| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
The Wistar Institute, Philadelphia, PA 19104; and
Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, PA 19111
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our studies have focused on understanding how the rules for tolerance that are evolving from model Ag systems apply to those Ags, such as dsDNA, that are typical of autoimmune disease. To do this, we have used the VH3H9 Ig transgenic (Tg) model (11). VH3H9 Tg mice allow us to track anti-dsDNA B cells in a diverse repertoire and compare their fate in both nonautoimmune (BALB/c) and autoimmune-prone (MRL-+/+ and MRL-lpr/lpr) mice. Similar to the HEL model, anergic anti-dsDNA B cells in nonautoimmune mice are excluded from the B cell follicle and have a rapid in vivo turnover rate. Additionally, and unlike the HEL model, anti-dsDNA B cells are developmentally arrested (12). Another striking difference from the HEL model is seen in the autoimmune background; lpr/lpr mice produce anti-dsDNA Abs (13). The VH3H9 Ig Tg model has been useful in that it allows us to follow anti-dsDNA B cells both in an environment where they are actively tolerized (BALB/c) and in one where their specificity is present in the serum (MRL-lpr/lpr). Using this approach, we have found that the manifestations of anergy seen in BALB/c mice are altered in MRL-+/+ and MRL-lpr/lpr mice, including changes in maturation status and follicular localization (12, 13).
In this study, we use a combination of in vivo and in vitro assays to determine the functional consequences of differences in developmental status and follicular localization. A comparison of the in vivo life span and proliferative status of anti-dsDNA B cells from BALB/c, MRL-+/+, and MRL-lpr/lpr mice indicates that the developmental arrest of a B cell influences its turnover rate. In vitro assays demonstrate that anti-dsDNA B cells from all three backgrounds are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40L and IL-4, suggesting their potential to respond to T cell help. In light of this, CD40L + IL-4 stimulation was used to generate hybridomas from a VH3H9 BALB/c mouse. In contrast to previous reports suggesting that anti-dsDNA B cells are deleted in the bone marrow (BM) (14, 15), we find multiple examples of anti-dsDNA B cells that use distinct Ig genes. Interestingly, the same Igs are used in anti-dsDNA+ B cell hybridomas generated from VH3H9 MRL-lpr/lpr mice (25).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c mice were purchased from Harlan Sprague-Dawley (Indianapolis, IN). MRL-lpr/lpr and MRL-+/+ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). VH3H9 Tg mice have been described previously (16). The VH3H9 Tg mice have been backcrossed onto the BALB/c and MRL backgrounds for at least 9 and 17 generations, respectively, and have been bred and maintained in a specific pathogen-free room at The Wistar Institute animal facility. BALB/c and MRL-+/+ mice were 2–12 mo old, and MRL-lpr/lpr mice were 6–20 wk old. In all cases, age-matched BALB/c mice and/or Tg- littermates were used as controls. The presence of the VH3H9 Tg was determined by PCR amplification of tail DNA with primers specific for VH3H9 (16).
Flow cytometry analysis
Cells (5 x 105) were surface stained
according to standard protocols (17). The following Abs
were used: 1D3-FITC or -biotin (anti-CD19), R26-46-FITC or biotin
(anti-Ig
total), R8-140-PE (anti-Ig
), GL1-PE
(anti-CD86, B7.2; PharMingen, San Diego, CA), LS136-biotin
(anti-Ig1, gift of G. Kelsoe, Duke University, Durham, NC),
JC5.1-PE (anti-Ig total, gift of J. Kearney, University of
Alabama, Birmingham, AL); and streptavidin-Red670 (Life Technologies,
Gaithersburg, MD). For B7-2 staining, FcRs were blocked with 2.4G2
(anti-FcR; grown as ascites) before staining. As we have shown
previously, the majority of Ig B cells are Ig1; therefore, we use
pan anti-Ig reagents to identify VH3H9/1 B cells (12, 13).
All samples were analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software. A total of 60,000 events, gated for live lymphocytes based on forward and side scatter, were collected for each sample.
Cell cycle analysis
Spleen cells were sorted into
CD19+Ig+ and
CD19+Ig- subsets using
a FACStarPlus (Becton Dickinson), and the sorted
cells were fixed in 95% EtOH at 4°C overnight. The cells were then
stained in a PBS containing 1% glucose, propidium iodide (PI) (10
µg/ml), and RNase A (50 µg/ml). Cell-cycle status was determined by
evaluating the DNA content of propidiumiodide-labeled B cells by flow
cytometry.
Detection of apoptotic cells in situ
Spleens were suspended in OCT, frozen in 2-Methyl-Butane cooled
with liquid nitrogen, sectioned, and fixed with acetone. The spleen
sections were stored at -20°C and then stained according to the
protocol described (18). Briefly, the sections were
blocked using PBS/5% normal goat serum (Sigma, St. Louis, MO)/0.1%
Tween 20 and then were stained with GK1.5-biotin (anti-CD4),
53-6.72-biotin (anti-CD8) (all grown as supernatants),
Cy34.1-biotin (anti-CD22) (PharMingen), and/or
anti-Ig-alkaline phosphatase (AP) (Southern Biotechnologies
Associates, Birmingham, AL). Streptavidin-HRP or AP (Southern
Biotechnologies Associates) were used as secondary Abs. Apoptosis was
determined using an in situ cell death detection kit (Boehringer
Mannheim, Indianapolis, IN) according to the manufacturer’s
instructions, with the exception that the slides were fixed with
acetone before fixing in 4% paraformaldehyde to optimize surface Ag
staining. TUNEL staining (dUTP-FITC) was detected with
anti-FITC-HRP (Chemicon, Temecula, CA). HRP and AP were developed
using the substrates 3-amino-9-ethyl-carbazole and Fast-Blue BB base
(Sigma), respectively. Apoptotic cells in germinal centers were used as
a positive control for TUNEL staining.
In vitro proliferation assay
Spleen cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) as described (19). CFSE-labeled spleen cells (2 x 106/ml) were then cultured in either media alone (RPMI 1640/10% FCS/5 x 10-5 M 2-ME), LPS (10 µg/ml; Sigma), or a combination of CD40L-CD8 fusion protein (gift of P. Lane, Basel Institute for Immunology, Basel, Switzerland) (20); and cross-linked with anti-CD8 (2.5 µg/ml) and baculovirus IL-4 (equivalent to 2 ng/ml recombinant protein; gift of E. Pure, The Wistar Institute, Philadelphia, PA). Cells were removed from culture after 1, 2, and 4 days to determine viability, as assessed by trypan blue exclusion under a light microscope, and proliferation. Proliferation was measured by flow cytometry as a decrease in CFSE intensity relative to unstimulated cells. Each cell division is accompanied by a decrease in CFSE intensity by one-half (21).
5-Bromo-2'-deoxyuridine (BrdU) labeling
Long-term labeling. Mice were injected i.p. with 200 µl of 3 mg/ml BrdU (Sigma) in PBS every 12 h for 4, 8, or 11 days. BrdU staining was performed essentially as described (22), with the exception that the cells were not fixed in ethanol. Briefly, spleen and BM cells from mice were isolated and surface stained to identify B cells as described above. The cells were then fixed and permeabilized with 1% paraformaldehyde containing 0.1% Tween 20. The DNA was denatured using 10 µM HCl and 100 U/ml DNase I. The incorporated BrdU was then detected using an anti-BrdU-FITC Ab (B44) from Becton Dickinson (Mountain View, CA).
Four-hour pulse. Mice were injected i.p. with 200 µl of 3 mg/ml BrdU in PBS. The spleen and BM cells were isolated 4 h later and stained by flow cytometry as described above. Proliferating B cells in the BM were used as a positive control for BrdU labeling.
Hybridoma generation
Spleen cells from a VH3H9 Tg mouse were cultured overnight in media (DMEM/10% FCS/5 x 10-5 M 2-ME) containing CD40L-CD8 fusion protein, cross-linked with anti-CD8 (2.5 µg/ml), and rIL-4 (2 ng/ml) (Genzyme Diagnostics, Cambridge, MA). The cells were then fused to the Ig- myeloma, Sp2/0. Cells were plated at limiting dilution and wells bearing single colonies were expanded for analysis.
Sequence analysis
The H- and L-chain V regions of the hybridoma Abs were sequenced
from mRNA according to the protocol described (23).
Briefly, cytoplasmic RNA was isolated and constant region-specific
primers were used to direct synthesis of cDNA copies of the H- (Cµ1)
and L- (C1 and C) chain V regions. The cDNA was then amplified
using the constant region primers in conjunction with VH5'1 for
H-chains and 1L or L5 primers for L-chains (23).
Amplification products were sequenced by automated analysis (The Wistar
Institute Nucleic Acid Facility). All hybridomas shown were found to
express a single L-chain. Sequence translation and comparison was
conducted using the Sequencher program (Gene Codes, Ann Arbor, MI) and
by searching EMBL/GenBank/DDBJ databases. The lowercase letter
designation was used to distinguish among individual genes as was
previously done for hybridoma Igs from VH3H9 Tg BALB/c
(24) and MRL-lpr/lpr (25) mice. A
sequence was designated the same gene and given the same lowercase
letter suffix if there were seven or fewer nucleotide differences
(97.5% nucleotide similarity), with no more than four of these in
framework regions.
Anti-nuclear Ab (ANA) assay
The presence of ANAs in hybridoma supernatants was detected
using permeabilized HEP-2 cells as the substrate following
manufacturer’s instructions (Antibodies, Inc., Davis, CA).
Supernatants giving a homogeneous nuclear (HN) staining pattern were
defined as ANA+. ANA binding was detected using
an anti-mouse IgM + IgG or Ig-FITC secondary Ab (Southern
Biotechnologies Associates). The samples were visualized under a
fluorescent microscope and scored in a blind fashion.
Crithidia luciliae assay
The presence of anti-dsDNA Abs in hybridoma supernatants was detected using permeabilized C. luciliae protozoans as the substrate following manufacturer’s instructions (Antibodies, Inc.). Abs were defined as anti-dsDNA+ if they stained the kinetoplast of the protozoan, an organelle containing deproteinated dsDNA (26).
Statistical significance
Statistical significance was determined using an unpaired Student’s t test and Instat software (GraphPad Software, San Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
anti-dsDNA model
The VH3H9 H-chain Tg pairs with endogenous L-chains to generate
both anti-DNA and non-DNA B cells (27). The
specificity of the Ig-bearing cells in VH3H9 mice is variable:
hybridoma analysis has identified Ig B cells that are
ANA+ and others that are
ANA- (25). ANAs are found in a high
frequency of systemic lupus erythematosus serum and correlate with the
presence of anti-dsDNA, anti-histone, and/or anti-chromatin
Abs (28). By flow cytometry, looking at cell-surface
phenotype as well as turnover rate, 70–80% of the Ig B cells in
VH3H9 Tg mice are analogous to Tg- B cells
(12). To analyze a defined population of anti-dsDNA B
cells, we rely on the fact that VH3H9 pairs with the endogenous Ig1
L-chain to generate an anti-dsDNA Ab (25, 27).
Therefore, we can use anti-Ig-specific reagents to follow
anti-dsDNA B cells in a diverse repertoire.
Previously, we have shown that the fate of VH3H9/ anti-dsDNA B
cells is different in nonautoimmune (BALB/c) vs autoimmune (MRL-+/+ and
MRL-lpr/lpr) mice (summarized in Table I
). Note that in all strains the level of
surface Ig is low, suggesting that in all backgrounds the
anti-dsDNA B cells have encountered Ag. In BALB/c mice, the
anti-dsDNA B cells are phenotypically immature, they are located at
the T/B interface in the splenic follicle, and their Ig is absent from
the serum (12). In MRL-+/+ mice, anti-dsDNA B cells
continue to localize to the T/B interface and their Ig is still not
found in the serum, but in this case they are developmentally mature
(13). In MRL-lpr/lpr mice, anti-dsDNA Ab is
present in the serum after 10 wk of age. However, even before this the
anti-dsDNA B cells are phenotypically mature and localize to the B
cell follicle (13).
|
To determine whether differences in developmental status and
splenic localization affect the turnover rate of anti-dsDNA B
cells, their in vivo life span was estimated. BALB/c, MRL-+/+, and
MRL-lpr/lpr mice were continuously labeled for 11 days with
the thymidine analogue BrdU. Mice were sacrificed on days 4, 8, and 11
of labeling and the incorporated BrdU in Ig vs Ig splenic B cells
was measured. Cells that are turning over more rapidly will be replaced
more quickly by their labeled precursors from the BM and, therefore, as
a population will have a higher percentage of
BrdU+ cells. Cells that are actively
proliferating in the spleen will also be labeled. To distinguish
between these possibilities, the in vivo proliferative status of the B
cells has also been assessed.
After 4, 8, and 11 days of labeling, a higher percentage of Ig B
cells in VH3H9 BALB/c mice is BrdU+ compared with
the Ig subset or B cells from Tg- BALB/c
mice, consistent with previous data (Fig. 1, A and B and Ref.
12). Furthermore, this increase in BrdU label was shown to
be due to an increased turnover of the Ig B cells and not due to
their proliferation because no BrdU label was detected in a short-term
BrdU pulse (12) and <5% of the Ig B cells were in
cycle (Table II). In contrast to VH3H9
BALB/c mice, the percentage of BrdU+ Ig cells
in VH3H9 MRL-+/+ mice is more similar to that in
Tg- MRL-+/+ mice (Fig. 1A).
Therefore, although VH3H9/ B cells in both BALB/c and MRL-+/+ mice
are located at the T/B interface, the turnover rate for the MRL-+/+
anti-dsDNA B cells is not as rapid as that in BALB/c mice. The
reduced life span of the VH3H9/ BALB/c cells appears to be a feature
of their immature status and not of their restricted localization.
Immature B cells from wild-type mice have also been shown to have an
increased turnover (22, 29).
|
|
B cells are both
mature and localize to the B cell follicle (13), a
location that has been correlated with a long-lived B cell
(29). However, in VH3H9 MRL-lpr/lpr mice, the
Ig B cells have a higher frequency of BrdU+
cells than in Tg- MRL-lpr/lpr mice or
VH3H9 MRL-+/+ mice (Fig. 1
A). One possibility is that the
anti-dsDNA B cells are turning over faster in
MRL-lpr/lpr mice than in MRL-+/+ mice, even though the
anti-dsDNA B cells are mature in both cases. Another possibility is
that the VH3H9/ B cells are proliferating.
To determine whether the increased frequency of BrdU label in the
MRL-lpr/lpr splenic B cells was due to proliferation, two
approaches were taken. First, mice were pulsed with BrdU for 4 h.
This time period is long enough to detect cells that are undergoing DNA
synthesis but short enough to exclude a contribution from exported BM
precursors. After a 4-h pulse, <5% of the anti-dsDNA B cells were
labeled in VH3H9 splenic B cells from BALB/c, MRL-+/+, and
MRL-lpr/lpr mice (Ref. 12 ; and data not shown).
Because BrdU will only label cells that are in S-phase, another
approach was taken to determine the percentage of cells in cycle.
VH3H9/ B cells were sorted and then analyzed for DNA content by
propidium iodide incorporation using flow cytometry. Again, very few
(<6%) of the B cells were found to be cycling (Table II). This shows
that the vast majority of anti-dsDNA B cells in BALB/c, MRL-+/+,
and MRL-lpr/lpr mice are not cycling. It is notable that a
higher percentage of Ig B cells in MRL-lpr/lpr mice than
in MRL-+/+ or BALB/c mice are cycling (Table II). Therefore, this makes
life span measurements by long-term BrdU labeling studies difficult to
interpret in MRL-lpr/lpr mice. The increased BrdU label of
MRL-lpr/lpr compared with MRL-+/+ Ig B cells could be
accounted for by this seemingly small difference in proliferation or by
a higher frequency of newly generated B cells from the BM. We are able
to compare the turnover rates of anti-dsDNA B cells from
MRL-lpr/lpr mice with those in BALB/c mice. Ig B cells in
MRL-lpr/lpr mice have a higher percentage in cycle and yet
have a decreased level of BrdU label compared with Ig B cells in
BALB/c mice. This indicates that the Ig B cells in
MRL-lpr/lpr mice are not turning over as rapidly as in
BALB/c mice.
Anti-dsDNA B cells do not die at the T/B interface
The increased turnover rate of anti-dsDNA B cells in BALB/c
mice suggests that they are rapidly dying in vivo, whereas Ig B
cells in VH3H9 MRL-+/+ mice do not show this accelerated turnover. In
both cases, the Ig B cells are located at the T/B interface. To
determine whether the anti-dsDNA B cells are dying at this
location, spleen sections were stained for apoptotic cells using the
TUNEL method. TUNEL+ cells were detected in the
spleens of both BALB/c and MRL-+/+ mice; however, the VH3H9/ B cells
found at the T/B interface were not TUNEL+ (data
not shown). This suggests that, although the anti-dsDNA B cells in
BALB/c mice have a decreased life span, they are not undergoing
apoptosis at the T/B interface. It is possible that the
apoptotic anti-dsDNA B cells are engulfed and cleared from this
site rapidly and, therefore, at any given time there are too few to be
detected. Alternatively, they may die at another location such as the
liver, as has been previously suggested for activated T cells
(30).
Proliferation to in vitro stimuli
One measure of the functional capacity of a B cell is its ability
to proliferate to in vitro stimuli, such as the B cell mitogen LPS, and
to CD40L + IL-4. To measure proliferation, anti-dsDNA B cells from
BALB/c, MRL-+/+, and MRL-lpr/lpr mice were labeled with the
vital dye CFSE before culture. CFSE incorporates into cells and
decreases in intensity by one-half with each cell division
(21). Therefore, proliferation can be measured on a per
cell level. After 4 days in culture, Ig and Ig B cells from
Tg- mice proliferate to both LPS and CD40L +
IL-4 (Fig. 2B). Little
proliferation is apparent in media alone (data not shown). On day 2 of
the LPS culture, B cells in MRL-+/+ and MRL-lpr/lpr mice but
not in BALB/c mice begin to proliferate (Fig. 2A). This may
be an indication that there is a population of B cells in MRL mice that
is hyperresponsive to LPS. In this regard, marginal-zone B cells have
been shown to have an increased proliferative response to LPS
(31), and MRL-+/+ and MRL-lpr/lpr mice have an
exaggerated population of
CD21/35highCD23lowIgMhighIgDlow
B cells (13), a phenotype that is reminiscent of
marginal-zone B cells (32). On day 3, B cells from
Tg- BALB/c mice also begin to proliferate to
LPS, and by day 4 they resemble those from MRL mice (Fig. 2B
and data not shown). Likewise, in VH3H9 Tg mice, the majority of Ig
B cells proliferate to both LPS and CD40L + IL-4, consistent with their
normal B cell phenotype (Fig. 2B).
|
B cells in VH3H9 mice, the anti-dsDNA VH3H9/ B cells do not
proliferate to LPS (Fig. 2B). This is true of VH3H9/ B
cells in BALB/c, MRL-+/+, and MRL-lpr/lpr mice. Therefore,
VH3H9/ B cells are functionally distinguished from naive B cells by
their inability to respond to LPS. It has been suggested that a
previous signal through Ig may dampen future responsiveness to mitogens
such as LPS (33). Therefore, this may be another
indication that anti-dsDNA B cells have previously encountered Ag.
In contrast to their lack of response to LPS, VH3H9/ anti-dsDNA
B cells do proliferate to CD40L + IL-4 (Fig. 2B). This
suggests that anti-dsDNA B cells may be able to be rescued by T
cell help. B cell survival to in vitro stimuli
On day 1 of culture, 
50% of the Ig B cells survived,
regardless of genotype or culture stimulus (Fig. 3A). By day 4, very few B
cells of any genotype survived in media alone. For the Ig B cells,
the addition of either LPS or CD40L + IL-4 promoted cell survival,
consistent with the proliferation data (data not shown). In
Tg- mice, the Ig B cells survived as well as
the Ig B cells to all stimuli. However, by day 4, <20% of the
Ig B cells from VH3H9 Tg mice survived in LPS (Fig. 3B).
Therefore, LPS does not foster proliferation or survival of VH3H9/ B
cells. Furthermore, even though the CD40L + IL-4 induced proliferation
of the VH3H9/ B cells, it rescued them to varying degrees. Ig B
cells from MRL-+/+ and MRL-lpr/lpr mice survived better in
CD40L + IL-4 (percent survival: 62.5 ± 25.5 and 67.5 ±
11.7, respectively) than did Ig B cells from BALB/c mice (33.8
± 24.6; p = 0.04) (Fig. 3B). This suggests
that being developmentally mature gives a survival advantage to the
anti-dsDNA MRL B cells.
|
Signals through CD28 on T cells from B7-2 on APCs, such as B
cells, are required for productive T/B collaboration
(34, 35, 36). B7-2 is up-regulated shortly after activation
through the B cell receptor and is only transiently expressed
(37, 38, 39). To determine whether there is a difference in
the ability of anti-dsDNA B cells from BALB/c, MRL-+/+, and
MRL-lpr/lpr mice to up-regulate B7-2, spleen cells were
cultured in vitro with CD40L + IL-4 for 24 h. Before culture, the
Ig B cells were primarily small by forward and side scatter and
expressed undetectable levels of B7-2 (Fig. 4). After culture, the Ig B cells
appeared blasted and increased their expression of B7-2 by 10- to
15-fold (Fig. 4). Importantly, the ability of anti-dsDNA B cells
from all three backgrounds to up-regulate the costimulatory molecule
B7-2 documents their potential to elicit productive T cell
help.
|
The fate of anti-dsDNA B cells in nonautoimmune mice has been
controversial. Although it has been reported, using an IgH+L Tg
(VH3H9/V4), that anti-dsDNA B cells are deleted in the BM
(15), we have documented another anti-dsDNA B cell,
VH3H9/1, that is not (12). In previous experiments,
anti-dsDNA B cells were absent from hybridoma panels from VH3H9
mice, generated after LPS stimulation (16). The data
presented in Figs. 2 and 3 may account for this; anti-dsDNA B cells
do not proliferate or survive in LPS cultures. To determine whether
additional anti-dsDNA B cells persist in the periphery, we took
advantage of the observation that CD40L + IL-4 induces proliferation of
anti-dsDNA B cells, and generated splenic hybridoma panels.
Hybridomas that use the VH3H9 Tg H-chain in combination solely with the
Ig1 L-chain were retrieved after CD40L + IL-4 stimulation (Ref.
12 and Table III).
Importantly,
ANA+/Crithidia+
hybridomas that use Ig L-chains were also recovered (Table III),
thus confirming that other anti-dsDNA B cells are also present in
the periphery of BALB/c mice. Two hybrids (7942-H26 and 7942-H46) using
the VH3H9 Tg H-chain in combination with the V12/13d L-chain were
isolated (Fig. 5). Although these hybrids
have similar staining patterns in the ANA and C. luciliae
assays, their use of distinct CDR3 (complementarity-determining
regions) and J segments indicates that the two are not clonally
related (Fig. 5 and Table III). Anti-dsDNA B cells using the VH3H9 Tg
H-chain with the V12/13d L-chain have previously been retrieved from
VH3H9 MRL-lpr/lpr-derived hybridomas (25). What
accounts for the deletion in nonautoimmune mice of VH3H9/V4
anti-dsDNA B cells but not of the anti-dsDNA B cells described
here has not been established. The ANA and Crithidia binding
patterns of these Abs are similar (Table III). Possibly there are
differences in specificity or affinity that the ANA and
Crithidia assays do not reveal or differences in when the
autoreactive Tg receptors are first expressed that dictate the fate of
the B cells.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The location of the VH3H9/ anti-dsDNA B cells in
MRL-lpr/lpr mice is reminiscent of Ag-activated Ig Tg
anti-HEL B cells given cognate T cell help (41). In
the first days of an immune response, the anti-HEL B cells were
reported to have undergone a proliferative burst and were scattered
throughout the B cell follicle. However, the anti-dsDNA B cells in
this study differ from the anti-HEL Ig Tg B cells in that the vast
majority of anti-dsDNA B cells are not proliferating.
Importantly, this study shows that anti-dsDNA B cells from BALB/c, MRL-+/+, and MRL-lpr/lpr mice all proliferate and up-regulate the critical costimulatory molecule B7-2 in response to CD40L + IL-4. In the anti-HEL B cell tolerance model, anergic anti-HEL B cells are impaired in their ability to up-regulate B7-2 in response to Ag and it is hypothesized that this prevents them from eliciting T cell help (42, 43). However, in another study they have been shown to up-regulate B7 molecules after anti-CD40 Ab treatment (44). Restoring B7-2 expression by a B7-2 Tg restored the ability of anergic anti-HEL B cells to respond to T cell help, emphasizing the importance of the block in B7-2 to the maintenance of B cell anergy (45). The ability of anti-dsDNA B cells to express B7-2 indicates that they may be able to elicit T cell help.
We suggest, based on the responsiveness of anti-dsDNA B cells to CD40L + IL-4, that one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ vs MRL-lpr/lpr mice may be the availability of T cell help. We have previously documented a CD4 T cell infiltration into the B cell follicle that accompanies the follicular localization of anti-dsDNA B cells in MRL-lpr/lpr mice (13). Additionally, when VH3H9 MRL-lpr/lpr mice are treated with anti-CD4 Abs, the anti-dsDNA B cells now localize to the T/B interface, suggesting a requirement for CD4 T cells in anti-dsDNA follicular localization (S. Seo, manuscript in preparation). Possibly these are autoreactive T cells, which are tolerized in Fas-sufficient mice but escape this fate in lpr/lpr mice. Experiments to characterize these T cells are underway.
In conclusion, this study has shown that alterations in developmental
maturity and splenic localization translate into differences in the
survival of anti-dsDNA B cells. Anti-dsDNA B cells from both BALB/c
and MRL-+/+ mice localize to the T/B interface; however, only those in
BALB/c mice have an increased turnover. This allows us to assign the
rapid turnover rate to their immature status and not to their
localization. Furthermore, because we do not detect apoptotic Ig
cells at the T/B interface, the anti-dsDNA B cells likely die at
another location. Importantly, we demonstrate that anti-dsDNA B
cells from all three backgrounds proliferate and up-regulate B7-2 in
response to CD40L and IL-4, suggesting that in vivo the anti-dsDNA
B cells may be reactivatable by bona fide T cell help. Finally, the
isolation of additional anti-dsDNA hybridomas from BALB/c mice that
use the same Igs as those found in MRL-lpr/lpr mice suggests
that the tolerance mechanisms we have described for VH3H9/1 may also
apply to other anti-dsDNA B cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jan Erikson, The Wistar Institute, Room 273, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: HEL, hen-egg lysozyme; CD40L, CD40 ligand; Tg, transgenic; BM, bone marrow; AP, alkaline phosphatase; ANA, anti-nuclear Ab; BrdU, 5-bromo-2'-deoxyuridine; CFSE, carboxyfluorescein diacetate succinimidyl ester; HN, homogenous nuclear.
Received for publication September 14, 1999. Accepted for publication November 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. J. Witsch and E. Bettelheim Allelic and Isotypic Light Chain Inclusion in Peripheral B Cells from Anti-DNA Antibody Transgenic C57BL/6 and BALB/c Mice J. Immunol., March 15, 2008; 180(6): 3708 - 3718. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Paul, A. Nelde, A. Verschoor, and M. C. Carroll Follicular exclusion of autoreactive B cells requires Fc{gamma}RIIb Int. Immunol., April 1, 2007; 19(4): 365 - 373. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. D. Hondowicz, S. T. Alexander, W. J. Quinn III, A. J. Pagan, M. H. Metzgar, M. P. Cancro, and J. Erikson The role of BLyS/BLyS receptors in anti-chromatin B cell regulation Int. Immunol., April 1, 2007; 19(4): 465 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Nijnik, H. Ferry, G. Lewis, E. Rapsomaniki, J. C. H. Leung, A. Daser, T. Lambe, C. C. Goodnow, and R. J. Cornall Spontaneous B cell hyperactivity in autoimmune-prone MRL mice Int. Immunol., July 1, 2006; 18(7): 1127 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Fields, M. H. Metzgar, B. D. Hondowicz, S.-A. Kang, S. T. Alexander, K. D. Hazard, A. C. Hsu, Y.-Z. Du, E. L. Prak, M. Monestier, et al. Exogenous and Endogenous TLR Ligands Activate Anti-Chromatin and Polyreactive B Cells. J. Immunol., June 1, 2006; 176(11): 6491 - 6502. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. William, C. Euler, N. Primarolo, and M. J. Shlomchik B Cell Tolerance Checkpoints That Restrict Pathways of Antigen-Driven Differentiation J. Immunol., February 15, 2006; 176(4): 2142 - 2151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C. Bosma, J. Oshinsky, K. Kiefer, P. B. Nakajima, D. Charan, C. Congelton, M. Radic, and M. J. Bosma Development of Functional B Cells in a Line of SCID Mice with Transgenes Coding for Anti-Double-Stranded DNA Antibody J. Immunol., January 15, 2006; 176(2): 889 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Fields, S. A. Nish, B. D. Hondowicz, M. H. Metzgar, G. N. Wharton, A. J. Caton, and J. Erikson The Influence of Effector T Cells and Fas Ligand on Lupus-Associated B Cells J. Immunol., July 1, 2005; 175(1): 104 - 111. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. William, C. Euler, E. Leadbetter, A. Marshak-Rothstein, and M. J. Shlomchik Visualizing the Onset and Evolution of an Autoantibody Response in Systemic Autoimmunity J. Immunol., June 1, 2005; 174(11): 6872 - 6878. [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]
|
|
|
|
|
|
E. H. Ekland, R. Forster, M. Lipp, and J. G. Cyster Requirements for Follicular Exclusion and Competitive Elimination of Autoantigen-Binding B Cells J. Immunol., April 15, 2004; 172(8): 4700 - 4708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Reichlin, A. Gazumyan, H. Nagaoka, K. H. Kirsch, M. Kraus, K. Rajewsky, and M. C. Nussenzweig A B Cell Receptor with Two Ig{alpha} Cytoplasmic Domains Supports Development of Mature But Anergic B Cells J. Exp. Med., March 15, 2004; 199(6): 855 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Paul, J. Lutz, J. Erikson, and M. C. Carroll Germinal center checkpoints in B cell tolerance in 3H9 transgenic mice Int. Immunol., February 1, 2004; 16(2): 377 - 384. [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]
|
|
|
|
|
|
R. R. Singh, F. M. Ebling, D. A. Albuquerque, V. Saxena, V. Kumar, E. H. Giannini, T. N. Marion, F. D. Finkelman, and B. H. Hahn Induction of Autoantibody Production Is Limited in Nonautoimmune Mice J. Immunol., July 1, 2002; 169(1): 587 - 594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. R. Sekiguchi, S. M. Jainandunsing, M. L. Fields, M. A. Maldonado, M. P. Madaio, J. Erikson, M. Weigert, and R. A. Eisenberg Chronic Graft-Versus-Host in Ig Knockin Transgenic Mice Abrogates B Cell Tolerance in Anti-Double-Stranded DNA B Cells J. Immunol., April 15, 2002; 168(8): 4142 - 4153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-P. Chu, D. Taylor, H.-G. Yan, B. Diamond, and L. Spatz Persistence of partially functional double-stranded (ds) DNA binding B cells in mice transgenic for the IgM heavy chain of an anti-dsDNA antibody Int. Immunol., January 1, 2002; 14(1): 45 - 54. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
C. M. Grimaldi, D. J. Michael, and B. Diamond Cutting Edge: Expansion and Activation of A Population of Autoreactive Marginal Zone B Cells in a Model of Estrogen-Induced Lupus J. Immunol., August 15, 2001; 167(4): 1886 - 1890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Mandik-Nayak, G. Huang, K. C. F. Sheehan, J. Erikson, and D. D. Chaplin Signaling Through TNF Receptor p55 in TNF-{alpha}-Deficient Mice Alters the CXCL13/CCL19/CCL21 Ratio in the Spleen and Induces Maturation and Migration of Anergic B Cells into the B Cell Follicle J. Immunol., August 15, 2001; 167(4): 1920 - 1928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Fields, C. L. Sokol, A. Eaton-Bassiri, S.-j. Seo, M. P. Madaio, and J. Erikson Fas/Fas Ligand Deficiency Results in Altered Localization of Anti-Double-Stranded DNA B Cells and Dendritic Cells J. Immunol., August 15, 2001; 167(4): 2370 - 2378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-j. Seo, J. Buckler, and J. Erikson Novel Roles for Lyn in B Cell Migration and Lipopolysaccharide Responsiveness Revealed Using Anti-Double-Stranded DNA Ig Transgenic Mice J. Immunol., March 15, 2001; 166(6): 3710 - 3716. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Rifkin, E. A. Leadbetter, B. C. Beaudette, C. Kiani, M. Monestier, M. J. Shlomchik, and A. Marshak-Rothstein Immune Complexes Present in the Sera of Autoimmune Mice Activate Rheumatoid Factor B Cells J. Immunol., August 1, 2000; 165(3): 1626 - 1633. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
