Hyperresponsiveness, Resistance to B-Cell
ReceptorⴚDependent Activation-Induced Cell Death,
and Accumulation of Hyperactivated B-Cells in Islets
Is Associated With the Onset of Insulitis but not
Type 1 Diabetes
Shabbir Hussain,1 Konstantin V. Salojin,1 and Terry L. Delovitch1,2
B-cells proliferate after B-cell receptor (BCR) stimulation and are deleted by activation-induced cell death
(AICD) during negative selection. We report that
B-cells from type 1 diabetesⴚsusceptible NOD and
type 1 diabetesⴚresistant but insulitis-prone congenic NOD.B6Idd4B and NOR mice, relative to B-cells
from nonautoimmune diseaseⴚprone C57BL/6 and
BALB/c mice, display a hyperproliferative response to
BCR stimulation and lower activation threshold in the
absence or presence of interleukin 4 (IL-4). This hyperproliferation is associated with an increased proportion
of NOD and NOR B-cells that enter into the S phase of
the cell cycle and undergo cell division. The relative
resistance to BCR-induced AICD of B-cells from NOD,
NOR, and NOD.B6Idd4B mice, all of which develop
insulitis, correlates with the presence of a higher
percentage of hyperactivated B-cells in the spleen
and islets of these mice than in nonautoimmune
diseaseⴚprone C57BL/6 and BALB/c mice. The NOD
islet-infiltrated activated B-cells are more responsive to
further stimulation by IL-4 than activated spleen Bcells. Our results suggest that resistance to AICD and
accumulation of hyperactivated B-cells in islets is associated with the onset of an inflammatory insulitis, but
not type 1 diabetes. Diabetes 53:2003–2011, 2004
A
utoimmune type 1 diabetes is characterized by
the T-cell⫺mediated destruction of insulin-producing B-cells in pancreatic islets. In type 1
diabetes⫺susceptible NOD mice, islet infiltration by T-cells and antigen-presenting cells (APCs), including B-cells, macrophages, and dendritic cells, begins at age
From the 1Autoimmunity/Diabetes Group, Robarts Research Institute, London, Ontario, Canada; and the 2Department of Microbiology and Immunology,
University of Western Ontario, London, Ontario, Canada.
Address correspondence and reprint requests to Dr. Terry L. Delovitch,
Director, Autoimmunity/Diabetes Group, Robarts Research Institute, 1400
Western Rd., London, Ontario N6G 2V4, Canada. E-mail: [email protected].
Received for publication 26 February 2004 and accepted in revised form 20
May 2004.
T.L.D. holds stock in Diabetogen Biosciences.
AICD, activation-induced cell death; APC, antigen-presenting cell; BCR,
B-cell receptor; CFSE, 5- (and 6-) carboxyfluorescein diacetate succinimidyl
ester; FasL, Fas ligand; FITC, fluorescein isothiocyanate; IAA, insulin autoantibody; IL, interleukin; MHC, major histocompatibility complex; PE, phycoerythrin; PLN, pancreatic lymph node; sIgM, surface IgM; TCR, T-cell receptor.
© 2004 by the American Diabetes Association.
DIABETES, VOL. 53, AUGUST 2004
3– 4 weeks (1,2). B-cells are highly efficient APCs due to
their ability to bind and internalize specific antigens
through their surface immunoglobulin B-cell antigen receptors (BCRs) (3). The presence of B-cells in islet infiltrates and the protection of B-cell⫺deficient NOD mice
from destructive insulitis and type 1 diabetes suggest a key
role for B-cells in the pathogenesis of type 1 diabetes (4,5).
This role is further supported by reports that NOD B-cells
are necessary for the priming (6,7) and optimal activation
(8,9) of autoantigen-specific CD4⫹ T-cells.
The low levels of B7-2 expression found on resting
B-cells are generally elevated after antigen stimulation in
activated B-cells (10), as was recently demonstrated for
lymph node⫺derived B-cells from NOD mice (11). B-cells
with increased B7-2 surface expression present antigen to
T-cells more efficiently and also elicit a hyperproliferative
response after antigen- or BCR-mediated stimulation, a
characteristic of self-reactive B-cells (12–14).
Self-reactive B-cells are usually deleted via negative
selection during B-cell development (15–17). However, a
proportion of self-reactive B-cells may escape this deletion
and migrate to secondary lymphoid organs, which can lead
to the onset of autoimmune disease (18). Extensive surface IgM (sIgM) cross-linking is required to initiate activation-induced cell death (AICD) in resting B-cells (19 –21).
This phenomenon may resemble antigen-mediated sIgM
cross-linking and AICD in the absence of costimulatory
signals, such as interleukin 4 (IL-4) and CD40-CD40L
interaction (19 –23).
Although hyperproliferation and resistance to AICD
after BCR stimulation are characteristics of self-reactive
B-cells (14,18), it is not known whether B-cells require
the presence of an inflammatory and autoimmunedisease⫺prone (e.g., type 1 diabetes⫺susceptible NOD
mice), inflammatory but autoimmune-disease⫺resistant
(e.g., type 1 diabetes⫺resistant congenic NOD.B6Idd4B
and NOR mice), or noninflammatory and nonautoimmunedisease⫺prone (e.g., C57BL/6 and BALB/c) environment
to possess these characteristics. NOR mice share ⬃88% of
their genome with NOD mice, including a diabetogenic
major histocompatibility complex (MHC)⫺associated
H-2g7 haplotype (24), exhibit periinsulitis (24), and develop
insulin autoantibodies (IAAs) (25). NOD.B6Idd4B mice
carry a ⬍5.2-cM C57BL/6-derived segment of chromosome
2003
B-CELL HYPERACTIVATION IN NOD MICE
11 (26). Although the majority of NOD.B6Idd4B mice (26)
and a proportion of NOR mice (25,27) develop an inflammatory invasive insulitis after age 25 weeks, these strains
are relatively resistant to the development of type 1
diabetes. In this study, we investigated which of the above
three types of environments—inflammatory and autoimmune disease prone, inflammatory but autoimmune disease resistant, or noninflammatory and nonautoimmune
disease prone— gives rise to the hyperproliferation, AICD
resistance, and islet infiltration of B-cells. Our results
suggest that B-cell hyperresponsiveness, AICD resistance,
and accumulation of hyperactivated B-cells in islets occur
during the development of an inflammatory insulitis, but
not type 1 diabetes.
RESEARCH DESIGN AND METHODS
NOD/Del, NOD.B6Idd4B, and NOR/Lt mice were bred in a specific pathogenfree barrier facility at the Robarts Research Institute (London, Canada). The
generation of congenic NOD.B6Idd4B mice has been previously described
(26). In female NOD mice, islet infiltration begins at age 3– 4 weeks and
progression to destructive insulitis and overt type 1 diabetes occurs by age
4 – 6 months. BALB/c and C57BL/6 (B6) mice were purchased from Charles
River Laboratories (Montreal, Canada). Female mice (age 4 –12 weeks) were
used in this study.
Cell preparation and flow cytometry. Splenocytes and pancreatic lymph
node (PLN) cells were prepared as previously described (28). Islets were
isolated by collagenase P (Roche Diagnostics, Laval, Canada) digestion of
pancreases (28). To obtain islet-infiltrating cells, islets were cultured overnight in complete RPMI medium; cells that migrated out from the islets were
harvested for flow cytometry. Spleen, PLN, and islet-infiltrating cells were
stained with fluorescein isothiocyanate (FITC) anti-B220, FITC anti-Fas,
phycoerythrin (PE) anti-FasL, and PE anti-CD69 mAbs (BD Biosciences,
Mississauga, Canada). The cells were then washed and analyzed by flow
cytometry using BD Cell Quest software.
B-cell proliferation. Spleen B-cells were purified (ⱖ98% purity) using a
StemCell Technologies (Vancouver, Canada) B-cell enrichment cocktail. Isletinfiltrated B-cells were purified (ⱖ95% purity) from overnight cultured islets
using magnetic B-cells separation beads (Miltenyi Biotec, Sunnyvale, CA).
B-cells (105/well) were cultured (37°C, 5% CO2) in quadruplicate in complete
RPMI 1640 supplemented with 10% heat-inactivated FCS, 10 mmol/l HEPES
buffer, 1 mmol/l sodium pyruvate, 2 mmol/l L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.05 ␮mol/l ␤-mercaptoethanol in 96-well
tissue culture plates. Cells were stimulated with a goat anti-mouse IgM F(ab⬘)2
antibody (0 –10 ␮g/ml; Jackson ImmunoResearch, Mississauga, Canada) or
anti-IgM F(ab⬘)2 antibody plus murine rIL-4 (10 ng/ml; BD Biosciences).
3
[H]-thymidine (1 ␮Ci/well) was added during the last 16 h of culture. The cells
were then harvested and assayed for 3[H]-thymidine incorporation (cpm).
Cell division analysis. Splenic B-cells were labeled with 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene,
OR), as previously described (8). CFSE-labeled cells were then cultured in the
presence or absence of anti-IgM F(ab⬘)2 (5 ␮g/ml) and anti-IgM F(ab⬘)2 plus
rIL-4 (10 ng/ml) at 37°C. Cells were harvested at the indicated time periods
and analyzed by flow cytometry.
Cell cycle analysis. Cell cycle analyses were performed as previously
described (29). Briefly, B-cells cultured at a density of 106 cells/ml in the
presence or absence of stimuli for the indicated times were harvested and
washed twice in sample buffer (Ca2⫹- and Mg⫹⫹-free PBS containing 0.1%
D-glucose). Cells were fixed in 70% ethanol while being vortexed and stored at
4°C for ⱖ24 h. Cells were then centrifuged, resuspended, and incubated in a
solution containing propidium iodide (10 ␮g/ml; Sigma, St. Louis, MO) and
RNase (250 ␮g/ml; Roche Diagnostics), respectively, at 37°C for ⱖ30 min.
Viable cells were analyzed to determine the proportion of cells in each phase
of cell cycle by Modfit LT software (Verity Software House, Topsham, ME).
Apoptosis. Apoptosis was determined by FITC-annexin V and propidium
iodide staining (BD Biosciences and Sigma, respectively) or by labeling the
free 3⬘-OH termini of fragmented DNA by fluorescein-dUTP using an in situ
cell detection kit (Roche Diagnostics).
Statistical analysis. Statistical analysis of the data was performed using
ANOVA and Mann-Whitney tests. Data are presented as means ⫾ SD.
2004
RESULTS
Early infiltration of islets with B-cells and protection of
B-cell⫺deficient NOD mice from insulitis and type 1
diabetes implies a role for these cells in the pathogenesis of type 1 diabetes (4,5). Type 1 diabetes⫺resistant
NOD.B6Idd4B mice develop an invasive insulitis after age
25 weeks (26). In type 1 diabetes⫺resistant NOR mice,
Ig␮ mRNA transcripts are found in islets at age 40 days
(27), serum IAAs are detectable at age 8 –20 weeks (25),
and periinsulitis develops (24), which in some mice
progresses to an invasive insulitis (25,27). These findings
suggest functional similarities between type 1 diabetes⫺
susceptible and ⫺resistant mice B-cells. To further examine
the role of B-cells in the development of type 1 diabetes, we
compared the B-cell phenotype and responsiveness to BCR
stimulation among type 1 diabetes⫺susceptible NOD, type 1
diabetes⫺resistant NOD.B6Idd4B and NOR, and nonautoimmune disease⫺prone B6 and BALB/c mice.
Splenic B-cells from NOD, NOD.B6Idd4B, and NOR
mice elicit a hyperproliferative response and lower
activation threshold after BCR-induced stimulation.
To determine whether B-cells from type 1 diabetes⫺susceptible NOD and type 1 diabetes⫺resistant NOD.B6Idd4B
and NOR mice are hyperproliferative upon activation,
splenic B-cells from NOD, NOD.B6Idd4B, NOR, and nonautoimmune disease⫺prone B6 and BALB/c mice were
stimulated via the BCR by an anti-IgM F(ab⬘)2 antibody in
the presence or absence of rIL-4. Stimulation in the presence of IL-4 was evaluated because IL-4 is a B-cell growth
factor (30,31). Kinetic analyses indicated that stimulation
of proliferation by anti-IgM F(ab⬘)2 (2.5 ␮g/ml) (Fig. 1A)
and anti-IgM F(ab⬘)2 (2.5 ␮g/ml) plus rIL-4 (10 ng/ml) (Fig.
1B) were optimal after 64 and 40 h, respectively. B-cells
from NOD, NOD.B6Idd4B, and NOR mice activated for 40
and 64 h with anti-IgM F(ab⬘)2 ⫾ rIL-4 yielded significantly
higher proliferation than B-cells from nonautoimmune
disease⫺prone B6 and BALB/c mice (P ⬍ 0.001).
To test whether the differences observed between the
proliferation of NOD, NOD.B6Idd4B, and NOR B-cells and
nonautoimmune disease⫺prone B6 and BALB/c B-cells
depend on the dosage of anti-IgM F(ab⬘)2 used, B-cells
were stimulated over a dosage range (0 –10 ␮g/ml) of
anti-IgM F(ab⬘)2. NOD, NOD.B6Idd4B, NOR, B6, and
BALB/c B-cells each showed a dosage-dependent increase
in proliferation after anti-IgM F(ab⬘)2 stimulation (Fig.
1C). NOD, NOD.B6Idd4B, and NOR B-cells displayed
significantly higher proliferation than B6 and BALB/c
B-cells (P ⬍ 0.001) at each concentration of anti-IgM
F(ab⬘)2 tested. IL-4 (10 ng/ml) in combination with antiIgM F(ab⬘)2 (2.5 ␮g/ml) enhanced B-cell proliferation in
all five mice strains analyzed (Fig. 1B). Thus, NOD,
NOD.B6Idd4B, and NOR spleen B-cells elicited a hyperproliferative response and lower activation threshold upon
BCR-induced stimulation.
A higher percentage of activated B-cells are present
in the spleen and islets of NOD, NOD.B6Idd4B, and
NOR mice. The BCR-stimulated hyperproliferative responsiveness observed in splenic B-cells from NOD,
NOD.B6Idd4B, and NOR mice raised the possibility that an
increased frequency of activated B-cells may be present
in these mice. Indeed, we found a significantly higher
percentage of CD69⫹ B-cells in spleens of NOD,
DIABETES, VOL. 53, AUGUST 2004
S. HUSSAIN, K.V. SALOJIN, AND T.L. DELOVITCH
FIG. 1. NOD, NOD.B6Idd4B, and NOR B-cells are
hyperproliferative. NOD, NOD.B6Idd4B, NOR, B6,
and BALB/c B-cells (105/well) were stimulated
with anti-IgM F(abⴕ)2 (2.5 ␮g/ml) (A), anti-IgM
F(abⴕ)2 (2.5 ␮g/ml) ⴙ IL-4 (10 ng/ml) (B), or
anti-IgM F(abⴕ)2 (0 –10 ␮g/ml) (C) for the indicated times (A and B) or 64 h (C). Background
proliferation of unstimulated spleen B-cells (400 –
600 3[H]-thymidine cpm) was subtracted from the
stimulated B-cell values. One of three independent and reproducible experiments is shown. P <
0.001 for NOD, NOD.B6Idd4B, and NOR vs. B6 and
BALB/c mice, for each time point and concentration of anti-IgM F(abⴕ)2 ⴞ IL-4 tested.
NOD.B6Idd4B, and NOR mice than in nonautoimmune
disease⫺prone B6 and BALB/c mice (P ⬍ 0.05) (Fig. 2A).
Consistent with the fact that B-cells are among the early
islet-infiltrating leukocytes (2,4,7,27), we found that B-cells
constitute 47.6% of the total islet infiltrate in NOD mice
(Fig. 2B). We next examined whether islet-infiltrated Bcells from NOD, NOD.B6Idd4B, and NOR mice also display an activated phenotype. Flow cytometric analyses
revealed the presence of a relatively high percentage of
CD69⫹ B-cells in the islets of NOD (22 ⫾ 1.6),
NOD.B6Idd4B (19 ⫾ 1.2), and NOR (20 ⫾ 1.1) mice (Fig.
2C). The more elevated expression of CD69 on isletinfiltrated B-cells than spleen B-cells observed suggests
that these islet-infiltrated B-cells are more highly activated, as might be expected after islet ␤-cell autoantigen
stimulation. To test whether islet-infiltrated B-cells are
also hyperproliferative, purified islet-infiltrated B-cells
from NOD mice were stimulated with anti-IgM F(ab⬘)2 for
64 h. These B-cells yielded a significantly greater (P ⬍
0.05) BCR-induced proliferative response than splenic
B-cells (Fig. 2D). It is interesting that the high proliferative
response of NOD PLN-derived B-cells was comparable
with that of islet-infiltrated B-cells. Thus, hyperactivated
B-cells are present at a greater frequency in the spleen and
islets of NOD, NOD.B6Idd4B, and NOR mice than in
nonautoimmune disease⫺prone mouse strains. It is possible that autoreactive B-cells activated in the PLNs migrate
to the pancreas where they infiltrate the islets and are
restimulated by islet ␤-cell autoantigens.
Islet-infiltrated B-cells are more responsive to IL-4
stimulation than splenic B-cells in NOD mice. We
investigated the functional relevance of the presence of an
increased frequency of activated B-cells in NOD pancreDIABETES, VOL. 53, AUGUST 2004
atic islets by taking advantage of the finding that IL-4 does
not enhance B-cell proliferation, but rather drives the
proliferation of preactivated B-cells (31). To determine
whether preactivated islet-infiltrated B-cells (Fig. 2C) respond better to IL-4 treatment than splenic B-cells, which
exhibit a lower frequency of activated B-cells (Fig. 2A), the
proliferative responses of IL-4⫺stimulated islet-infiltrated
B-cells and splenic B-cells were compared. IL-4⫺stimulated islet-infiltrated B-cells elicited a significantly higher
response than splenic B-cells (P ⬍ 0.05) (Fig. 2E). Thus,
islet-infiltrated B-cells are more responsive to further
stimulation than splenic B-cells.
A higher percentage of BCR-activated B-cells from
NOD and NOR mice enter into the S phase of the cell
cycle. Next we examined whether the hyperproliferative
responsiveness of NOD and NOR B-cells promotes their
early cell division and an increased frequency of entry into
the S phase of the cell cycle. The number of cell divisions
was quantitated in CFSE-labeled NOD, NOR, and B6
B-cells after stimulation with anti-IgM F(ab⬘)2 ⫾ IL-4 for
24, 48, and 72 h. B-cells from all mouse strains tested did
not undergo any cell division for the first 48 h after
anti-IgM F(ab⬘)2 stimulation. Anti-IgM F(ab⬘)2 ⫹ IL-4 stimulation induced one cell division (Fig. 3). At 72 h poststimulation, as many as three cell divisions were observed
in NOD, NOR, and B6 B-cells stimulated with anti-IgM
F(ab⬘)2 with and without IL-4. However, under both conditions of stimulation, a lower proportion of B6 B-cells
underwent this number of divisions. NOD and NOR B-cells
also underwent cell division more rapidly than did B6
B-cells.
To determine in which phase of the cell cycle B-cell
division is arrested after activation, anti-IgM F(ab⬘)2–
2005
B-CELL HYPERACTIVATION IN NOD MICE
FIG. 2. BCR stimulation elicits an activated phenotype and hyperproliferative response by NOD islet-infiltrated
B-cells. Flow cytometric analyses show
the percentages of NOD, NOD.B6Idd4B,
NOR, B6, and BALB/c CD69ⴙ spleen
B-cells (A; *P < 0.05); NOD isletinfiltrated B-cells (B); and NOD,
NOD.B6Idd4B, and NOR CD69ⴙ isletinfiltrated B-cells (C). D and E: Spleen,
PLN, and islet-infiltrated B-cells (105)
were stimulated with anti-IgM F(abⴕ)2
(5 ␮g/ml) (D) or IL-4 (10 ng/ml) (E) for
64 h. B-cell proliferation was quantitated as in Fig. 1, and the background
proliferation values subtracted were
similar for unstimulated islet-derived
and spleen-derived B-cells (526 ⴞ 69 vs.
481 ⴞ 68 cpm). One of three independent and reproducible experiments is
shown. *P < 0.05.
stimulated NOD, NOR, and B6 B-cells were harvested for
cell cycle analysis at different times after stimulation. Cell
cycle analyses revealed no significant change in different
phases of the cell cycle among NOD, NOR, and B6 B-cells
24 h after anti-IgM F(ab⬘)2 ⫾ IL-4 stimulation. However, at
48 h of stimulation with anti-IgM F(ab⬘)2, a higher percentage of NOD and NOR B-cells entered into the S and G2/M
phases of the cell cycle compared with B6 B-cells and a
lower percentage of NOD B-cells remained in the G0/G1
phase (P ⬍ 0.05) (Table 1). No significant differences were
evident among the different phases of the cell cycle among
NOD, NOR, and B6 B-cells at 72 h poststimulation with
anti-IgM F(ab⬘)2 ⫾ IL-4. Exogenous IL-4 further increased
the percentage of NOD, NOR, and B6 B-cells that entered
into the S phase after stimulation with anti-IgM F(ab⬘)2 for
48 and 72 h (Table 1). However, similar to anti-IgM F(ab⬘)2
stimulation, the percentage of NOD and NOR B-cells that
entered into the S phase of the cell cycle was higher than
that of B6 B-cells at 48 h after stimulation with anti-IgM
F(ab⬘)2 ⫹ IL-4 stimulation (P ⬍ 0.05) (Table 1). Thus, the
detection of an increased percentage of NOD and NOR Bcells that enter the S phase compared with B6 B-cells in the
absence or presence of IL-4 indicates that both NOD and
NOR B-cells are hyperresponsive upon BCR-stimulation.
B-cells from NOD and NOR mice are resistant to
AICD. The percentages of freshly isolated NOD, NOR, and
B6 apoptotic B-cells do not differ, as determined by FITC⫺
annexin V/propidium iodide staining (S.H., T.L.D., unpub2006
lished observations), and when cultured in complete
RPMI without any stimulus, also do not differ in their
level of spontaneous apoptosis (S.H., T.L.D., unpublished observations). To determine whether B-cells from
type 1 diabetes⫺susceptible NOD and type 1 diabetes⫺
resistant NOR mice differ from those of nonautoimmune
disease⫺prone B6 mice in their level of BCR-induced
AICD, the percentage of AICD among purified B-cells from
these mice stimulated with anti-IgM F(ab⬘)2 for 48 h was
measured by FITC⫺annexin V/propidium iodide staining.
B6 B-cells exhibited a significantly higher (P ⬍ 0.05) AICD
than NOD and NOR B-cells (Fig. 4A). To confirm that NOD
and NOR B-cells are resistant to not only the initial round
of BCR stimulation, FITC⫺annexin V binding was detected on a large population of B-cells (gated by forward
and side scatter), some of which could still have been in
the initial stages of AICD (Fig. 4B). The frequency of
FITC/annexin V–stained large B-cells (blasts) from B6
mice (18.6%) was greater than that observed for large NOD
(13.5%) and NOR (11.8%) B-cells, indicating that NOD and
NOR B-cells are relatively resistant to BCR-induced AICD.
Finally, we confirmed that NOD (48%) and NOR (53%)
B-cells are more resistant than B6 B-cells (72%) to BCRinduced AICD as determined by TUNEL assay (Fig. 4C),
which provides a more accurate quantitation of the frequency of apoptotic cells. To investigate whether IL-4
reduces the level of anti-IgM F(ab⬘)2–induced AICD, Bcells were stimulated with anti-IgM F(ab⬘)2 ⫾ IL-4 for 48 h
DIABETES, VOL. 53, AUGUST 2004
S. HUSSAIN, K.V. SALOJIN, AND T.L. DELOVITCH
FIG. 3. Higher percentages of NOD and NOR than B6 B-cells undergo cell division. CFSE-labeled NOD, NOR, and B6 B-cells (106/ml) were
stimulated with anti-IgM F(abⴕ)2 (5 ␮g/ml) ⴞ IL-4 (10 ng/ml) for 48 or 72 h and analyzed by flow cytometry. The number of cell divisions in NOD,
NOR, and B6 B-cells stimulated with anti-IgM F(abⴕ)2 ⴞ IL-4 and the percentage of cells present at each peak of division are indicated. One of
three independent and reproducible experiments is shown.
before AICD was determined by FITC⫺annexin V staining.
IL-4 reduced the levels of AICD in NOD, NOR, and B6
B-cells (P ⬍ 0.05) (Fig. 4D). Thus, stimulation by IL-4
lowered the percentage of apoptotic B-cells in type 1
diabetes⫺susceptible and ⫺resistant mice.
Finally, we investigated whether the relative resistance
of NOD and NOR B-cells to ACID is associated with a
change in Fas and Fas ligand (FasL) surface expression.
Flow cytometric analyses of B-cells stained with anti-Fas and
anti-FasL mAbs revealed a low level of Fas expression
(10–13%) and no FasL expression on NOD, NOR, or B6 Bcells (Fig. 5). Anti-IgM F(ab⬘)2 stimulation for 48 h did not
increase Fas or FasL expression on these B-cells (Fig. 5).
DISCUSSION
In this study, we demonstrated that B-cells from type 1
diabetes⫺susceptible NOD mice as well as type 1
diabetes⫺resistant NOD.B6Idd4B and NOR mice exhibit a
hyperproliferative responsiveness after BCR stimulation.
DIABETES, VOL. 53, AUGUST 2004
This BCR-induced B-cell hyperproliferation is accompanied by a resistance to BCR-dependent AICD and an
increased frequency of activated B-cells in the S phase of
the cell cycle in these mouse strains. The observations that
1) NOD, NOD.B6Idd4B, and NOR mice each develop an
invasive insulitis during which activated B-cells accumulate in pancreatic islets, and 2) this insulitis progresses to
the onset of type 1 diabetes only in NOD mice suggest that
the expression of the B-cell phenotypes of hyperproliferation and resistance to AICD requires the development of
an inflammatory response in pancreatic islets, but not the
onset of autoimmune disease. This notion is further supported by our findings that these B-cell phenotypes were
not observed in the inflammation-free, nonautoimmune
disease⫺prone B6 and BALB/c mice. Although we do not
presently know the autoantigen specificities of the
BCRs expressed by the islet-infiltrated B-cells in NOD,
NOD.B6Idd4B, and NOR mice, our data agree with previ2007
B-CELL HYPERACTIVATION IN NOD MICE
TABLE 1
More NOD and NOR than B6 B-cells enter into the S-phase of the cell cycle
Stimulation
Cell cycle
Mice
24 h
48 h*†
72 h
NOD
NOR
B6
94.1 ⫾ 4.8
93.4 ⫾ 5.2
94.8 ⫾ 2.2
78.1 ⫾ 5.9
77.5 ⫾ 4.8
87.4 ⫾ 2.3
85.0 ⫾ 5.9
86.7 ⫾ 6.1
84.5 ⫾ 5.9
NOD
NOR
B6
2.41 ⫾ 0.2
2.50 ⫾ 0.4
1.50 ⫾ 0.7
13.5 ⫾ 1.1
14.1 ⫾ 1.3
6.90 ⫾ 1.2
11.6 ⫾ 4.7
10.3 ⫾ 4.2
12.0 ⫾ 4.7
NOD
NOR
B6
3.70 ⫾ 1.8
3.50 ⫾ 1.9
3.50 ⫾ 2.1
9.30 ⫾ 1.2
8.80 ⫾ 1.2
6.20 ⫾ 1.1
4.00 ⫾ 1.5
3.80 ⫾ 1.8
4.40 ⫾ 1.5
NOD
NOR
B6
93.5 ⫾ 1.8
92.9 ⫾ 6.1
94.1 ⫾ 1.3
54.5 ⫾ 4.3
55.1 ⫾ 3.6
65.8 ⫾ 0.9
82.6 ⫾ 4.6
80.5 ⫾ 5.2
83.9 ⫾ 2.7
NOD
NOR
B6
4.60 ⫾ 1.4
3.30 ⫾ 0.3
3.70 ⫾ 0.7
39.0 ⫾ 5.1
38.4 ⫾ 4.9
29.0 ⫾ 1.1
14.0 ⫾ 4.8
15.8 ⫾ 3.9
10.9 ⫾ 3.7
NOD
NOR
B6
2.60 ⫾ 0.9
3.60 ⫾ 1.7
2.30 ⫾ 1.7
6.30 ⫾ 0.8
6.10 ⫾ 0.6
4.30 ⫾ 0.9
3.80 ⫾ 1.2
3.60 ⫾ 1.4
5.80 ⫾ 2.4
Anti-IgM
G0/G1
S
G2/M
Anti-IgM ⫹ IL-4
G0/G1
S
G2/M
Data are means ⫾ SD and represent the results from four independent and reproducible experiments. *P ⬍ 0.05, NOD and NOR vs. B6 at
48 h of anti-IgM F(ab⬘)2 ⫾ IL-4 stimulation; differences existed at all stages of the cell cycle. †P ⬍ 0.05, 48 h of anti-IgM F(ab⬘)2 vs. anti-IgM
F(ab⬘)2 ⫹ IL-4 stimulation.
ous reports that hyperproliferation and resistance to AICD
after BCR stimulation are characteristics of self-reactive
B-cells (14,18). Moreover, the presence of autoantibodies
against islet ␤-cell autoantigens in the sera of NOD and
NOR mice indicates that self-reactive B-cells are indeed
present in these mice (25).
The differences we observed in the proliferative responses of NOD, NOR, and B6 B-cells correlate directly
with the detection of an increased proportion of BCRactivated NOD and NOR B-cells that enter into the S phase
of the cell cycle. It is notable that the percentage of NOD
and NOR B-cells in the S phase exceeded that of B6
B-cells. These results, coupled with our finding that stimulation with IL-4 ⫹ anti-IgM F(ab⬘)2 enhances the proliferation of both NOD and NOR spleen B-cells, demonstrate
that IL-4 can prime B-cells to enter the S phase and
undergo cell division in a type 1 diabetes⫺susceptible and
⫺resistant environment. This finding is perhaps not surprising, as similar data were previously reported for resting B-cells from nonautoimmune disease⫺prone mice
(30,31). Nonetheless, the observed hyperproliferation of
NOD and NOR B-cells might be due to the increased
percentage of NOD and NOR B-cells that enter the S phase
after stimulation by anti-IgM F(ab⬘)2 ⫾ IL-4. NOD and NOR
B-cell hyperproliferation potentiated by anti-IgM F(ab⬘)2 ⫾
IL-4 stimulation is also consistent with an increased
percentage of NOD and NOR B-cells that undergo cell
division.
It is interesting to note that we observed that ⬃48% (Fig.
2B) of the islet-infiltrated cells in 10-week-old NOD mice
were B-cells and that ⬃25% (Fig. 2C) of these B-cells
2008
displayed an activated phenotype (CD69⫹) and a hyperproliferative response upon BCR stimulation by anti-IgM
F(ab⬘)2. This hyperproliferation phenotype of PLN-derived
and islet-infiltrated B-cells relative to spleen B-cells in
NOD mice suggests that B-cells may be activated in the
regional draining lymph node before their migration to the
site of inflammation (i.e., pancreatic islets).
B-cell hyperresponsiveness to BCR stimulation of type 1
diabetes⫺susceptible NOD and type 1 diabetes⫺resistant
NOD.B6Idd4B and NOR mice is consistent with the presence of an increased percentage of CD69⫹ B-cells in the
spleen and islet infiltrates of these mice. The presence of
an increased percentage of CD69⫹ B-cells in the PLN of
NOD mice may establish a link between a B-cell activation
phenotype and the NOD MHC haplotype (11). Detection of
IAAs in the sera of NOD and NOR mice at age 8 –20 weeks
(25) also supports the notion that NOD and NOR B-cells
are functionally similar. Thus, B-cells from NOD and NOR
mice appear to share both phenotypic and functional
properties. Accordingly, resistance to type 1 diabetes in
NOR mice may be controlled by genes in the 11.6% of the
C57BL/KsJ genome expressed by NOR but not NOD mice
(24).
Our finding that activated B-cells are present in inflamed
islets of NOD mice during the development of invasive
insulitis is consistent with the recent report that autoreactive B-cells are impaired in their ability to enter follicles
and form germinal centers in lymphoid tissues (32,33).
Rather, autoreactive B-cells seem to migrate into extrafollicular T-cell zones, where the persistent expression of
autoantigen may lead to the activation and expansion of
DIABETES, VOL. 53, AUGUST 2004
S. HUSSAIN, K.V. SALOJIN, AND T.L. DELOVITCH
FIG. 4. NOD and NOR B-cells are resistant to BCR-induced AICD. NOD, NOR, and B6 B-cells were stimulated for 48 h with anti-IgM F(abⴕ)2 (5
␮g/ml) (AⴚC) or anti-IgM F(abⴕ)2 (5 ␮g/ml) ⴞ IL-4 (10 ng/ml) (D). The percent apoptosis was determined by FITCⴚannexin V/propidium iodide
staining (A), annexin V binding (B and D), or TUNEL assay (C). A: Results from four independent and reproducible experiments are presented.
*P < 0.05. B: Annexin V binding was determined on activated B-cells blasts gated as R1. FSC, forward scatter; SSC, side scatter. C: The areas
representative of viable and apoptotic cells as well the percentage and mean fluorescence intensity (MFI) of apoptotic cells are shown. Data in
B and C are representative of three independent and reproducible experiments. D: Annexin V binding was determined on all cells. Results from
four independent experiments are presented. *P < 0.05 for anti-IgM vs. anti-IgM ⴙ IL-4 stimulation; **P < 0.05, NOD and NOR vs. B6 mice.
autoreactive B-cells (32,33). Persistent expression of an
autoantigen(s) and the infiltration of T-cells into the PLN
and islets may provide the antigen- and T-cell⫺derived
signals that stimulate the hyperproliferation and increased
number of cell divisions of NOD B-cells observed here.
The BCR-induced hyperproliferation of NOD and NOR
B-cells observed is characteristic of self-reactive B-cells
that are normally deleted from the periphery by AICD
(14 –17). Thus, the BCR-stimulated hyperresponsiveness of
NOD B-cells may result from their inability to reach the
threshold of activation required to undergo apoptosis. In
the absence of apoptosis-mediated feedback mechanisms
that control and limit excessive expansion of potentially
DIABETES, VOL. 53, AUGUST 2004
autoreactive B-cells, these B-cells may drive the ongoing
autoimmune process in NOD islets by providing costimulation to autoreactive T-cells. We (34) and others (35)
have shown that NOD T-cells are resistant to T-cell
receptor (TCR)-induced AICD. Our current results show
that in addition to T-cells, B-cells from NOD and NOR mice
are also resistant to AICD. It is important to note that
although NOD T-cells are hyporesponsive to TCR stimulation, NOD and NOR B-cells are hyperresponsive to BCR
stimulation. A previous report that NOD T- and B-cells are
both resistant to cyclophosphamide-induced apoptosis
supports these findings (35). T-cell interaction with B-cells
that present cognate self or foreign peptides via MHC class
2009
B-CELL HYPERACTIVATION IN NOD MICE
FIG. 5. Spleen B-cells express low levels of Fas, but do not express
FasL. Fas and FasL expression were examined by flow cytometry of
stained NOD, NOR, and B6 B-cells that were unstimulated (UNST) or
stimulated (48 h) by anti-IgM F(abⴕ)2 (5 ␮g/ml). The percentages of
Fasⴙ and FasLⴙ cells ⴞ SD from three experiments are shown.
II molecules results in the activation of both cell types
(36). Such activated T- and B-cells express Fas and FasL,
and reciprocally receive an apoptotic signal unless an
additional survival signal such as IL-4 is provided (37,38).
In support of this notion, we found that exposure of B-cells
to exogenous IL-4 enhanced the B-cell proliferative response and reduced the frequency of B-cells that underwent BCR-induced apoptosis. The presence of increased
insulitis and a higher B-cell number in the islets and
spleens of rIL-4 –treated NOD mice also suggests a role for
IL-4 in B-cell survival and insulitis (39).
The resistance to apoptosis of NOD and NOR B-cells
compared with B6 B-cells observed in our study was not
due to the differential expression of Fas and FasL between
insulitis-prone (NOD and NOR) and insulitis-free B6 Bcells, as a similar level of Fas expression was observed on
B-cells from the three strains tested. Furthermore, Fas-toFasL interaction was not feasible in our in vitro study due
to a lack of FasL expression on B-cells, even after anti-IgM
F(ab⬘)2 stimulation (Fig. 5). Previous reports (40,41) indicating an absence of FasL expression by B-cells both
constitutively and after anti-IgM F(ab⬘)2 stimulation also
support our data.
In conclusion, a B-cell hyperresponsiveness and resistance to AICD observed in spleen B-cells from NOD, NOR,
and NOD.B6Idd4B mice is associated with the development of an inflammatory environment, but not type 1
diabetes.
ACKNOWLEDGMENTS
This work was supported by grants from the Canadian
Diabetes Association (T.L.D.) and the Ontario Research
and Development Challenge Fund (T.L.D.). S.H. is the
recipient of a Canadian Diabetes Association postdoctoral
fellowship in honor of the late Flora I. Nichol. T.L.D. is the
Sheldon H. Weinstein Professor in Diabetes.
We thank all members of our laboratory for their advice
and encouragement.
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