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Department of Medicine, Division of Rheumatology and Immunology, Vanderbilt University, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In some circumstances, self Ag is found to have no measurable impact on the development and function of autoreactive B cells. Low levels of lysozyme (1 ng/ml) in high affinity anti-HEL transgenic mice and soluble H-2Kb in anti-H-2Kb transgenics failed to show any change in B cell development or differentiation (14, 15). These observations led to the conclusion that at low concentrations of Ag, the usual mechanisms that maintain B cell tolerance are ineffective, a state referred to as clonal ignorance (16). Immune responses to many self Ags present in low concentrations, such as growth factors, cytokines, or hormones, are often attributed to clonal ignorance. The high frequency of organ-specific autoimmune disorders associated with immune responses to such Ags suggests that clonal ignorance may provide a reservoir for expansion of autoreactivity in pathological states. Therefore, understanding the fate and function of clonally ignorant B cells is an important first step in discerning their contribution to autoimmune disease.
The immune response to insulin is an ideal tool for investigating
clonally ignorant repertoires. The small molecule (5800 Da) is produced
by the pancreatic 
cells of all mammals, and it circulates
postprandially in low concentrations as part of the physiological
response to glucose. Abs that bind autologous hormone are routinely
produced after administration of the hormone in the treatment of
diabetes mellitus, and spontaneous Abs are detected following viral
infections, hypersensitivity states, and in the prodrome of autoimmune
endocrine disease (17, 18, 19, 20, 21). To directly investigate the
nature of tolerance in the B cell repertoire for insulin, we introduced
the V region genes from mAb125 into the germline of C57BL/6 mice as an
IgMa transgene (125Tg). IgG mAb125 is derived from the primary response
of a BALB/c mouse immunized with human insulin and uses a mutated
VH-IX gene (22, 23). mAb125 binds
human insulin with an affinity of 3 x 10-8
L/M, and it binds autologous rodent insulin at
10-7 L/M (24). Based on the
HEL/anti-HEL model system (10-9 L/M), the
peak and basal levels of endogenous insulin (25) and the
affinity of mAb125 are anticipated to generate B cells whose receptor
affinities reside well within a range of clonal ignorance. In contrast
to this expectation, most B cells from 125Tg mice are effectively
silenced by endogenous insulin, as demonstrated by adoptive transfer
and immunization with heterologous insulin and proinsulin. Although the
majority of B cells in 125Tg mice are profoundly anergic to Ag
challenge, some insulin Ab is autonomously produced in the absence of T
cells or immunization as part of the natural autoantibody repertoire of
125Tg mice. Circulating insulin Abs in 125Tg mice are effectively
depleted by hypotonic peritoneal lysis, suggesting that some B cells
may have escaped tolerance by entering a B1-like compartment. Overall,
the data indicate that for conventional B cells induction of tolerance
may occur at intermittent low levels of physiological autoantigen, and
continuous Ag production is not required to maintain the anergic state.
These data do not support clonal ignorance as a central mechanism for
insulin autoimmunity. We conclude that regulation within B cell
compartments, such as the germinal centers from which mAb125 arose and
the peritoneal cavity that supports constitutive insulin Ab production
in 125Tg mice, is responsible for maintaining self-reactivity to
insulin in the repertoire.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Conventional C57BL/6 mice used in these studies were obtained
from The Jackson Laboratory (Bar Harbor, ME). The
VH10-µ heavy chain and V
10-C light chain
constructs previously used to express anti-HEL transgenes in the
pSVG-gpt vector were the gift of C. C. Goodnow (John Curtin School
of Medical Research, Canberra City, Australia) (26). The
VH and V regions derived from the HyHEL10 mAb
were removed and replaced with the 5' untranslated and L-VDJ or L-VJ
DNA segments cloned from anti-insulin mAb125. The nucleotide
sequences of the engineered 125VH and 125V
chains were confirmed and the constructs tested for expression by in
vitro transfection of B cells lines, as previously described
(23). For microinjection, gene constructs were linearized
with SalI and purified chromatographically. To avoid the
possible complications of anti-insulin Abs on gestation,
VH125-µ heavy chain and V125-C founder
mice were initially generated individually from microinjection of
B6 x DBA2 single cell oocytes (27). Founders were
originally identified by Southern blot hybridization using cDNA probes
on tail DNA digested with BglII and Pst for 125VH and with
HindIII for 125V genes. Based on hybridization signals
relative to endogenous V genes, the number of transgene copies is
estimated to be low (<3). Founder mice were backcrossed to C57BL/6
mice, and subsequent progeny were monitored for transgene transmission
using PCR to identify VH and V transgenes or
by flow cytometry to identify IgMa+ B cells. Mice
were intercrossed to obtain offspring that express both
125VH and 125V transgenes, 125Tg. 125Tg mice
were subsequently found to mate and reproduce normally. All animals
used in the current study were backcrossed >10 times to C57BL/6.
mAbs and flow cytometry
mAbs used in this study include: RS-3.1 (IgMa), AF6 (IgMb), 6B2 (B220), 57-3 (CD5), and M1/70 (Mac1) (BD PharMingen, San Diego, CA). B220 and isotype-specific reagents were biotin modified, and detection was with FITC avidin (Boehringer Mannheim, Indianapolis, IN) or PE streptavidin (Sigma, St. Louis, MO). FITC-labeled goat anti-mouse IgG was used for indirect detection of binding with some reagents (Cappel Laboratories, Cochranville, PA). Anti-insulin mAb123 was biotinylated on an insulin column to protect the binding site, as previously described (28). Pork insulin was biotinylated at its single lysine residue on the insulin B chain using normal human serum biotin (Pierce, Rockford, IL). Binding of insulin to the cell surface was detected using PE or FITC avidin. For flow cytometry, single-cell suspensions of spleens were prepared by lysis of RBC in NH4Cl and were stained with the indicated Ab for 1 h at 4°C. For two-color analysis, cells were incubated sequentially with biotinylated reagents, followed by either PE or FITC conjugates. FACScan fluorescence data (Becton Dickinson, San Diego, CA) were displayed in two-dimensional dot plots.
Ab determinations
ELISA for detection of anti-insulin Abs were done using 96-well Immulon II plates (Dynatech Laboratories, Alexandria, VA) coated with biosynthetic human or pork insulin (Lilly, Indianapolis, IN) (1 µg/ml in borate-buffered saline, pH 8). Sera were diluted (1/100–1/1000) in PBS, pH 7.4, containing 0.5% Tween-20, and binding was measured at OD405 using an alkaline phosphatase-conjugated second Ab and para-nitrophenyl phosphate (Sigma) as substrate (17). For insulin-specific binding, results of binding to insulin-coated plates were reported as inhibitable OD405 U in the presence of 50 µg/ml of insulin. For detection of IgM allotypes in serum, microtiter wells were coated with goat anti-mouse sera (Cappel), and biotinylated isotype-specific mAbs were used to measure capture of IgMa and IgMb by OD405. Control reagents for these studies include normal C57BL/6 and BALB/c sera, insulin-specific IgMa mAb301 (29), and polyclonal IgM anti-insulin obtained from B6 or BALB/c mice immunized with Brucella insulin (30).
Immunization and adoptive transfer
To provide maximum insulin-specific T cell help in H-2b mice, animals were immunized i.p. with 0.1 cc of CFA containing 50 µg/ml of a mixture of beef insulin and pork proinsulin. Preliminary studies using trinitrophenyl-keyhole limpet hemocyanin-primed B cells and beef insulin-primed T cells were conducted to determine optimal kinetics for insulin-specific help. For adoptive transfer, B cells were purified from B6 or 125Tg mice by magnetic bead depletion of non-B cells (MACS; Miltenyi Biotec, Auburn, CA), and 2 x 107 B cells were transferred i.v. into irradiated (650 rad) recipients. Immune T cells were obtained from nontransgenic B6 mice by negative selection (MACS), and 107 T cells were used per recipient. The purity of these B and T cell populations as assessed by flow cytometry was >90%. After cell transfer, recipients were boosted with the same insulins in CFA. In some experiment cells, animals received a second injection of Ags in immunofluorescence assay. Insulin Ab in sera was measured by ELISA before and following immunization.
Glucose tolerance testing and pancreatic histology
To examine cell function mice, a glucose tolerance test was
performed after removing food for an overnight fast. Animals were
administered i.p. glucose (1.8 g/kg) in PBS and blood sugars at 20- to
40-min intervals. Blood sugars were measured by the glucose oxidase
method with an Elite Glucometer (Bayer, Elkhart, IN). For pancreatic
histology, Formalin-fixed pancreata were embedded in paraffin, and
10–12 nonsequential sections were obtained from each gland. Slides
were stained with hematoxylin and eosin and examined by light
microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the role of clonal ignorance in Ab responses to a
physiological autoantigen expressed at low levels, transgenic mice that
express the V regions of anti-insulin mAb125 were produced, as
described in Materials and Methods. The expression of
transgenic Ig was first examined using B220 and IgM allotype-specific
mAbs (Fig. 1
). Mice that express only the
heavy chain V region demonstrate high level of IgMa expression and
effectively exclude >95% of endogenous IgMb on the surface of
B220+ spleen cells (A and
B). Highly efficient allelic exclusion is also observed in
mice when the VH125 IgMa heavy chain is expressed
with its V partner from mAb125 (125Tg mice). Effective allelic
exclusion in these mice is maintained for long periods (>40 wk). The
finding that nearly normal numbers of B cells are present in 125Tg mice
indicates that clonal deletion has not taken place. However, 125Tg mice
(VH + V) routinely demonstrate a 10–20%
reduction in mean fluorescent intensity of surface of IgMa compared
with heavy chain-only transgenics (D). These findings
suggest that acquisition of insulin binding through pairing of the IgMa
heavy chain transgene with its V125 partner may result in a modest
down-regulation of surface BCR expression.
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To examine Ag binding by the BCR in mice that express
anti-insulin transgenes, FITC avidin and biotinylated insulin were
used to examine interactions on the surface of 125Tg B cells. Using a
range of insulin concentrations between 0.1 and 1000 ng/ml, spleen
cells from normal mice have no detectable insulin-binding
B220+ B cells above that of background on avidin
FITC alone (Fig. 2A). In
addition, insulin-binding B cells are not detected in mice that express
only the heavy chain transgene (identical with A, not
shown). In contrast, essentially all B cells from mice that express H
and L anti-insulin transgenes (125Tg mice) bind insulin (Fig. 2, B–H). The concentration of insulin that occupies 
50% of
BCR (5 ng or 1 µM) is outside the physiological range. However, at nM
concentrations of hormone, 5–15% of 125Tg B cells are observed to
bind insulin (B and C). These concentrations are
close to physiological levels of insulin in the postprandial period,
and suggest that endogenous insulin may engage the Ag receptors on
125Tg mice.
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To determine whether anti-insulin B cells encounter and bind
endogenous insulin under physiological conditions, a biotinylated
second anti-insulin mAb (mAb123) was used to detect insulin
epitopes on the surface of B lymphocytes. mAb123 is structurally
different from mAb125, and the binding sites of the two do not overlap
(22, 24). Therefore, we used avidin FITC and biotinylated
mAb123 to determine whether insulin epitopes are displayed on the
surface of 125Tg and nontransgenic B cells. In these studies, mice
received only normal laboratory chow, and manipulation of carbohydrate
loads to increase endogenous insulin production was not necessary. As
shown in Fig. 3, B cells from normal B6
mice (A) are not bound by mAb123. Furthermore, no binding of
mAb123 is observed in B cells from mice expressing only the heavy chain
transgene (identical with A, not shown). In contrast, a
large majority of B cells from 125Tg mice are bound by mAb123 (Fig. 3B). All binding of mAb123 to 125Tg B cells is inhibited by
excess soluble insulin (50 µg/ml). These findings indicate that most
BCR in 125Tg mice are occupied by insulin as a consequence of exposure
to hormone under physiological conditions. As discussed below, the
function of insulin as a molten globule may contribute to its
persistent association with 125Tg B cells, despite low affinity for
rodent insulin (10-7 L/M) and limited Ag
exposure.
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The data shown above suggest that anti-insulin B cells are not
ignorant of endogenous insulin, but they encounter hormone and may be
subject to functional silencing. Accordingly, relative binding in ELISA
was used to measure circulating IgM allotypes, and competitive
inhibition was used to detect insulin-specific Abs. The results show
that in contrast to the effective allelic exclusion seen in B cells
from 125Tg mice, endogenous IgMb accounts for greater than 98% of
circulating IgM (Fig. 4A). The
relative amount of IgMb is similar to that found in C57BL/6 mice (Fig. 4B). Although the preponderance of circulating IgMb suggests
silencing of most transgenic B cells, some insulin-specific Ab of the
IgMa allotype is detected (Fig. 5A). Insulin-specific binding
is never observed in nontransgenic B6 mice (Fig. 5B). To
further test the function of transgenic B cells, 125Tg mice were
immunized with combinations of beef insulin and pork proinsulin that
are known to provide optimal T cell help for anti-insulin responses
in H-2b mice (31, 32, 33). The results
show that in contrast to normal B6 mice (square), 125Tg mice (circles)
fail to increase anti-insulin levels following immunization with a
combination of beef insulin and pork proinsulin in adjuvant (Fig. 6A). Further evidence for the
anergic state of 125Tg B cells is observed in adoptive transfer
experiments using 125Tg B cells and insulin-immune T cells, followed by
boosting with Ag (Fig. 6B). In these studies, transfer of
125Tg B cells alone results in the development of measurable levels of
insulin Abs (closed circles). When immunized and boosted (arrows) in
the presence of insulin-immune T cells (open circles), Ab levels do not
exceed those reached in the absence of T cell help. The slight
differences in Ab levels seen at early time points (day 5) are also
observed in the absence of immunization and appear to reflect
reconstitution differences and not Ag-specific response (unpublished
observations). When naive B cells and insulin-primed T cells are
transferred, a strong anti-insulin response develops after a single
immunization (filled square) even though the precursor frequency of
anti-insulin B cells in this population is <10-5.
Transfer of naive B cells alone (open square) does not yield measurable
insulin-specific binding. Together, these findings indicate that large
numbers of 125Tg B cells are functionally silenced by physiological
concentrations of insulin. However, the presence of circulating insulin
Abs of transgene origin (IgMa) indicates that some transgenic B cells
may differentiate and produce insulin Abs as part of an endogenous
repertoire of natural autoantibodies.
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Autoantibodies present in the natural repertoire are often
attributed to the B1 subset of B lymphocytes that can replicate
autonomously in peritoneal and pleural spaces (34). The
immunization-independent production of insulin Abs that we observe in
anti-insulin transgenics is characteristic of B1 type B cells. In a
transgenic model of autoimmune hemolytic anemia, depletion of B1 B
cells by distilled water lysis abolished autoantibody production and
disease (12, 35). Therefore, to determine whether
peritoneal B cells contribute to insulin autoantibody production in
125Tg mice, 125Tg mice were treated with i.p. injection of sterile
water or PBS as a control (Fig. 7).
Injection of the mice with PBS did not decrease the levels of IgMa
anti-insulin, while hypotonic treatment anticipated to lyse
peritoneal B cells resulted in a sharp and sustained decline in insulin
binding. Examination of the peritoneal B cell population shows only
small numbers (3%) of CD5+ or
Mac1+ B cells in 125Tg mice compared with
nontransgenic mice (30%). The majority of this small population is
eliminated with the peritoneal injections (data not shown). Although
125Tg mice do not have an expanded population of B1 B cells, the
findings are consistent with the interpretation that most circulating
insulin Ab in 125Tg mice arise from a B1-like population, and suggest
that B2 or conventional B cells are functionally silenced.
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Several autoimmune syndromes are associated with insulin Abs, and
older studies suggest that passive transfer of insulin Abs may induce
insulitis. To determine whether circulating insulin Abs have
pathological effects on transgenic mice, we initially examined the
pancreata of 125Tg mice for the presence of insulitis. None of a dozen
mice ages 12–24 wk of age showed evidence of cellular infiltrates in
the islets of Langerhans or in pancreatic tissue. In addition, random
blood sugar determinations in larger number of animals have not shown
hyperglycemia. To specifically examine cell function, we measured
fasting blood sugars and blood sugars 90 min following a glucose load
(Fig. 8). The results suggest that these
Abs may influence carbohydrate metabolism, as glucose levels after an
overnight fast were significantly reduced in the 125Tg mice compared
with control B6 mice. However, the cell response to glucose
challenge in 125Tg mice was not different from that of normal B6 mice.
These data indicate that a large population of insulin-binding B cells
and circulating insulin Abs in the repertoire of 125Tg mice do not
alter cell function. However, insulin Abs in 125Tg mice can
interfere with the delivery of insulin to the periphery and result in
lower blood sugars in fasting 125Tg mice. These findings are consistent
with a phenotype of hypoglycemia and insulin autoimmunity recognized
clinically as the insulin autoimmune syndrome (20).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cell
destruction or insulitis. These findings indicate that self-reactivity
to self insulin does not emerge unimpeded from the preimmune B cell
repertoire. Rather, unique functions of microenvironments, such as the
peritoneal cavity that may foster natural autoantibodies and immune
follicles from which this anti-insulin specificity originated,
maintain immune reactivity to insulin in the repertoire.
The transgene BCR used in this study (125Tg) expresses
VH and V from mAb125 that binds human insulin
with an affinity of 10-8 L/M and rodent insulin
at 10-7 L/M. In the anti-HEL system, when
the circulating concentration of the facultative autoantigen (HEL) was
comparable with a physiological hormone (1 ng/ml), B cells were
clonally ignorant and responded to immunization, even when the BCR
affinity was high (>10-9 L/M)
(39). Thus, our anti-insulin transgene falls well
below the affinity range expected for clonal ignorance. However, the
bulk of 125Tg B cells are functionally silenced or anergic, as defined
by their inability to differentiate and secrete Ab. The features of
anergy include: preponderance of circulating IgMb derived from a small
percentage (<5%) of residual endogenous B cells, failure to increase
insulin Ab following immunization of 125Tg mice with heterologous
insulin and proinsulin, and failure of insulin-primed T cells to induce
responses in adoptive transfer with anti-insulin 125Tg B cells. In
contrast to other models in which autoantibody transgenes are derived
from animals with autoimmune disease, the anti-insulin specificity
use by 125Tg mice is derived from a normal BALB/c mouse
(23). Thus, an autoimmune specificity for insulin that is
permitted in the immune repertoire of an adult mouse is effectively
silenced when introduced into the germline. These observations question
the concept of clonal ignorance and suggest that tolerance for insulin
is effective in the preimmune repertoire at ranges of receptor affinity
far below that in other transgenic models. This raises the issue of why
the specificity of mAb125 was not silenced in the immune response from
which it was generated. One potential mechanism may reside in the
selection process that is part of germinal center reactions in T
cell-dependent immune responses. Our previous studies on the origins of
mAb125 suggest that it may arise from precursors that are not overtly
insulin specific (23). Thus, B cell selection in follicles
may be initiated with BCRs that somatically evolve to include insulin
binding, and self-reactivity is permitted because of the unique
properties (costimulation, cytokines, cell interactions, etc.) of the
germinal center microenvironment. In the future, experiments using mice
that express transgenes for the germline precursors of mAb125 may help
resolve this issue.
The effective maintenance anergy in 125Tg mice correlates with the
unexpectedly high level of BCR occupancy by endogenous insulin (Fig. 3
). Sustained BCR occupancy occurs even though physiological exposure
to insulin is not continuous and is limited to the postprandial period.
The efficiency of tolerance induction to this natural Ag further
contrasts other transgenic autoantigen systems (HEL, dsDNA, and RBC) in
which Ag is present continuously. One possible mechanism is related to
the structure of insulin as a molten globule that may change its
conformation after receptor binding (40, 41). Accordingly,
we hypothesize that the insulin molecule may reside in a more stable
configuration once bound by the BCR. The induced fit between insulin
and the Tg125 BCR may sustain a slow off rate and favor maintenance of
tolerance especially when these interactions occur in the preimmune
repertoire. Recent studies on the function of B cells expressing a
rheumatoid factor transgene also indicate that features of BCR
interaction with Ag other than affinity for ligand may determine the
threshold for induction of tolerance (42).
Not all anti-insulin B cells are functionally silenced in 125Tg mice. Although greater than 98% of the circulating IgM detected in these transgenics is from the endogenous IgMb allotype, every transgenic mouse tested had measurable anti-insulin Abs of the IgMa allotype. Insulin-specific binding is not detected in normal B6 mice, in heavy chain-only transgenic mice, nor in the endogenous IgMb in transgenics. Although some anti-insulin IgMa is present, immunization does not increase these anti-insulin levels in 125Tg mice or in immunized recipients following adoptive transfers of 125Tg B cells with nontransgenic insulin-immune T cells. In adoptive transfer experiments, recipients of 125Tg B cells alone produce circulating insulin Ab independent of immune T cells. This self-regenerating capacity of B cells that produce insulin Ab suggests a relationship to the B1 B cell subset (34). Using the approach that was successful in anti-RBC transgenic mice (12, 35), we find that i.p. lysis reduces the level of insulin Ab in 125Tg mice. This finding is consistent with a B1-like origin for those anti-insulin B cells that escape silencing. The anti-insulin transgenes are not preferentially expanded in the B1 subset. Specifically, we do not observe increases in CD5+ B cells in spleen compared with B6 mice. In the peritoneum, the numbers of CD5+ and Mac1+ B cells are low in our transgenic mice; however, both B1a and B1b B cells in the peritoneum are decreased after sterile water treatment (unpublished observations). These findings suggest that while the bulk of B2 B cells are anergic in 125Tg mice, a residual B1-like population may be responsible for the presence of circulating IgMa Abs detected in most transgenic mice. Recent data on anti-RBC transgenics in germ-free conditions indicate that environment signals may help maintain autoantibody expression in this compartment (13). Thus, physiological exposure to endogenous insulin in the context of an environment that favors B1-like differentiation pathways may permit some 125Tg B cells to contribute to the natural autoantibody repertoire. Anti-insulin B cells that have structural characteristics of a natural autoantibody are reported in human insulin-dependent diabetes mellitus, suggesting that similar B cells may contribute to an autoimmune process (43). Studies are in progress to determine whether differences in compartmental silencing of B cells in normal and in insulin-dependent diabetes mellitus-prone mice alter the fate and function of 125Tg B cells in autoimmune diabetes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James W. Thomas, Department of Medicine, T-3219 Medical Center North, Vanderbilt University, Nashville, TN 37232-2681.
3 Abbreviations used in this paper: BCR, B cell receptor; HEL, hen egg lysozyme; 125Tg, mice that express V region genes from mAb125 as an IgMa transgene.
Received for publication October 23, 2000. Accepted for publication December 29, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and transgene (IgMa, 
) Ig
in serum (1/100) are compared in individual 125Tg mice
(A, top) and in normal B6 mice
(B, bottom). Data are reported as binding
in ELISA (OD405) with allotype-specific reagents, as
described in Materials and Methods.
). The
response of nontransgenic B6 mice (n = 4) on day 18
is shown (closed square). B, 125Tg B cells produce
insulin Ab independent of T cell help in adoptive transfer. Irradiated
recipients received 10-7 125Tg B cells in the presence
(
