 (MMP-9) secretion in B lymphopoiesis Int. Immunol., September 1, 2006; 18(9): 1355 - 1362. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Roy, N.-H. Chang, Y. Cai, G. Bonventi, and J. Wither Aberrant IgM Signaling Promotes Survival of Transitional T1 B Cells and Prevents Tolerance Induction in Lupus-Prone New Zealand Black Mice J. Immunol., December 1, 2005; 175(11): 7363 - 7371. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hillion, A. Saraux, P. Youinou, and C. Jamin Expression of RAGs in Peripheral B Cells outside Germinal Centers Is Associated with the Expression of CD5 J. Immunol., May 1, 2005; 174(9): 5553 - 5561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Donahue, K. L. Hess, K. L. Ng, and D. A. Fruman Altered splenic B cell subset development in mice lacking phosphoinositide 3-kinase p85{alpha} Int. Immunol., December 1, 2004; 16(12): 1789 - 1798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Oliver, M. Wang, Y. Zhu, J. White, J. Kappler, and P. Marrack Loss of Bim Allows Precursor B Cell Survival But Not Precursor B Cell Differentiation in the Absence of Interleukin 7 J. Exp. Med., November 1, 2004; 200(9): 1179 - 1187. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Edry and D. Melamed Receptor Editing in Positive and Negative Selection of B Lymphopoiesis J. Immunol., October 1, 2004; 173(7): 4265 - 4271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, M. Holm, X.-Q. Xie, M. Wolf-Watz, and T. Grundstrom AML1/Runx1 Recruits Calcineurin to Regulate Granulocyte Macrophage Colony-stimulating Factor by Ets1 Activation J. Biol. Chem., July 9, 2004; 279(28): 29398 - 29408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Erman, T. I. Guinter, and A. Singer Defined {alpha}{beta} T Cell Receptors with Distinct Ligand Specificities Do Not Require Those Ligands to Signal Double Negative Thymocyte Differentiation J. Exp. Med., June 21, 2004; 199(12): 1719 - 1724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D. Wines, H. M. Trist, R. C. Monteiro, C. van Kooten, and P. M. Hogarth Fc Receptor {gamma} Chain Residues at the Interface of the Cytoplasmic and Transmembrane Domains Affect Association with Fc{alpha}RI, Surface Expression, and Function J. Biol. Chem., June 18, 2004; 279(25): 26339 - 26345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Whyburn, K. E. Halcomb, C. M. Contreras, C. A. Lowell, O. N. Witte, and A. B. Satterthwaite Reduced Dosage of Bruton's Tyrosine Kinase Uncouples B Cell Hyperresponsiveness from Autoimmunity in lyn-/- Mice J. Immunol., August 15, 2003; 171(4): 1850 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Mehr, G. Shahaf, A. Sah, and M. Cancro Asynchronous differentiation models explain bone marrow labeling kinetics and predict reflux between the pre- and immature B cell pools Int. Immunol., March 1, 2003; 15(3): 301 - 312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Whyburn, K. E. Halcomb, C. M. Contreras, R. Pappu, O. N. Witte, A. C. Chan, and A. B. Satterthwaite Haploinsufficiency of B cell linker protein enhances B cell signaling defects in mice expressing a limiting dosage of Bruton's tyrosine kinase Int. Immunol., March 1, 2003; 15(3): 383 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Gary-Gouy, J. Harriague, G. Bismuth, C. Platzer, C. Schmitt, and A. H. Dalloul Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production Blood, December 15, 2002; 100(13): 4537 - 4543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. B. Petro, R. M. Gerstein, J. Lowe, R. S. Carter, N. Shinners, and W. N. Khan Transitional Type 1 and 2 B Lymphocyte Subsets Are Differentially Responsive to Antigen Receptor Signaling J. Biol. Chem., December 6, 2002; 277(50): 48009 - 48019. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu and T. Grundstrom Calcium Regulation of GM-CSF by Calmodulin-Dependent Kinase II Phosphorylation of Ets1 Mol. Biol. Cell, December 1, 2002; 13(12): 4497 - 4507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shivtiel, N. Leider, O. Sadeh, Z. Kraiem, and D. Melamed Impaired Light Chain Allelic Exclusion and Lack of Positive Selection in Immature B Cells Expressing Incompetent Receptor Deficient of CD19 J. Immunol., June 1, 2002; 168(11): 5596 - 5604. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
