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Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado School of Medicine, Denver, CO 80206
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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9 Ig transgenic
mouse, with a specificity prototypic of the subset, we have explored
conditions responsible for the previously reported Ag
hyporesponsiveness of these cells. We report that peritoneal
VH11V9 B cells exhibit typical B-1 behavior
with high basal intracellular free Ca2+ and negligible
receptor-mediated calcium mobilization. However, splenic B cells from
this mouse, while phenotypically similar to their peritoneal
counterparts, including expression of CD5, mount robust B-2-like
responses to Ag as measured by calcium influx and altered tyrosine
phosphorylation responses. When these splenic cells are adoptively
transferred to the peritoneal cavity and encounter their cognate
self-Ag, they acquire a B-1 signaling phenotype. The ensuing
hyporesponsiveness is characterized by increases in both basal
intracellular calcium and resting tyrosyl phosphorylation levels and is
highlighted by a marked abrogation of B cell receptor-mediated calcium
mobilization. Thus, we show that self-Ag recognition in specific
microenvironments such as the peritoneum, and we would propose other
privileged sites, confers a unique form of anergy on activated B cells.
This may explain how autoreactive B-1 cells can exist while
autoimmunity is avoided. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In wild-type mice, B-1 cells develop predominantly during fetal hemopoiesis (12, 17); however, we and others have shown that cells with a B-1 phenotype can be produced from adult precursors in several model transgenic systems (18, 19, 20, 21, 22). In these studies, B-1 cell development appeared to correlate with Ag receptor specificity. Recognition of self Ag, either endogenously or transgenically expressed, was shown to be necessary for the generation and expansion of the B-1 cell population (18, 21, 23). The pleural and peripheral compartments of these mice become replete with cells bearing the B-1 phenotype. The tenets of tolerance induction might predict that although these autoreactive cells may escape deletion in the bone marrow, peripheral mechanisms should render these cells anergic and/or short-lived. Indeed, while the surface characteristic of these cells has been well documented, little is known regarding the forces that drive their altered responsiveness to Ag, and less about how the responsiveness of these cells may differ depending on their anatomical location.
Among the known BCR specificities typical of B-1 cells are
autoantigens, such as plasma membrane phospholipids, and conserved
epitopes present on common pathogens, such as polysaccharide moieties.
One highly represented example is the specificity encoded by germline
VH11 and V9 Ig genes.
This rearrangement is expressed by 5–15% of the peritoneal B-1 cells
in normal mice and reacts with phosphatidylcholine (PtC), a normal
component of the cell membrane (24). We recently reported
that B cells constrained by a
VH11V9 Ig transgene
generated CD5+, PtC-specific B cells in both the
spleen and peritoneum (22). Interestingly, splenic B cells
from VH11V9 mice exhibit
low basal intracellular free Ca2+
([Ca2+]i) levels, and
mobilize Ca2+ following BCR aggregation,
suggesting that they are functionally equivalent to nonautoreactive,
conventional B-2 cells. However, we also found that transgenic B cells
isolated from the peritoneum of these mice display an elevated basal
[Ca2+]i and a greatly
reduced ability to mobilize Ca2+ following BCR
aggregation, typical of B-1 cells. Thus, a dichotomy exists in the
signaling phenotype of B cells with identical receptor specificity by
virtue of their microenvironments.
Deficits in peritoneal B-1 cell BCR signaling, notably calcium influx,
have been reported previously. While aggregation of the BCR on these
cells results in tyrosyl phosphorylation of many BCR proximal proteins,
including phospholipase C-
, the activation of this enzyme, measured
by phosphatidylinositol 4,5P2 hydrolysis, is
markedly diminished compared with splenic B cells (25).
And, unlike splenic B cells, translocation of the transcription factor
NF-B to the nucleus does not occur following BCR aggregation on
peritoneal B-1 cells (9). Interestingly, similar changes
in BCR signal transduction have been reported in anergic B cells from
the hen egg lysozyme (HEL)/anti-HEL model in which the peripheral B
cells are constantly exposed to their cognate soluble Ag
(26). However, in the HEL model, the BCR exhibits a
greatly diminished ability to transduce signals leading to tyrosyl
phosphorylation of virtually all receptor proximal signaling
intermediaries (27) and calcium mobilization
(28). Recently, another model of B cell tolerance has been
developed in which an unknown autoantigen, with much lower affinity
than the HEL/anti-HEL model, is capable of inducing a similar
anergic state (29). Here again, peripheral B cells display
elevated basal
[Ca2+]i and BCR
signaling defects similar to the HEL/anti-HEL model. Taken
together, these data suggest that CD5+ B-1 cells
may display a unique hyporesponsive phenotype that is distinct from
anergy. Additionally, the hyporesponsiveness of B-1 cells may not be a
simple consequence of Ag exposure, as the site of exposure, such as the
peritoneum and other pleural cavities, appears to play a significant
contributing role.
In this study, we describe the phenotypic and functional differences
between VH11V9
receptor-bearing splenic and peritoneal B cells. Although both subsets
would be classified as B-1 cells by cell surface phenotype, only the
peritoneal B cells are hyporesponsive to BCR ligation. We further show
that the splenic population becomes hyporesponsive upon movement to the
peritoneal milieu and that this transition is a consequence of cognate
signaling through the BCR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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VH11V9 transgenic
mice, originally obtained from R. R. Hardy (Fox Chase Cancer
Center, Philadelphia, PA) on the CB17 background, were backcrossed at
least six times on the B10.D2nSn/J background. The 3-83 µ
transgenic mice were maintained on the autoantigen-free, nondeleting
(H-2Kd) background of B10.D2nSn/J (The Jackson
Laboratory, Bar Harbor, ME). Nontransgenic (NTg) control mice were
comprised of VH11V9
littermates negative for both H and L chain transgenes. Mice were
housed and bred at the National Jewish Medical and Research Center,
Biological Research facility and used at 8–12 wk of age.
Cell isolation and tissue culture
Splenic and peritoneal B cells were prepared as previously described (22). Briefly, purified splenic B cells were obtained by Percoll gradient separation after RBC and T cell lysis; peritoneal B cells were purified from peritoneal lavage by removal of adherent accessory cells and magnetic bead depletion of T cells. Where noted, splenic and peritoneal B cells were treated with CFSE (Molecular Probes, Eugene, OR), as described elsewhere (30). Labeled cells were washed twice with IMDM (Life Technologies, Gaithersburg, MD) and placed in culture at concentrations ranging from 5 x 105 to 2 x 106 cells/ml in complete IMDM at 37°C, or in cell-free peritoneal exudate (PEx). Cell-free PEx was obtained by lavage of several NTg mice with 4 ml each of IMDM (Life Technologies). The combined washes were then centrifuged at 3000 RPM for 10 min; the supernatant was 0.2 µm filtered, and supplemented with FCS. Where noted, PtC-containing liposomes were added to the cultures.
Phenotypic analysis
Cells were resuspended in PBS containing 1% BSA and 0.1% sodium azide and incubated with an optimal amount of biotinylated or directly fluoresceinated Ab. mAbs directed against the following mouse cell surface molecules were used: B220 (RA3-6B2; American Type Culture Collection (ATCC), Manassas, VA), CD23 (B3B4; BD PharMingen, San Diego, CA), CD19 (1D3; BD PharMingen), CD5 (53-7.313; BD PharMingen), IgMa (RS3.1; ATCC), CD69 (H1.2F3; BD PharMingen), CD80 (16-10A1; BD PharMingen), CD86 (GL-1; BD PharMingen). Fluorescent reagents were either purchased or labeled in-house using a standard protocol and fluoresceindiamine tetraacetic acid or N-hydroxysuccinimido-biotin (Sigma-Aldrich, St. Louis, MO). Cells were incubated for 30 min at 4°C and washed twice in PBS/BSA/azide. Biotinylated Abs were visualized with streptavidin coupled to TriColor, PE, or allophycocyanin (Caltag Laboratories, Burlingame, CA) for analysis on a FACSCalibur (BD Biosciences, San Jose, CA). Forward and side scatter gates were adjusted to include only nucleated cells, and dead cells were excluded based on 7-amino actinomycin D (7-AAD) incorporation (Via-Probe; BD PharMingen). Postexperiment analysis was performed using CellQuest Pro software (BD PharMingen).
Cell proliferation
Before all analyses of cell proliferation, cells were counted on a hemocytometer using trypan blue exclusion, and the frequency of B220+ cells was determined by immunofluorescence. For proliferation, 5 x 105 B cells were cultured in triplicate or quadruplicate wells of a 96-well microtiter plate for 24 or 48 h. Culture conditions included the addition of LPS (Sigma-Aldrich), anti-mIgM (b76), PMA (Sigma-Aldrich), and ionomycin (Sigma-Aldrich). Next, 1 µCi [3H]thymidine (DuPont NEN, Boston, MA) was added per well, and an additional 12–16 h of culture was performed. Cells were harvested onto filter paper, and radioactivity was counted.
Immunoblotting
Cells (2 x 106) were treated for 2
min at 37°C with either medium or rabbit anti-mouse IgM mAb
F(ab')2 (Zymed Laboratories, San Francisco, CA)
and lysed in 0.5% CHAPS 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, then centrifugation was performed at 14,000 rpm for 10 min
at 4°C. Supernatants were mixed with SDS reducing sample buffer and
boiled for 5 min. Proteins were separated by SDS-PAGE, transferred to
polyvinylidene difluoride membranes and visualized using specific Abs
in conjunction with ECL (DuPont NEN). To detect tyrosine
phosphorylation, the AB-2 anti-phosphotyrosine Ab was used
(Oncogene, Boston, MA). Polyvinylidene difluoride membrane was then
stripped and reprobed for Syk as a loading control. Polyclonal rabbit
anti-Syk antisera was prepared in our laboratory.
PtC liposomes
Liposomes were prepared by a method similar to that used by Mercolino et al. (24). Briefly, a liposome preparation was made using 25% distearoylphosphatidylcholine (Avanti Polar Lipids, Birmingham, AL), 30% cholesterol (Avanti Polar Lipids), 30% sphingomyelin (Avanti Polar Lipids), and 15% distearoylphasphatidylethanolamine in a 90:10 mixture of chloroform/methanol, and the organic solvent was removed by evaporation under nitrogen gas. PBS (pH 7.1) was added to the preparation to a final concentration of 10 µM. The lipids were emulsified by sonication and extruded through a 0.2-µm filter. For 95% PtC liposomes, a 95% distearoylphosphatidylcholine and 5% distearoylphosphatidylethanolamine preparation was extracted as above. All samples were stored under nitrogen gas.
Analysis of calcium mobilization
Purified splenic and peritoneal B cells were loaded with Indo-1AM (Molecular Probes) for 45 min at 37°C. Cells were washed twice, resuspended at a concentration of 2 x 106 cells/ml in IMDM and 5% FCS, and stimulated with F(ab')2 rabbit anti-mouse Ig (RamIg; Zymed Laboratories) or PtC liposomes. Mean fluorescence was evaluated using a BD-LSR (BD Biosciences), which allows for 7-parameter flow cytometry including real time analysis of changes in UV fluorescence on gated populations. Data analysis was performed using CellQuest (BD PharMingen) and FloJo (TreeStar, San Carlos, CA) software. In separate experiments, splenic B cells were isolated as previously mentioned, labeled with CFSE, and either adoptively transferred into NTg recipients or in vitro cultured. Calcium analysis was then performed on isolated, B220+, 7-AAD-, and Indo-1AM-loaded peritoneal cells from these recipients, or from cultures.
PtC adsorption
Anti-PtC Abs produced from the 10E8 hybridoma were obtained from
R. R. Hardy (Fox Chase Cancer Center). Originally derived from
CD5+ B cells that recognize bromelain-treated
mouse RBCs, the 10E8 hybridoma secretes
VH11V9 Abs that
cross-react with PtC. Purified 10E8 or isotype-matched control
anti-trinitrophenol (TNP) Abs were biotinylated, as
previously mentioned. The biotinylated Abs were then prebound to
streptavidin-coated magnetic beads (Dynal Biotech, Lake Success, NY) at
100 µg Ab/mg beads. The Ab-coated beads were then washed five times
with PBS and added to the cell-free PEx at 5 mg beads/ml. Ag was
adsorbed by rotating the mixture for 45 min at 4°C. Beads were then
removed by sequential rounds of magnetic separation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To begin to address the molecular mechanisms underlying the
functional differences between the clonally related
VH11V9 splenic and
peritoneal B-1 cells, we compared their expression of a number of
phenotypic markers. As shown in Fig. 1
A, the most striking
differences included the lower level of B220 and higher level of
surface IgM on peritoneal vs splenic
VH11V9 B cells. These
phenotypic differences are generally used to differentiate B-1
(B220lowIgMhigh) from
conventional B-2
(B220highIgMlow) cells, the
latter represented by NTg splenic B cells (Fig. 1A). Yet the
expression levels of other cell surface markers used to differentiate
B-1 from B-2 cells, including the presence of CD5 and CD43 and absence
of CD23, were similar on splenic and peritoneal
VH11V9 B cells,
suggesting a B-1 classification. And, despite the higher B220 and
lower IgM expression levels in the splenic cells, they do not match the
levels of B220 and IgM expressed on normal splenic B-2 cells from NTg
or 3-83µ Ig transgenic control mice (22).
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9 splenic and
peritoneal B cells (Fig. 1
A) suggests that both these cell
populations have previously encountered Ag (29). This is
not surprising given a model for positive selection in the development
of B-1 cells; however, the somewhat higher CD80 expression on the
VH11V9 peritoneal
population could represent a more recent or continuous exposure within
the peritoneal cavity. In support of this hypothesis, several other
hallmarks of Ag recognition were evident in these peritoneal B cells,
including an elevated basal
[Ca2+]i and reduced
ability to further mobilize Ca2+ following BCR
ligation (Fig. 1, B and C, respectively), as well
as an elevated basal level of cellular protein tyrosine
phosphorylation. This phenotypic transition is a feature of
most, if not all peritoneal
VH11V9 B cells (Fig. 1, A and C). These data suggest that the peritoneal
cavity may harbor factors that drive low-level activation and BCR
desensitization in the
VH11V9 B cells.
Interestingly, unlike the receptor desensitization seen in the
HEL/anti-HEL anergy model (27, 28), the disruption in
BCR signaling in the
VH11V9 peritoneal B
cells is downstream from the most proximal events, as BCR aggregation
induces similar and substantial tyrosine phosphorylation of cellular
substrates in both splenic and peritoneal
VH11V9 cells (Fig. 3D and unpublished
data).
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), with the
exception of PMA. Presumably, the elevation in basal
[Ca2+]i allows these
cells to respond to the phorbol ester in the absence of a
Ca2+ ionophore. Importantly,
VH11V9 as well as NTg
peritoneal B cells were less responsive than their splenic
counterparts, supporting the possibility that residence in the
peritoneal cavity results in B cell hyporesponsiveness. These results
suggest that two signaling defects exist in peritoneal B-1 cells: a
defect in an intermediary signaling event between the BCR and
Ca2+ mobilization, and a defect that affects
mitogenic signaling via multiple receptors, e.g., LPS.
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9 splenic B cells can
survive in the peritoneal cavity following adoptive transfer
The differences in phenotypic and functional responses seen
between splenic and peritoneal
VH11V9 B cells prompted
our hypothesis that the peritoneum offers a unique microenvironment for
VH11V9 B cells, leading
to the altered phenotype of these resident B cells. To address this
hypothesis, we transferred
VH11V9 splenic B cells
into the peritoneal cavity and assessed their phenotype at various time
points. However, because the survival of splenic
VH11V9 B cells in the
peritoneum had not been established, we performed i.p. or i.v. adoptive
transfers of CFSE-labeled splenic
VH11V9 and 3-83µ B
cells into age- and allotype-matched NTg littermates and assessed their
presence in the spleen and peritoneum of recipients 24 and 72 h
later. Fig. 2 shows that
VH11V9, but not 3-83,
splenic B cells can reside in the peritoneal cavity for at least
72 h when transferred i.p. In fact, even when transferred i.v.,
splenic VH11V9
B cells have the capacity to migrate to and populate the
peritoneum. As expected, both 3-83 and
VH11V9 splenic B cells
can be found in small numbers in the spleen following i.v. transfer.
The inability of splenic 3-83 µ B cells to populate the peritoneum
in numbers comparable with
VH11V9 B cells following
i.p. transfer was also anticipated and may reflect cell death or
migration to sites other than the spleen (Fig. 2). These data
demonstrate the ability of splenic
VH11V9+
B cells to survive in either the splenic or peritoneal
microenvironment.
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To address the mechanisms underlying the apparent locale-specific
phenotype of VH11V9
peritoneal cells, we analyzed the phenotype of
VH11V9 splenic B cells
transferred into the peritoneal cavity of NTg recipient mice. We have
previously determined that splenic
VH11V9 B cells not only
survive long-term in vitro (22), but can also survive
long-term in the peritoneum when transferred i.p. into NTg recipient
mice (Fig. 2). Importantly, after 72 h, the i.p. transferred
splenic VH11V9 B cells
(Fig. 3B) exhibited elevated
basal [Ca2+]i and poor
Ca2+ mobilization responses to BCR aggregation
similar to resident peritoneal cells (Fig. 3B). Furthermore,
these transferred splenic B cells acquired other characteristics of
peritoneal B-1 cells, such as elevated surface IgM (Fig. 3C)
and elevated basal whole cell tyrosine phosphorylation (Fig. 3D, lane 5 vs lane 3). Once again,
the site of disruption of the BCR signaling cascade must lie
downstream from the initial tyrosine phosphorylation events as the i.p.
transferred B cells showed substantial tyrosine phosphorylation
following BCR ligation (Fig. 3D, lane 6). It is
noteworthy, however, that the pattern of proteins phosphorylated is
partially distinct from splenic
VH11V9 B cells. To this
point, studies are ongoing to elucidate individual effector molecules
that may differ between splenic B-2 and peritoneal B-1 cells and that
could contribute to the B-1 hyporesponsiveness. Further analysis
revealed that i.p. transferred
VH11V9 splenic B cells
proliferate less well in response to BCR ligation and LPS stimulation,
and more in response to PMA stimulation, than their splenic
counterparts (Table II), a characteristic
of peritoneal B-1 cells. Regardless of route of transfer,
VH11V9 B cells recovered
from the spleens of recipient animals did not display the B-1 phenotype
by either increased surface IgM (Fig. 3C) or increased basal
tyrosyl phosphorylation (data not shown).
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We next sought to define the factor(s) within the microenvironment
that drives acquisition of the peritoneal B-1 phenotype. We first
cultured VH11V9 splenic
B cells in cell-free PEx to determine whether a soluble factor is
responsible for the locale-dependent changes in Ag receptor signaling.
As shown in Fig. 4A, splenic
VH11V9 B cells underwent
a time-dependent increase in basal
[Ca2+]i following culture
in PEx. Concomitant with this elevation was a decrease in the number of
cells responsive to BCR aggregation (Fig. 4B). Splenic B
cells from NTg littermates or another Ig transgenic (3-83µ) mouse
line treated equivalently did not show this change. Interestingly,
VH11V9 peritoneal B-1
cells cultured in medium for as little as 24 h demonstrated a
significant reduction in basal
[Ca2+]i. However, this
decrease never reached the resting
[Ca2+]i levels observed
in ex vivo splenic
VH11V9 B cells.
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9 B cells is
driven by Ag in the PEx. To assess whether Ag can elicit similar
changes in vitro, we prepared liposomes containing physiologically
relevant levels of PtC (
25%) and assessed their ability to modulate
[Ca2+]i in
VH11V9 splenic B cells.
Fig. 5A illustrates the
inability of NTg B cells to respond to PtC liposome stimulation.
Interestingly, VH11V9
splenic B cells, which responded normally to anti-µ stimulation,
displayed only a gradual rise in
[Ca2+]i following 25%
PtC liposome stimulation. Liposomes containing very high levels (95%)
of PtC were capable of inducing a Ca2+
mobilization response similar to anti-µ in splenic (Fig. 5A), but not peritoneal
VH11V9 B cells (data not
shown). It is likely that the reduced avidity for liposomes containing
a more physiologic proportion of PtC accounts for the differences in
splenic VH11V9 B cell
responses. Following 24 h cultured with 25% PtC liposomes,
splenic VH11V9, but not
NTg, B cells exhibited increased basal
[Ca2+]i and a reduced
ability to respond to further BCR ligation, similar to the
hyporesponsiveness shown for peritoneal B cells, and splenic B cells
cultured in PEx (Fig. 5B).
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9 B cells in the
peritoneum. To test this hypothesis, we depleted PEx of PtC by
adsorption using magnetic beads coated with the anti-PtC mAb 10E8,
encoded by the same
VH11V9 pairing. As shown
in Fig. 6, VH11V9 splenic B cells
cultured for as little as 24 h in PEx or PEx mock depleted, using
an isotype-matched control Ab to TNP, exhibited an increase in basal
[Ca2+]i and showed a
diminished capacity for BCR-mediated
[Ca2+]i mobilization. By
contrast, cells cultured with PEx precleared with 10E8 Ab did not
elevate basal [Ca2+]i and
retained their ability to mobilize calcium following BCR aggregation.
Acquisition of unresponsiveness was complete by 72 h in cultures
using PEx or mock-adsorbed PEx, with resting basal
[Ca2+]i reaching levels
similar to that observed in peritoneal B-1 cells. After 72 h,
10E8-adsorbed cultures remained capable of responding to BCR
stimulation; however, a slight increase in resting
[Ca2+]i was observable at
this time point (Fig. 6). This marginal increase in resting
[Ca2+]i of splenic
VH11V9 B cells may
reflect other components of the PEx, but we cannot exclude the
possibility that antigenic forms of PtC reappear in prolonged culture
because of cell death or normal cell blebbing. Thus, PtC appears
necessary to cause the conversion of signaling-competent splenic
VH11V9 B cells to cells
with a peritoneal B-1 signaling phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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9
anti-PtC transgenic mice display a surface phenotype commonly
associated with B-1 cells, including the expression of the inhibitory
coreceptor CD5, yet exhibit B-2-like responses to Ag. However,
peritoneal B cells from the same animal, displaying a very similar
surface phenotype, exhibit a typical B-1-like unresponsiveness to BCR
aggregation. This hyporesponsiveness can be induced in splenic
VH11V9 B cells by
exposure to the microenvironment of the peritoneum, either through
adoptive transfer or in vitro culture. After 72 h under such
conditions, these cells exhibit an increase in resting
[Ca2+]i and basal tyrosyl
phosphorylation levels and a striking abrogation of
[Ca2+]i mobilization
following BCR aggregation. This transition is predicated on receptor
specificity, as NTg and 3-83µ splenic B cells do not acquire this
phenotype when similarly treated, and is strongly influenced by the
microenvironment, the latter serving minimally as a source of
cognate Ag.
It is surprising that specificity for a self epitope as common as PtC
does not affect all B cells uniformly. One possible interpretation of
these results is that differentiation to signaling-incompetent B-1
occurs only in specific sites such as the peritoneum. This seems
inconsistent with the findings of Liou et al. (32), who
found that peritoneal B-1 and splenic B-1-like cells from Ig transgenic
mice were similarly resistant to experimental tolerance induction.
These results suggested that B-1 cells were phenotypically and
functionally identical regardless of locale. Importantly, the cells in
these experiments were similar, but not identical to
VH11V9 B cells in having
a predisposition, by virtue of their Ag specificity, to become B-1. As
mentioned previously, subtle differences in the concentration of Ag
and/or its availability could explain these discrepancies. In addition,
we would contend that the experimental tolerance procedures used in
these studies would, in fact, approximate our hyporesponsive
conditioning of VH11V9
splenic B cells in the medium with PtC (Fig. 6). We propose that
peritoneal B-1 cells already exhibit a form of tolerance, albeit far
from that classically defined as anergy. Perhaps a more appropriate and
a more sensitive indication of unresponsiveness would be entry into
cell cycle, i.e., proliferation (Tables I and II).
In classic examples of peripheral tolerance such as the soluble
HEL/anti-HEL model (33, 34), self-Ag encounter renders
B cells functionally unresponsive to subsequent BCR
challenge with notable increases in basal
[Ca2+]i and significantly
diminished receptor-mediated tyrosyl phosphorylation. It is again
important to mention in this study that this anergic unresponsiveness
is qualitatively distinct from the B-1 hyporesponsiveness we observe,
because induced phosphorylation of virtually all substrates is lost.
Interestingly, it has been demonstrated that low levels of CD5
expression on anergic cells in the HEL model may prevent their deletion
at earlier stages of development and their inappropriate activation in
the periphery (35). And, indeed,
VH11V9 splenic B cells
display indications of prior Ag exposure, such as expression of CD5 and
CD80. Yet, we observed no deficit in the signaling capability of
VH11V9 splenic B cells
as compared with conventional splenic B-2 cells. Our data would support
the conclusion that the periphery (i.e., secondary lymphoid organs) of
the mice does not promote the acquisition of hyporesponsiveness in
VH11V9 B cells. This
could be due to a paucity of accessible Ag or the lack of other
factor(s) vital to this transition.
While we have provided evidence that a BCR signal is obligatory for the
induction of this hyporesponsive phenotype, we do not believe this is a
result of receptor desensitization, as these B cells are still capable
of eliciting signals in the form of tyrosyl phosphorylation via their
BCR. Instead, it is possible that the uniqueness of the
microenvironment in which these cells encounter Ag also contributes to
this transition. This is at once apparent in the increased
longevity and the incremental, yet significant, rise in basal
[Ca2+]i of even control B
cells cultured in cell-free PEx (Fig. 4 and data not shown) and may
reflect soluble factors present in this milieu. Several recent studies
point to attractive candidate molecules, such as, IL-10, Tall-1
(BAFF), and TGF-
, which are produced by various peritoneal
cell populations and have prosurvival and inflammatory effects on B
cells (10, 36, 37, 38, 39). As hyporesponsiveness is not immediate
in our system, such local factors might increase survival
and facilitate the acquisition of this phenotype through, for example,
de novo synthesis of novel effector molecules. We are currently
investigating the individual factors of the peritoneal microenvironment
that may support or augment the transition to hyporesponsiveness.
Additionally, chemokine receptor expression and proper chemotaxis via
these receptors have been shown to be important for the establishment
of B-1 peritoneal populations (36, 39). BCR signaling can
influence the response to chemokines and may explain, in part, the
propensity for i.p. transferred splenic
VH11V9, but not
3-83µ, B cells to remain in peritoneal cavity (Fig. 2).
Interestingly, i.p. transfer experiments using CFSE-labeled
VH11V9 B cells also
showed little indication of cell proliferation despite the obvious
presence of PtC in this milieu. It is likely, therefore, that the
acquisition of B-1 hyporesponsiveness occurs via a complex pathway in
which antigenic signaling through the BCR induces independent
phenotypic and functional alterations, possibly based on the strength
of BCR signal. Such a model may explain the conversion of bone marrow
VH11V9 cells to a B-1
phenotype in the absence of receptor editing (22), the
maintenance of the signaling-competent B-1 phenotype in the spleen, and
the full conversion to signaling-incompetent B-1 cells in the
peritoneum.
Based on our results, we suggest that the microenvironment of the peritoneum, and perhaps other pleural cavities, provides a unique milieu conducive to the induction of the hyporesponsiveness observed in our studies. Important elements of this environment include autoantigens, and perhaps cytokines and chemokines not present in the peripheral lymphoid organs. In support of our studies, Qian et al. (20) demonstrated the ability of splenic B cells to differentiate into B-1 cells in vivo. In a BCR transgenic model specific for ribonucleoprotein Sm, transgenic B cells populating the peritoneum expressed a B-1 phenotype, while those in the spleen expressed a B-2 phenotype. Interestingly, it was demonstrated that the strength of BCR signal influenced the ability of anti-Sm B cells to either differentiate into B-1 or remain B-2. The authors concluded that differentiation to B-1 helps maintain tolerance to Sm through the expression of negative regulators of BCR signaling such as CD5. Thus, microenvironment-specific events may determine the likelihood that a given B cell, either adult or fetal derived, enters this pathway. CD5 expression and possibly localization to the peritoneum appear to provide some protection to autoreactive cells otherwise destined for elimination.
A number of findings in this study support the concept that the BCR on
CD5+ peritoneal B-1 cells exhibits a
qualitatively altered signal-transducing ability. First, BCR
aggregation causes substantial protein tyrosine phosphorylation in
peritoneal B cells, but no Ca2+ mobilization,
indicating that these cells are not simply anergic. Previous studies
(25) and preliminary evidence in our lab (J.
M. Dal Porto, manuscript in preparation) indicate that signaling
defects may lie in the coupling of the BCR to phospholipase C-
activation, most likely resulting in the lack of BCR-mediated inositol
1,4,5-triphosphate production previously reported in peritoneal B
cells. Second, we demonstrate that surface IgM levels increase, while
B220 levels decrease following localization of
VH11V9 B cells to the
peritoneum. Increases in mIgM expression can also be induced in vitro
by culturing splenic
VH11V9 B cells with PEx,
and more specifically IL-10 (data not shown). These alterations in B220
to mIgM ratio may shift the equilibrium of Src family kinases toward
repression, thereby restricting participation of certain Src family
kinases in BCR signaling. Finally, expression of unique effector
molecules (Ref. 40 and manuscript in preparation)
in peritoneal B-1 cells could cause alterations in the BCR signaling
axis. These changes suggest that the normal BCR signaling cascade has
been modified as a consequence of exposure to Ag in the peritoneum.
The data presented in this study also suggest an unexpected plasticity
in B cells. This plasticity allows cells to maintain particular
functional characteristics despite the presence of autoantigen under
certain circumstances, while in others they are either deleted or
anergized. One advantage of this would be the ability to generate a
population that produces biologically important low affinity,
polyreactive Abs, while preventing these cells from participating in
immune responses that result in class switching and affinity
maturation. Stimulation of such a response by endogenous or
cross-reactive exogenous Ag could result in the production of
pathogenic, high affinity IgG Abs. And, while B-1 cells do not seem to
enter germinal centers (11), it is of critical importance
that these B-1 cells remain functional and produce natural Abs as a
first line of defense against potential pathogens. Boes et al.
(13) demonstrated the importance of Abs with PtC
specificity by showing that a mutant mouse strain deficient in secreted
IgM exhibited increased susceptibility to septic peritonitis induced by
cecal ligation and puncture. Most importantly, resistance could be
rescued by passive administration of an anti-PtC mAb. Therefore, we
propose that the effect of low affinity autoantigen on our PtC-specific
VH11V9 B cells is to
establish a signaling set point wherein the BCR transduces
qualitatively distinct signals that support survival and Ab production,
while not allowing other responses, such as participation in
thymus-dependent immune responses. It is appealing to hypothesize that
the low number of BCR specificities found within the B-1 population may
reflect the availability of Ags at specific sites.
|
Acknowledgments |
|---|
9 mice as well as purified 10E8 Abs. We
also thank John Stolpa, Robert Benschop, and Paula Oliver for helpful
advice, and William Townsend and Molly Houseman for technical
assistance. |
Footnotes |
|---|
2 Current address: Department of Pharmacology, University of Texas Health Science Center, Dallas, TX 75390.
3 M.J.C. and J.M.D.P. contributed equally to this study.
4 Address correspondence and reprint requests to Dr. John C. Cambier, Department of Immunology, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: cambierj{at}njc.org
5 Abbreviations used in this paper: BCR, B cell receptor; 7-AAD, 7-amino actinomycin D; [Ca2+]i, intracellular free Ca2+; HEL, hen egg lysozyme; NTg, nontransgenic; PEx, peritoneal exudate; PtC, phosphatidylcholine; TNP, trinitrophenol.
Received for publication August 13, 2001. Accepted for publication June 10, 2002.
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