| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
8 transgene. In
contrast to 2-12H transgenic mice, in which anti-Sm B cells are
predominantly splenic transitional, and peritoneal B-1, low-affinity
anti-Sm B cells are long-lived B-2 cells and are found in the
spleen, lymph nodes, and peritoneum. However, they are unresponsive to
LPS in vitro, indicating that they are anergic, although they do not
down-regulate IgM and are not excluded from follicles even in the
presence of nonautoreactive B cells. Thus, low-affinity anti-Sm B
cells appear to have a partial form of anergy. Interestingly, these
cells have elevated levels of MHC class II and CD95, but not CD40,
CD80, or CD86, suggesting that they are poised to undergo deletion
rather than activation upon T cell encounter. These data identify
anergy as a mechanism involved in anti-Sm B cell
regulation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anergy is of particular interest because this mechanism retains autoreactive B cells in the repertoire, offering a large window of opportunity for the loss of tolerance. Not all anergic B cells are alike. For example, at one extreme are anergic anti-hen egg lysozyme (HEL) B cells. These B cells have down-regulated surface IgM and have a short half-life due to exclusion from B cell follicles (1, 4). In addition, the BCR is uncoupled from the signal transduction pathways in these cells (13). This contrasts with anergic anti-dsDNA B cells. Although similar to anti-HEL B cells in that they down-regulate surface IgM and are excluded from B cell follicles, they differ in that they developmentally arrest at the transitional B cell stage (7, 14). At the other extreme are anti-ssDNA B cells. They are long-lived B-2 cells that do not down-regulate surface IgM and can occupy splenic follicles (7, 15). Moreover, they retain at least some signal transduction potential (7, 15). Notwithstanding these differences, all anergic B cells share an impaired ability to differentiate to Ab-secreting cells (ASC) in response to Ag and LPS. Relevant to the development of autoimmunity is the fact that anergy is reversible and, thus, under the appropriate circumstances, anergic B cells could provide a source of autoreactive B cells that contribute to autoimmune disease (7, 16).
We have characterized the anti-Smith Ag (Sm) response in autoimmune
MRL/lpr mice because it occurs in human and murine lupus
(17, 18, 19). These mice spontaneously develop a systemic
lupus erythematosus-like disease beginning at 
5 mo of age. These
mice develop anti-Sm responses at a prevalence of 25%, similar
to that in human systemic lupus erythematosus (20, 21). To
understand how anti-Sm B cells are regulated, we generated Ig H
chain transgenic (Tg) mice (2-12H) (22). This H chain
rearrangement derives from the anti-Sm hybridoma 2-12 of
MRL/lpr origin (17, 18). Abs using 2-12H chains
can bind Sm, ssDNA, both, or neither, depending on the L chain with
which it is paired. Moreover, the L chain determines the affinity for
these autoantigens, which ranges over 2–3 orders of magnitude
(23).
2-12H Tg mice have large numbers of anti-Sm and anti-ssDNA B
cells. In the spleen most of these cells are transitional, suggesting a
block of differentiation to the mature B-2 cell stage
(22). Interestingly, anti-Sm B cells also constitute
30% of the peritoneal B-1 cell subset in these mice
(24), yet the serum levels of anti-Sm Abs are low and
not different from those of non-Tg mice. However, these cells appear to
be functional, because anti-Sm Abs are secreted upon immunization
with murine small nuclear ribonucleoproteins, and both transitional and
B-1 anti-Sm B cells can be activated by LPS (22, 24).
Hence, the absence of elevated levels of serum anti-Sm is a
paradox, particularly due to the large number of peritoneal anti-Sm
B-1 cells present in these mice. Differentiation to B-1 may be involved
in the maintenance of anti-Sm B cell tolerance, possibly through
the migration of anti-Sm B cells to the peritoneum and away from
self-Ag or through the expression of CD5, a negative regulator of BCR
signaling (24). Thus, anti-Sm B cells in 2-12H Tg mice
appear to be regulated by developmental arrest and differentiation to
B-1. While 2-12H Tg mice have been instrumental in the characterization
of anti-Sm B cell regulation in nonautoimmune mice, the diversity
of affinities that are conferred by the use of multiple endogenous L
chains does not allow us to exclude the possibility that other
mechanisms regulate cells of this specificity.
To elucidate the role of BCR affinity in anti-Sm B cell regulation,
we interbred 2-12H and V8 Tg mice. Abs
composed of 2-12H and V8 L chains have a low
affinity for Sm and do not bind ss- or dsDNA (23). In
contrast to anti-Sm B cells of 2-12H Tg mice, low-affinity
anti-Sm B cells become mature long-lived B-2 cells and do not
differentiate to B-1. They occupy B cell follicles in the spleen even
when nonautoreactive B cells are a majority of the B cell repertoire.
These cells are not spontaneously activated, as indicated by the low
levels of transgene-encoded Ab in the serum, and, most significantly,
they do not differentiate to ASC upon in vitro stimulation. Therefore,
we conclude that they are anergic. Furthermore, they have up-regulated
MHC class II and CD95 expression, suggesting that they are poised to
undergo deletion, rather than activation, upon T cell encounter.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2-12H Tg mice were created as previously described
(22). V8 Tg mice
(25) were provided by Dr. M. Weigert (Princeton
University, Princeton, NJ). Both 2-12H and V8
Tg mice were bred with C-/-
mice (26) (provided by GenPharm International, San Jose,
CA) to limit the endogenous light chain use to 
. 2-12H
C-/- mice were then bred to
V8
C-/- to create
2-12H/V8 double-Tg mice
(V8 Dbl Tg). Experiments were performed on
F1 mice of this cross. All mice were 2–5 mo of
age at the time of analysis. Animals were housed and bred in a
conventional facility at University of North Carolina (Chapel Hill,
NC).
Screening of 2-12 and V8 transgenes was
performed by PCR as previously described (22, 25).
C-/- mice were detected by
PCR using primers that amplified the J2 and
J3 regions of the wild-type locus. A
649-bp product was produced in mice that had one or more copies of the
wild-type locus. Primers sequences are: J
forward, 5'-CGGTGCTCAGACCATGCTCAG-3'; and
J reverse, 5'-GCCTGAGGCTGATCCAGTTAGAT-3'.
The PCR included 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM
MgCl2, 1 µM of each primer, and 2.5 U
Taq DNA Polymerase (Life Technologies, Gaithersburg, MD).
Product amplification was for 5 min at 94°C, 30 s at 94°C, 1
min at 62°C, and 1 min at 70°C. A total of 30 cycles of
amplification were performed. The final extension was for 7 min at
70°C.
Flow cytometric analysis
Spleens and lymph nodes were crushed between the frosted ends of two slides to make single-cell suspensions. Bone marrow cells were extracted from the femur and tibia of the two hind legs, and peritoneal cells were isolated by lavage. After cell suspensions were made in HBSS, RBCs were lysed using a solution that contained 0.15 M ammonium chloride, 1.0 mM potassium bicarbonate, and 0.1 mM EDTA. FcR were blocked using mAb 2.4G2. Cells were incubated for at least 10 min at 4°C. Staining was performed in HBSS supplemented with 3% FBS (Life Technologies).
The Abs used for flow cytometry were against IgMa
(DS-1), IgMb (AF6-78), (R5-240), CD5
(53-7.3), CD45R/B220 (RA3-6B2), CD23 (B3B4), CD24 (M1/69), CD43 (S7),
CD21/35 (7G6), CD40, CD95, CD80, CD86, CD22, CD44, and
IAb/d and were obtained from BD PharMingen (San
Diego, CA). These Abs were biotinylated or conjugated to
allophycocyanin, PE, FITC, or CyChrome. For the identification
of anti-Sm B cells we used Sm (SMA-3000; Immunovision, Springdale,
AR) that was biotinylated in our laboratory as described previously
(22). For the detection of biotinylated probes we used
streptavidin-FITC (Jackson ImmunoResearch Laboratories, West Grove,
PA), streptavidin-PerCP (BD PharMingen), or streptavidin-Cy5
(Calbiochem, La Jolla, CA). Three- and four-color analyses were
performed at the University of North Carolina Flow Cytometry Facility
(Chapel Hill, NC) using a FACScan and a FACSCalibur (BD Biosciences,
Mountain View, CA), respectively. Analysis software was purchased from
Cytomation (Fort Collins, CO).
BrdU labeling
Mice were given 0.5 mg/ml 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) with 1 mg/ml dextrose in their drinking water continuously for 8 wk. At different time points mice were sacrificed and spleens were isolated for analysis. Cell preparations and surface staining of 1 x 107 cells was done as described above. Staining for BrdU was done as described by Allman et al. (27) using anti-BrdU-FITC (BD Biosciences, San Jose, CA).
ELISA
Anti-Sm Abs in serum or tissue culture supernatants were measured using the protocol described by Eisenberg et al. (28). Briefly, 96-well flat-bottom polyvinylchloride plates were coated with Sm protein (Immunovision) for at least 5 h in borate-buffered saline. All washes were performed with borate-buffered saline. Plates were blocked with borate-buffered saline, 0.5% BSA, and 0.4% Tween 80 for a minimum of 1 h before adding the samples. All samples were analyzed in duplicate. To develop the assays we used either goat anti-mouse IgM-alkaline phosphatase (AP; Southern Biotechnology Associates, Birmingham, AL) or anti-IgMa-biotin followed by streptavidin-AP (BD PharMingen). Paranitrophenyl phosphate (1 mg/ml; Sigma-Aldrich) in 0.01 M diethanolamine was used to develop the assays. OD readings were taken at 405/600 nm.
For the detection of 2-12H/V8-encoded Ab,
plates were coated with rat anti-mouse (BD PharMingen),
and detection was made using biotinylated rat
F(ab')2 anti-mouse IgMa
(HB100, Bet-1; American Type Culture Collection, Manassas, VA) with
streptavidin-AP. Total IgM was detected by coating with goat
anti-mouse IgM and developing with the same reagent coupled to AP
(Southern Biotechnology Associates). To measure total
IgMa or IgMb, plates were
coated with purified anti-IgMa or
anti-IgMb (BD PharMingen) and developed with
anti-IgM-AP. Purified mouse IgMa (TEPC 183;
Sigma-Aldrich) was used as a standard for total IgM and
IgMa ELISAs. The IgMb
standard (CBPC112) was a gift from Dr. P. Cohen (University of
Pennsylvania, Philadelphia, PA).
LPS stimulation
Spleen cells were harvested, and RBC were lysed using RBC lysis buffer described in the flow cytometry section. Cells were resuspended in DMEM-high glucose at 1 x 106 cells/ml. Triplicate cultures were set for 72 h in the presence or the absence of 50 µg/ml LPS. Supernatants were analyzed by ELISA.
Immunofluorescence
Freshly isolated spleens were imbedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) and frozen in 2-methyl-butane and liquid nitrogen. Five-micrometer spleen sections were prepared and acetone-fixed before staining. Sections were blocked for 30 min with 6% BSA in PBS. Staining was performed for 1 h at room temperature with anti-CD3-FITC, anti-B220-allophycocyanin, and anti-IgMa or IgMb-biotin (BD PharMingen). After the slides were rinsed with PBS, the sections were stained with streptavidin-Cy3 (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Fluorescein signal was enhanced using the Alexa Fluor 488 signal amplification kit for FITC-conjugated probes (Molecular Probes, Eugene, OR) as described by the manufacturer, and the slides were then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Analysis was performed at the Physiology Imaging and Neuroscience Confocal Facility at University of North Carolina, using a Leica TCS-NT laser scanning confocal microscope (Leica, Deerfield, IL).
Bone marrow chimeras
Hematopoietic stem cells from 2- to 3-mo-old
V8 Dbl Tg mice and B6 non-Tg mice were
isolated as described by Goodell et al. (29). Briefly,
bone marrow cells were extracted, and RBCs were lysed as described for
flow cytometric analysis. Cells were resuspended at 1 x
106 cells/ml in RPMI 1640 medium (HyClone
Laboratories, Logan, UT) with 2% FBS and 5 µg/ml Hoechst 33342.
Staining was performed at 37°C for 90 min. Propidium iodide at 2
µg/ml was added just before analysis. Analysis and sorting of side
population cells, which exclude the Hoechst dye and are enriched for
hemopoietic stem cells, were performed with a MoFLo high speed sorter
(Cytomation). Cells (2 x 103) in PBS were
injected via tail vein into B6 RAG-1-/- mice
(The Jackson Laboratory, Bar Harbor, ME) that had been given 600 rad
24 h previously. Analysis of recipients was performed at least 8
wk after reconstitution.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously demonstrated that 2-12H chains paired with
V8L chains bind Sm with low affinity
(23). Thus, to generate low-affinity anti-Sm Tg mice,
2-12H and V8 Tg mice were bred to generate
double-Tg mice (V8 Dbl Tg). The L chain
encoded by the V8 transgene is identical with
the V8 L chain used in the earlier in vitro
analysis (23, 25). Thus, the anti-Sm Abs produced by
V8 Dbl Tg mice will be low affinity. These
mice were also bred to C-/-
to ensure that the all L chains are of transgene origin. Unless
otherwise indicated, all V8 Dbl and
V8 Tg mice used in this study are
-/-, and all control 2-12H Tg and non-Tg
mice are +/+. As shown in Table I
, total spleen cell numbers in
V8 Dbl Tg mice were not significantly
different from those in 2-12H, V8 Tg, and
non-Tg mice, although, as is typical of Ig Tg mice, the total number of
B cells were significantly lower than those in non-Tg mice
(p < 0.005).
|
8 Dbl Tg mice, spleen cells were stained with
Abs specific for IgHa (the 2-12H allotype) and
. As shown in Fig. 1A,
>93% of splenic B220+ cells of
V8 Dbl Tg mice express
IgMa. The remaining B cells express b
allotype H chains or -chains (data not shown). Nearly all (>95%)
lymph node B cells are also IgMa (data not
shown). Thus, by virtue of expression of both transgenes the majority
of splenic and lymph node B cells in V8 Dbl Tg
mice are anti-Sm. The ability of these B cells to bind Sm was
directly assessed by flow cytometry using biotinylated Sm. As shown in
Fig. 1B, only the IgMhigh B cells
exhibit Sm staining, despite the fact that nearly all splenic B cells
of V8 Dbl Tg mice are anti-Sm. Thus, the
lack of Sm staining by the IgMlow B cells must be
due to their low surface IgM level. As previously demonstrated, 30%
of 2-12H splenic B cells stain with biotinylated Sm (22)
(Fig. 1B). Anti-IgHa-specific Abs are used to
follow the development and regulation of these low-affinity anti-Sm
B cells, because essentially all IgHa B cells
express the V8 transgene and thus are
low-affinity anti-Sm B cells.
|
79%) from
V8 Dbl Tg mice have a mature B-2 cell
phenotype. They are CD23+,
HSAlow, CD21/35low,
CD5-, and CD43- (Fig. 1
C and data not shown). In addition, some (6%) are
HSAhighCD21/35-
transitional 1 cells, and a few (1%) are
HSAhighCD21/35low
transitional 2 cells (30). Essentially no
CD21/35highCD23- or
CD5+CD43+ cells are
detectable, indicating that marginal zone (MZ) B cells and B-1 cells
contribute little or not at all to the splenic repertoire. In contrast,
MZ (Fig. 1C) and B-1 cells (data not shown) are present in
the spleens of V8 Tg mice.
Anti-Sm B cells of 2-12H Tg mice differentiate to B-1 and constitute
30% of the peritoneal B-1 repertoire (24). To
determine whether low-affinity anti-Sm B cells differentiate to
B-1, we examined peritoneal cells of V8 Dbl Tg
mice for evidence of differentiation to B-1. As shown in Fig. 2, low-affinity anti-Sm B cells in
the peritoneum have a mature B-2 cell phenotype. They are
CD23+IgMlowB220high
and do not express the B-1 cell markers CD5 and CD43 (Fig. 2). Thus, in
contrast to anti-Sm B cells in 2-12H Tg mice, low-affinity
anti-Sm B cells do not differentiate to B-1.
|
the turnover rate of transitional (HSAhigh) B
cells of V8 Dbl Tg mice suggests they have a
half-life of 3 days, significantly shorter
(p < 0.0001) than the 2-wk half-life of their
non-Tg C-/- counterparts.
The half-life of mature low-affinity anti-Sm B cells is estimated
to be 5 wk, not significantly different from the 7 wk estimated for
B cells of non-Tg C-/-
littermates. Thus, peripheral deletion or developmental arrest do not
appear to have a role in the regulation of low-affinity anti-Sm B
cells, as they acquire a mature phenotype and are long-lived.
|
To determine whether low-affinity anti-Sm B cells are
activated in vivo, we examined the levels of transgene-encoded
(IgMa) Ab in circulation. Assaying for
IgMa Abs in the serum of
V8 Dbl Tg mice is more sensitive than
determining the presence of anti-Sm Abs due to the low affinity of
2-12H/V8 Abs for Sm. As shown in Fig. 4A, the total serum IgM Ab
level in V8 Dbl Tg mice, which includes both
IgMa and endogenous IgMb
Abs, is 100-fold lower than that in non-Tg
C-/- littermates
(p = 0.0019) and non-Tg BALB/c mice
(p < 0.0001). It is 3-fold lower than that
in 2-12H Tg mice (p = 0.0057). More
significantly, IgMa Ab in
V8 Dbl Tg mice, which in these mice is
anti-Sm, is 10-fold lower than that in 2-12H Tg
(p = 0.0005) and 100-fold lower
(p < 0.0001) than that in BALB/c mice despite
the fact that the nearly all V8 Dbl Tg B cells
are IgMa (Figs. 4B and
1B). Thus, V8 Dbl Tg mice have very
little anti-Sm Ab in the circulation, suggesting that either
low-affinity anti-Sm B cells have not encountered Ag and are
ignorant or that they have encountered Ag and are anergic.
|
8 Dbl Tg B
cells were examined. As shown in Fig. 5, the expression levels of CD40, CD80 (B7-1), and CD86 (B7-2) on
V8 Dbl Tg B cells are slightly lower than or
equal to those on V8 Tg control B cells. In
addition, the level of IgM is somewhat higher than it is on B cells of
V8 Tg mice, indicating that there is no
down-regulation of IgM on these autoreactive B cells (Fig. 5). However,
V8 Dbl Tg B cells have higher levels of MHC
class II and CD95 than V8 Tg littermates.
Because both MHC class II and CD95 expression levels can be elevated in
response to Ag stimulation (32, 33), the elevated
expression of these markers on low-affinity anti-Sm B cells
suggests that they have encountered Ag in vivo.
|
8 Dbl Tg
mice were cultured with LPS for 3 days and Ab production was measured
by ELISA. As shown in Fig. 6, low-affinity anti-Sm B cells from V8 Dbl
Tg mice do not secrete IgM upon LPS stimulation. This was assessed by
an IgM-specific ELISA that detects both IgMa and
IgMb Abs and by an anti-Sm ELISA. This result
contrasts with the production of IgM by B cells of
nonautoreactive control mice and with the production of anti-Sm Abs
by B cells from 2-12H Tg mice (Fig. 6). Similar results
were obtained with supernatants of 7-day LPS cultures (data not shown).
Thus, low-affinity anti-Sm B cells of V8
Dbl Tg mice are unresponsive to mitogen stimulation and therefore are
anergic.
|
Exclusion from B cell follicles can contribute to the regulation
of autoreactive B cells. Both anti-HEL B cells and anti-dsDNA B
cells are excluded from B cell follicles (1, 14). To
determine whether follicular exclusion plays a role in the regulation
of anergic anti-Sm B cells in V8 Dbl Tg
mice, the localization of low-affinity anti-Sm B cells was
determined. Spleen sections of V8 Dbl Tg mice
were stained with fluorescent Abs against CD3 and B220 to identify T
cell and B cell areas, respectively. Transgenic
IgMa B cells were identified using a
biotin-labeled anti-IgMa Ab and
streptavidin-Cy3 (red) for detection. As shown in Fig. 7A,
IgMa+ B cells of V8 Dbl
Tg mice are located in follicles alongside T cell-rich periarteriolar
lymphoid sheaths.
|
8 Dbl Tg and non-Tg mice were enriched
and sorted as described by Goodell et al. (29), mixed at
various ratios, and injected i.v. into sublethally irradiated B6
RAG-1-/- mice. Chimeric mice were analyzed 8 wk
later. Immunofluorescence of spleen sections shows that low-affinity
anti-Sm B cells identified as
IgMaB220+ cells are
intermixed among
B220+IgMa- B cells in
follicles (Fig. 7B). Thus, nonautoreactive B cells have no
effect on the follicular localization of low-affinity anti-Sm B
cells.
The presence of nonautoreactive B cells also does not alter the
differentiation of low-affinity anti-Sm B cells. Analysis of
IgMa and IgMb B cells in
chimeric mice confirms that the mice are chimeric (Fig. 8A). The majority of low-affinity anti-Sm B cells
(IgMa cells) have a mature B-2 cell phenotype
(CD23+HSAlow) regardless of
the ratio of Tg to non-Tg stem cells used for reconstitution (Fig. 8B). The same results were obtained with lymph nodes and
peritoneal B cells (data not shown). Moreover, the anti-Sm B cells
of chimeric mice do not contribute significantly to serum Ab levels;
IgMb Abs are present in circulation even in mice
that were reconstituted with mostly IgMa stem
cells, but IgMa Abs are not detectable (limit of
detection, 2.5 ng/ml) regardless of the stem cell ratio (Fig. 8C). Finally, IgMa B cells in chimeric
mice do not respond to LPS (Fig. 8D), indicating that they
remain anergic. Thus, the presence of nonautoreactive B cells does not
alter the trafficking and regulation of low-affinity anti-Sm B
cells.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The range of phenotypes that has been characterized for anergic B cells
correlates with changes in BCR signaling capabilities. A weak
tolerogenic signal appears to affect only some signaling capabilities,
whereas a stronger signal appears to have a more significant effect on
signaling. Anergic anti-HEL B cells, which receive a strong
tolerogenic signal (38), show evidence of disruption in
early signaling events, such as the partition of the BCR into lipid
rafts (13). Anergic anti-ssDNA B cells of
3H9/V8 Tg mice, which presumably receive a
weak tolerogenic signal (7), flux
Ca2+ and phosphorylate a number of intracellular
proteins upon BCR cross-linking. Neither a Ca2+
flux nor protein phosphorylation is seen in anergic anti-dsDNA or
anti-HEL B cells following IgM cross-linking (7, 38).
These biochemical differences in BCR signaling capabilities may be
significant to the etiology of lupus, because anergic B cells specific
for lupus-associated Ags have biochemical changes resulting from weak
tolerogenic signals that may be more easily overridden. In this regard
the anergy of anti-ssDNA B cells is overcome in MRL/lpr
mice (36). We are currently examining the signaling
capability of these anergic anti-Sm B cells.
The induction of anergy in V8 Dbl Tg mice
contrasts with the regulation of anti-Sm B cells in 2-12H Tg mice.
Most splenic anti-Sm B cells of 2-12H Tg mice appear to
developmentally arrest at the transitional B cell stage
(22). However, other anti-Sm B cells differentiate to
B-1, constituting 30% of the peritoneal B-1 population
(24). There is no evidence of developmental arrest or
differentiation to B-1 in V8 Dbl Tg mice.
Thus, anergy is a third pathway for the regulation of anti-Sm B
cells. There are likely to be anergic anti-Sm B-2 cells in 2-12H Tg
mice, because transgene-expressing B cells are free to use the
available L chain repertoire. However, they are likely to be difficult
to detect by flow cytometry, because, like V8
Dbl Tg B cells, they probably do not stain well with biotinylated Sm.
However, such cells may be detectable with biotinylated Sm as they pass
through the IgMhigh transitional B cell stage. In
this regard, we note that 2-12H Tg mice have an
IgMhigh intermediate Sm-binding population (Fig. 1), which may consist of low-affinity anti-Sm B cells of the type
described here.
The strength of the tolerogenic signal is likely to be the determining
factor in how anti-Sm B cells are regulated. Because the
availability of Ag in 2-12H and V8 Dbl Tg mice
is unlikely to be different, the affinity of the BCR for Sm must
determine the mechanism of regulation. Both the transitional and B-1
anti-Sm B cells of 2-12H Tg mice appear to be of higher affinity
than those of V8 Dbl Tg mice, as the cells of
both populations from 2-12H Tg mice stain brighter with biotinylated Sm
than do those from V8 Dbl Tg mice (Fig. 1B). This difference cannot be due to the level of surface
IgM, because the transitional anti-Sm B cells of
V8 Dbl Tg mice have the same or a higher
surface IgM level as many transitional anti-Sm B cells of 2-12H Tg
mice (Fig. 1, A and B). Thus, we suggest that
low-affinity interactions with Sm allow differentiation to an anergic
B-2 cell, while higher-affinity interactions induce either
developmental arrest or differentiation to B-1. Presumably, the
affinity of the BCR for Sm also determines which cells differentiate to
B-1, because, as shown in this work, low-affinity anti-Sm B cells
do not become B-1. However, other features of anti-Sm binding may
play a role, such as the ability to bind other autoantigens, such as
DNA (17, 19, 22, 23).
Consistent with the idea that the fate of anti-Sm B cells is dependent on the strength of the tolerogenic signal, modulation of the BCR coreceptors CD19 and CD22 affects anti-Sm B cell differentiation in 2-12H Tg mice (24). Decreasing the strength of the tolerogenic signal by eliminating CD19 results in a detectable anti-Sm B-2 population in the spleens of 2-12H Tg mice, suggesting that cells that would normally be excluded from B-2 are now included (24). Conversely, increasing the tolerogenic signal results in an increase in the number of anti-Sm B-1 cells in 2-12H Tg mice (24).
That anergy is induced in low-affinity anti-Sm B cells while higher-affinity anti-Sm B cells are positively selected into the B-1 subset seems paradoxical. We have suggested that migration of B-1 cells to the peritoneum may decrease the exposure to self-Ag or that the expression of CD5, which is induced upon differentiation to a B-1 cell, raises the threshold of activation for these cells. Thus, selection into the B-1 subset may establish ignorance to Sm in nonautoimmune mice, which, like anergy, prevents the activation of anti-Sm B cells. Perhaps anti-Sm Abs have a protective function under certain conditions, and retaining some as functional B-1 cells has survival value. Alternatively, these cells could be selected into the B-1 subset as a consequence of a mechanism evolutionarily selected for B cells of other specificities that have a protective role, and the presence of anti-Sm B cells does no harm.
The elevated level of CD95, a proapoptosis receptor, suggests that
low-affinity anti-Sm B cells will be eliminated upon T cell
encounter. Elimination of anergic B cells via interaction with CD95 by
T cells expressing CD95 ligand induces apoptosis of anergic
anti-HEL B cells (39, 40), and engagement of CD95 is
implicated in the regulation of anti-DNA B cells based on findings
from CD95-deficient (lpr) mice (36, 41, 42, 43). Interestingly, while anergic anti-Sm B cells
up-regulate CD95 and MHC class II, they show no evidence of up
regulation of other cell surface proteins involved in B cell activation
by T cells, such as CD40, CD80, CD86, and CD44 (Fig. 5 and data not
shown). This contrasts with anti-Sm B cells of 2-12H Tg mice that
show up-regulation of CD40, CD44, and CD80, but not CD95
(44). Thus, low-affinity anti-Sm B cells may undergo
apoptosis rather than activation upon interaction with T cells. This
predicts that in the event of the loss of T cell tolerance to Sm, these
anergic B-2 cells would be eliminated rather than activated. If so,
then the activation of low-affinity anti-Sm B cells in lupus
would require not only a loss of T cell tolerance, but also a change in
the B cell to prevent cell death upon T cell interaction. This is
consistent with the findings of others that there are defects in both B
and T cells in MRL/lpr mice (20, 45, 46, 47). Such
a change in the B cell could be the expression of an activation marker,
especially B7.2, because signals provided by B7.2 lead to activation
and proliferation of anergic B cells rather than deletion
(48). Interestingly, splenic anti-Sm B cells of
2-12H/MRL/lpr mice, in which tolerance to Sm is lost,
express elevated levels of B7.2 consistent with this possibility
(44).
Although no general defects in central and peripheral tolerance in MRL/lpr (42, 49) or lpr-deficient mice (50) have been identified using Tg mouse models specific for the neo-Ags H2K and HEL, the regulation of lupus Ag-specific lymphocytes in autoimmune mice has shown different results. Anti-dsDNA Tg B cells in MRL/lpr mice no longer developmentally arrest and enter B cell follicles (41). Furthermore, Brard et al. (36) have demonstrated anti-ssDNA B cells that would otherwise be anergic can develop to ASC in MRL/lpr mice. Moreover, once activated, these cells acquired pathogenic specificities via somatic mutation and receptor editing. We have shown that tolerance to Sm is also lost in 2-12H MRL/lpr mice (44). The anti-Sm response is accelerated and increased in prevalence in these mice. Which anti-Sm B cells are involved in this response is not yet known, but the similarities between anti-ssDNA and low-affinity anti-Sm B cells suggest that it could involve the breakdown of anergy in low-affinity anti-Sm B cells. However, differentiation of anti-Sm B cells to B-1 cells is blocked in 2-12H MRL/lpr mice (44), suggesting that cells that would otherwise be induced to differentiate to B-1 may be diverted toward activation.
In summary, anergy, developmental arrest, and differentiation to B-1 are involved in the regulation of anti-Sm B cells. Which mechanism is used to regulate a given anti-Sm B cell depends at least in part on the affinity of their BCR. Low-affinity anti-Sm B cells appear to have a partial form of anergy, similar to anti-ssDNA B cells (7, 15). In addition, they have up-regulated the cell death receptor CD95, but not activation molecules important in B-T cell collaboration. Thus, anergic anti-Sm B cells appear poised to undergo cell death in the presence of cognate T cell help. The long half-life of anergic anti-Sm B cells suggests that they are an important population in the loss of tolerance to Sm in murine lupus.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department of Microbiology and Immunology, University of North Carolina, CB#7290, 804 MEJB, Chapel Hill, NC 27599. E-mail address: shl{at}med.unc.edu
3 Abbreviations used in this paper: BCR, B cell receptor; AP, alkaline phosphatase; ASC, Ab-secreting cell; BrdU, 5-bromo-2'-deoxyuridine; HEL, hen egg lysozyme; HSA, heat-stable Ag; MZ, marginal zone; Sm, Smith Ag; Tg, transgenic; V8 Dbl Tg, 2-12H/V8 double-Tg.
Received for publication July 13, 2001. Accepted for publication October 22, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and VHCDR3 structures. J. Immunol. 155:2248.[Abstract]
8 L chain transgene on endogenous rearrangements and the immune response to the HA(Sb) determinant on influenza virus. J. Immunol. 147:2024.[Abstract]
light chain genes. EMBO J. 12:821.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. T. Miller, D. R. Beisner, D. Liu, and M. P. Cooke Inositol 1,4,5-Trisphosphate 3-Kinase B Is a Negative Regulator of BCR Signaling That Controls B Cell Selection and Tolerance Induction J. Immunol., April 15, 2009; 182(8): 4696 - 4704. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Duty, P. Szodoray, N.-Y. Zheng, K. A. Koelsch, Q. Zhang, M. Swiatkowski, M. Mathias, L. Garman, C. Helms, B. Nakken, et al. Functional anergy in a subpopulation of naive B cells from healthy humans that express autoreactive immunoglobulin receptors J. Exp. Med., January 16, 2009; 206(1): 139 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Diz, S. K. McCray, and S. H. Clarke B Cell Receptor Affinity and B Cell Subset Identity Integrate to Define the Effectiveness, Affinity Threshold, and Mechanism of Anergy J. Immunol., September 15, 2008; 181(6): 3834 - 3840. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Liu, L. Li, and C. Mohan The Role of Rearrangement at the Second Ig Heavy Chain Locus in Maintaining B Cell Tolerance to DNA J. Immunol., June 1, 2008; 180(11): 7721 - 7727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Kim, J. A. Rutan, and B. J. Vilen The transmembrane tyrosine of {micro}-heavy chain is required for BCR destabilization and entry of antigen into clathrin-coated vesicles Int. Immunol., December 1, 2007; 19(12): 1403 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Kilmon, N. J. Wagner, A. L. Garland, L. Lin, K. Aviszus, L. J. Wysocki, and B. J. Vilen Macrophages prevent the differentiation of autoreactive B cells by secreting CD40 ligand and interleukin-6 Blood, September 1, 2007; 110(5): 1595 - 1602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Gilbert, D. G. Carnathan, P. C. Cogswell, L. Lin, A. S. Baldwin Jr, and B. J. Vilen Dendritic Cells from Lupus-Prone Mice Are Defective in Repressing Immunoglobulin Secretion J. Immunol., April 15, 2007; 178(8): 4803 - 4810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Wang, M. W. Nicholas, K. L. Conway, P. Sen, R. Diz, R. M. Tisch, and S. H. Clarke EBV Latent Membrane Protein 2A Induces Autoreactive B Cell Activation and TLR Hypersensitivity. J. Immunol., September 1, 2006; 177(5): 2793 - 2802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Acevedo-Suarez, D. M. Kilkenny, M. B. Reich, and J. W. Thomas Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells J. Immunol., August 15, 2006; 177(4): 2234 - 2241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Mandik-Nayak, J. Racz, B. P. Sleckman, and P. M. Allen Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help J. Exp. Med., August 7, 2006; 203(8): 1985 - 1998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Qian, K. L. Conway, X. Lu, H. M. Seitz, G. K. Matsushima, and S. H. Clarke Autoreactive MZ and B-1 B-cell activation by Faslpr is coincident with an increased frequency of apoptotic lymphocytes and a defect in macrophage clearance Blood, August 1, 2006; 108(3): 974 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sirac, C. Carrion, S. Duchez, I. Comte, and M. Cogne Light chain inclusion permits terminal B cell differentiation and does not necessarily result in autoreactivity PNAS, May 16, 2006; 103(20): 7747 - 7752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. Oliver, T. Vass, J. Kappler, and P. Marrack Loss of the proapoptotic protein, Bim, breaks B cell anergy J. Exp. Med., March 20, 2006; 203(3): 731 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Culton, B. P. O'Conner, K. L. Conway, R. Diz, J. Rutan, B. J. Vilen, and S. H. Clarke Early Preplasma Cells Define a Tolerance Checkpoint for Autoreactive B Cells J. Immunol., January 15, 2006; 176(2): 790 - 802. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Kilmon, J. A. Rutan, S. H. Clarke, and B. J. Vilen Cutting Edge: Low-Affinity, Smith Antigen-Specific B Cells Are Tolerized by Dendritic Cells and Macrophages J. Immunol., July 1, 2005; 175(1): 37 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Birrell, L. Kulik, B. P. Morgan, V. M. Holers, and K. J. Marchbank B Cells from Mice Prematurely Expressing Human Complement Receptor Type 2 Are Unresponsive to T-Dependent Antigens J. Immunol., June 1, 2005; 174(11): 6974 - 6982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Liu and T. Manser Antinuclear Antigen B Cells That Down-Regulate Surface B Cell Receptor during Development to Mature, Follicular Phenotype Do Not Display Features of Anergy In Vitro J. Immunol., April 15, 2005; 174(8): 4505 - 4515. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. F. Widhopf II, D. C. Brinson, T. J. Kipps, and H. Tighe Transgenic Expression of a Human Polyreactive Ig Expressed in Chronic Lymphocytic Leukemia Generates Memory-Type B Cells That Respond to Nonspecific Immune Activation J. Immunol., February 15, 2004; 172(4): 2092 - 2099. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
L. M. Heltemes and T. Manser Level of B Cell Antigen Receptor Surface Expression Influences Both Positive and Negative Selection of B Cells During Primary Development J. Immunol., August 1, 2002; 169(3): 1283 - 1292. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and IgMb
(
) B cells that were recovered from chimeric mice is shown. The
percentage of V