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*
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599;
Department of Biology, University of Puerto Rico, Rio Piedras, Puerto Rico; and
Department of Immunology, Duke University, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B-1 cells in normal mice are frequently autoreactive and include cells specific for the lupus Ags DNA and IgG (11, 12). B-1 cells are enriched in the peritoneal cavity and are responsible for the production of most of the circulating IgM in normal mice (13). The physiological functions of these cells are still unknown, but several studies suggest that they are a first line of defense against infection (14, 15, 16, 17, 18). How autoreactive B cells like those that bind DNA fit into this picture is not known. B-1 cells may also contribute to autoantibody production in disease (13, 19, 20, 21, 22). For example, B-1 cells are responsible for autoantibody secretion causing hemolytic anemia in anti-RBC Tg mice (23), and autoimmune NZB and motheaten mice have expanded B-1 cell populations (19, 20) that produce autoantibodies. Thus, autoreactive B-1 cells can contribute to autoimmune disease.
The regulation of autoreactive B cells, including differentiation to B-1, is controlled by many factors. Ag concentration, the nature of the Ag, the location of the Ag, and the affinity of the B cell receptor (BCR) for Ag can all affect B cell signaling and hence B cell regulation (24, 25, 26, 27, 28, 29, 30). B cell coreceptors modulate the strength of the signal through the BCR and can also affect regulation. Of particular importance are CD19 and CD22. CD19 amplifies BCR signaling by increasing the protein tyrosine kinase activity of Src family protein tyrosine kinases (31). CD22 is able to associate with a tyrosine-specific phosphatase, SHP-1, to negatively regulate BCR signaling (32). Coligation of CD19 and BCR decreases the threshold for BCR-dependent stimulation (33). Conversely, ligation of CD22 raises the threshold for which BCR signaling activates a B cell (32). Thus, these coreceptors serve to raise and lower the Ag concentration required for B cell triggering. They also have a role in regulating the size of the B-1 cell subset. B-1 cell numbers are increased in mice that overexpress CD19 (34) and in CD22-deficient mice (35, 36). Conversely, their numbers are significantly decreased in CD19-deficient mice (34, 37). The fate of B cells destined for differentiation to B-1 in CD19-deficient mice is not known. Do they undergo cell death or do they follow a different differentiative pathway?
In this report, we examine the regulation of B cells specific for the RNP Sm, a target of the immune system in human and mouse lupus. We demonstrate that anti-Sm B cells are regulated in nonautoimmune mice by differentiation to B-1. Our studies of the regulation of anti-Sm B cells are facilitated by µ-chain Tg mice using an unmutated, rearranged VHJ558 gene from the MRL-Mp/lpr/lpr anti-Sm hybridoma 2-12 (38). 2-12H Tg mice generate substantial numbers of B cells specific for Sm and ssDNA (39). Most of the autoreactive B cells in 2-12H Tg spleens are transitional. The remainder are mature B-2 cells. This dominance of transitional anti-Sm B cells suggests an inability of anti-Sm B cells to become mature B-2 cells or to persist as B-2 cells. Despite the large number of anti-Sm B cells in these mice, they have serum anti-Sm levels that are equal to those of non-Tg littermate mice. However, immunization with mouse small nuclear RNPs (snRNPs) induces an anti-Sm response, indicating that anti-Sm B cells are functional and ignorant of endogenous levels of Sm (39). Here, we report that 2-12H Tg mice have a high frequency of peritoneal anti-Sm B-1 cells. The transfer of splenic B cells from 2-12H Tg mice to irradiated non-Tg littermates indicates that splenic anti-Sm B cells can differentiate to B-1. The differentiation of anti-Sm B cells to B-1 is sensitive to signal strength, as decreasing signal strength through the elimination of CD19 expression directs differentiation to B-2 and inhibits differentiation to B-1. Serum anti-Sm levels are also affected by changes in CD19 and CD22 expression; increasing the level of CD19 or eliminating CD22 increases serum anti-Sm levels. We suggest that differentiation to B-1 helps maintain ignorance to Sm by raising their activation threshold.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-12H Tg mice have been described previously (39) and are maintained in our colony at the University of North Carolina by backcrossing to C.B-17 mice. Offspring are identified by PCR analysis of tail genomic DNA as previously described (39). CD19-/-, and CD22-/- mice, and hCD19 Tg mice were described previously (34, 37) and bred with 2-12H Tg mice.
CD19-/- and CD22-/- mice were identified by PCR analysis of tail genomic DNA. The primer pairs for the PCR were designed to flank the portion of the CD19 and CD22 genes that contained the neo insertion (34, 37). The PCR products from wild-type mice result in a 758-bp product for CD19 and 444 bp for CD22, whereas knockout mice result in no product. Forward primer GGT GGA TGG ATA GTC CCA GTG and reverse primer CGA CTT GAA AGC CTT TCA AGG C were used for identification of CD19-/- mice. Forward primer GTC CCA CCC AGA CGA GAC ACC and reverse primer CCG AGA CAT TGA GGT GAA TGG G were used for identification of CD22-/- mice.
hCD19 Tg mice were identified by analysis of PBLs. After lysis of RBC, the cells were incubated with anti-human CD19 PE-conjugated Ab for 30 min on ice and washed. The stained cells were analyzed by flow cytometry. hCD19 Tg mice were determined by positive staining with anti-human CD19 Ab using non-hCD19 Tg mice as control for background staining. Mean fluorescence intensity of hCD19+/+ mice was twice as much as that of hCD19+/- mice.
Flow cytometry
The Abs used specific for IgMa (DS-1), IgMb (AF6-78), B220 (RA3-6B2), HAS (30-F1), CD23 (B3B4), CD43 (S7), and CD5 (53-7.3) were obtained from PharMingen (San Diego, CA) and were fluoresceinated, biotinylated, or conjugated to PE. In three-color experiments, directly fluoresceinated and PE-conjugated Abs were combined with a biotinylated Sm, which was revealed with streptavidin-PerCP. In four-color experiments, biotinylated ssDNA (Life Technologies, Grand Island, NY) was revealed with streptavidin-PerCP, and Sm was directly Cy5 (Molecular Probes, Eugene, OR) labeled and combined with directly fluoresceinated and PE-conjugated Abs. To detect membrane expression of various molecules, single-cell suspensions were prepared in RPMI 1640 medium (HyClone, Logan, UT) containing 0.1% sodium azide and 3.0% bovine calf serum (HyClone). Cells were incubated with previously determined optimal amounts of Ab in 50 µl of buffer for 20 min, after which they were washed three times with buffer and incubated with second-step reagents. After washing as before, the cells were analyzed using a FACScan and FACSCalibur (Becton Dickinson, Mountain View, CA). Data were analyzed using WinMDI (The Scripps Institute, La Jolla, CA). All data represent cells that fall within the lymphocyte gate determined by forward and 90° light scatter. One to 5 x 105 cells per sample were analyzed. All contour plots are 5% probability.
Adoptive transfer
Splenic B cells from 2-12 mice were enriched using a mouse B cell recovery column kit (Cedarlane Laboratories, Hornby, Ontario, Canada) according to the manufacturer’s instructions. Approximately 2 x 107 cells were transferred i.v. into sublethally (500 rad) irradiated non-Tg littermates. Spleen and peritoneal cells were taken after 5 days for flow cytometry analysis. For cell division analysis, spleen cells were labeled with CFSE (Molecular Probes) as described by Lyons and Parish (40) before adoptive transfer. Briefly, the CFSE stock solution was prepared at 5 mM in DMSO. Spleen cells were harvested and washed with PBS three times and resuspended at 2x 107 cells/ml in PBS. Immediately before labeling, the CFSE was thawed and diluted to 10 µM in a volume of PBS equal to that in which the cells were suspended. The two volumes were mixed together and incubated at room temperature for 10 min. An equal volume of calf serum was added to quench the labeling. The labeled cells were then washed three times with PBS and transferred i.v.
Serum ELISA
Quantitation of anti-Sm Abs and total IgM in mouse serum was done by ELISA as previously described (39). Briefly, 96-well PVC plates were coated with either Sm or goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL) in borate-buffered saline, washed, and blocked with borate-buffered saline/BSA/Tween 20. Mouse sera were diluted serially in borate-buffered saline/BSA/Tween 20 and added to Ag-coated plates in duplicate and then incubated overnight. After washing, goat anti-mouse IgM alkaline phosphatase (Southern Biotechnology Associates) was added and the anti-mouse IgM was revealed by the addition of 1 mg/ml p-nitrophenyl phosphate (Sigma, St. Louis, MO) in 0.01 M diethanolamine buffer. OD405 was determined after 90–120 min of incubation. Serum titers of anti-Sm were determined by generation of a standard curve for each assay using serial dilutions of mouse IgM of a known concentration.
LPS stimulation
Splenic and peritoneal exudate cells were harvested, washed, and resuspended at 1x 106 cells/ml in complete RPMI 1640 and incubated for 72 h in 5% CO2 in the presence or absence of 50 µg/ml LPS. Supernatants from cultures were tested by ELISA for anti-Sm and total IgM production as described above.
Statistical analysis
Statistical analysis was performed using Student’s t test. A value of p < 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously demonstrated that 15–35% of splenic B cells in
nonautoimmune 2-12H Tg mice are anti-Sm (Table I
and Ref. 39).
Immunofluorescence studies indicate that most of these B cells are
transitional, as they are IgMhigh,
HSAhigh, CD23-,
CD43-, and CD5- (Fig. 1A and Ref. 39).
They also express intermediate levels of CD21 consistent with
transitional B cells, indicating that they are not marginal zone B
cells. However, some appear to be mature B cells, since an
IgMlow, HSAlow,
CD23+, CD43-, and
CD5- populations can be detected. We have not
detected anti-Sm B cells in the spleen that have a B-1 phenotype.
However, Fig. 1 shows that there is a large population of anti-Sm
B-1 cells in the peritoneum. These cells were missed in the previous
analysis (39) because they were inadvertently gated out.
They are IgMhigh, CD23-,
CD43+, and CD5+ (Fig. 1A), and they are larger and more granular than the
CD23+ B cells of the peritoneum (Fig. 1B). They constitute 
30% of the B-1 subset (Table I).
Nearly all of the peritoneal anti-Sm B cells express CD5,
indicating that they are predominately B-1a. Interestingly, 6% of
the B-1 repertoire of non-Tg mice are anti-Sm (Table I and Fig. 1A), indicating that they are a significant component of the
normal B-1 repertoire in mice.
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shows that
peritoneal anti-Sm B cells of 2-12H Tg mice are responsive to LPS.
IgM levels are similar to those of non-Tg littermates and the level of
anti-Sm is 5-fold higher in 2-12H Tg cultures than in non-Tg
cultures. In contrast, LPS stimulation of non-Tg mouse peritoneal B
cells induces IgM Ab secretion, but little anti-Sm autoantibody
secretion. Thus, by this criterion peritoneal anti-Sm B-1 cells are
functional.
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To investigate the relationship between the predominantly
transitional splenic anti-Sm B cells and the mature peritoneal
anti-Sm B-1 cells, we transferred splenic B cells from 2-12H Tg
mice to sublethally irradiated littermate mice. Both spleen and
peritoneal B cells were examined 5 days later. Donor B cells were
identified by IgMa staining, as endogenous B
cells are IgMb. In the spleen, donor anti-Sm
B cells are CD23-, CD43-,
and CD5- and thus appear to be transitional
(Fig. 3A). Donor B cells are
also found in the peritoneum, including many that bind Sm.
Interestingly, here the majority of anti-Sm B cells are B-1 (i.e.,
IgMhigh, CD23-,
CD43+, and CD5+) (Fig. 3A).
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B shows that 5 days after transfer, nearly
all of the donor peritoneal anti-Sm B cells, identified by their
CFSE staining, have undergone only one or two divisions. These findings
indicate that expansion of an undetected population of contaminating
B-1 cells of donor origin cannot be responsible for the peritoneal
anti-Sm B-1 cell population in recipient mice. Thus, splenic
anti-Sm B cells have differentiated to B-1 in recipient mice. We
also note that there is a large population of B-1 cells of donor origin
that do not bind Sm, indicating that B cells of other specificities in
2-12H Tg spleens are selected into the B-1 subset. Altered B cell coreceptor expression affects anti-Sm B cell differentiation
To investigate the effect of signal strength on anti-Sm B cell
differentiation, we varied the levels of two B cell coreceptors, CD19
and CD22. 2-12H Tg/CD19-/- mice have 40% fewer
B cells in the spleen than 2-12H Tg mice (p <
0.05, Table I), consistent with previous findings (37).
The absence of CD19 has a similar effect on non-Tg mice. However, the
frequency of anti-Sm B cells increases from 30% in 2-12H Tg
mice to 47% in 2-12H Tg/CD19-/- mice
(p < 0.05), a 57% increase, indicating that
anti-Sm B cell development is favored in the absence of CD19. In
contrast to 2-12H Tg mice in which splenic anti-Sm B cells
are mostly CD23- transitional B cells, splenic
anti-Sm B cells of 2-12H Tg/CD19-/- mice
have a mature B-2 cell phenotype (i.e., CD23+,
CD43-, CD5-, and
HSAlow) (Fig. 4).
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and Fig. 4), consistent with the finding that CD19-/-
mice are deficient in B-1 cell differentiation (37, 44).
Moreover, the proportion of anti-Sm B cells decreases from 32.5%
to 5% (Table I), and thus the total number of peritoneal anti-Sm B
cells decreases dramatically (Table I). Like splenic anti-Sm B
cells, peritoneal anti-Sm B cells of 2-12H
Tg/CD19-/- mice are B-2 (i.e.,
IgMhigh, CD23+,
CD43-, and CD5-) (Fig. 4). Thus, in the absence of CD19 anti-Sm B cells differentiate to
B-2 rather than to B-1.
Overexpression of CD19 increases the number of B-1 cells. The level of
CD19 can be varied using mice hemizygous for the human CD19 transgene
(hCD19+/-) and mice that are homozygous for the
hCD19 transgene (hCD19+/+) (34). We
have examined anti-Sm B cells in 2-12H Tg mice that are either
hCD19+/- or hCD19+/+. The
total number of splenic lymphocytes is reduced in 2-12H
Tg/hCD19+/+ mice and to a lesser degree in 2-12H
Tg/hCD19+/- mice. This is consistent with
previous reports of overexpression of CD19 (37). 2-12H
Tg/hCD19+/- and 2-12H
Tg/hCD19+/+ mice have 38 and 58% fewer B cells
than 2-12H Tg mice, respectively (Table I). Oddly, the frequency of
anti-Sm B cells is lower in 2-12H
Tg/hCD19+/- mice (18.1%) than in 2-12H
Tg/hCD19+/+ mice (25.8%) (Table I). The latter
frequency is not different from that in 2-12H Tg mice. As in 2-12H Tg
mice, the anti-Sm B cells in hCD19 Tg mice are mostly transitional
(i.e., IgMhigh, CD23-,
CD43-, and CD5-)
(Fig. 4).
The number and frequency of the peritoneal B cells of 2-12H Tg/hCD19 Tg
mice is similar to that of 2-12H Tg mice (Table I). However, the number
of peritoneal B-1 cells increases as the dose of hCD19 increases. Thus,
90% of 2-12H Tg/hCD19+/- and 96% of 2-12H
Tg/hCD19+/+ peritoneal B cells exhibit a B-1
phenotype (Fig. 4), compared with 72% in 2-12H Tg mice. This is
consistent with the finding of Sato et al. (34) that
increased CD19 expression results in an increased frequency of B-1
cells in the peritoneum. In addition, the frequency of anti-Sm B
cells increases by 2-fold in hCD19+/+ mice
relative to 2-12H Tg mice (Table I). Phenotypic analysis indicates that
all of the anti-Sm B cells are B-1, as they are
IgMhigh, CD23-,
CD43+, and CD5+ (Fig. 4).
As in 2-12H Tg mice, all anti-Sm B-1 cells in hCD19 Tg mice
carrying the 2-12H transgene are CD5+ and
therefore B-1a.
CD22 is a negative regulator of B cell signaling through its
association with SHP-1 (32). To determine how CD22
influences differentiation of anti-Sm B-1 and transitional B cells,
we bred 2-12H Tg/CD22-/- mice. Although the
frequency of splenic B cells in both 2-12H Tg and 2-12H
Tg/CD22-/- mice is the same, the absence of
CD22 decreases by 48% the number of splenic lymphocytes. A lack of
CD22 also results in a decrease in the total number and frequency of
anti-Sm B cells in 2-12H Tg mice (Table I). However, the
anti-Sm B cells remain transitional (i.e.,
IgMhigh, CD23-,
CD43-, and CD5-)
(Fig. 4).
In the peritoneum the number of lymphocytes increases 4-fold over
CD22 intact mice, and the frequency of the B-1 cells increases from 78
to 98% in 2-12H Tg mice (Table I). Although the frequency of
anti-Sm B cells decreases in 2-12H Tg as a result of the absence of
CD22, the total number of anti-Sm B cells increases as a result of
the increase in the total number of peritoneal B cells in
CD22-/- mice (Table I). However, in
CD22-/- mice they are B-1 (i.e.,
IgMhigh, CD23-, and
CD43+, and CD5+) (Fig. 4).
Serum anti-Sm levels in 2-12H Tg mice are affected by changes in coreceptor expression
Serum anti-Sm levels in 2-12H Tg mice were evaluated by ELISA.
As we showed previously (39), serum anti-Sm levels in
2-12H Tg mice are not significantly different from those of non-Tg
littermate mice, despite the substantially higher number of anti-Sm
B cells in the spleens and peritoneums of 2-12H Tg mice (Fig. 5). However,
2-12H/CD22-/-,
2-12H/hCD19+/+, and 2-12H
Tg/hCD19+/- mice have levels of anti-Sm that
are significantly higher than those of 2-12H Tg mice
(p < 0.05) and their non-Tg counterparts
(p < 0.05) (Fig. 5). These levels are
comparable to those of 2-12H Tg MRL-Mp/lpr/lpr
mice (Fig. 5). Conversely, 2-12H Tg/CD19-/-
mice, which have no anti-Sm B-1 cells, have significantly decreased
serum anti-Sm levels (p < 0.05) compared
with 2-12H Tg mice. This suggests that changes in B-1 cell sensitivity
to signaling affects serum anti-Sm levels.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30% of the B-1 repertoire. This is in addition to the anti-Sm B
cells in the spleens of these mice, the majority of which are
transitional. We also show a direct relationship between the splenic
and peritoneal anti-Sm B cells; adoptive transfer of splenic
anti-Sm B cells results in their differentiation to B-1 and
movement to the peritoneum. Thus, splenic anti-Sm B cells are
capable of differentiation to B-1.
A B-2 to B-1 differentiative pathway was originally proposed by Wortis
and colleagues (45), and we have demonstrated that B cells
specific for phosphatidyl choline (PtC), which are exclusively B-1 in
normal mice, follow this pathway of differentiation in vivo (46, 47). It remains to be determined whether differentiation to B-1
occurs throughout adult life. The peritoneal B-1 cell population
develops early in life and maintenance of the B-1 population occurs by
the self-renewal capacity of these cells (48, 49). It does
not require continuous infusion of new B cells, as is required for the
B-2 population (48, 49). However, this does not preclude
that some cells enter the B-1 subset in adult life since not all B-1
cells may have the same half-life. Because anti-Sm transitional B
cells are continuously generated by the adult bone marrow, there may be
a constant flow of anti-Sm B cells to the B-1 subset throughout
life. Alternatively, their differentiation may be blocked and they may
instead undergo apoptosis, despite their capacity to differentiate to
B-1. Such a block might be due to a negative feedback from already
differentiated B-1 cells. In this regard, we note that there are 30
times as many anti-Sm transitional B cells as anti-Sm B-1 cells
in 2-12H Tg mice (Table I). If the anti-Sm transitional B cells all
differentiate to B-1, then by necessity the anti-Sm B-1 cells must
be short-lived. Preliminary experiments suggest that the half-life of
anti-Sm B-1 cells is not short and is equal to that of non-Tg B-1
cells. Thus, many transitional anti-Sm B cells probably undergo
apoptosis in the spleen as previously suggested (39).
Experiments are currently underway to resolve this issue.
Differentiation of anti-Sm B cells to B-1 appears to be governed by
the strength of the signal delivered by Ag. Decreasing the strength of
Ag receptor signaling by CD19 elimination resulted in B cell
differentiation into B-2 cells rather than into B-1 cells. Thus, weak
signals generated through the Ag receptor may result in transitional B
cell differentiation into B-2 cells rather than undergoing apoptosis or
differentiation to B-1. Since B-1 cell numbers are greatly reduced in
CD19-deficient mice, the assumption was that signal strength was
insufficient to support the long-term survival of B-1 cells
(34). However, the current study suggests that appropriate
signals generated through the Ag receptor are required for B cell
development into B-1 cells. In support of this, overexpression of CD19
or elimination of CD22 significantly increased the number of 2-12H B-1
B cells, while the numbers of 2-12H B-2 cells in these mice were
decreased (Table I and Fig. 4). Furthermore, consistent with B-1 as the
origin of circulating IgM, serum anti-Sm autoantibody levels in
2-12H Tg/hCD19+/+ or +/- and 2-12H
Tg/CD22-/- mice were 4- to 5-fold higher than
the levels in 2-12H Tg mice and non-Tg mice (Fig. 5). This is
comparable to the levels in 2-12H Tg
MRL-Mp/lpr/lpr mice (Fig. 5). Thus, activation
thresholds regulate B-1 cell differentiation in addition to regulating
tolerance and the levels of serum anti-Sm.
The differentiation of anti-Sm B cells to B-1 and the absence
of higher serum anti-Sm in 2-12H Tg mice relative to non-Tg mice is
paradoxical (Fig. 5). B cells are selected by Ag for recruitment into
the B-1 cell subset, and B-1 cells are responsible for most of the
circulating IgM in unmanipulated mice (13, 50). Indeed,
anti-PtC Tg mice have 100-fold more serum anti-PtC than
non-Tg littermates due to the increase in anti-PtC B-1 cell numbers
(51). Why then do 2-12H Tg mice not have higher serum
anti-Sm levels than non-Tg littermates, given the higher number of
anti-Sm B-1 cells? One possibility is that migration to the
peritoneum removes these cells from contact with other cells or
molecules required for activation and differentiation to Ab-secreting
cells. For example, exposure to the activating Ag in the peritoneum may
not be sufficient to drive differentiation of anti-Sm B-1 cells to
Ab-secreting cells. Another possibility is that differentiation to B-1
is accompanied by changes in the B cell itself that decrease its
sensitivity to Ag. One change induced by differentiation to B-1
relevant to this is the expression of CD5, a negative
regulator of BCR signaling through its association with the phosphatase
SHP-1 (52, 53). This association would increase the amount
of Ag required for activation. Hippen et al. (54) have
recently demonstrated that CD5 is expressed by anergic anti-hen egg
lysozyme (HEL) B cells and that it is responsible for the very low
levels of circulating anti-HEL. In the absence of CD5, anergic
anti-HEL B cells produce high levels of circulating anti-HEL
similar to those found in mice with functional anti-HEL B cells.
Thus, CD5 is instrumental in blocking activation of anergic B cells in
this model. In an analogous way, CD5 expression could inhibit
activation of anti-Sm B cells and account for the low levels of
anti-Sm in unimmunized 2-12H Tg mice (Fig. 5 and Ref.
39). Thus, differentiation to B-1 may be an essential
component in maintaining ignorance to Sm. Increasing the level of Sm Ag
could overcome the negative effects of CD5 on BCR signaling and account
for the ability to activate these cells upon immunization with snRNPs
(39). This may relate to the finding that all anti-Sm
B-1 cells of 2-12H Tg mice express CD5, as do the peritoneal
anti-Sm B-1 cells of non-Tg littermates. Thus, regulation of CD5
expression may control the ability of B-1 cells to be activated and
hence the autoantibodies secreted by B-1 cells.
In summary, we have demonstrated that anti-Sm B cells differentiate to B-1 and that the strength of the signal provided by Ag determines whether an anti-Sm B cell differentiates to B-2 or to B-1. In addition, alteration of the activation threshold increases or decreases the level of serum anti-Sm, indicating that the activation threshold setting determines the level of circulating Ab. We suggest that differentiation to B-1 helps maintain ignorance to Sm in nonautoimmune mice through the expression of negative regulators of BCR signaling such as CD5. Alterations in the threshold setting or ability of B cells to differentiate to B-1 could be a predisposing factor in production of autoantibodies in disease. Thus, anti-Sm B cells provide a new perspective on maintaining tolerance to self-Ags targeted in lupus.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department of Microbiology and Immunology, CB 7290, 804 MEJB, University of North Carolina, Chapel Hill, NC 27599.
3 Abbreviations used in the paper: RNP, ribonucleoprotein; Tg, transgenic; BCR, B cell receptor; PtC, phosphatidyl choline; HEL, hen egg lysozyme.
Received for publication October 5, 2000. Accepted for publication December 4, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. C. Poe, K. M. Haas, J. Uchida, Y. Lee, M. Fujimoto, and T. F. Tedder Severely Impaired B Lymphocyte Proliferation, Survival, and Induction of the c-Myc:Cullin 1 Ubiquitin Ligase Pathway Resulting from CD22 Deficiency on the C57BL/6 Genetic Background J. Immunol., February 15, 2004; 172(4): 2100 - 2110. [Abstract] [Full Text] [PDF]
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Y. Qian, H. Wang, and S. H. Clarke Impaired Clearance of Apoptotic Cells Induces the Activation of Autoreactive Anti-Sm Marginal Zone and B-1 B Cells J. Immunol., January 1, 2004; 172(1): 625 - 635. [Abstract] [Full Text] [PDF]
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H. Wang and S. H. Clarke Evidence for a Ligand-Mediated Positive Selection Signal in Differentiation to a Mature B Cell J. Immunol., December 15, 2003; 171(12): 6381 - 6388. [Abstract] [Full Text] [PDF]
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B. D. Aplin, C. L. Keech, A. L. de Kauwe, T. P. Gordon, D. Cavill, and J. McCluskey Tolerance through Indifference: Autoreactive B Cells to the Nuclear Antigen La Show No Evidence of Tolerance in a Transgenic Model J. Immunol., December 1, 2003; 171(11): 5890 - 5900. [Abstract] [Full Text] [PDF]
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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]
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 P. L. Cohen Autoimmunity and Lymphoproliferation: Two Genes are Worse than One Mol. Interv., November 1, 2002; 2(7): 427 - 430. [Abstract] [Full Text]
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 S.-C. Wong, W.-K. Chew, J. E.-L. Tan, A. J. Melendez, F. Francis, and K.-P. Lam Peritoneal CD5+ B-1 Cells Have Signaling Properties Similar to Tolerant B Cells J. Biol. Chem., August 16, 2002; 277(34): 30707 - 30715. [Abstract] [Full Text] [PDF]
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M. J. Chumley, J. M. Dal Porto, and J. C. Cambier The Unique Antigen Receptor Signaling Phenotype of B-1 Cells Is Influenced by Locale but Induced by Antigen J. Immunol., August 15, 2002; 169(4): 1735 - 1743. [Abstract] [Full Text] [PDF]
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 C. H. Nielsen and R. G. Q. Leslie Complement's participation in acquired immunity J. Leukoc. Biol., August 1, 2002; 72(2): 249 - 261. [Abstract] [Full Text] [PDF]
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D. Yin, L. Ma, A. Varghese, J. Shen, and A. S.-F. Chong Intact Active Bone Transplantation Synergizes with Anti-CD40 Ligand Therapy to Induce B Cell Tolerance J. Immunol., May 15, 2002; 168(10): 5352 - 5358. [Abstract] [Full Text] [PDF]
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H. Wardemann, T. Boehm, N. Dear, and R. Carsetti B-1a B Cells that Link the Innate and Adaptive Immune Responses Are Lacking in the Absence of the Spleen J. Exp. Med., March 18, 2002; 195(6): 771 - 780. [Abstract] [Full Text] [PDF]
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 R. Ceredig The ontogeny of B cells in the thymus of normal, CD3{varepsilon} knockout (KO), RAG-2 KO and IL-7 transgenic mice Int. Immunol., January 1, 2002; 14(1): 87 - 99. [Abstract] [Full Text] [PDF]
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M. Borrero and S. H. Clarke Low-Affinity Anti-Smith Antigen B Cells Are Regulated by Anergy as Opposed to Developmental Arrest or Differentiation to B-1 J. Immunol., January 1, 2002; 168(1): 13 - 21. [Abstract] [Full Text] [PDF]
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 P. HASLER and M. ZOUALI B cell receptor signaling and autoimmunity FASEB J, October 1, 2001; 15(12): 2085 - 2098. [Abstract] [Full Text] [PDF]
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 P Youinou and P M Lydyard CD5/ B cells in nonorgan-specific autoimmune diseases: a fresh look Lupus, August 1, 2001; 10(8): 523 - 525. [PDF]
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S. Santulli-Marotto, Y. Qian, S. Ferguson, and S. H. Clarke Anti-Sm B Cell Differentiation in Ig Transgenic MRL/Mp-lpr/lpr Mice: Altered Differentiation and an Accelerated Response J. Immunol., April 15, 2001; 166(8): 5292 - 5299. [Abstract] [Full Text] [PDF]
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H. H. Wortis and R. Berland Cutting Edge Commentary: Origins of B-1 Cells J. Immunol., February 15, 2001; 166(4): 2163 - 2166. [Abstract] [Full Text] [PDF]
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) and anti-Sm (
)
concentrations (in µg/ml) were measured. A total of eight 2-12H Tg
and eight non-Tg littermates were analyzed in two experiments. All
LPS-stimulated cultures were significantly different from their
unstimulated counterparts. *, significant difference
(p < 0.001) between LPS-stimulated 2-12H Tg and
non-Tg peritoneal B cells.
